JP2002531194A - In vivo biosensor device and method of use - Google Patents

Abstract

  (57) [Summary] Disclosed are bioluminescent bioreporter integrated circuit devices that detect a selected analyte in a fluid when implanted in an animal body. The device includes a bioreporter, the bioreporter being genetically engineered to include a nucleic acid segment comprising a cis-activation response element, wherein the cis-activation response element encodes a bioluminescent reporter polypeptide. Responsive to a selected substance that is operatively linked to the gene. In a preferred embodiment, the target analyte is glucose, glucagon, or insulin. By exposing the biosensor to a target substance, a response element upregulates the nucleic acid sequence encoding the reporter polypeptide, producing a luminescent response that is detected and quantified.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is related to US Provisional Application No. 60 / 110,68, filed December 2, 1998.
This is a continuation application claiming priority of No. 4. The entire contents of that application are specifically incorporated herein by reference.

[0002] The United States Government has certain rights in the invention according to Assignment Number R21RR14169-01 from the National Institutes of Health.

1.1 Field of the Invention The present invention generally relates to implantable diagnostic devices (ie, devices located within an animal) that monitor one or more substances, analytes or metabolites of an animal of interest. ) Field. More specifically, the present invention provides an implantable biosensor device for monitoring and controlling levels of analytes in human tissue and the circulatory system. In an exemplary embodiment, the device includes a biosensor used to monitor blood glucose levels in a diabetic or hypoglycemic patient. The disclosed sensors can also be used to control or regulate the delivery of drugs or other pharmaceutical media from external or implantable drug delivery systems. For example, the device may form part of an artificial pancreas that controls insulin dosage in response to glucose levels detected in situ.

(1.2 Description of Related Art) (1.2.1 Biosensor) A biosensor is a hybrid device that combines an analytical measurement element and a biological component. The biological component reacts and / or interacts with the target analyte (s), producing a response that can be measured by an electronic, optical or mechanical transducer. The most common configurations currently available use immobilized macromolecules such as enzymes or antibodies to form biological components. Examples of analytes and immobilized polymers include:
Examples include the following: Glucose and immobilized glucose oxidase (e.g., Wilkins et al., 1995); nitrate and immobilized nitrate reductase (e.g., Wu et al., 1997); hydrogen peroxide, 2.3-dichlorophenoxyacetic acid, and immobilized horseradish peroxidase (e.g., Ru in 1998
btsova et al.); aspartic acid and immobilized L-aspartic acid (1995
Campanella et al.).

[0005] 1.2.2 Whole-Cell Biosensors [0005] In recent years, further improvements have been made to biosensors. It utilizes intact living cells (eg, microbial or eukaryotic cells) or cell cultures that replace immobilized enzymes. Microbial cells are particularly well suited for biosensor technology. For example, microbial cells are physically robust and can exist under very harsh and widely varying environmental conditions. Microbial cells have an extensive repertoire that responds to their environment. And they can be genetically engineered to generate reporter systems that are very sensitive to these environmental responses. A polynucleotide sequence, including a particular promoter sequence, is operably linked to one or more genes encoding the desired reporter enzyme (s), and is then introduced into living cells and into living cells. Is maintained in. When the target analyte is present, the reporter gene is expressed, producing the enzyme (s) resulting from the production of the measured signal. Commonly used reporter systems include β-galactosidase (lacZ) or catechol-2,3-dioxygenase (x
yIE) enzyme (Kricka, 1993).

[0006] Unfortunately, the limitations of these systems are that after the system is exposed to the target substance (s), the cells must be lysed destructively and the enzyme (s) It must be isolated. The lysate is then subsequently supplemented with one or more secondary metabolites to obtain a colorimetric signal proportional to the concentration of enzyme (s) in the solution. As a result, a means for quantifying the concentration of the original target substance is provided.

[0007] The most recent improvement of such a sensor utilizes the green fluorescent protein as a reporter system, with the significant advantage that cells do not require destructive assay techniques to generate colorimetric signals. I have. However, these systems are rather complex and have little advantage for the detection of analytes in situ, since the substrate must first be added to the green fluorescent protein constituent to elicit a light response ( (Prasher, 1995).

1.2.3 In Vivo Sensors The development of an integrated in vivo implantable glucose monitor was first reported by Wilkins and Atanasov (1995). This system uses glucose oxidase immobilized in a microbioreactor. This enzyme catalyzes the oxidation of β-D-glucose by molecular oxygen, resulting in O ₂
Glucose concentration which is proportional to the product of the consumption or of H ₂ O ₂ occurs gluconolactone and hydrogen peroxide. Unfortunately, the presence of glucose oxidase inhibitory molecules in the human bloodstream tends to subtract the proportionality constant, creating insufficient and inaccurate devices to monitor and control accurate glucose (1997). Goug
h et al.). Also, the relatively large size of the device (about 5 x 7 cm) is a limitation,
The device was denied its utility as an implantable device.

Subsequently, a number of smaller needle-type and microdialysis glucose sensors were developed to avoid size limitations (Gough et al., 1997,
Despite the 1997 Selam), the reliability of their glucose oxidase enzyme-based systems limits their overall efficacy and reliability.

[0010] Some unspecified electrochemical sensors have also been studied as potential glucose sensors in vivo (eg, Yao et al., 199, 1994).
Lager et al. For 4 years). However, problems including limited sensitivity, instability, and limited long-term reliability have prevented their widespread use (1995).
Patzer et al.). According to Atanasov et al. (1997), a continuously functioning, implantable glucose biosensor with long-term stability has not yet been achieved.

1.3 Defects of the Prior Art Despite the significant miniaturization of biosensors in the past decades, they are still relatively large and stand out to serve as ideal implantable devices. Current methods using mammalian bioluminescent reporter cells require cell lysis and the addition of exogenous substrates to produce a measurable response. Consequently, these cells cannot serve as continuous online monitoring devices.

[0012] Thus, a small, implantable, monolithic (ie, durable, inexpensive, wireless, and telecommunications drug delivery system that can provide controlled delivery of therapeutic agents such as insulin) There remains a need for the development of bioelectronic monitors (including both biological and electrical components configured on a single substrate layer).

2.0 SUMMARY OF THE INVENTION [0013] The present invention provides these and other unique techniques in the prior art by providing an implantable device and method for detecting and quantifying a particular analyte in an animal's body. Overcome limitations. In particular, the present invention provides devices for the in vivo detection and quantification of microorganisms such as metabolites, drugs, hormones, toxins, or viruses in humans or animals. In an exemplary embodiment, the invention provides a BBIC device useful for detecting glucose in a human. Such devices initially provide an accurate on-line detector for glucose monitoring and provide the ability to control the administration of a pharmaceutical agent via an external or implantable drug delivery system. Also disclosed are BBIC devices for detecting the concentration of signature molecules (ie, proteins released from cancer cells, etc.), clotting factors, enzymes, etc., as well as other analytes present in the bloodstream or interstitial fluid. Is done. In the field of oncology, biosensor devices find utility in monitoring both early and remission, online measurement of chemotherapy efficacy, and stimulation / activity of the immune system. Similarly, biosensor devices are useful in other fields of medicine, including online monitoring for enzymes associated with blood clot appearance (stroke, heart attack, etc.), detection and quantification of coagulation factors (maintenance) level),
Hormone replacement, continuous drug monitoring (tests for controlled substances in defendants, military personnel, etc.), monitoring of military personnel exposed to sublethal exposure to nerve agents and other debilitating drugs, effects on mental illness And the monitor level of the compound.

In one embodiment, an implantable monolithic bioelectronic device for detecting an analyte in an animal body is provided. In a general sense, the device comprises a bioreporter operably positioned on a material present on the integrated circuit. The bioreporter can metabolize the target analyte and, when contacted with the analyte, emits light following the metabolite. The device further comprises a sensor closely located to the integrated circuit that detects the emitted light, and generates an electrical signal in proportion to the amount of light generated by the bioreporter. Preferably, the entire implantable device is contained in a biocompatible container, which is implanted in the animal body where detection of the analyte is desired.

[0015] The biocompatible container may be composed of silicon nitride, silicon oxide, or a suitable polymeric matrix, with exemplary materials such as polyvinyl alcohol, poly L-lysine, and arginine being particularly preferred. The polymeric matrix can also further include a porous hydrogel, a mesh-reinforced hydrogel, or a filter-supported hydrogel.

In certain embodiments, it may also be desirable to provide a transparent, biocompatible, bio-resistant separator, wherein the separator is operably disposed between the light converter and the bioreporter.

The bioreporter preferably provides a plurality of eukaryotic or prokaryotic cells, which produce a bioluminescent reporter polypeptide in response to the presence of the target analyte. Prokaryotic cells, such as one or more bacterial strains, and eukaryotic cells, such as mammalian cells, are particularly preferred. An exemplary mammalian cell is pancreatic islet β
Human cells, such as cells, immortalized stem cells, or liver cells, with immortalized stem cells being particularly preferred.

These cells preferably contain one or more nucleic acid segments, which encode luciferase polypeptides or green fluorescent proteins produced by the cells in response to the presence of the analyte. Preferably, the nucleic acid segment is an Aqueorea Victoria, Renilla renifo
rmis, or humanized green fluorescent protein, or more preferably, bacterial L
ux polypeptide (eg, LuxA polypeptide, LuxB polypeptide, L
uxC polypeptide, LuxD polypeptide, or LuxE polypeptide)
Or LuxAB or L fused to a polypeptide described herein.
uxCDE code.

Exemplary bacterial lux gene sequences that can be used to prepare the genetic construct include Virio fischerii, or more preferably, Xenor
habdus luminescens luxA gene, luxB gene, l
uxC gene, luxD gene, luxE gene, luxAB gene or l
uxCDE gene.

An exemplary l that can be used for the preparation of the genetic constructs described herein
The ux gene sequence includes the gene sequence disclosed in SEQ ID NO: 1. Exemplary Lux polypeptide sequences are disclosed in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

The Lux polypeptide preferably comprises at least 10 contiguous amino acid sequences from one or more of the polypeptide sequences disclosed in SEQ ID NO: 2 through SEQ ID NO: 6. More preferably, the Lux polypeptide comprises at least 15 contiguous amino acid sequences from one or more of the polypeptides disclosed in SEQ ID NO: 2 through SEQ ID NO: 6, and more preferably still further comprises SEQ ID NO: 2 through SEQ ID NO: 6. At least 20 contiguous amino acid sequences from one or more of the polypeptide sequences disclosed in US Pat.

Such a polypeptide is preferably at least 20 amino acids from SEQ ID NO: 1.
, At least 25, at least 30, at least 35, at least 40, or at least 45 or more contiguous nucleotides.

[0023] Expression of a Lux-encoding nucleic acid segment is preferably regulated by a nucleic acid regulatory sequence operably linked to the Lux-encoding segment. Preferably, the regulatory sequence includes a cis-acting element that responds to the presence of the target analyte. Exemplary cis-acting response elements are selected from the group consisting of: S14 gene sequence, liver L-pyruvate kinase gene sequence, liver 6-phosphofructo-2-kinase gene sequence, islet β cell insulin gene sequence, Glomerular stromal transforming growth factor-β gene sequence, and acetyl-Coenzyme A carboxylase gene sequence.

In an exemplary embodiment, the cis-acting response element comprises a contiguous nucleotide sequence from the islet β-cell insulin gene sequence or liver L-pyruvate kinase gene sequence. The expression of the nucleic acid sequence is preferably regulated by a promoter sequence, such as from the L-pyruvate kinase encoding gene described herein.

The device may further comprise a wireless transmitter, an antenna and a source of nutrients capable of maintaining the bioreporter cells. Similarly, a biocompatible container containing a bioreporter can further comprise a membrane that is permeable to the analyte, but not the bioreporter cells themselves. Such a semipermeable membrane allows analytes to flow freely from bodily fluids to the detector device, from which bioreporters to the surrounding tissues or circulatory system of the body into which the device is implanted. Cell migration is restricted.

In one embodiment, the integrated circuit is a complementary metal oxide semiconductor (CMOS) integrated circuit. The integrated circuit may comprise one or more light converters, the light converters themselves comprising one or more light converters.
It can be composed of the above photodiodes. Similarly, the integrated circuit may further comprise a photodiode, a current-to-frequency converter, a digital counter, and / or a transmitter capable of transmitting either digital or analog data.

[0027] The present invention also provides an implantable controlled drug delivery system comprising a BBI
A CBI device and an implantable drug delivery pump, the pump comprising a BBI
C can be operatively controlled and can deliver a drug to the animal's body in response to control by the device. The invention also relates to a method of providing a controlled supply of a drug to a patient in need thereof. The method generally involves implanting a controlled drug delivery system into the patient's body.

The present invention also provides a method of determining the amount of a drug required for a patient in need of the drug (eg, when giving an appropriate amount of insulin to a diabetic patient). The method generally comprises in the body of the diabetic one or more BBICs responsive to either glucose, glucagon, insulin, or another glucose metabolite.
Implanting the device and determining the amount of insulin required by the patient based on the level of analyte detected in the body fluid by the device. If the device indicates that high levels of insulin are needed, an appropriate control signal can be sent to the drug delivery system, and additional insulin is injected into the body. If the device indicates that low levels of insulin are needed, then an appropriate control signal can be sent to the drug delivery system and less insulin can be administered. Such "real-time" monitoring of glucose in the animal's body allows for controlled release of insulin throughout the day and daily or more of insulin, which may be either too much or too little at a particular time of administration. Eliminates the need for frequent injections. This gives a more cost effective administration of the drug, and also "on demand (
Provide more stable dosing of insulin to the patient based on "as needed" criteria.

The present invention also provides kits for the detection of an analyte, and such kits generally comprise one or more of the disclosed one or more in combination with appropriate instructions for using the detection device. BBIC device. Such kits may also routinely comprise one or more standard reference solutions for the construction of the device, and also, during either storage or single use implanted into the animal's body, Appropriate storage or nutrient media may be provided to maintain bioreporter cells. In the case of therapeutic kits, such kits also generally include one or more controlled delivery systems for administration of the drug to the animal's body.

The present invention also provides a method of regulating blood glucose levels in an animal in need thereof. The method generally comprises using a BBIC device to monitor the level of glucose in the bloodstream or interstitial fluid of the patient, and an effective amount of insulin composition sufficient to regulate blood glucose levels. Administering the article to the patient.

