CN1664560A - Multiple channel surface plasma resonant image sensor based on-chip PCR - Google Patents

Abstract

The invention relates to a biosensor based on the surface plasma resonance principle, in particular to use the biology PCR technology combined with it. Which including the following steps: a) combining the SPR sample pond with the PCR reflecting pond and formatting the sensor reflecting pond; (b) the top of the sensor body is a rectangular reflecting sensor pond made up of the silicon chip, the lower is a cylinder prism, the two sides of the sensor body are set with the incident path, exit path and the computer plate connected with the end of the exit path;(c) the homogeneous light incidences, using the single color surface CCD to collect SPR picture and analyze through it, measuring the resonance and the change of it, which means the response of each channel. The invention has for characters: (a) measuring several samples once (b) measuring different characters of a sample once; (c) setting the consulting channel in the multi-channel and avoiding the effect of non-specific; (d) measuring the DNA molecule of low concentration.

Description

Multi-channel surface plasma resonance image sensor based on-chip PCR

Technical Field

The invention relates to a biosensor based on the surface plasma resonance principle, in particular to a biosensor manufactured by combining the biological PCR technology and the plasma resonance principle, which can detect a plurality of biological signals in parallel.

Background

The 21 st century is an era of biological development, and particularly bioinformatics, which is generated by the fusion of bioscience and computer science, will be developed vigorously. The sensing technology is one of the main technologies of information science and is a means for acquiring information. The use of sensing technology to obtain information from biological samples is an important aspect of the development of biological detection technology.

Among them, optical methods are considered to be the most mature and best biosensing technology due to their non-destructive and high sensitivity characteristics. Since the first discovery of the Surface Plasmon Resonance (SPR) phenomenon in optical experiments by Wood in 1902, studies of SPR instruments and SPR biosensors have been receiving attention. Surface Plasmon Resonance (SPR) is a plasmon oscillation of free electrons present at the edge of a metal. These oscillations are influenced by the refractive index of the material adjacent to the metal surface. This phenomenon is used to detect subtle changes in the surface refractive index and forms the basis of various sensor mechanisms. One of the requirements for the formation of SPR phenomenon is the presence of an interface between the metal and the dielectric. When the incident light is incident at a specific angle, the reflectivity thereof is significantly reduced, and the incident angle is referred to as the SPR angle. The substances attached to the metal surface have different SPR angles, and the same substance has different SPR angles depending on the amount of the substance attached to the metal surface. According to the above principle, the SPR biosensor can immobilize a known biomolecule on a surface of a metal film several tens nm thick, when it is bound to a complementary target biomolecule, the SPR angle is changed due to a change in the surface structure, and the kind and concentration of the bound target biomolecule can be known from the changed value of the SPR angle.

SPR was visualized by imaging, which was first proposed by yeastman in 1987. Bengt Ivarsson proposes a further idea: a multi-wavelength asynchronous SPR imager is used. SPR with biosensors was used first in 1983 and was applied to image analysis in 1987. With the advent of SPR image analysis, a number of new, label-free, real-time, multi-point biochemical analysis devices have been developed for increasing the amount of detection per unit time. There are three methods for measuring SPR images. The first is achieved by measuring the intensity of reflected light at a specific wavelength and resonance angle on the SPR tilt angle side (i.e. reflectance method); the second, fixed angle of incidence, detects the resonant wavelength of polychromatic light (wavelength interrogation method). The third method is the detection of polarization state and phase (phase interrogation method).

In recent years, SPR sensors have been widely used in a variety of fields such as vaccine development, disease diagnosis, drug target and drug development, case detection, environmental monitoring, food safety inspection, and stimulant detection due to their advantages of real-time, label-free detection, high sensitivity and miniaturization. In particular in biosensors, the use of SPR to detect antibodies and their response to antigens is a major concern in biomedical diagnostics, where the presence of antibodies associated with bacteria or viruses is an important indicator of infection. SPR can also be used for gene probes where deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) linked to a defined sequence in the target analyte can be used.