[0031] This new type of bioluminescence-based bioreporter can monitor target substances without the disadvantageous requirement that cells be disrupted to produce a measurable signal. This, at the same time, allows monitoring to occur online and in real time (Simpson et al., 1998a, 1998b). These cells rely on the luciferase gene for the reporter enzyme system (called lux in prokaryotes and luc in eukaryotes). US Patent Application No. 08 / 978,439 and International Patent Application No. PCT
/ US98 / 25295, each of which is specifically incorporated by reference herein in its entirety, is a self-contained miniature designed to detect specific molecular targets ex vivo or ex vivo. A bioluminescent bioreporter integrated circuit ("BBIC") is disclosed.

The present invention relates to an implantable BBIC device or an in situ or in vivo BBIC device that can be implanted in an animal body and detects the concentration of one or more analytes present in the animal. I can do it. The implantable monolithic bioelectronic device of the present invention generally comprises a substrate, a bioreporter capable of responding to a particular substance by emission of light, a container attached to the substance capable of holding the bioreoporter, a light, An integrated circuit on a substrate that includes a light transducer operable to generate an electrical signal in response to the signal, wherein the signal indicates the concentration of the substance; and a biocompatible implantable body in an animal. A housing, wherein a portion of the housing is converted to a bioreporter container comprising a semi-permeable membrane, which allows the passage of the analyte from the animal body to contact the biosensor. Limit this bioreporter molecule from spreading into the body of the animal, including the implanted device. The bioreporter can be in solution (ie, a cell suspension) and is captured in a container by a semi-permeable membrane, or the bioreporter can be selectively permeable to a polymer matrix, (Which may allow selected substances in solution to reach the bioreporter).
Preferably, the matrix is optically clear.

The device may further comprise a layer of a bioresistant / biocompatible material (eg, a layer of silicon nitride) between the substrate and the container. The integrated circuit is preferably a CMOS integrated circuit, and the light converter is preferably a photodiode.

The integrated circuit may also include a current-to-frequency converter and / or a digital counter. Further, the integrated circuit may also include one or more transmitters. Such a transmitter may be wireless or conventional wired. In a preferred embodiment, the apparatus also comprises a drug delivery device capable of receiving the transmission from the transmitter.

[0035] A further embodiment of the present invention is an implantable device for the detection of a selected substance in a solution. The device comprises: a light transducer adapted to input an electrical signal into the circuit in response to light; a bioreporter capable of reacting to selected substances in solution by radiation; An integrated circuit having a reporter adapted to contact a substance; and a transparent, light-transmitting and bio-reporter disposed between the bio-reporter for striking the light converter with light emitted from the bio-reporter. Biocompatible and bioresistant separator. In a preferred embodiment of the invention, the substance selected is glucose. The bioreporter can be a mammalian cell that contains a nucleotide sequence that encodes one or more luminescent reporter molecules. Such a nucleotide sequence may include one or more lux genes. In a preferred embodiment, the lux gene comprises both the luxCDE gene and the fused luxAB gene. In one embodiment, these lux genes are from Xenorhabdus luminescens. These lux genes can be regulated by nucleic acid sequences that include one or more cis-acting glucose response elements. In an exemplary embodiment, the glucose responsive element can be derived from a β-islet gene or a liver L-pyruvate kinase gene. In a very preferred embodiment, to drive the expression of one or more lux genes, p. LPK. The Luc _FF plasmid is used to provide one or more glucose responsive elements and the L-pyruvate quinase promoter. The cells that make up the bioreporter can be suspended and placed on the IC by a semipermeable membrane.
Alternatively, the cells that make up the bioreporter may be encapsulated in a polymer matrix attached to the IC. Such a matrix can be permeable to the selected substance in solution.

A further embodiment of the present invention relates to an implantable monolithic bioelectronic device for detecting a selected substance in a body fluid. The device generally comprises: a biocompatible housing; a bioreporter capable of responding to a selected substance by radiation; and a sensor capable of generating an electrical signal in response to receiving emitted light. Such a device may also include a transparent, bioresistant and biocompatible separator disposed between the bioreporter and the sensor, as well as a semi-permeable membrane disposed in the biocompatible housing. May be accessible to the bioreporter.

A standard integrated circuit (IC) is coated with a layer of an insulating material (eg, silicon dioxide or silicon nitride). This process involves passivation.
) And serves to protect the surface of the chip from humidity, contamination and mechanical damage. BBIC requires a second coating. This coating must be biocompatible and bioresistant and must be free of OASI from chemical stress.
C must be protected, optically tuned to efficiently transmit light from the material under test, must adhere to the oxide coating, and Must not be, and must be able to be patterned to form openings across the binding pad and any structures that may be required to maintain or collect the sample.

The present invention contemplates that the components of the biosensor can be packaged in kit form. The kit may comprise, in suitable container means, an integrated circuit comprising one or more bioreporters and light converters. The kit may further include a drug delivery device.

The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood with reference to one or more of these figures in combination with the detailed description of the specific embodiments set forth herein. Illustrative embodiments of the present invention are shown in the figures, using the same numbers to reference the same and corresponding parts of the various drawings.

DESCRIPTION OF ILLUSTRATIVE EXAMPLES The luciferase system has been adapted for use in in vivo biosensors. In prokaryotes, the lux system comprises the genes luxA and luxA
Consists of a luciferase composed of two subunits encoded by xB. These genes luxA and luxB oxidize long-chain fatty aldebids to the corresponding fatty acids, resulting in blue-green emission at a wavelength of about 490 nm (Tu and Mager, 1995). The system also comprises a multiprotein fatty acid reductase consisting of three proteins; a reductase encoded by luxC; a transferase encoded by luxD; And synthetase. These genes are contained within one operon, which allows complete cloning of the lux gene cassette downstream from a user-specific promoter to monitor gene expression using bioluminescence. Becomes The majority of bioluminescent bioreporters consist of gram-negative organisms engineered to detect and monitor critical chemical and environmental stressors (Ramanathan et al., 1997).
, Steinberg et al., 1995). Luciferase fusions in Gram-positive bacteria and yeast cell lines have also been performed successfully (Andrew and Ro).
berts, 1993, Srikantha et al., 1996).

The eukaryotic luciferase gene, cloned into a bacterial reporter, contains 5
Includes firefly luciferase (luc) emitting near 60 nm and click beetle luciferase (lucOR) emitting near 595 nm (Cebolla)
Et al., 1995, Hastings, 1996). Eukaryotic bioreporters
Glucose concentration in rat islet β cells (Kennedy et al., 1997);
Steroid activity in HeLa cells (Gagne et al., 1994), the effect of ultraviolet light in mouse fibroblasts (Filatov et al., 1996), the toxic effect in human liver cancer cells (Anderson et al., 1995), and in breast cancer cell lines. Estrogen compounds and antiestrogenic compounds (Demirence)
1995) and erythropoietin gene induction in human liver cancer cell lines (
Gupta and Goldwasser, 1996). To date, most eukaryotic bioluminescent reporters rely on cell destruction and extraneous substrates (usually luciferin) to produce a measurable luminescent response.
Need to be added.

Green fluorescent protein (“GFP”) is also routinely used as a reporter system and has the significant advantage that cells to generate colorimetric signals do not require disruptive assay techniques ( Hanakam et al., 1996; Gryg
orczyk et al., 1996; Siegel and Isacoff, 1997; B
iondi et al., 1998). However, the substrate must first be added to the GFP construct to initiate a light response (Prasher, 1995). Humanized GFP cDNAs have been developed which are particularly adapted for high level expression in mammalian cells, especially cells of human origin (Zolotukhi).
n, 1996). Humanized GFP can be efficiently inserted into mammalian cells using viral vectors (Levy et al., 1996; Gram et al., 1998).

Detection of a bioluminescent signal from a reporter organism can be achieved by using an optical transducer (eg,
This is achieved through the use of photomultipliers, photodiodes, microchannel plates, photographic films, charge-coupled devices, etc. Light collection and transfer to the transducer can be through lenses, fiber optic cables, or liquid light guides. However, for applications requiring small capacity, remote sensing or multiple simultaneous sensing, a new class of instrumentation is needed that is small and portable, yet retains a high level of sensitivity. 4.1 System Overview The present invention describes an implantable BBIC that detects selected substances. A bioreporter is a genetically engineered cell line in which a nucleic acid sequence contains a cis activation response element that responds to a selected agent. In a preferred embodiment, the selected substance is glucose. By exposing the bioreporter to a selected agent, a response element up-regulates a nucleic acid sequence encoding one or more polypeptides that produces a luminescent response. In a preferred embodiment, the luminescence response is
Produced by the prokaryotic lux system.

The function of the IC part of the BBIC is to detect, filter, amplify, digitize and report on the bioluminescence signal. Substantially this I
C functions as a complete on-chip laboratory instrument, a microluminometer.

Silicon-based ICs use PN, which is commonly used to form transistors
The conjugation can be used to detect optical signals in the near ultraviolet, visible, and near infrared regions (
Simpson et al., 1999a). Approximately 66% quantum efficiency was measured at 490 nm bioluminescence wavelength by using an n-well / p-substrate photodiode in a 0.5 μm bulk CMOS IC process (Sim).
pson et al., 1999b). Various signal processing procedures can be used. However, by counting the pulses from the current-to-frequency converter circuit, a long time constant integrator is formed, which is the decisive factor of a matched filter for low-level bioluminescence signals in white noise. Part. Using the photodiode described above with this signal processing procedure, an rms noise level of 175 electrons / sec was measured for an integration time of 13 minutes. This corresponds to a detection limit of about 500 photons / second (Simpson et al., 1999b).

The toluene-sensitive bioreporter P. By placing putida TVA8 on a custom integrated microluminometer, a prototype BBI
C was constructed. FIG. 6 shows the prototype BBIC (including the bioreporter enclosure) used for the characterization study (Simpson et al., 1998b: S
impson et al., 1998c; Simpson et al., 1998d).

With no luminescence signal from the cells, the integration time was set to one minute and a plurality of measurements were performed. The leakage current produced a signal of about 6 counts / minute with a standard deviation (σ) of 0.22 counts / minute. As expected, σ decreased with the square root of the integration time. A longer integration time was created off-line by summing the measurements every minute.

[0048] Bioluminescence was induced in BBIC cells and in control samples of cells by exposure to toluene vapor. From measurements on control samples, the toluene concentration was estimated to be only 1 ppm. A signal of 12 counts / minute (6 counts / minute above background) was measured.
By prior measurement, P.I. putida TVA8 until the concentration is saturated (
It has been found that it has a linear response to toluene concentration when it reaches a level of about 10 ppm). The minimum detectable toluene concentration of this BBIC as a function of integration time is shown in FIG. Generally, the minimum detectable concentration is also a function of the number of bioreporter cells and the area of the photodiode.

The microluminometer and bioreporter P. fluoresce
Naphthalene sensitive BBICs were made using ns 5RL. Using the same experimental procedure described above, the BBIC was exposed to naphthalene vapor at a concentration of about 10 ppm.
A signal of 240 counts / min was recorded.

To eliminate the need for the addition of exogenous substrates, the cells themselves must supply the appropriate substrates for luciferase. In bacterial systems, the substrate is lux
Produced by the fatty acid reductase complex encoded by the CDE gene.
This enzyme complex reduces short chain fatty acids to the corresponding aldehyde. Luciferase then oxidizes the aldehyde to the corresponding fatty acid. A preferred fatty acid for this reaction is myristic acid, which is present in eukaryotes (Rudnick et al., 199).
3). Myristic acid is usually involved in amino-terminal myristoylation associated with membrane binding (Borgese et al., 1996; Brand et al., 1996).

In a preferred embodiment, the bioreporter for glucose monitoring comprises:
A mammalian bioluminescent reporter cell line that has been genetically engineered to exhibit luminescence in response to glucose concentration on a continuous substrate without the need for cell disruption and the addition of exogenous substrates. Current methodology using mammalian bioluminescent reporter cells requires cell lysis and the addition of exogenous substrates to elicit a measurable response. Consequently, these cells cannot serve as continuous online monitoring devices. In a preferred embodiment, the new cell line is an X. lactis integrated in a plasmid-based system (designated p.LPK.Luc _FF ) containing a eukaryotic luc gene capable of responding to glucose concentrations. luxAB and l from luminescens
Utilizing the uxCDE gene, it is constructed using a bioluminescent reporter. lux
Replacement of the luc gene with the AB gene allows bioluminescence measurements to occur in real time with glucose concentration, eliminating the need for cell disruption and the addition of substrate.

To form an implantable glucose monitoring BBIC, a bioreporter can be captured in a container behind the semi-permeable membrane that holds the bioreporter in place on an IC photodetector. Alternatively, the bioreporter can be encapsulated within a polymer matrix. The BBIC is encapsulated in a biocompatible housing with a semi-permeable membrane covering the bioreporter area. This membrane allows the passage of glucose to the bioreporter, but prevents the passage of larger molecules that can interfere with glucose measurements. When the glucose reaches the bioreporter, it is metabolized and the cells emit visible light. The IC detects the light, amplifies and filters the signal, and then reports the measurement. This measurement may be reported to the patient (eg, a wristwatch receiver) or to an insulin pump in a closed loop system that functions much like the pancreas.

FIG. 1 shows a perspective view of the present invention. The glucose 10 to be detected enters the BBIC 11 through the semipermeable membrane 12 covering the bioreporter.

FIG. 2 shows a side view of the present invention. The BBIC is encapsulated in a biocompatible housing 20 having a semi-permeable membrane 21 covering a bioreporter held in a container 22. The cells that make up the bioreporter can be in suspension or can be encapsulated in a polymer matrix. The bioreporter is separated from the photodetector 23 by a protective coating 24. A single substrate 25 includes additional circuitry 26 for processing and transmitting signals and a photodetector.

FIG. 3 shows a block diagram of one embodiment of an integrated circuit (“IC”). The light detector is a photodiode 33 connected to the current-frequency converter 30. The photodiode responds to light by sinking a current. The current is converted into a series of pulses stored in digital counter 31. The number of counts in the counter over a period of time is directly proportional to the amount of light collected by the photodiode, which is directly proportional to the glucose concentration. Digital processing circuitry within the digital counter determines the appropriate next step for the insulin pump based on the measured glucose level. The measured concentration, or the next indication to the insulin pump, may be reported via wireless transmitter 32.
All of these circuits (photodiode, signal processing, and radio transmission)
It can be manufactured on one IC.

FIG. 4 shows a bioreporter supplied with water and nutrients. Since the fluid and nutrient reservoir 141 is connected to the microfluidic pump 142, the nutrient and fluid 144 can be used to encapsulate the bioreporter in the polymer matrix 1.
43. Each of these components can be configured on a single substrate 140.

FIG. 5A shows a high quality photodetector made using standard N-well CMOS processing. This photodetector consists of two parallel reverse bias diodes.
The upper diode is formed between the P + active layer 45 and the N well 46, and the lower diode is formed between the N well 46 and the P substrate 47. The upper diode has good short wavelength light s
The lower diode provides good long-wavelength photosensitivity (500-1100 nm), while having an activity (400-550 nm). Therefore, the entire diode has photosensitivity over the range of 400 to 1100 nm. The luminescent compound 41 under test is separated from the photodetector by the Si ₃ N ₄ layer 40 and the SiO ₂ layer 42.

FIGS. 10A, 10B, and 10C show schematic representations of an implantable biosensor including two separate photodetectors with a bioreporter responsive to either increasing or decreasing glucose concentration. Show.

FIG. 10B shows a side view of a biosensor showing a silastic coating.

FIG. 10C shows a schematic representation of the use of a selectively permeable membrane to protect a bioreporter from an immune response.

(4.2 Photodetector) The first element of the microluminometer signal processing system is a photodetector. The main requirements for the photodetector are as follows.

• sensitivity to the wavelength of light emitted by the bioluminescent or chemiluminescent compound under test; • low background signal (ie, leakage current) due to parasitic reverse biased diodes; A suitable coating that prevents the material from interfering with the bioluminescent or chemiluminescent process under test and prevents the process under test from degrading the performance of the microluminometer; and Compatibility with the manufacturing process used to fabricate Two photodetector configurations that meet these requirements are described below. However, it should be understood that alternative ways of constructing such a photodetector may be used by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims. is there.

[0063] In a first embodiment, the photodetector is manufactured in a standard N-well CMOS process. As shown in FIG. 5A, the detector connects the PN junction between the PMOS active region and the N-well in parallel with the PN junction between the N-well and the P-type substrate. It is formed. The resulting detector is approximately 400 nm
Sensitive to light in the range of about 1100 nm. This range includes the emission range of 450-600 nm for the most commonly used bioluminescent or chemiluminescent compounds or organisms. To meet the requirement that the device have a low background signal, the device operates at zero bias and the operating voltage of the diode is set equal to the substrate voltage. The photodiode coating may be formed from a deposited silicon nitride layer or other material compatible with semiconductor processing technology.

In a second embodiment of the photodetector, the detector comprises silicon on insulator (SOI)
It is manufactured by a CMOS process. The internal leakage current in the SOI process is due to the presence of a buried oxide insulating layer between the active layer and the substrate, resulting in a standard CMO
2 to 3 orders of magnitude lower than the internal leakage current in S. Two photodetector structures are envisioned in the SOI process. The first structure shown on the left side of FIG.
Consists of a detector. In this PIN detector, the P- layer is formed by a P + contact layer, the I (intrinsic) region is formed by a lightly doped active layer,
The N region is formed from an N + contact layer in an SOI CMOS process. The spectral sensitivity of this sideways detector is set by the thickness of the active layer and can be tailored to a particular bioluminescent or chemiluminescent compound.

The second structure, shown on the right side of FIG. 3B, shows that the junction is deposited cobalt silicide (Co
Similar to the first structure, except that it is formed from a Schottky junction between a Si ₂ ) or other suitable material layer and the lightly doped active layer.

The inventors take into account that other photodetector structures can be envisioned in silicon or semiconductor processing that meet the above criteria.

(4.3 Low-Noise Electronic Component) The low-noise electronic component is the second element of the micro illuminometer signal processing system. The requirements for low-noise electronics are: sensitivity to the very low signal levels provided by the photodetector; immunity or compensation to electronic noise in the signal processing system; minimum sensitivity to temperature variations; power supply voltage (battery Minimum sensitivity to changes (for drive applications); for some applications, the electronics must have sufficient linearity and dynamic range to accurately record the detected signal level; and In electronic applications, electronic components must easily detect the presence of a signal in the presence of electronic and environmental noise.

Three embodiments meeting these requirements are described below. However, it should be understood that alternative methods of detecting small signals while meeting these requirements may be used without departing from the spirit and scope of the invention as defined by the claims.

FIG. 6A schematically illustrates a first approach for detecting very small signals. This device has a structure that is compatible with standard CMOS IC processes.
Using a diffuse / N-well photodiode in open circuit mode with a readout amplifier (manufactured on the same IC with the photodiode) The luminescent signal creates electron-hole pairs in the P-diffusion and N-wells. The photo-generated electrons during P-diffusion are injected into the N-well, while the photo-generated holes in the N-well are
Injected during diffusion. Since the N-well is coupled to ground potential, no charge accumulates in this region. However, since the P-diffusion is only attached to the input impedance of the CMOS amplifier (approaching infinity at low frequencies), positive charges accumulate in this region. Thus, the voltage on the P-diffusion node begins to rise.

As the P-diffusion voltage begins to rise, the P-diffusion / N-well photodetector is forward biased, thereby producing a current in the opposite direction to the photogenerated current. When the system reaches steady state, the voltage across the P-diffusion node produces a forward bias current of magnitude (but opposite polarity) exactly equal to the photocurrent. If the PN junction is not deviated from the ideal diode equation, the output voltage is given by the following _{equation: V out = V t ln (} I p / (AI s) +1) ( Equation 1) Here, V _t is The thermal voltage (approximately 26 mV at room temperature), I _p is the photocurrent, A is the cross-sectional area of this PN junction, and I _s is the reverse saturation current for a PN junction having a unit cross-sectional area. The value of I _s is highly dependent on the IC process and material parameters.

Two main error currents are present at PN junctions operating at low current densities (recombination and generated currents). Other than at very low temperatures, free carriers are generated randomly in the PN junction space charge region. Since this region has a high electric field, these thermally excited carriers are immediately swept across the junction and form a current component (generated current) in the same direction as the photocurrent. Carriers that traverse the space charge region also have a finite recombination probability. As a result, another current component (recombination current) is generated in the direction opposite to the photocurrent. Therefore, considering these fault current, equation 1 becomes _{V out = V t ln ((} I p + I g -I r) / (AI s) +1) ( Equation 2).

This output voltage is generally a function of a parameter that we cannot control. However, we control A over the junction area. Unfortunately, we need a small A to make our output signal larger, while we need a large A for high quantum efficiency (QE).

FIG. 6B shows a second microluminometer embodiment that satisfies both these requirements. This circuit uses a large area photodetector for efficient light collection,
A small area diode is used in the feedback loop to provide a forward bias current that cancels the photocurrent. Again, the amplifier and feedback diode are manufactured on the same IC as the photodetector. For this circuit, V _out = 3 V _t ln ((I _p + I _g −I _r ) / (A _fb I _s ) +1) (Equation 3) where A _fb is the small cross-sectional area of the feedback diode. . More than one diode is used in the feedback path, and the DC of any subsequent amplifier amplifier stage
Generates a large output signal compared to the offset. This method allows for efficient collection of light using a large area photodetector, while still producing a large output voltage with a small area diode in the feedback path.

The feedback circuit of FIG. 6B maintains the photo detector at zero bias. If no potential is applied, the recombination and generated currents should cancel. Equation 3 becomes V _out = 3V _t ln ((I _p / (A _fb I _s )) + 1) (Equation 4) when the smaller recombination current and generated current in the smaller feedback diode are negligible.

The main advantages of the second microluminometer embodiment shown in FIG. 6B include:

The SNR is entirely determined by the photodiode, with negligible noise from small diodes and amplifiers.

A diode can be added to the feedback path until the signal level at the output of the amplifier is significant compared to the next stage offset voltage (and offset voltage drift).

The method is fully compatible with standard CMOS processes without adding additional masks, materials and manufacturing steps.

This detection scheme can be manufactured on the same IC as analog and digital signal processing circuits and RF communication circuits.

• Measurements can be made without applying power to the circuit. Power must be applied before the measurement can be read, but the measurement can be obtained without applying power.

The third microluminometer implementation shown in FIG. 6C was correlated to minimize the effects of low frequency (flicker) amplifier noise and time or temperature dependent changes in amplifier offset voltage. Double sampling (CDS) is used. FIG. 6C
As shown, a photodiode having a capacitance C _d and a noise power spectral density S _i is coupled through a set of switches controlled by the logic level of the flip-flop output to a feedback capacitance C _f and an input noise power spectral density S _i. Connected to an integrating preamplifier with _v . The flip-flop output is L
When OW, the switch is positioned so that the photocurrent flows out of the preamplifier, thereby increasing the output voltage of the integrator. When the output voltage of the integrator, filtered by the low-pass filter, exceeds the threshold V _HI , the upper comparator “wakes up”, sets the flip-flop, and sets its output to HIGH. The detector switches the change position to allow current to flow into the integrating amplifier. As a result, the output voltage of the amplifier decreases. When the output of the integrator falls below the second threshold value V _LO , the lower comparator outputs “
Activate "to reset the flip-flop and bring its output low again. The above process is repeated as long as the photocurrent is present.

The average period Δt of the output pulse is given by the following equation.

[0083]

(Equation 1) Here, V _HI and V _LO are the threshold voltages of the comparator, and I _p is the photocurrent of the diode. Two noise sources cause errors in the measurement of At. S _i is the input noise current power spectral density mainly associated with the photodiode, and S _v
Is the input noise voltage power spectral density mainly related to the preamplifier. The diode noise is given by the following equation.

[0084]

(Equation 2) Here, I _s is the photodiode reverse saturation current, I _p is the photocurrent. As the photocurrent approaches zero, the noise power spectral density approaches a finite value of 4qI _s A ² / Hz. The noise voltage S _{v of the} preamplifier is determined by its design and has a unit of V ² / Hz.

The transfer function from the point at which diode noise is introduced at the output of the integrator is approximately given by:

[0086]

(Equation 3) Here, ω ₁ is the corner frequency of the integrating amplifier, and s = jω.
Here, ignoring the effect of the switch, the transfer function from the point where amplifier noise is introduced into the output of the integrator is given by approximately:

[0087]

(Equation 4) The switch attenuates noise that appears below the switching frequency of the output pulse train,
Performs a correlated double sampling function. The transfer function of the correlated double sampling circuit is approximated to linear by the following equation:

[0088]

(Equation 5) Here, Δt is the average period of the output pulse train. Thus, considering the switch, the transfer function from the point at which amplifier noise is introduced at the output of the integrator is approximately given by:

[0089]

(Equation 6) This is an important result. This is because the effect of flicker noise of the amplifier is reduced by the effective zero value incorporated in the noise voltage transfer function. This is particularly useful in CMOS implementations of microluminometers where flicker noise can have a major effect.

The mean square output noise of the output of the integrator is given by the following equation:

[0091]

(Equation 7) The RMS noise voltage is then given by the following equation:

[0092]

(Equation 8) The RMS error during the measurement period is determined by the slope of the integrated signal and the noise at the output of the integrator and follows the relationship:

[0093]

(Equation 9) Or, approximately, follow the formula:

[0094]

(Equation 10) The error in measuring Δt can be reduced by collecting many output pulses and obtaining an averaging period. The error in the measured average pulse duration improves in proportion to the square root of the number of pulses collected,

[0095]

[Equation 11] Becomes Here, t _meas is the total measurement time.

Thus, the implementation of the microluminometer has the following advantages: The low-frequency "flicker" noise of the amplifier is reduced by the correlated double sampling process; and, ideally, the measured The accuracy of the photocurrent can be improved endlessly by acquiring data over time.

It will be appreciated that the substantial limitations imposed by the lifetime and stability of the signal generated by the luminescent compound under test ultimately determine the resolution of this implementation.

4.4 Readout Electronics Several methods of communicating data from the BBIC to an external receiver or in vivo drug delivery system are envisioned. In a preferred embodiment, the communication method is an on-chip wireless communication system that communicates the level of the photocurrent to a computing circuit included in an in vivo drug delivery system or an external receiver. In a closed loop system, the computation circuit determines the amount of drug to be delivered by the in vivo drug delivery system. When using an external receiver, data from the BBIC along with user input is used to determine the amount of drug to be administered. The external receiver may include a wireless transmission circuit in communication with the in vivo drug delivery system, or may administer the drug manually. Other methods of communicating BBIC data include: Wiring to an in vivo drug delivery system to generate a DC voltage level proportional to the photocurrent Wiring to the in vivo drug delivery system and proportional to the photocurrent Wiring to an in vivo drug delivery system to generate a logic pulse string whose rate is proportional to the photocurrent Wiring to an in vivo drug delivery system to convey a numerical value proportional to the photocurrent On-chip implementation of an analog-to-digital converter On-chip implementation of a serial or parallel communication port hardwired to the in vivo drug delivery system and delivering a number proportional to the photocurrent Hardwired to the in vivo drug delivery system If the photocurrent exceeds a predetermined level, Generate radio frequency signals or beacons when photocurrent exceeds a predetermined level.In vivo wireless communication is limited by signal attenuation due to health fluid, tissue, and health restrictions on RF signal levels. Can be done. This may not be the optimal configuration for all cases, but may require that the BBIC and in vivo drug delivery system be placed in close proximity. In such a case, B
The BIC may be able to communicate with an external receiver located ex vivo but closer to the BBIC. The receiver may be connected (wired or wireless) to a transmitter located ex vivo but closer to the in vivo drug delivery system.

[0099] Numerous algorithms are envisioned to control the in vivo drug delivery system with the BBIC. These include, but are not limited to: A simple look-up table that administers a given drug level determined only by one BBIC data point Algorithm for determining drug dosage by increasing or decreasing rate. Algorithm for determining drug dosage by matching BBIC data points to data point patterns stored in memory. Using BBIC data point history and user input. Learning algorithms that predict the correct drug dosage to achieve the desired result Some of these algorithms may require two-way communication between the BBIC and the in vivo drug delivery system. In this case, the receiver is included on the BBIC.

4.5 Biocompatible Housing and Semipermeable Membrane The BBIC is included in a biocompatible housing with a semipermeable membrane covering the bioreporter area. The preparation of biocompatible covers for implants and prosthetic devices to minimize encapsulation and physiological rejection is an extensive area of research. For example, U.S. Patent Nos. 5,370,684 and 5,387,247, each specifically incorporated herein by reference, describe biocompatible bioprosthetic devices. Disclosed is the application of a carbon film. Two-dimensional fabric (woven) or woven fabric (fab) of organic fibers
ric) is disclosed in US Pat. No. 5,711,96.
No. 0, which is specifically incorporated herein in its entirety.
Other covers for implants constructed to present the biocompatible surface of the present invention to a body are disclosed in US Pat. No. 5,653,755, US Pat. No. 5,779,7.
No. 34 and U.S. Pat. No. 5,814,091 each of which is specifically incorporated herein by reference. Furthermore, collagen and albumin coatings have been shown to improve the biocompatibility of implants and prosthetic devices (Marios et al., 1996; Ksa
nder, 1988). The present invention contemplates the use of any suitable biocompatible material for either coating or forming the housing.