Biosensor products based on the SPR principle are available in the world, for example, the Biacore series biosensor of Biacore AB, Sweden, which is prepared by fixing a 100nm thick gold film on a glass substrate, embedding the glass sheet in a plastic plate holder, and coupling the chip to a glass prism using a polymer with an index of refraction matching the prism. The detection of single-point SPR is only completed, only one substance can be detected at one time, and the detection capability of the single-point SPR is not provided with the image detection capability, so that the capability of screening various DNA sequences in one test is severely limited, and the application of the single-point SPR is limited to a certain extent. Moreover, the equipment is large in size and expensive, which limits the use in China. Therefore, the development of SPR sensors with independent intellectual property rights, compact structure and reasonable price is a common pursuit target of engineering technicians in the field in China.

It has been found by literature search that US 5,313,264 describes an SPR sensorcomprising a network of sample fluid manipulation modules formed of micro-conduits and valves for moving an appropriate volume of fluid containing a detection substance over the surface of the conductors forming the detection layer SPR and then to a target fluid located above the detection layer. The SPR sensor is also composed of a monochromatic light source and an optical system. The optical system generates light provided by the light source to form a wedge-shaped converging light beam and directs the light beam to the sensor surface. The wedge-shaped beam impinges on the detection layer along a spatially fixed, relatively narrow sensor surface within a range of resonance angles. The range of the resonance angle is determined by the convergence angle of the wedge-shaped light beam. The reflected light is imaged on a two-dimensional photodetector. The signal provided on the photodetector is a measure of the change in the refractive index of the surface of the detection layer, which is caused by the interaction of the substance attached to the detection layer with the solution to be measured flowing through the surface of the detection layer through the sample liquid treatment module.

Many conventional methods and instruments for forming probe layer SPR require the sample liquid to flow through the detection layer and optically scan the sensor surface, and these methods are relatively complex, uneconomical and time consuming. The sensor is complex in design, large in size and not beneficial to miniaturization of instruments. There is a need for a sensor with selective SPR that can have multiple levels of analytical probes while pumping sample fluid through several channel detection layers.

The US 2003048452 patent describes a two-dimensional image SPR instrument based on optical analysis of a sample area by a sensor surface. The instrument contains a sensor surface layer made of a conductive material capable of responding to SPR, such as a number of free-charged metals: gold, silver, aluminum, and the like. A beam of electromagnetic waves of two or more wavelengths is irradiated from the front end or the rear end of a two-dimensional surface area of the sensor, and at the same time, the reflection intensities of the two or more wavelengths on the sensor surface are detected, and the detection means provides a two-dimensional or multi-dimensional surface image in which the effective refractive index of each point of the surface is displayed as a two-dimensional image constituting a color image. However, the SPR sensor apparatus employs an operation mode in which the incident light angle is fixed and the wavelength is variable. The sensitivity of these devices is limited by the spectrometer resolution, temperature variation, and wavelength of the incident light, and the use of polychromatic light together can achieve high sensitivity and accuracy over a relatively large dynamic range.

The successful use of nucleic acid array technology in life science research has saved time over the traditional methods for quantitative detection of complex biological systems, but this technology has been limited due to the high cost of quantitative detection of biomolecules, their radioactivity, and the need for fluorescent labeling and detection. US 20030049639 describes a method for resonance SPR image detection based on nucleic acid microarray for large scale identification of biomolecules. The corrected polychromatic light irradiates on the nucleic acid molecule membrane on the surface of the chemically modified gold membrane in the SPR angle range, and the reflected light is collected by the CCD through a band-pass filter (830nm) with the width of 10 nm. The signal collected by CCD can detect the different nucleic acid molecule reflectivity on the surface of gold film. Since different probes on the surface of the gold film bind different target molecules, biomolecules such as DNA and RNA can be detected quickly.

CN 1421699 describes a biosensor which can detect multiple biological signals in parallel based on the principle of Surface Plasmon Resonance (SPR). The biosensor consists of a substrate, a metal film, a chemical modification layer, a cross-linking agent layer and a biological monomolecular layer in sequence, and is characterized in that various biological molecules are combined at different positions of the biological monomolecular layer, and the concentration of specific target molecules in a biological sample can be quantitatively detected in parallel. The disadvantage of this design is that the measurement accuracy is not sufficient. The factors influencing the measurement accuracy of the SPR sensor are many, and the components, concentration and temperature of the sample solution and the functions of the molecules to be detected and the sensitive membrane in the sample solution are main factors influencing the detection accuracy. Except that the interaction between the molecules to be detected and the probe molecules on the sensitive film can change the dielectric constant of the sensitive film, so as to change the resonance angle or the resonance wavelength (specific response), the change of the dielectric constant of the sample liquid can be caused by the change of other components and the concentration thereof and the temperature change in the sample liquid, and the interaction between the molecules not to be detected in the sample liquid and the sensitive film can directly change the dielectric constant of the sensitive film; these changes will eventually cause a change in the resonance angle or resonance wavelength (non-specific response).