The semi-permeable membrane comprises a portion of the BBIC housing that covers the bioreporter and captures it on an integrated circuit. The membrane prevents the passage of larger molecules, but allows the selected substance (eg, glucose) to pass to the bioreporter. Membranes designed for use with glucose oxidase-based biosensors can also be used in preferred embodiments of the present invention. Membranes that have been studied and designed for use with glucose oxidase-based biosensors include, but are not limited to:
Polytetrafluoroethylene membranes (Vaidaya and Wilkins, 199
3); perfluorinated ionomer membranes (Moussy et al., 1994); charged and uncharged polycarbonate membranes (Va
diya and Wilkins 1994); and cellulose acetate membranes (
Wang and Yuan, 1995; Sternberg et al., 1988). In addition, other membranes have been developed for use in transplanting islet cells or other cells that have been bioengineered to produce insulin. This membrane must be permeable to glucose and other metabolites, while eliminating elements of the host immune system. Such membranes can be adapted for use with the present invention, and include, but are not limited to: asymmetric poly (vinyl alcohol) membranes (Young et al., 1996); poly (L-lysine) ) Membrane (Tziampaz)
is and Sambanis, 1995); Polyurethane (Zondervan)
Et al., 1992); nucleopore membranes (Ohgawara).
Et al., 1998); and agarose gels (Taniguchi et al., 1997).
Biocompatible semipermeable membranes for encapsulation of cells to form artificial organs are disclosed in US Pat. Nos. 5,795,790 and 5,620,883, each of which is hereby incorporated by reference in its entirety. Which is specifically incorporated in this document). Biocompatible semi-permeable segmented block polyurethane copolymer membranes and their use to permeate molecules of a given molecular weight range are disclosed in US Pat.
No. 28,123, which is specifically incorporated herein by reference in its entirety. The present invention contemplates the use of any suitable semi-permeable membrane that allows the selected substance to access the bioreporter, but prevents the passage of larger molecules.

4.6 Drug Delivery Devices A number of drug delivery devices (implantable and external devices) have been described previously that can be controlled by wireless telemetry. For example, U.S. Pat.
No. 44,659, which is specifically incorporated herein by reference in its entirety,
An implantable piezoelectric pump for drug delivery in an outpatient is described. U.S. Patent No. 5,474,552, specifically incorporated herein in its entirety, discloses a glucose sensor that can deliver multiple activators, such as glucose, glucagon, or insulin, if needed. An implantable pump for use in combination is described. Separate pumps can be used to deliver each factor, or a single pump switchable between them can be used. US Patent 5,
No. 569,186, which is specifically incorporated herein in its entirety, describes a closed loop infusion pump system controlled by a glucose sensor. U.S. Pat. No. 4,637,391, which is specifically incorporated herein in its entirety, describes an implantable remotely operated micropump for the delivery of a pharmaceutical agent. The use of an external drug delivery system is contemplated in other embodiments of the present invention. For example, US Pat. No. 5,800,420, which is specifically incorporated herein in its entirety, delivers a liquid drug (eg, insulin) via a hollow delivery needle extending into the dermis. Disclose the pump location relative to the skin surface topically. In other embodiments of the invention, the drug delivery system may interface with the biosensor device and be controlled directly, as opposed to telemetry control from the BBIC.

The pump delivery system described above is an example to facilitate use of the present invention. Drug delivery devices other than pump systems are also contemplated by the present invention. For example, US Pat. No. 5,421,816, which is specifically incorporated herein in its entirety, describes an ultrasonic transdermal drug delivery system. The ultrasonic energy is used to release the stored drug and force the drug through the skin of the organism and into the bloodstream. Thus, the present invention contemplates the use of any suitable drug delivery system that can be controlled by a BBIC glucose monitor. BBIC
The factors that determine the choice of such a drug delivery system and its use with glucose monitor use are known to those of skill in the art in light of the present disclosure.

4.7 Bioluminescent Bioreporter In a preferred embodiment of the present invention, the bioreporter for glucose monitoring is a mammalian bioluminescent reporter cell line. This cell line is genetically engineered and emits light continuously in response to glucose concentration. Implantable bioluminescent sensors require a bioluminescent reporter that can function without exogenous addition of a substrate to the luciferase reaction. Current eukaryotic luciferase systems used in molecular biology require the addition of exogenous substrates due to the complex nature of eukaryotic luciferin production. Cells must either be permeabilized or lysed and then treated with a luciferin-containing assay solution. Thus, current eukaryotic luciferase systems are not preferred candidates for online monitoring.

The need for the addition of exogenous substrates can be eliminated by the use of bacterial lux genes.
In a preferred embodiment of the present invention, The lux genes of luminescens, luxAB and luxCDE, are used as bioluminescent reporter systems. This X. The luminescens luxAB gene encodes the α and β subunits of the luciferase enzyme, exhibiting maximum temperature stability at 37 ° C. (although other bacterial luciferases lose significant activity above 30 ° C.). . This luxCDE gene is required to eliminate the need for the addition of exogenous substrates. The aldehyde substrate of luciferase encoded by the luxAB gene is produced by the fatty acid reductase complex encoded by the luxCDE gene. The preferred fatty acid for this reaction is myristic acid present in eukaryotic organisms (Rudnick et al., 1993), and eukaryotic cells are therefore suitable host cells for this receptor. The enzyme complex reduces fatty acids for the corresponding aldehyde. Luciferase then oxidizes the aldehyde back to fatty acids.

Other bioluminescent nucleic acid segments are available from Vibrio fischerii, lux
The lux gene for luciferase from CDABE, or other bioluminescent organisms that can be adapted so as not to require the addition of exogenous substrates, can be included. In another embodiment of the invention, the nucleic acid segment is an Aqueorea victory.
a or the green fluorescent protein of Renilla reniformis.

4.8. Recombinant Vectors Expressing Bioluminescent Genes One important embodiment of the present invention is a recombinant vector comprising one or more nucleic acid segments encoding one or more bioluminescent polypeptides. Vector. Such vectors can be transferred to eukaryotic or prokaryotic hosts and replicated in those hosts.

[0108] The coding DNA segment is intended to be under the control of a recombinant or heterologous promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding a crystal protein or peptide in its natural environment. In nature, it is important to use a promoter that effectively directs the expression of the DNA segment in the cell type, organism or even animal chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology (eg, Sambrook).
Et al., 1989). In this preferred embodiment, such a promoter is directed by a cis-operated glucose response element. In one preferred embodiment, the glucose response element is an L4 box that directs the L-pyruvate kinase ("L-PK") promoter in liver cells and islet β cells. The L4 box consists of a tandem repeat of the non-standard E box (Kennedy et al., 1997). Glucose enhances liver and pancreatic β-cells by modifying the transactivation ability of upstream stimulating factor (“USF”) binding to the L4 box (Kennedy et al., 1997; Do).
iron et al., 1996).

[0109] The exact mechanism by which glucose controls the transactivating ability of the USF protein is unclear. One possibility is the reversible phosphorylation of the USF protein. Glucose can change the phosphorylation state through pentose phosphate shunt via xylose 5-phosphate (Dorion et al., 1996). Another mechanism is through the intracellular concentration of glucose 6-phosphate (Foufelle et al.,
1992). Other glucose metabolites may be contemplated. Phosphorylated glucose metabolites include, but are not limited to: fructose 6-
Phosphate, 6-phosphogluconic acid, 6-phosphoglucono-δ-lactone, ribulose 5-phosphate, ribose 5-phosphate, erythrose 4-phosphate, sedoheptulose 7-phosphate, glyceraldehyde 3-phosphate, and dihydroxyacetone phosphate. Non-phosphorylated glucose metabolites include, but are not limited to, citric acid, cis-aconitic acid, threo-isocitric acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, pyruvic acid, and lactic acid.

Another glucose response element (similar to the arrangement for the L4 box of the L-PK gene) is a regulatory sequence involved in the induction of transcription of the rat S14 gene (Sh
ih et al., 1995). Other glucose response elements described include liver 6
-Phosphofructo-2-kinase gene (Dupriez and Roussea)
u, 1997), β-islet insulin gene (German and Wang, 199).
4), mesangial transforming growth factor-β gene (Hoffman et al., 1998), and acetyl coenzyme A carboxylase (Girard et al., 1).
997), but is not limited thereto. The present invention contemplates the use of any glucose response element that can efficiently direct a promoter or otherwise control the expression of a reporter protein in response to glucose.

[0111] In a preferred embodiment, the recombinant vector comprises a nucleic acid segment encoding one or more bioluminescent polypeptides. Highly preferred nucleic acid segments are X
. The lux genes are luminescens luxAB and luxCDE. Bacterial luciferase may need to be modified to optimize expression in eukaryotic cells. (1990) described the removal of the TAA stop codon from luxA without disrupting the reading frame. The luxAB gene from harveyi, the intervening region between the two genes, and the first methionine from luxB were fused. This fusion is based on Saccharomyces cerevisiae and Drosophila
It has been successfully expressed in la melanogaster. The same strategy is described in X. Used in luxAB from luminescens. The resulting constructs were sequenced to confirm genetic changes, generate fusions, and confirmed them. The sequence of the fusion region is as follows:

[0112]

Embedded image This fusion is E. coli. The fusion protein was successfully expressed in E. coli and successfully cloned into a mammalian vector as described in section 5.1.2.

In a further embodiment, we contemplate a recombinant vector comprising a nucleic acid segment encoding one or more enzymes capable of effecting a reaction that produces a luminescent product or a product that can be directly converted to a luminescent signal. . For example, commonly used β-galactosidase or alkaline phosphatase (alkaline phosphatase)
The substrates of the enzymes are commercially available and emit light when converted by the respective enzyme (chemiluminescence).

[0114] In another important embodiment, a biosensor comprises at least a first transformed cell expressing one or more recombinant expression vectors. The host cell can be either a prokaryotic or eukaryotic cell. In a preferred embodiment, the host cell is a mammalian cell. Host cells can include stem cells, beta islet cells, or liver cells. In a preferred embodiment, the host cell is an allogeneic cell (ie, a cell taken from a patient that has been cultured, genetically manipulated, and then integrated into the BBIC). Particularly preferred host cells are lumi
A host cell that expresses a nucleic acid segment containing a recombinant vector encoding the lux gene, luxAB and luxCDE of nescens. These sequences correspond to X. The luminescens lux transcription protein is particularly preferred because it has the ability to function at 37 ° C. (around human body temperature).

A wide variety of methods are available for introducing nucleic acid segments that express a polypeptide that can provide bioluminescence or chemiluminescence to a microbial host under conditions that allow for stable maintenance and expression of the gene. It is. Those skilled in the art will recognize translational and transcriptional regulatory signals for the expression of this nucleic acid segment, the nucleic acid segments under their regulatory control, and DNA sequences homologous to sequences in the host organism (whereby integration occurs) or the host. Including a replication system that is functional in E. coli, thereby resulting in integration and / or stable maintenance, or both.
A construct is provided.

[0116] Transcription initiation signals include a promoter and start site for transcription initiation. In preferred cases, it is desirable to provide regulated expression of a nucleic acid segment that can provide bioluminescence or chemiluminescence, where expression of the nucleic acid segment only occurs after release into a suitable environment. This can be achieved using an operator, or a region that binds to an activator or enhancer. These allow for induction upon changes in the physical or chemical environment of the microorganism. For translation initiation, there is a ribosome binding site and an initiation codon.

A variety of manipulations can be utilized to enhance messenger RNA expression, particularly by using active promoters and by using sequences that enhance messenger RNA stability. Transcription and translation termination regions include stop codons, terminator regions, and optionally a polyadenylation signal (if used in eukaryotic systems).

In the direction of transcription, ie, 5 ′ to 3 ′ of the coding or sense sequence, the construct comprises a transcription regulatory region (if present) and a promoter (where
This regulatory region can be on either the 5 'or 3' side of the promoter)
Ribosome binding site, initiation codon, structural gene having an open reading frame in phase with the initiation codon, termination codon, polyadenylation signal (if present), and terminator region. This sequence as a double strand may itself be used for transformation of a microbial host, but will usually be included with the DNA sequence containing the marker. Here, the second DNA sequence can be joined to the expression construct during the introduction of the DNA into the host.

By “marker”, the invention refers to a structural gene that provides for the selection of modified or transformed hosts. This marker usually provides a selective advantage (eg, biocide resistance (eg, resistance to antibiotics or heavy metals); provides complementation to provide prototrophy to an auxotrophic host, etc.). Do). One or more markers can be utilized for construct development and host modification.

In the absence of a functional replication system, the construct will also have at least 50 base pairs (bp
), Preferably a sequence of at least about 100 bp, more preferably a sequence of at least about 1000 bp, and usually about 2000 of sequences homologous to sequences in the host.
bp or less. In this way, the likelihood of legitimate recombination is increased so that the gene is integrated into the host and is stably maintained by the host. Desirably, the nucleic acid segment capable of providing bioluminescence or chemiluminescence is proximal to the gene providing for complementation and the gene providing competitive advantage. Thus, in the event of loss of a nucleic acid segment capable of providing bioluminescence or chemiluminescence, the resulting organism will likely have lost the complementing gene and / or the gene that provides the competitive advantage.

A large number of transcription regulatory regions are available from a wide variety of microbial hosts, such as bacteria, bacteriophages, cyanobacteria, algae, fungi, and the like. Various transcription regulatory regions include those associated with the trp gene, lac gene, gal gene, λ _L and λ _R promoters, and the tac promoter. For example,
U.S. Pat. Nos. 4,332,898; 4,342,832;
356,270, each of which is specifically incorporated herein by reference in its entirety. The termination region may be a transcription initiation region or a termination region normally associated with a different transcription initiation region, so long as the transcription initiation region is compatible and functional with the host. In a preferred embodiment of the present invention,
As described in Kennedy et al. (1997), a fragment of the L-PK gene containing the L-pyruvate kinase promoter and the L4-box glucose responsive element is used. In a very preferred embodiment, the luc gene encoding firefly luciferase has been deleted and the fused X. luminesc
ens luxAB gene, except that p. LPK. The Luc _FF plasmid is used (Kennedy et al., 1997).

When stable episomal maintenance or integration is desired, a plasmid is utilized (a plasmid uses a replication system that is functional in the host). This replication system
It may be from a chromosome, an episomal element normally present in the host or a different host, or a replication system derived from a virus that is stable in the host. A number of plasmids are available, for example, pBR322, pACYC184, RSF1010, p
R01614 and the like. See, for example, Olsen et al., 1982; Bagda.
Sarian et al., 1981 and U.S. Pat. Nos. 4,356,270;
Nos. 62,817, 4,371,625, and 5,441,884, each of which is specifically incorporated herein by reference.