As mentioned above, these methods have three major drawbacks: the response time is low and the angular resolution is limited, and the measurement accuracy of the SPR sensor is not high enough. In addition, the minimum coverage of the surface of the biomolecule material which can be detected by the SPR sensing system at present is 1 multiplied by 10^-12g/mm²This is not sufficient for detecting low concentration, small molecular weight samples, and thus the sample concentration can be increased by PCR amplification, thereby improving the resolution of SPR detection.

Therefore, the combination of PCR technology and SPR technology can greatly improve the detection range and effect of SPR technology. Meanwhile, to improve the detection accuracy of specific responses, it is necessary to eliminate non-specific responses from actually measured responses.

Disclosure of Invention

The method adopted by the invention is as follows: monochromatic light is incident, the reaction tank adopts an MEMS (micro-electromechanical systems) process, and a PCR (polymerase chain reaction) reaction tank and an SPR (surface plasmon resonance) reaction tank are integrally manufactured, so that the amplification and the detection of DNA (deoxyribonucleic acid) molecules can be finished on one chip; the monochromatic area array CCD is used for collecting SPR images, resonance angles of all channels can be detected simultaneously through SPR image analysis, real-time and dynamic detection of SPR response curves is achieved, detection precision and speed are improved, use is convenient, and development trend of SPR sensors is met. The light path design of the sensing system enables incident light to simultaneously irradiate each channel at different angles within the angle range where resonance is likely to occur, and reflected light intensities of the incident light rays at different angles can be simultaneously detected through SPR images.

It is understood from the principles of the present invention that SPR techniques are particularly useful for the study of interactions between biomolecules, since no labeling or purification of the reactants is required. An advantage of SPR sensors is the ability to monitor the interaction of substance molecules, particularly biomolecules, in real time without interference. The advantage of using PCR is high sensitivity (the amount of PCR product generated is increased exponentially, the picogram-sized initial template to be detected can be amplified to microgram level, and the minimum detectable rate in bacteriology is 3 bacteria); simple and quick; the purity requirement of the specimen is low (without separating virus or bacteria and culturing cells, the crude product of DNA and total RNA can be used as an amplification model, and crude DNA amplification detection of clinical specimens such as blood, coelomic fluid, rinsing fluid, hair, cells, living tissues and the like can be directly used).

The technical scheme of the invention is as follows: the multichannel surface plasma resonance image sensor based on-chip PCR comprises a resonance SPR sample pool and a PCR reaction pool, and is characterized in that: it combines the resonance SPR sample pool and the PCR reaction pool) together to form a sensor reaction pool; the upper part of the sensor main body is a cuboid-shaped sensor reaction tank formed by silicon wafers, the lower part of the sensor main body is a cylindrical prism, a glass substrate is arranged between the sensor reaction tank and the sensor reaction tank, a sensitive film is arranged on the glass substrate, a sensor reaction tank of the silicon wafers is arranged on the sensitive film, a resonance SPR sample tank and a PCR reaction tank are arranged in the silicon wafer reaction tank, and a sample inlet and a sample outlet of sample liquid are arranged at the upper part of the silicon wafer sensor reaction tank;

an incident light path, an emergent light path and a computer platform (19) connected with the tail end of the emergent light path are arranged on two sides of the sensor main body; light emitted by a light emitting diode in an incident light path passes through a spherical lens, is converged at a pinhole, is converted into parallel light through a plano-convex lens, passes through an optical filter and a polarizing film in sequence, adjusts the position of the polarizing film to generate P polarized light, and finally carries out angular convergence on the parallel light by utilizing a cylindrical lens so that the parallel light is converged into a straight line at a sensitive film of the cylindrical prism and is emitted to the bottom surface of the cylindrical prism;

the emergent light path consists of a converging lens and an area array CCD, the incident light becomes parallel light after being totally reflected on the bottom surface of the cylindrical prism through the converging lens, the area array CCD finishes the collection of optical signals and then sends the parallel light to a computer for processing.

The detection method of the multi-channel surface plasma resonance image sensor based on-chip PCR comprises 10 steps.

The invention firstly provides a detection mode combining the PCR technology and the SPR technology, solves the problem that the SPR technology is difficult to measure low-concentration DNA molecules, simplifies the relatively complicated detection process after PCR amplification and improves the detection efficiency.

The invention overcomes the influence of nonspecific response on the measurement accuracy of the SPR sensor, and provides the multi-channel image sensor so as to well overcome the nonspecific response and obtain specific response, thereby realizing the detection of the biomolecules to be detected and the interaction thereof and improving the performance of the sensor on the whole.

The invention adopts the angular convergent incident light, so that the incident light cansimultaneously irradiate each channel at different angles within the angle range which can generate resonance, and the error possibly brought by mechanical adjustment is avoided.

The invention uses SPR image detection means, can simultaneously detect the resonance angle of each channel, realizes the real-time and dynamic detection of the SPR response curve, and improves the detection precision and speed.

Still another aspect of the present invention is the ability to miniaturize SPR instruments and SPR-based sensors, increase thermal and mechanical stability of the instrument, and provide a compact configuration.

The related principle of the invention is also applicable to a method for detecting the resonance wavelength by incident polychromatic light.

The invention has the following four characteristics: (a) the detection of a plurality of samples can be completed at one time, and the specific response caused by the interaction of the biomolecules is detected in real sense in real time and dynamically, which is necessary in the drug screening; (b) the detection of different characteristics of the same sample can be completed at one time, so that the probability of false positive in disease detection can be effectively reduced; (c) the reference channel is arranged in multiple channels, so that the influence of non-specific response can be eliminated. (d) Can measure DNA molecules with low concentration, and simplifies the relatively complex detection process after PCR amplification.

Drawings

FIG. 1: sensor system structure diagram

FIG. 2: top view of reaction tank

FIG. 3: sectional view of reaction tank

FIG. 4: structure of sensor chip

FIG. 5: A. b, C channel total response graph

FIG. 6: c channel specific response plots

Wherein: 1. a sample inlet; 2. a sample outlet; 3. a silicon wafer reaction tank; 4. a resonance SPR sample cell; 5. a sensitive film; 6. a light emitting diode; 7. a spherical lens; 8. a pinhole; 9. a plano-convex lens; 10. an optical filter; 11. a polarizing plate; 12. a cylindrical lens; 13. a cylindrical prism; 14. an area array CCD; 15. an incident light path; 16. an emergent light path; 17. a converging lens; 18. a glass substrate; 19. a computer; 20. a sensor body; 21. a PCR reaction tank; 22. a prism placement groove; 23. a channel A; 24. a channel B; 25. a channel C; 26. cleaning an inlet of the channel; 27. cleaning channel outlet

Detailed Description

For better understanding of the present invention, the basic structure and operation principle of the on-chip PCR multichannel image SPR sensor and how to overcome the non-specific response and obtain the specific response to detect the biomolecule and the interaction thereof will now be described with reference to the drawings and examples.

The multichannel surface plasma resonance image sensor based on-chip PCR comprises a resonance SPR sample pool and a PCR reaction pool, and is characterized in that: the resonance SPR sample pool 4 and the PCR reaction pool 21 are combined together to form a sensor silicon wafer reaction pool 3; the upper part of the sensor main body 20 is a cuboid-shaped sensor silicon chip reaction tank 3 formed by silicon chips, the lower part is a cylindrical prism 13, a glass substrate 18 is arranged between the sensor silicon chip reaction tank and the sensor silicon chip reaction tank, a sensitive film 5 is arrangedon the glass substrate, the sensor silicon chip reaction tank 3 of the silicon chips is arranged on the sensitive film, a resonance SPR sample tank and a PCR reaction tank are arranged in the silicon chip reaction tank, and a sample inlet 1 and a sample outlet 2 of sample liquid are arranged at the upper part of the silicon chip sensor reaction tank;