A desired gene can be introduced between a transcription initiation and translation initiation region and a transcription termination and translation termination region such that it is under regulatory control of the initiation region. The construct is included in a plasmid, which contains at least one replication system, but can be more than one. Here, one replication system is used for cloning during the development of the plasmid, and a second replication system is required to function in the final host. In addition, one or more markers may be present and have been described previously. If integration is desired, the plasmid desirably contains sequences homologous to the host genome.

[0124] Transformants can be isolated according to conventional methods and usually employ selection techniques. This allows the unmodified organism, if present, or being transposed (transf
erring, as well as for the organism, the selection of the desired organism is possible. The transformants can then be tested for bioluminescent or chemiluminescent activity. If desired, unwanted or auxiliary DNA sequences can be introduced into site-specific recombination systems (eg,
US Patent No. 5,441, which is specifically incorporated herein by reference in its entirety.
No. 884) can be selectively removed from recombinant bacteria.

4.9 Assembly and Storage of In Vivo Biosensors When a biosensor consists of bioengineered cells that are captured in suspension behind a semi-permeable membrane, they are encapsulated in a matrix. In contrast, the cells can be added to the BBIC at any time from the time of biosensor implantation to several hours prior to implantation. Alternatively, a biosensor may consist of cells encapsulated in a polymer matrix. The matrix includes materials previously shown to be successful in encapsulating live cells, including polyvinyl alcohol, sol-
Gels, and alginates (Cassidy et al., 1996). Prior to encapsulation, the prokaryotic cell line may be lyophilized in a lyophilization system (eg, Savant) according to the manufacturer's protocol. Lyophilization allows for long-term storage (several years) and a simple rehydration protocol makes BBIC
Required for cell resuscitation before use (Malik et al., 1993). S. cere
visiae eukaryotic cells can be lyophilized as well. Eukaryotic cell lines, preferably consisting of islet β cells, stem cells, or hepatocytes, can be encapsulated on IC in a polyvinyl alcohol mesh reinforced or microporous filter supported hydrogel. Previously performed successfully in these types of cell encapsulation (
Baker et al., 1997; Burczak et al., 1996; Gu et al., 1994;
noue et al., 1991).

However, in the case of mammalian cell lines, lyophilization is not an alternative. In such a case, the mammalian cells can be encapsulated in a sol-gel or another immobilized matrix and attached to the BBIC as previously described. The encapsulated BBIC is then stored in serum or another suitable maintenance medium or maintained until use. The advantages of using immortalized stem cell lines are evident for long-term use and storage. Implantation can be performed according to the particular application. In the case of glucose detection, the area where interstitial fluid is accessible is most appropriate. However, hormones or other blood-borne molecules
Implantable devices with specific applications to detect emolecules must be accessible to the bloodstream. Synthetic vein or catheter systems may need to be used to allow for continuous monitoring of blood levels of target molecules. A particular example other than glucose is the use of in vivo biosensor devices to detect molecules associated with colon cancer. In this case, the biosensor is implanted in the colon.

The integrated circuits can be individually packaged in sterile, static-proof bags. Prokaryotic-based and yeast eukaryotic biosensors consisting of lyophilized cells can be stored individually in sterile, static-proof, vacuum-sealed bags for about several years. Cells are typically rehydrated in minimal nutrient media before use. Mammalian cell lines, either individually on the BBIC, or frozen or refrigerated if captured, remain frozen for extended periods of time (up to 7 years at -150 ° C). Or refrigerated for short term (several days) storage. In all cases, cell viability can be checked by exposing the BBIC to a known concentration of the analyte of interest, thus generating a quantitative bioluminescent signal of known magnitude. One or more control vials of the analyte or reference "standard" can be included as part of the diagnostic kit or provided for proper calibration of the implantable device.

4.10 Implanting and Using Biosensor Devices In a preferred embodiment of the present invention, a BBIC analyte biosensor is implanted in contact with the animal's interstitial fluid. For example, in the case of a glucose biosensor, it has been shown that glucose kinetics in interstitial fluid can be predicted by compartmental modeling (Gastaldelli et al., 1997).
In particular, subcutaneous placement of glucose sensors has been demonstrated (Schmidt et al., 1993).
Poitout et al., 1993; Ward et al., 1994; Steenberg et al.
1995; Bantle and Thomas, 1997). Other potential analyte biosensor tissue implant sites include the peritoneum, pleura and pericardium (W
olfson et al., 1982). In fact, we have found that depending on the particular analyte or metabolite being detected, the implantable biosensor can be placed in any convenient location in the body using conventional surgical and implantation methodologies. It is intended that it can be arranged in For example, the device can be implanted in such a way that it is contacted with interstitial fluid, lymph, blood, serum, synovial fluid or bursa-spinal fluid, depending on the particular analyte to be detected.

[0129] In certain embodiments, the implantable device can be placed in contact with a particular tissue, organ, or a particular organ system. Similarly, it may be desirable to implant the biosensor so that it is in contact with a particular, intracellular, intercellular, or any other bodily fluid whose target substance may be monitored.

[0130] The present invention also contemplates the use of multiple biosensors for the detection of multiple different analytes. For example, in the case of glucose monitoring, one or more devices may be used to monitor various glucose or glucose metabolites, glucagon, insulin, and the like. Similarly, one or more biosensor devices can be
It can be used in controlled drug delivery systems. As such, the device may be controlled by the biosensor and a certain amount (determined by the concentration of one or more analytes detected by the BBIC device) of a particular drug, hormone,
It can be operably connected to a drug delivery pump or device that can introduce a protein, peptide or other pharmaceutical composition into the body of an animal. Thus, as in the case of chronic or long-term medical conditions or when symptoms persist for a long period of time,
A controlled drug delivery diameter that is particularly desirable in providing long term administration of the drug to the animal is contemplated by the inventors. Controlled long-term delivery of pain treatment, cardiac or other cardiac regulators, diuretics or drugs such as hormones or peptides (eg, insulin), or metabolites such as glucagon or glucose,
It can be facilitated by such a biosensor / pump system. Multiple drug delivery systems or a single interchangeable drug delivery diameter are contemplated to be particularly useful where it is necessary to deliver more than one drug or metabolite to the animal.

[0131] Host rejection effects can be minimized by immunoisolation techniques. Previous studies have shown that viable non-host cells surrounded by a hydrogel membrane are protected from post-transplant immune rejection (Baker et al., 1997; Burczak et al., 1996: I.
noue et al., 1991). However, hydrogel block access by the humoral and cellular components of the host immune system remains permeable to the target substance glucose. The BBIC can be completely encapsulated using a mesh reinforced polyvinyl alcohol hydrogel bag developed by Gu et al. (1994), which allows for implantation of an immunosuppressive response.

[0132] Host rejection of an implanted biosensor is not a problem if cells from that host are used for the biosensor construction. However, if other cell lines are used, the passage between the cell and its appropriate body fluid, allowing passage of signature molecules or analytes but not of bioreporter cells or somatic cells (such as leukocytes) May need to be provided with a barrier. Immunosuppressed patients are not affected. Because the implant does not contain any kind of pathogen affecting the patient. In all cases, the surgical methods involved in implanting the disclosed BBIC devices are well known to those skilled in the surgical arts.

[0133] In an exemplary embodiment, a BBIC glucose sensor may be used to monitor glucose in diabetes. However, such sensors can also be used in other situations where glucose concentration is an issue, such as in endurance sports or other situations involving hypoglycemia or hyperglycemia. Such a measurement can be a long chain or an endpoint for diagnostic purposes, or the sensor can be coupled to the drug delivery system via telemetry or directly.

The use of implantable BBIC for substances other than glucose can be used in a range of therapeutic situations. With the incorporation of the appropriate cis-activated response element, BBI
C can monitor a number of substances and find use in chronic pain treatment, cancer therapy, chronic immunosuppression, hormonal therapy, cholesterol management and lactate threshold in heart attack patients. For example, Section 5.7 describes the use of BBIC in the detection and diagnosis of cancer.

[0135] Individual biosensors can be calibrated to check for cell viability and performance. The calibration is performed by exposing the sensor to solutions containing various concentrations of the analyte of interest. The bioreporter can be calibrated for a particular application after its initial construction with a range of standard analyte concentrations.
The overall online performance can be monitored using microfluidics using the reservoir of the assay, which systematically provides a known concentration to the cells, which allows both calibration and testing for viability .

The luminescence response is then correlated to the concentration and its parameter set. Viability can also be continuously monitored by the bioengineered cells. In this cell, the reporter shows continuous luminescence. Loss of survival results in reduced luminescence. This technique was used to detect multinucleated cell viability.
Thus, the BBIC contains two bioreporters, which detect the selected substance, and a second bioreporter that emits light proportional to cell viability. Measuring the ratio of the signals from the two bioreporters provides a detection method that automatically corrects for any loss of viability.

Once prepared, the bioreporter can be stored in a suitable maintenance medium, such as standard tissue medium, serum or other suitable growth or nutrient formulation, and then Can be calibrated. The viability of this device is checked by bioluminescence using microfluidics or by quantification of a known standard or other reference solution to confirm the viability and integrity of the system before or after implantation.

In certain embodiments of the present invention, the monolithic biosensor device can be used outside the body of the individual being monitored. In some clinical settings, the monitor may be used to monitor glucose in body fluids in an extracorporeal manner. This device can be used even in the field of pathology or forensics to detect the amount of a particular analyte, such as in body tissue or fluid. Similarly, the present invention also contemplates the use of biosensor devices in veterinary medicine. For monitoring hormone levels in the blood (ie, to optimize milk production), for monitoring the onset of estrus (fever) in a large number of animals to maximize the efficiency of artificial insemination, and for the milk produced Implantation of such devices into animals, such as for monitoring hormone levels online (in the breast) in animals, has certain benefits for commercial livestock operations, the livestock industry, and for use by those skilled in veterinary medicine. It is intended to provide

4.11 Diagnostic Kits Including In Vivo Biosensors Although the individual components of the invention described herein can be obtained and assembled individually, we have identified, for simplicity, It is contemplated that the components of the biosensor can be packaged in kit form. The kit may comprise, in a suitable container means, an IC comprising one or more bioreporters and a light converter. The kit also preferably includes instructions for using the biosensor device,
And further, if desired, may include a drug delivery device or a second biosensor device. The kit may comprise a single container means comprising one or more bioreporters and an IC comprising a light converter and a drug delivery device. Alternatively, the kit of the invention may comprise a different container means for each component. In such a case, one container may contain one or more bioreporters, either pre-encapsulated in a suitable medium or in a polymer matrix, and another container may contain one or more bioreporters.
C, and another container may include the drug delivery device. If the bioreporter is pre-encapsulated, the kit may include one or more encapsulation media. The use of different container means for each component allows for adjustment of the various components of the kit. For example, some bioreporters may be available to select, depending on the substance that one wants to detect. Replacing the bioreporter may allow the remaining components of the kit to be utilized for entirely different purposes, thus permitting re-use of the components.

The container means may be a container, such as a vial, test tube, packet, sleeve, shrink wrap, or other container means, in which the components of the kit may be located. The bioreporter or other reagent may also be split into smaller containers or delivery vehicles if desired.

The kits of the present invention may also include means for containing individual sealed containers for marketing, such as, for example, injection or blown plastic containers.
The desired components of the kit are held in the container.

[0143] Regardless of the number of containers, the kits of the invention may also include or be packaged with devices to assist in placing the bioreporter on the IC. Such an apparatus may be a syringe, pipette, tweezers, or any other similar surgical or implantable device. The kit also
One or more stents, catheters or other surgical devices may be provided to facilitate implantation into the body of the target animal. Such a kit may also include a device for telemetry or a device for data storage or long-term recording of data obtained from the monitoring device. Similarly, in the case of a controlled drug delivery system, the kit may include one or more drug delivery pumps, as described above, and may also include one or more pharmaceutical agents themselves for administration. . By way of example, in the case of a glucose monitoring system, the system typically comprises a glucose sensitive BBIC device, a drug delivery pump, instructions for implantation and / or use of the system, and optionally insulin, glucagon. Alternatively, a reference standard or pharmaceutical formulation of another pharmaceutical composition is provided. The system can also optionally
Growth media and / or storage media may be included to support the nutritional needs of the bioreporter cells contained in the BBIC device.

5.0 Examples The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art will recognize that the techniques disclosed in the examples below are representative of the techniques discovered by the inventors that work well in the practice of the present invention and therefore constitute preferred embodiments for the practice thereof. It should be understood that you get. However, one of ordinary skill in the art, in light of the present disclosure, that many changes may be made in the particular embodiments disclosed,
It is still to be understood that similar or similar results can be obtained without departing from the spirit and scope of the invention.

5.1. Example 1: Construction of a Bioluminescent Receptor for Mammalian Cell Lines To facilitate the construction of an implantable bioluminescent glucose sensor, the externalization of a substrate for the luciferase reaction There is a need to create a bioluminescent reporter system that can function without addition. This external addition results from the complex nature of luciferin production for various eukaryotic luciferases. The cells must be either permeabilized or lysed and then treated with an assay solution containing luciferin. Thus, this state of the bioluminescent reporter used in eukaryotic molecular biology makes "on-line" monitoring unsuitable. Firefly luciferase has been used to test the regulation of L-pyruvate kinase promoter activity in single living rat islet β cells (Kenned
y et al., 1997). However, these cells do not produce Be
Ele luciferin had to be perfused.

To eliminate this limitation, preferred bioluminescent reporter systems for the present invention are those that do not require the addition of an external substrate. In the case of a bacterial luciferase-based detection system, This can be achieved using a bioluminescent gene from luminescens. In this organism, the luxA and luxB genes (or a single fused luxAluxB gene) encode the α and β subunits of the luciferase enzyme, respectively (Meighen
Et al., 1991). This luciferase exhibits maximum thermostability at 37 ° C, while other bacterial luciferases lose significant activity above 30 ° C. Thus, these bacterial luciferases can be expressed in eukaryotic cells with minor modifications. Almashanu et al. (1990) describe in V.S. The luxAB gene from harvey was fused by removing the TAA stop codon from luxA, the intervening region between the two genes and the starting methionine from luxB without disrupting the reading frame. This fusion was successfully prepared by S.M. cerevisiae
And D. It was expressed in melanogaster. Using the same strategy,
The fused luxAB gene sequence has the X. Developed using genes from luminescens.