an incident light path 15, an emergent light path 16 and a computer platform 19 connected with the tail end of the emergent light path are arranged on two sides of the sensor main body;

light emitted by a light emitting diode 6 in an incident light path passes through a spherical lens 7, is converged at a pinhole 8, is converted into parallel light through a plano-convex lens 9, passes through an optical filter 10 and a polarizing film 11 in sequence, is adjusted in position to generate P polarized light, and is finally subjected to angular convergence by using a cylindrical lens 12 to be converged into a straight line at a sensitive film 5 of a cylindrical prism 13 and is emitted to the bottom surface of the cylindrical prism;

the emergent light path 16 consists of a convergent lens 17 and an area array CCD14, incident light becomes parallel light after being totally reflected on the bottom surface of the cylindrical prism through the convergent lens, the area array CCD finishes the collection of optical signals and then sends the optical signals to a computer for processing.

The angle of incidence of the light rays incident on the optical path must be between 40 and 90.

The aperture phi converged at the pinhole (8) is 0.1-0.3 mm.

The filter is used for improving the monochromaticity of incident light, and the peak wavelength of the filter is 632.8 nm.

The detection method of the multi-channel surface plasma resonance image sensorbased on-chip PCR comprises the following steps:

1) firstly, simultaneously introducing a mixture phosphate PBS buffer solution of biotinylated thiol derivatives and OH radical thiol diluent with the molar ratio of 1: 9 into A, B, C three channels, reacting for 16-30 hours for a long time, generating a self-assembled monomolecular film SAM tightly covered on the surface of each channel, and then washing with the phosphate PBS buffer solution;

2) then sequentially introducing phosphate PBS buffer solution and Streptavidin with high enough concentration into each channel, reacting for 5-20 minutes, and simultaneously detecting the resonance angle of A, B channel in real time;

3) calculating the difference value of resonance angles before and after reaction, and calculating the surface sensitivity;

4) washing the three channels by using Phosphate Buffered Saline (PBS), and introducing phosphate PBS buffer containing the rat liver f protein gene probe into the channel C to complete the fixation of the probe molecules;

5) and (3) introducing phosphate PBS buffer solution to clean the C channel, then cleaning the three channels by purified water, then sequentially introducing purified water and ethylene glycol into the three channels simultaneously, calculating the difference value of the resonance angle of each channel when purified water and ethylene glycol are introduced, and calculating the body sensitivity and K_C；

6) The reactants are injected into a PCR amplification reaction pool by using a peristaltic pump, the reaction volume is about 20 mu l, and the PCR amplification sample is rat liver f protein gene and has the concentration of 1.3 mu g/mu l. The reaction buffer solution contains 20pmol each of the upstream and downstream primers, 1.25u of Taq enzyme and 0.2mmol each of dNTP;

7) stabilizing the temperature of reactants at 95 ℃, 55 ℃ and 72 ℃ in sequence, and performing 30 temperature cycles to achieve the purpose of DNA amplification;

8) before introducing sample liquid to be detected into the SPR sample pool for detection, PBS is introduced to clean each channel of the SPR sample pool;

9) injecting the amplified product in the PCR reaction pool into the SPR sample pool by using a peristaltic pump, controlling the hybridization temperature, and detecting the resonance angle of each channel in real time, wherein the result is shown in FIG. 5;

10) and (3) obtaining the response of each channel at any time t by taking the resonance angle of the PBS as a reference: delta theta_A(t)、Δθ_B(t)、Δθ_C(t), the specific dynamic response curve of the C channel can be obtained from the obtained parameters.

The filter is used for improving the monochromaticity of incident light, and the peak wavelength of the filter is 632.8.

The preparation method of the silicon wafer reaction tank by using the MEMS process comprises the following steps:

1. cleaning: selecting a required monocrystalline silicon wafer (determining crystal orientation, resistivity and the like), and cleaning the surface by using a cleaning solution and a large amount of cold and hot deionized water.

2. And (3) oxidation: growing a layer of SiO on the surface of monocrystalline silicon₂The film protects and passivates the surface of the silicon wafer and is used as a masking layer for selective corrosion, and the quality of the oxide film has great influence on the subsequent process.

3. Gluing: in SiO₂The film is coated with a layer of photoresist.

4. Photoetching: photoetching is a precise surface processing technology combining graph copying and chemical corrosion, and a designed system structure is copied on a photoresist layer by utilizing ultraviolet light through a mask.

5. And (3) developing: and (3) putting the photoetched silicon wafer into a developing solution to dissolve the exposed photoresist, so as to display a system structure pattern.

6.SiO₂And (3) corrosion: SiO coated with no adhesive film using hydrofluoric acid (HF) etchant₂And etching off to obtain a system structure graph. The proportion of the corrosive liquid is hydrofluoric acid (48 percent), ammonium fluoride (the pH value is about 4-5) and deionized water is 3 ml, 6 g and 10 ml. Etching of SiO by hydrofluoric acid₂Is in a mechanism with SiO₂A complex reaction is carried out to generate a complex compound which is soluble in water,

the reaction formula is as follows:

the overall reaction formula is:

7. removing the photoresist: and removing the residual photoresist on the surface of the corroded silicon wafer.

8. Anisotropic etching of silicon: based on the anisotropic property of silicon, the etching solution is alkaline and free of SiO₂The monocrystalline silicon protected by the film is corroded to form a V-shaped groove or a U-shaped groove.

9. Removal of SiO₂Protection ofFilm formation: removing residual SiO by hydrofluoric acid corrosive liquid₂And protecting the layer to obtain the substrate silicon wafer with the etched system structure.

10. Bonding: according to the requirement, a plurality of silicon chips with manufactured microstructures are combined together by utilizing a bonding process to form a closed three-dimensional system, and the required functions are completed.

Fig. 4 is a structure of a sensor chip: 23. a channel, 24, B channel, 25, C channel. There were A, B, C three channels in total on the glass substrate. Each channel first formed a uniform 50nm thick gold film simultaneously, and then a high refractive index dielectric layer (Ta) with a thickness of about 15nm was formed on the gold film of the B channel₂O₅Or TiO₂Etc.). Before testing, a sensitive membrane containing probe molecules is generated on the gold membrane of the C channel. The substrate of the sensing chip is made of the same material as the cylindrical prism, and the substrate and the cylindrical prism are combined into a complete semi-cylindrical prism through optical coupling liquid. The preparation of various films on the sensing chip adopts sputtering and photoetching processes. The angular convergent monochromatic P-polarized light beam generated by the incident light path is just converged into a straight line orthogonal to the three channels on the surface of the gold film of the sensing chip. The monochromatic area array CCD and the computer together complete the real-time acquisition of the SPR image. Different pixel rows in the SPR image correspond to different incident angles, and the pixel values are the reflected light intensities of the light rays corresponding to the incident angles. The sample introduction system consists of a PCR-SPR reaction pool, a temperature control system (consisting of a semiconductor refrigeration sheet and a temperature control circuit) and a micro peristaltic pump.

The volume response is the change of the resonance angle caused by the in-vivo change of the components, concentration, temperature and the like of the sample solution to be measured. A. B, C the responses of the three channels may be expressed as:

Δθ_A(t)＝b_A(t)+s_A(t)＝B_AΔn(t)+S_AΔN_n(t) (1)

Δθ_B(t)＝b_B(t)+s_B(t)＝B_BΔn(t)+S_BΔN_n(t) (2)

Δθ_C(t)＝b_C(t)+s_C(t) (3)

＝B_CΔn(t)+S_C(K_CΔN_n(t)+ΔN_SC(t))

Δθ_A(t)、Δθ_B(t)、Δθ_C(t) represents the response (i.e., change in resonance angle) of the A, B, C channels, respectively, b_A(t)、b_B(t)、b_C(t) represents the response, s, due to the change in refractive index in the bulk of the A, B, C channel_A(t)、s_B(t)、s_C(t) represents the response due to the surface index change in the A, B, C channel. B is_A、S_A、B_B、S_B、B_C、S_CIndicating the bulk and surface sensitivities of the A, B, C channels, respectively. Δ N (t), Δ N_n(t)、ΔN_SC(t) shows the change in refractive index in vivo,The surface refractive index change due to nonspecific effects and the surface refractive index change due to specific effects, and t represents time.