[0146] To eliminate the need for the addition of exogenous substrates, the cells must themselves provide a suitable substrate for luciferase. In bacterial systems, this substrate is produced by the fatty acid reductase complex encoded by the luxCDE gene. This enzyme complex reduces short chain fatty acids to the corresponding aldehyde. The luciferase then oxidizes the aldehyde to the corresponding fatty acid. The preferred fatty acid for this reaction is myristic acid, which is present in eukaryotes (Rudnick et al., 1993). Myristic acid is commonly involved in myristoylation of the amino terminus associated with membrane binding (Borgese et al., 1).
996, Brand et al., 1996). Thus, to eliminate the need for an exogenous supply of luciferase substrate, the biosensor also preferably has three lus
Includes nucleic acid sequences encoding subunits encoded by xC, luxD and luxE. As in the case of the luxAluxB gene fusion, this luxC, lu
The xD and luxE genes were fused to generate a single luxCDE gene fusion, encoding the three subunits of this enzyme complex. A method for preparing such a gene fusion is described below: (5.1.1 Fusion of luxAB and luxCDE genes) The luxAB gene can be fused using conventional molecular biology techniques. For example, the polymerase chain reaction can be conventionally used for this purpose. 5 'primer (this sequence starts with ATG for the start codon of the luxA gene)
And by synthesizing a 3 'primer (terminating at the codon immediately before the ATT stop codon). These primers were then used in the amplification reaction and the product could be gel purified. The luxB gene can also be amplified as described above using primers that eliminate the ATG initiation methionine codon but preserve its reading frame. The PCR ^™ reaction is a thermostable polymerase (eg, Stra
tagene (Pfu ^™ polymerase from La Jolla, CA) is used. This polymerase has no terminal deoxytransferase activity and therefore produces blunt ends. These resulting PCR ^™ products were ligated at blunt ends (bl
The ligation was then subjected to PCR ^™ using the 5 ′ primer from luxA and the 3 ′ primer from luxB using Taq polymerase and TA cloning (Invitrogen, S.I.
an Diego, CA). Only ligations with the correct fragment orientation are amplified. The luxAB amplicon is then gel purified and the appropriate vector (eg, PCRI
^I.TM. vector) and cloned into E. coli. E. coli.

Transformants are screened for light production by the addition of n-decanal. This n-decanal produces bioluminescence when oxidized by luciferase. Only colonies that emit light are selected. Because these are
This is because it exists in an appropriate direction for further genetic manipulation. The luxCDE fusion is generated using the same strategy as described above, except that the transformants are screened by miniprep followed by restriction digest analysis to determine orientation. The plasmid is E. coli. Amplify in E. coli, recover, and purify twice on a CsCl gradient.

5.1.2 Expression of luxAB and luxCDE in HeLa cells To determine the relative activity of this fused bacterial luciferase component,
The cloned fragment containing xAB is cloned into a suitable mammalian expression vector (eg, pcDNA3.1) and the luxCDE-containing fragment is cloned into a suitable mammalian expression vector (eg, pcDNA / Zeo3.1) (
(Invitrogen, Faraday, CA). Both vectors constitutively express the inserted gene. HeLa cells were then luxed according to the manufacturer's protocol (Promega, Madison, WI).
Transfected with AB or both luxAB and luxCDE and selected using appropriate antibiotics. Cells bearing the luxAB fusion are exposed to n-decanal and examined for bioluminescence. These cells co-transfected with luxCDE were then tested for bioluminescence and lux
Confirm the relative expression of the CDE fusion. This allows for a comparison of bioluminescence via addition of exogenous aldehyde versus endogenously generated aldehyde.

An alternative strategy to enhance bioluminescence expression is pcDNA3.1 (S
(Tratagene, La Jolla, Calif.) to manipulate a vector containing three copies of the eukaryotic expression machinery. This is luxCDE
Enables the expression of individual components of This is because the fused luciferase has already been shown to be expressed in eukaryotic cell lines (Almashhanu et al., 1990).

5.2 Example 2-Construction of a Glucose Bioluminescence Biosensor Firefly luciferase has been used in testing the regulation of L-pyruvate kinase promoter activity in single living islet β cells. (Kennedy
Et al., 1997). A glucose response element, termed the L4 box, has been determined to be in the proximal promoter. A 200 bp fragment containing this region was ligated with firefly luciferase (luc) in plasmid pGL3Basic.
), Which allows p. LPK. A glucose reporter plasmid named Luc _FF was generated. The results resulted in the detection of single cells exposed to 16 mM glucose, but not at 3 mM glucose. However, these cells must be perfused with beetle luciferin, which makes them unacceptable for online biosensors. Therefore,
A bioluminescence sensor for glucose is available at p. LPK. Firefly luciferase in Luc _FF was constructed by replacing the fused luxAB gene described below.

5.3 Example 3-Bioluminescent Reporter Construct and Transfection of Rat Islet β Cells The bioluminescent reporter plasmid was prepared as described in p. LPK. It was constructed by removing the Luc gene encoding firefly luciferase from Luc _FF and replacing it with the fusion luxAB gene. This is p. LPK. Lu from Luc _FF
This was achieved by truncating the c gene and cloning the luxAB gene. The resulting plasmid was transformed into E. coli. E. coli and the plasmid DNA was extracted and purified twice on a CsCl gradient.

Islet cells were prepared as previously described (German et al., 1990) and electroporated using this bioluminescent reporter construct and a plasmid containing the constitutively expressed luxCDE construct. Transfected. This configuration results in the cell maintaining a pool of aldehyde substrates available for this reporter gene (luxAB). Cells are grown from 3 mM to 30
Screened for light production in the mM glucose concentration range. The transfected cells were washed, concentrated, and placed in microwells in a light-tight cell, which was then connected to an integrated circuit. Different concentrations of glucose and assay medium (Kennedy et al., 1997) were added to these cells to test the sensitivity and response time of glucose BBIC.

5.4 Example 4—Preparation of Bioluminescent Reporter Constructs Reporter gene technology has been widely used in the study of gene regulation in both eukaryotic and prokaryotic systems. Various genes are used depending on the cell line being investigated. However, using BBIC technology requires the use of a reporter gene that produces light emission. Therefore, a reporter gene encoding bioluminescence is used. All previously developed reporters that utilize other reporter genes (eg, the gene encoding β-galactosidase (lacZ)) can be converted to a bioluminescent version using standard molecular techniques, and This reporter gene may be utilized for this particular application (modified lux system). Thus, any existing reporter cell line for testing gene expression in a mammalian cell line may be adapted for use as a bioreporter when converted to this lux reporter. Implantable systems simply include the appropriate reporter cell line. Table 1 shows a list of examples of eukaryotic reporter cell lines that can be utilized in implantable biosensors.

[0154]

[Table 1] 5.5 Example 5-Construction and Implantation of a Glucose Biosensor and an Insulin Delivery Pump In one embodiment, a pair of bioluminescences that are in series and specifically respond to deviations in glucose concentration A reporter may be used. One bioreporter is p. LPK. X. integrated into a plasmid-based system named Luc _FF . luxAB and IuxCDE from luminescens
Utilize genes. This p. LPK. Luc _FF is a eukaryotic luc gene that can respond to glucose concentration (increased bioluminescence corresponds to increased glucose concentration)
including. This second bioreporter utilizes a plasmid construct containing the promoter of the phosphoenolpyruvate carboxylase gene (PEPCK),
This promoter is also responsive to glucose concentration, but an increase in bioluminescence corresponds to a decrease in glucose levels. LuxAB and luxCDE for each construct
Gene incorporation allows bioluminescence measurements to occur in real time with deviations in glucose concentration, which eliminates cell destruction and the need for substrate addition.

In this embodiment, the integrated circuit comprises a separate photodetector unit for each bioreporter (FIGS. 9A, 9B and 9C). The bioluminescence response from each construct can be monitored independently, as the signal processing circuit switches between one bioreporter response to increasing glucose concentration and a second bioreporter response to decreasing glucose concentration. Allow to distinguish. This signal processing circuit processes the signal from the photodetector,
It is converted to digital form and this information is relayed to the implanted insulin pump (FIG. 10). This in-line set of bioreporters allows for more accurate signal and redundancy at the detector. Due to the occasional fatality of overcoming hypoglycemia, this tandem system also allows for more careful monitoring and alerting of the onset of hypoglycemia.

The cells used in this in-line bioreporter system can be connected to each photodetector either directly by mounting or encapsulation in a hydrogel (
Prevost et al., 1997). It is necessary to separate these bioreporters using a semi-permeable membrane to allow the transport of small molecules (eg, glucose and insulin) across the membrane and the inhibition of the flux of immune effector cells and antibodies. (Monaco et al., 1993; Suzuki et al., 1998). However, small molecules such as cytokines can still enter the selective membrane and interfere with these bioluminescent reporter cell lines. This approach has been used at high cost by those skilled in the art.

When applicable, bioluminescent reporter cell lines can be constructed from cells taken directly from the patient undergoing the transplant. This approach is particularly desirable for long-term implants (eg, implantable insulin delivery). Cells can be obtained from patients, genetically engineered for appropriate monitoring functions, grown in cell culture, evaluated, and stored for prolonged storage. The use of cell lines generated from the patient's own cells is particularly desirable. This is because it reduces the chance of host rejection and the generation of an immune response to the implanted device. Preferably, where appropriate, stem cells (if achievable, immortalized stem cells) can be used and maintained and cultured in a suitable culture medium. Such pluripotent, totipotent or other immortalized cell lines offer certain advantages in making suitable long-term implantable devices.

Prior to implantation, the biosensor can be calibrated by injecting various concentrations of glucose delivered from the insulin delivery pump and the auxiliary pump on the reservoir into the chamber containing the cell (FIG. 10). This allows the appropriate parameters to be determined and allows the appropriate dose of insulin to be delivered. Once the parameters are set, the pump can be evaluated for insulin delivery. Systematically, the glucose biosensor is recalibrated in the patient utilizing a glucose standard contained in the delivery pump.

For a drug delivery system, the glucose biosensor can be operably connected to the delivery pump via a hardwired or wireless connection. The biochip provides digital data that can be input directly into the signal processing network of the pump and proportionally distribute insulin. Alternatively, the digital data can be converted to analog data and used to control the pump. If wireless capability is added to the bioreporter device, remote monitoring of this sensor is possible. For example, in this configuration, the patient may place the wireless transmitter / receiver outside the body near the implanted device and transmit data from the implanted device to a remote station. In some embodiments, the wireless transmitter / receiver may be coupled to a computer programmed to transfer data to a remote station over a network (eg, a local area network, a wide area network, or even the Internet). . Such wireless applications allow for remote monitoring and maintenance of the patient. There are currently several pumps on the market that are candidates for connecting to biosensors. In one embodiment,
The Medtronic Synchronized infusion system is widely used in drug delivery and can be used to program the pump from outside the body using a portable computer (www.asri.edu/n).
euro / brochure / pain6. htm). The pump may also be refilled through the skin via a self-sealing septum. The pump is 1 inch thick, 3 inches in diameter, and weighs about 6 ounces. This biosensor can be integrated into existing electronic networks and utilize in vitro programming with portable computers. The tip can be powered using a battery to power the delivery pump.

The biosensor / insulin pump device can be surgically implanted in the abdominal cavity using local anesthesia. Both the sensor and the pump can be implanted in the abdominal peritoneal space for simplicity and to avoid complications of placing the catheter directly in the bloodstream. The glucose concentration is monitored and insulin is delivered to the peritoneum as required by the patient (FIG. 10).

5.6 Example 6-Bioluminescent Reporter Construct and Transfection of Rat Islet β-Cells and H4IIE Hepatoma Cells Modulation of the PEPCK gene is utilized to construct a bioluminescent reporter to detect decreased glucose levels Is done. This system is highly regulated since phosphoenolpyruvate carboxylase is the rate-limiting enzyme in gluconeogenesis. PEP
CK gene expression is increased in the presence of glucocorticoids and cAMP and decreased in the presence of insulin (Sasaki et al., 1984; Short et al., 1986). In both rat liver and H4IIE hepatoma cells, the insulin effect is dominant, and glucocorticoid and cAMP are additive.
The promoter region of PEPCK is cloned before the fusion luxAB.
The resulting construct then produces increased bioluminescence in the presence of low glucose concentrations.

A bioluminescent reporter plasmid for detecting an increase in glucose concentration
. LPK. It can be constructed by removing the Luc gene encoding firefly luciferase from Luc _FF and replacing it with the fusion luxAB. This is called p.
LPK. This is achieved by cutting the luc gene from Luc _FF and cloning into the luxAB gene. A bioluminescent reporter plasmid for detecting low glucose concentrations was prepared using the previously constructed PEPCK promoter CA.
It is constructed by replacing the chloramphenicol transferase (CAT) gene in the T fusion (Petersen et al., 1988; Quinn et al., 1988) with the luxAB gene. The resulting plasmid was transformed into E. coli. E. coli,
The plasmid DNA was then extracted and purified twice on a CsCl gradient.

Pancreatic islet cells and hepatoma cells have been described previously (German et al., 1990;
(Petersen, 1988) and can be co-transfected with the bioluminescent reporter construct and a plasmid containing the constitutively expressed luxCDE gene constructed in the plasmid of interest. This configuration results in cells maintaining a pool of aldehyde substrates available for the reporter gene (luxAB). Cells are grown at glucose concentrations ranging from 3 mM to 30 mM,
Screen for light generation. The transfected cells are washed, concentrated, and placed in a microwell in a light-tight chamber, which is then connected to an integrated circuit. Different concentrations of glucose and assay medium (Kennedy et al., 1997) are added to the cells to test the sensitivity and response time of glucose BBIC. After initial characterization, the bioluminescent glucose reporter can also be tested in a flow cell. Cells are placed in encapsulated media on an integrated circuit, and media containing different concentrations of glucose (3-30 mM) is perfused through these cells to test for a dramatic response.

5.7 Example 7-BBIC and Cancer Detection During Diabetes Colon cancer is the leading cause of cancer death, second only to lung cancer in the United States, and the incidence increases with age However, 97% of colon cancers occur in people over the age of 40 (Coppola and Karl, 1998). Although most cases of colon cancer are sporadic, in 15% of patients there is a strong familial history of similar tumors in first-degree relatives (Coppola and Karl,
1998). These familial cancers (eg, hereditary non-polyposis colon cancer (HNP)
CC) and familial adenomatous polyposis (FAP)) result from autosomal dominant inherited mutations in putative tumor suppressor genes, and the spectrum of lesions
Hyperplasia-dysplasia-adenomas-arising from cancer (Coppola and Karl, 19
98). As many of the early molecular pathologies are known for hereditary colon cancer, they represent a useful model for developing new biosensor strategies for early clinical detection. Biosensors are hybrid devices that combine biological components with a computerized measurement transducer.