For channel C, the response elicited by the specific effect is:

Δθ_SC＝S_CΔN_SC(t)

(4)

＝Δθ_C(t)-B_CΔn(t)-S_CK_CΔN_n(t)

to determine K_CThe value of (4) is 0, and the sample solution containing no target molecule is introduced to the sensor surface, and the response of the C channel does not include the response due to specific absorption:

$K_C=\frac{{\mathrm{\θ}}_C\left(t\right)-B_C\mathrm{\Δn}\left(t\right)}{S_C\mathrm{\Δ}{N}_n\left(t\right)}=\frac{{\mathrm{\θ}}_C\left(t\right)-\frac{B_C}{B_A}{b}_A}{\frac{S_C}{S_A}{S}_A}---\left(5\right)$

then, the expressions (1) and (2) are used to obtain Δ N (t) and Δ N_n(t) and K_CThe non-specific response of the C channel can be obtained by substituting the above components into the formula (4).

The incident light path can provide incident light in a certain angle range, and the detection of the reflected light intensity in the certain angle range can be completed at one time by matching with the area array CCD. And an optical filter and a polaroid are additionally arranged in front of the cylindrical lens. The filter is used to improve monochromaticity of incident light, and the polarizer is used to generate P-polarized light.

The specific parameters and descriptions of the optical path are as follows:

① LED Toshiba TLRH190P LED with the parameters as described above.

② spherical lens, diameter phi is 10mm, focal length f is 10mm, LED is 10 times of focal length (about 100mm, approximately parallel incidence) away from spherical lens.

③ plano-convex lens, diameter phi is 20mm and focal length f is 35 mm.

④ cylindrical lens, the focal length f is 40mm, and the size is 15 mm.

⑤ optical filter (interference filter) with peak wavelength of 632.8nm and transmittance of 60%.

⑥ polarizer with diameter phi of 25.4mm, transmittance>60%, polarization direction in the plane of figure, and uniform polarizer and cylindrical lens orientation.

⑦ all the components of the optical path can be easily disassembled and assembled and replaced, and the space between the components is designed according to the above parameters and can be finely adjusted.

⑧ all optical elements are packaged in the metal tube, which can weaken the stray light and improve the detection precision.

⑨ the whole light path is designed to make full use of the energy of the light source and to obtain a convergent line with a small width.

The light-passing aperture of the cylindrical lens is 12mm multiplied by 12mm, the focal length f is 40mm, the estimated incidence angle range is about α which is 2 multiplied by arctg [12/(2 multiplied by 40)]≈ 17 degrees, the CCD is about 120mm away from the metal film, the width of a light spot formed on the surface of the CCD by the reflected light is about 36mm, and the effective length of the CCD is about 37.4 mm.

Example 1

The following experimental procedure was used to determine the surface sensitivity of the A, B, C channel while completing the fabrication of the sensing membrane. When determining the sensitivity of each channel, using the sample cell (3, silicon chip substrate, 22, prism placement groove, 23, A channel sample cell, 24, B channel sample cell, 25, C channel sample cell) as shown in FIG. 2, firstly introducing a mixture of biotinylated thiol derivative and OH group thiol diluent (PBS buffer solution environment) with a molar ratio of 1: 9 into A, B, C three channels simultaneously, reacting for a sufficient time (more than 16 hours), generating a tightly covered self-assembled monomolecular film (SAM) on the surface of each channel, and then washing with PBS buffer solution. Then, surface plasmon PBS buffer, Streptavidin (Streptavidin) with a sufficiently high concentration (reaction time about 5 to 20 minutes), phosphate PBS buffer were sequentially introduced into each channel, and the resonance angle of A, B channels was detected in real time, and the difference between the resonance angles before and after the reaction was calculated. The surface sensitivity was calculated from the above-mentioned equations (1) to (3).

Immobilization of the C channel probe molecules was accomplished by the following experiment and the ratio of the body sensitivities could be determined. And (3) washing the three channels by using PBS (phosphate buffered saline) buffer, and then introducing phosphate PBS buffer containing the rat liver f protein gene probe into the C channel to finish the fixation of the probe molecules. And (3) introducing phosphate PBS buffer solution to clean the C channel, then cleaning the three channels by purified water, then sequentially introducing purified water and ethylene glycol into the three channels simultaneously, and calculating the difference value of the resonance angle of each channel when the purified water and the ethylene glycol are introduced. And calculating the body sensitivity and K according to the formulas (1) - (3) and (5)_C. At this point, the sensor chip is completed, as shown in fig. 4.