This example describes the adaptation of an implantable biosensor device, allows for early detection of cancer, and enables means for monitoring remission and cancer recurrence. Because the minimized biosensor of the present invention is small enough to be implantable and can be used in conjunction with a reporter system engineered to generate light without the need for cell lysates or additional substrates, an implantable device A powerful tool for early diagnosis of colon cancer in the form of is now possible for the first time.

As described above with respect to glucose and other metabolite biosensors, the inducible reporter system utilized is based on the use of an X. coli in a eukaryotic reporter cell. l
It is based on the luxAB gene and luxCDE gene from luminesceus. Thus, the expression of specific genes or their products can be detected by the expression of bioluminescence by the BBIC device. When eukaryotic reporter cells are treated with mitomycin C, the cells are unable to divide but can still respond metabolically,
Then, a quantitative bioluminescence signal is generated.

[0167] Colon cancer is the leading cause of the second cancer death in the United States, with at least 50% of this population developing colorectal tumors by age 70 (Kinzler and Vo).
gelstein, 1996). Although most cases of colorectal cancer are sporadic, 15% have inherited cancer syndrome, familial adenomatous polyposis (FAP).
) And hereditary non-polyposis colorectal cancer (HNPCC) (Kin
zler and Vogelstein, 1996). Familial adenomatous polyposis is a syndrome characterized by the development of hundreds to thousands of adenomas or polyps in the colon and rectum, only a few develop into invasive cancers (Kinzle
r and Vogelstein, 1996). adenomatous pol
Loss of function of both alleles of the yposis coli (APC) tumor suppressor gene predisposes humans to developing malignant cancers (Coppola and Mar)
ks, 1998). In addition, most sporadic colon cancers are also found to contain specific mutations in the APC gene (Kinzer and Vogelstei).
n, 1996). In hereditary non-polyposis colorectal cancer, there is significant microsatellite instability secondary to mutations in DNA mismatch repair genes (eg, hMSH2 and hMSH1); single, advanced tumors develop in young And is usually restricted to the right colon (Coppola and Karl, 1
996; Smyrk, 1994). Cells with mutations in the APC are generally aneuploid from loss of all compartments of the chromosome, whereas hMSH2 or hMSH2.
Cells having a mutation in H1 are euploid.

The molecular phenomena that cause the development of colon neoplasia are fairly well understood (Kin
zler and Vogelstein, 1996). Humans who are completely deficient in APC develop lesions of the colon called dysplastic crypt foci that lead to early adenoma progression (Kinzler and Vogelstein, 1996). Other mutations (eg, mutations in K-Ras or p53) begin to accumulate and tumors progress to late adenomas, cancers and metastatic cancers (Kinzler and Vo)
gelstein, 1996). A similar progression is seen in HNPCC as well. Because the order of this genetic event is fairly well understood for both these types of cancer, these genetic events can be used to detect in vitro one or more modified cells. Represents an excellent model for the development of specific diagnostic tests with sensitivity.

The APC gene encodes a cytoplasmic protein localized at the end of microtubules in the focal adhesion complex (Kinzler and Vogelstein, 199).
6). As cells move through the crypts, the expression of APCs becomes terminally differentiated,
And increase until the localized colon epithelial cells undergo apoptosis (Ki
nzler and Vogelstein, 1996). Cadherin is a transmembrane protein that is localized to focal adhesion plaques in most epithelial cells (Ap
lin et al., 1998). The carboxy terminus of each cadherin catenins to interact with known cytoplasmic structural proteins (Aplin et al., 1998). There are three types of catenin: β-catenin binds to the cytoplasmic domain of cadherin;-catenin binds to β-catenin and actin cytoskeleton via actinin; Function in place of β-catenin (Aplin et al., 1998). β-catenin is also part of a signaling pathway that includes the secreted glycoprotein Wnt and glycogen synthase kinase 3 (GSK3) (Aplin et al., 1998). APC is a component of the Wnt-β-catenin-GSK3 pathway (β-catenin and γ-catenin, GSK
3 (including Tubulin) (Aplin et al., 1998).
Most of the mutations in colorectal cancer are in the carboxy-terminal region of APC,
As a result, APC no longer binds β-catenin (Aplin et al., 1998).
). In fact, -catenin is downstream of the APC and is important for its function as a tumor suppressor gene (Aplin et al., 1998). When the Wnt pathway is inactivated, GSK3 phosphorylates the N-terminus of β-catenin and targets it for degradation by the ubiquitin pathway (Munemitsu et al., 1996).
When β-catenin accumulates, it activates gene transcription via the transcription factor Lef-1 / TCF (Morin et al., 1997). APC works in concert with GSK3 to inhibit -catenin-mediated transcriptional activity (Kinzler and Vo)
gelstein, 1996).

In hereditary non-polyposis colon cancer, microsatellite instability is the result of mutations in one or more DNA mismatch repair genes (Jirnicy).
1998; Nicholides et al., 1994). At least 90% HNP
CC tumors have microsatellite instability (Karran 1996;
Smyrk 1994). One potential marker for microsatellite instability in colorectal tumors is the inactivation of the type II receptor for TGF-β (Markowitz et al., 1995). Decreased function of βRII is due to HNPC
C associated with decreased colorectal adenoma growth regulation and tumor progression in C (
Wang et al., 1995). Other signaling components of TGF-β involved in colorectal tumorigenesis include mutations in Smad3 and Smad4, both of which result in the development of colorectal adenocarcinoma in mice (Zhu et al., 1998)
Takaku et al., 1998). Decreased function of βRII is a useful marker of early foci in HNPCC (Markowitz et al., 1995).

The mutations in APC are the most common mutations in colorectal cancer,
A reporter construct for cellular transcription factor (Tcf) was devised to screen multiple colon cancer cell lines for activation of transcriptional activity. Mutations in either APC or β-catenin result in activation of Tcf-responsive transcription via accumulation of non-phosphorylated cytoplasmic β-catenin (Morin et al., 1997), and detection of reporter construct activation Useful as markers for mutations in any of these genes. Vector pDISPLAY (Invitro
gen) allows the expression of the promoter of Tcf on the surface of bioreporter cells; this construct consists of the following tandem set of Tcf promoters: one upstream of the luxAB gene and the other upstream of luxCDE. Excess β-
In the presence of catenin, this promoter construct stimulates reporter activity and bioluminescence occurs.

[0172] Once the HepG2 and HeLa cells had the pcx encoding the luxAB gene
When transfected with DNA3, these cells are attached to a biosensor chip. It is necessary to ensure that these cells cannot divide, so that after transcription and sorting, these cells are given 6,000 rad from a ⁶⁰ Co source.
Γ-radiation (UT College of Veterinary M)
editine). In some embodiments, it may be necessary to attach cells to the biochip before irradiation so that efficient attachment can occur. Alternatively, the cells are treated with mitomycin C to prevent further mitosis. Biochips can be coated with Matrigel, a basement membrane material that promotes epithelial cell attachment. An alternative approach is to suspend these cells in Matrigel and form a gel on the surface of the biochip. These cells are then
It is immobilized in the basement membrane material and is not subjected to frictional removal. Optionally, the surface of the chip can be modified by adding a net charge (eg, poly-L-lysine), coating the surface with a surgical tissue adhesive, or allowing the biopolymer to adhere to the surface. It can be modified by adding some other surface modification that can be attached. Since mutations in APC are the most common mutations in colorectal cancer, reporter constructs for T cell transcription factor (Tcf) can be devised to screen multiple colorectal cancer cell lines for activation of transcriptional activity. .

The present invention also provides biosensors that can be used for endoscopic screening of colonic mucosa to detect the presence of mutant cells before the onset of global morphological changes. For the sensitivity required to detect small foci of malignant transformation, it may be necessary to attempt simultaneous detection of more than one abnormality. For example, many colon tumors,
In particular, tumors with mutations in APC are cyclooxygenase-2 (COX-2
) And secretes large amounts of prostaglandins (Kutcher
a et al., 1996; Sheng et al., 1997; Coffey et al., 1997; Kin.
zler and Vogelstein, 1996). Cyclooxygenase-2
Is an early response gene that is not constitutively expressed but is stimulated in colon epithelial cells by growth factors and tumor promoters (Kutchera et al., 1996)
Sheng et al., 1997; Coffey et al., 1997). In the intestinal lumen,
It is possible to bioengineer the reporter cells to bioluminescent in the presence of increased levels of prostaglandins. Prostaglandins pass freely through the cell membrane and enter the cytoplasm of the reporter cell,
The reporter construct can be activated. Engineering reporter cells to detect increased levels of prostaglandins by use of the cyclooxygenase-2 promoter fused to the luxAB gene may also be advantageous for early detection of colon cancer. Since prostaglandin levels can increase in inflammation as well as neoplasia, this approach lacks the appropriate specificity for cancer diagnosis. However, this approach is useful in determining which patients will benefit from treatment with a specific cyclooxygenase inhibitor.

6.0 References The following references, to the extent that these references provide illustrative procedures or other detailed supplements to those described herein, are provided. In detail for reference.

US Pat. No. 4,332,898 (issued June 1, 1982) US Pat. No. 4,342,832 (issued August 3, 1982) US Pat. No. 4,356,270 (1982) U.S. Pat. No. 4,362,817 (promulgated on December 7, 1982) U.S. Pat. No. 4,371,625 (promulgated Feb. 1, 1983) U.S. Pat. No. 884 (issued on Aug. 15, 1995) US Pat. No. 4,637,391 (issued on Jun. 16, 1987) US Pat. No. 4,944,659 (issued on Jul. 31, 1990) US Pat. U.S. Pat. No. 5,387,247 (issued on Feb. 7, 1995) U.S. Pat. No. 5,421,816 (issued on Jul. 6, 1995) US Patent No. 5,4 US Patent No. 5,474,552 (promulgated on December 12, 1995) US Patent No. 5,569,186 (promulgated on October 29, 1996) US Patent No. 5,620,883 (issued on April 15, 1997) US Patent No. 5,653,755 (issued on August 5, 1997) US Patent No. 5,711,960 (on June 27, 1998) U.S. Pat. No. 5,779,734 (promulgated on July 14, 1998) U.S. Pat. No. 5,795,790 (promulgated on Aug. 18, 1998) U.S. Pat. No. 5,800,420 (1998) U.S. Pat. No. 5,814,091 (promulgated on September 29, 1998)

[0176]

(Equation 12) All compositions, methods, devices, apparatus and systems disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the methods, devices, apparatus and systems of the present invention have been described with reference to preferred embodiments, changes may be made in these methods, devices, apparatuses and systems, and without departing from the spirit, scope and scope of the present invention. It will be apparent to one skilled in the art that the steps or sequence of steps of the methods described herein may be applied. More specifically, it is clear that certain reagents, both chemically and physiologically relevant, can replace the reagents described herein, but achieve the same or similar results. . All such similar substitutions and modifications apparent to those skilled in the art are
It is considered to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the exclusive right claiming a patent is as set forth in the following claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a perspective view of an exemplary embodiment of the present invention.

FIG. 2 is a side view of an exemplary embodiment of the present invention.

FIG. 3 shows a block diagram of an exemplary embodiment of an integrated circuit.

FIG. 4A illustrates a high quality photodetector that can be fabricated using a standard N-well CMOS process.

FIG. 4B shows two photodetector structures fabricated in a CMOS process on silicon on an insulating layer. The left is a lateral PIN detector and the right is a device similar to the left except that the bond is formed by a Schottky junction.

FIG. 5A shows a single photodiode comprising a P diffusion layer, an N well, and a P substrate.

FIG. 5B illustrates a circuit that provides a forward bias current to cancel photocurrent using a large area photodiode for efficient light collection and a small area diode for a feedback loop.

FIG. 5C illustrates a circuit that uses correlated double sampling (CDS) to minimize the effects of low frequency (flicker) amplifier noise as well as time or temperature dependent variations in amplifier offset voltage.

FIG. 6 shows the current-to-frequency converter architecture of the device.

FIG. 7 shows a prototype BBIC biosensor.

FIG. 8 shows the minimum detectable concentration of toluene as a function of the integration time of a prototype BBIC using Pseudomonas putida TVA8.

FIG. 9 shows a schematic of a peritoneal glucose biosensor and an insulin pump.

FIG. 10A shows a schematic of an implantable biosensor that includes two additional photodetectors with a bioreporter responsive to either increasing or decreasing glucose concentration. FIG. 10B shows a side view of the biosensor showing the silastic cover.
FIG. 10C shows a schematic of the use of a selectable dialysis membrane to protect the bioreporter from the immune response.

[Procedure amendment] [Date of submission] May 1, 2002 (2002.5.1) [Procedure amendment 1] [Document name to be amended] Description [Item name to be amended] Claims [Amendment method] Change [Content of amendment] [Claims] [Procedure amendment 2] [Document name to be amended] Specification [Item name to be amended] 0027 [Correction method] Change [Details of amendment] The present invention is also portable. Provides a controlled drug delivery system, comprising both a self-contained miniature bioluminescent bioreporter integrated circuit ("BBIC") device and an implantable drug delivery pump, the pump being operable by the BBIC. It can be controlled and can deliver the drug to the animal's body in response to control by the device. The invention also relates to a method of providing a controlled supply of a drug to a patient in need thereof. The method generally involves implanting a controlled drug delivery system into the patient's body. [Amendment 3] [corrected document name] specification [corrected item name] 0057 [correction method] change [Correction contents] [0057] Figure 4 A is made using a standard N-well CMOS process , A high quality photodetector. This photodetector consists of two parallel reverse bias diodes.
The upper diode is formed between the P + active layer 45 and the N well 46, and the lower diode is formed between the N well 46 and the P substrate 47. The upper diode has good short wavelength light s
The lower diode provides good long-wavelength photosensitivity (500-1100 nm), while having an activity (400-550 nm). Therefore, the entire diode has photosensitivity over the range of 400 to 1100 nm. The luminescent compound 41 under test is separated from the photodetector by the Si ₃ N ₄ layer 40 and the SiO ₂ layer 42. [Amendment 4] [corrected document name] second structure shown on the right side of the specification [corrected item name] 0065 [correction method] change [Correction contents] [0065] Figure 4 B, the bonding is deposited Cobalt silicide (Co
Similar to the first structure, except that it is formed from a Schottky junction between a Si ₂ ) or other suitable material layer and the lightly doped active layer. [Amendment 5] [corrected document name] specification [corrected item name] 0069 [correction method] change [Correction contents] [0069] Figure 5 A is a first approach to detect very small signals Is schematically shown. This device has a structure that is compatible with standard CMOS IC processes.
Using a diffuse / N-well photodiode in open circuit mode with a readout amplifier (manufactured on the same IC with the photodiode) The luminescent signal creates electron-hole pairs in the P-diffusion and N-wells. The photo-generated electrons during P-diffusion are injected into the N-well, while the photo-generated holes in the N-well are
Injected during diffusion. Since the N-well is coupled to ground potential, no charge accumulates in this region. However, since the P-diffusion is only attached to the input impedance of the CMOS amplifier (approaching infinity at low frequencies), positive charges accumulate in this region. Thus, the voltage on the P-diffusion node begins to rise. [Amendment 6] [corrected document name] specification [corrected item name] 0073 [correction method] change [Correction contents] [0073] Figure 5 B, a second micro-luminometer satisfy both these requirements An embodiment will be described. This circuit uses a large area photodetector for efficient light collection,
A small area diode is used in the feedback loop to provide a forward bias current that cancels the photocurrent. Again, the amplifier and feedback diode are manufactured on the same IC as the photodetector. For this circuit, V _out = 3 V _t ln ((I _p + I _g −I _r ) / (A _fb I _s ) +1) (Equation 3) where A _fb is the small cross-sectional area of the feedback diode. . More than one diode is used in the feedback path, and the DC of any subsequent amplifier amplifier stage
Generates a large output signal compared to the offset. This method allows for efficient collection of light using a large area photodetector, while still producing a large output voltage with a small area diode in the feedback path. [Amendment 7] [corrected document name] specification [corrected item name] 0074 [correction method] change [Correction contents] [0074] Feedback circuit of FIG. 5 B maintains photodetector at zero bias. If no potential is applied, the recombination and generated currents should cancel. Equation 3 becomes V _out = 3V _t ln ((I _p / (A _fb I _s )) + 1) (Equation 4) when the smaller recombination current and generated current in the smaller feedback diode are negligible. [Amendment 8] [corrected document name] specification [corrected item name] 0075 [Correction Procedure] The main advantage of embodiments of the changes [correction contents] [0075] The second micro-luminometer shown in FIG. 5 B Includes: [Amendment 9] [corrected document name] specification [corrected item name] 0081 [correction method] change [Correction contents] [0081] The third micro luminometer shown in FIG. 5 C implementation, a low frequency ( Flicker) Use correlated double sampling (CDS) to minimize the effects of amplifier noise and time or temperature dependent changes in amplifier offset voltage. Figure 5 C
As shown, a photodiode having a capacitance C _d and a noise power spectral density S _i is coupled through a set of switches controlled by the logic level of the flip-flop output to a feedback capacitance C _f and an input noise power spectral density S _i. Connected to an integrating preamplifier with _v . The flip-flop output is L
When OW, the switch is positioned so that the photocurrent flows out of the preamplifier, thereby increasing the output voltage of the integrator. When the output voltage of the integrator, filtered by the low-pass filter, exceeds the threshold V _HI , the upper comparator “wakes up”, sets the flip-flop, and sets its output to HIGH. The detector switches the change position to allow current to flow into the integrating amplifier. As a result, the output voltage of the amplifier decreases. When the output of the integrator falls below the second threshold value V _LO , the lower comparator outputs “
Activate "to reset the flip-flop and bring its output low again. The above process is repeated as long as the photocurrent is present. [Procedure amendment 10] [Document name to be amended] Description [Item name to be amended] Brief description of drawings [Correction method] Change [Contents of amendment] [Brief description of drawings] [FIG. 1] FIG. FIG. 2 is a perspective view of an exemplary embodiment with a stub. FIG. 2 is a side view of an exemplary embodiment of the present invention. FIG. 3 shows a block diagram of an exemplary embodiment of an integrated circuit. FIG. 4 shows a bioreporter supplied with water and nutrients. FIG. 4A illustrates a high quality photodetector that can be fabricated using a standard N-well CMOS process. FIG. 4B shows two photodetector structures fabricated in a CMOS process on silicon on an insulating layer. The left is a lateral PIN detector and the right is a device similar to the left except that the bond is formed by a Schottky junction. FIG. 5A shows a single photodiode comprising a P diffusion layer, an N well, and a P substrate. FIG. 5B illustrates a circuit that provides a forward bias current to cancel photocurrent using a large area photodiode for efficient light collection and a small area diode for a feedback loop. FIG. 5C illustrates a circuit that uses correlated double sampling (CDS) to minimize the effects of low frequency (flicker) amplifier noise as well as time or temperature dependent variations in amplifier offset voltage. FIG. 6 shows the current-to-frequency converter architecture of the device. FIG. 7 shows a prototype BBIC biosensor. FIG. 8 shows the minimum detectable concentration of toluene as a function of the integration time of a prototype BBIC using Pseudomonas putida TVA8. FIG. 9 shows a schematic of a peritoneal glucose biosensor and an insulin pump. FIG. 10A shows a schematic of an implantable biosensor that includes two additional photodetectors with a bioreporter responsive to either increasing or decreasing glucose concentration. FIG. 10B shows a side view of the biosensor showing the silastic cover.
FIG. 10C shows a schematic of the use of a selectable dialysis membrane to protect the bioreporter from the immune response. [Procedure amendment 11] [Document name to be amended] Drawing [Item name to be amended] Fig. 4 [Correction method] Added [Content of amendment] [Fig. 4] [Procedure amendment 12] [Document name to be amended] Drawing [Item name to be amended] Fig. 9 [Correction method] Change [Content of amendment] [Fig. 9] [Procedure amendment 13] [Document name to be amended] Drawing [Item name to be amended] Fig. 10 [Correction method] Change [Content of amendment] [Fig. 10]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/10 G01N 27/30 A G01N 27/30 33/483 F 27/414 A61B 5/14 310 33 / 483 301U (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ) , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO , RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Sailor, Gary S . United States Tennessee 37709, Blaine, McKinney Road, Box 60, Route 2 (72) Inventors Simpson, Michael El. United States Tennessee 37919, Knoxville, Nuvin Ridge Road 7745 (72) Inventor Applegate, Bruce M. United States Tennessee 37919, Knoxville, Sutherland Avenue 3700 (72) Inventor Rip, Steven A. United States Tennessee 37906, Knoxville, No. 137, Sunbeam Lane 6020 F-term (reference) 2G045 AA13 CA25 CA26 DA31 FB05 4C038 KK10 KL01 KL09 KX01 KY10 4C084 AA16 MA65 NA13 ZC351 4C085 HH11 HH20 JJ11 KA26 KB82 JJ11 KA26 KB82 KB82 EJ11 KB99 KA11 GG16

Claims (45)

[Claims] 1. An implantable monolithic bioelectronic device for detecting an analyte in an animal body, the device comprising: (a) a bioreporter on top of an integrated circuit substrate. Wherein the bioreporter is capable of metabolizing the analyte when in contact with the analyte and emitting light as a result of the metabolism; and (b) in proximity to the integrated circuit. A sensor arranged to generate an electrical signal in response to receiving the luminescence, wherein the device is disposed within a biocompatible container implanted within the body of the animal. Contained, implantable, monolithic bioelectronic device. 2. The implantable monolithic bioelectronic device of claim 1, wherein said biocompatible container comprises a polymer matrix. 3. The implantable monolithic bioelectronic device of claim 2, wherein said polymer matrix comprises polyvinyl alcohol, poly-L-lysine, or alginate. 4. The implantable monolithic bioelectronic device of claim 2, wherein the polymer matrix further comprises a pore hydrogel, a mesh reinforced hydrogel, or a filter supported hydrogel. 5. The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit comprises a light converter. 6. The implantable monolithic bioelectronic of claim 5, further comprising a transparent biocompatible bioresistant separator operatively disposed between said light converter and said bioreporter. Engineering device. 7. The implantable monolithic of claim 1, wherein the bioreporter comprises a plurality of eukaryotic or prokaryotic cells that produce a bioluminescent reporter polypeptide in response to the presence of the analyte. Bioelectronic devices. 8. The implantable monolithic bioelectronic device of claim 7, wherein said plurality of prokaryotic cells comprises bacteria. 9. The implantable monolithic bioelectronic device of claim 7, wherein said plurality of eukaryotic cells comprises mammalian cells. 10. The implantable monolithic bioelectronic device of claim 9, wherein said plurality of eukaryotic cells comprises islet β cells, immortal stem cells, or hepatocytes. 11. The implantable monolithic bioelectronic device of claim 10, wherein said plurality of eukaryotic cells comprises recombinant human immortal stem cells. 12. The implantable cell of claim 7, wherein said plurality of cells comprises a nucleic acid segment encoding a luciferase polypeptide or green fluorescent protein produced by said cells in response to the presence of said analyte. Monolithic bioelectronic device. 13. The nucleic acid segment according to claim 1, wherein the nucleic acid segment is Aqueorea Victor.
13. The implantable monolithic bioelectronic device of claim 12, wherein the device encodes ia or Renilla reniformis green fluorescent protein. 14. The implantable monolithic bioelectronic device of claim 12, wherein said nucleic acid segment encodes a humanized green fluorescent protein. 15. The implantable monolithic bioelectronic device of claim 12, wherein the nucleic acid segment encodes a bacterial Lux polypeptide. 16. The method according to claim 16, wherein the nucleic acid segment is a bacterial LuxA polypeptide, L
uxB polypeptide, LuxC polypeptide, LuxD polypeptide, or LuxE polypeptide, or LuxAB fusion polypeptide or LuxCD
16. The implantable monolithic bioelectronic device of claim 15, which encodes an E-fusion polypeptide. 17. The method according to claim 17, wherein the nucleic acid segment is Vibrio fischeri.
i or Xenorhabdus luminescens LuxA polypeptide, LuxB polypeptide, LuxC polypeptide, LuxD polypeptide,
Or LuxE polypeptide, or LuxAB fusion polypeptide or L
17. The implantable monolithic bioelectronic device of claim 16, which encodes a uxCDE fusion polypeptide. 18. The method according to claim 18, wherein the nucleic acid is Xenorhabdus luminesc.
ens LuxA polypeptide, LuxB polypeptide, LuxC polypeptide, LuxD polypeptide, or LuxE polypeptide, or LuxAB
2. The fusion polypeptide or encodes a LuxCDE fusion polypeptide.
An implantable monolithic bioelectronic device according to claim 7. 19. The implantable monolithic bioelectronic device of claim 18, wherein said polypeptide is encoded by a sequence comprising at least 25 contiguous nucleotides from SEQ ID NO: 1. 20. The implantable monolithic bioelectronic device of claim 19, wherein said polypeptide is encoded by a sequence comprising at least 30 contiguous nucleotides from SEQ ID NO: 1. 21. The implantable monolithic bioelectronic device of claim 20, wherein said polypeptide is encoded by a sequence comprising at least 35 contiguous nucleotides from SEQ ID NO: 1. 22. The implantable monolithic bioelectronic device of claim 16, wherein expression of said nucleic acid segment is regulated by a nucleic acid sequence comprising a cis-activating element responsive to the presence of said analyte. 23. The cis-activation responsive element is S14 gene sequence, liver L-pyruvate kinase gene sequence, liver 6-phosphofructo-2-kinase gene sequence, β-islet insulin gene sequence, mesangial transforming growth factor. 23. The implantable monolithic bioelectronic device of claim 22, which is a nucleotide sequence selected from the group consisting of a -β gene sequence and an acetyl coenzyme A carboxylase gene sequence. 24. The implantable implant of claim 23, wherein said cis-activation responsive element comprises a contiguous nucleotide sequence from a β-islet insulin gene sequence or from a liver L-pyruvate kinase gene sequence. Monolithic bioelectronic device. 25. The implantable monolithic bioelectronic device of claim 12, wherein expression of said nucleic acid sequence is regulated by a promoter sequence derived from an L-pyruvate kinase encoding gene. 26. The implantable monolithic bioelectronic device of claim 1, wherein the analyte is glucose, glucagon or insulin. 27. The method further comprising a source of a nutrient capable of sustaining the cells.
An implantable monolithic bioelectronic device according to claim 7. 28. The implantable monolithic bioelectronic device of claim 1, further comprising a wireless transmitter. 29. The implantable monolithic bioelectronic device of claim 1, further comprising an antenna. 30. The implantable device of claim 1, further comprising an implantable drug delivery pump, wherein the pump can be controlled by the device and deliver the drug to the body of the animal. Monolithic bioelectronic device. 31. The biocompatible container further comprises a membrane permeable to the analyte but not permeable to the bioreporter.
An implantable monolithic bioelectronic device according to claim 1. 32. The implantable monolithic bioelectronic device of claim 1, wherein said bioreporter expresses said light-emitting polypeptide following metabolism of said analyte by said bioreporter. 33. The implantable monolithic bioelectronic device of claim 2, wherein said biocompatible container comprises silicon nitride or silicon oxide. 34. The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit is a complementary metal oxide semiconductor (CMOS) integrated circuit. 35. The implantable monolithic bioelectronic device according to claim 5, wherein the light converter comprises a photodiode. 36. The implantable monolithic bioelectronic device of claim 1, wherein said integrated circuit further comprises a photodiode and a current-to-frequency converter. 37. The implantable monolithic bioelectronic device of claim 1, wherein the integrated circuit further comprises a current-to-frequency converter and a digital counter. 38. The implantable monolithic bioelectronic device of claim 1, further comprising a transmitter. 39. The implantable monolithic bioelectronic device of claim 38, wherein said transmitter is capable of transmitting digital data. 40. An implantable controlled drug delivery system comprising the device of claim 1 and an implantable drug delivery pump that can be operatively controlled by the device. Drug delivery system. 41. A method of providing a controlled delivery of a drug to a patient in need thereof, wherein the controlled delivery system of claim 40 is implanted in the body of the patient. A method comprising the steps of: 42. A method for determining the amount of a drug required by a patient in need of the drug, the method comprising: implanting the device of claim 1 in the body of the patient; Determining the amount of drug required by the patient based on the output. 43. A kit for detecting an analyte, comprising a device according to claim 1 and instructions for using the device. 44. The kit of claim 43, further comprising a standardized reference solution. 45. A method of regulating blood glucose levels in an animal in need of regulating blood glucose levels, said method comprising using a device according to claim 1 or a kit according to claim 43 to treat a patient with a blood glucose level. Monitoring the level of glucose in the bloodstream or interstitial fluid of the subject, and administering to the patient an effective amount of an insulin composition sufficient to regulate the blood glucose level.