The following experiment can realize direct detection of PCR amplification products by using an SPR detection method, and simplifies the relatively complicated detection process after PCR amplification.

The PCR amplification sample is rat liver f protein gene, and the concentration is 1.3 mug/mul. The reaction buffer contained 20pmol each of the upstream and downstream primers, 1.25u of Taq enzyme and 0.2mmol dNTP, and the reaction volume was about 20. mu.l by injecting the reaction mixture into the PCR amplification reaction cell using a peristaltic pump.

One thermal cycle of PCR amplification includes the following three steps:

①, when the sample is heated to about 95 deg.C, the DNA molecule is decomposed into two single strands from the double-stranded structure.

②, cooling to about 55 deg.C to make the decomposed single strand combine with the primer according to the base complementary principle.

③, heating to 72 deg.C again to make the oligonucleotide grow along the primer according to the base pairing principle, and completing the primary DNA replication.

In the experiment, a semiconductor refrigerating sheet temperature control system is adopted to control the temperature of the reaction tank, the temperature of reactants is stabilized at 95 ℃, 55 ℃ and 72 ℃ in sequence, and 30 temperature cycles are carried out to achieve the purpose of DNA amplification and prepare for subsequent SPR detection.

Before introducing a sample solution to be detected into the SPR sample cell for detection, PBS should be introduced to clean each channel of the SPR sample cell, then an amplification product containing a sample to be detected is introduced, the hybridization temperature is controlled, and the resonance angle of each channel is detected in real time, with the result shown in FIG. 8. The response of each channel at any time t (based on the resonance angle of the PBS) can be determined from the graph: delta theta_A(t)、Δθ_B(t)、Δθ_C(t), the specific dynamic response curve of C channel can be determined by using the equations (1) to (5) and the determined parameters in combination with the calibration results, as shown in FIG. 6.

Claims (5)

1. The utility model provides a multichannel surface plasmon resonance image sensor based on PCR on piece, includes resonance SPR sample cell and PCR reaction tank, its characterized in that: the resonance SPR sample pool (4) and the PCR reaction pool (21) are combined together to form a sensor reaction pool (3); the upper part of the sensor main body (20) is a cuboid-shaped sensor reaction tank (3) formed by a silicon wafer, the lower part is a cylindrical prism (13), a glass substrate (18) is arranged between the sensor reaction tank and the sensor reaction tank, a sensitive film (5) is arranged on the glass substrate, the sensor reaction tank (3) of the silicon wafer is arranged on the sensitive film, a resonance SPR sample pool and a PCR reaction pool are arranged in the silicon wafer reaction tank, and a sample inlet (1) and a sample outlet (2) of sample liquid are arranged at the upper part of the silicon wafer sensor reaction tank;

an incident light path (15), an emergent light path (16) and a computer platform (19) connected with the tail end of the emergent light path are arranged on two sides of the sensor main body;

light emitted by a light emitting diode (6) in an incident light path passes through a spherical lens (7), is converged at a pinhole (8), is converted into parallel light through a plano-convex lens (9), the parallel light passes through an optical filter (10) and a polarizing film (11) in sequence, the position of the polarizing film is adjusted to generate P polarized light, and finally, the parallel light is angularly converged by a cylindrical lens (12) to be converged into a straight line at a sensitive film (5) of a cylindrical prism (13) and is emitted to the bottom surface of the cylindrical prism;

the emergent light path (16) is composed of a convergent lens (17) and an area array CCD (14), incident light becomes parallel light through the convergent lens after being totally reflected on the bottom surface of the cylindrical prism, the area array CCD finishes the collection of optical signals and then sends the parallel light into a computer for processing.

2. The on-chip PCR-based multi-channel surface plasmon resonance image sensor of claim 1, wherein: the angle of incidence of the light rays incident on the optical path must be between 40 and 90.

3. The on-chip PCR-based multi-channel surface plasmon resonance image sensor of claim 1, wherein: the aperture phi converged at the pinhole (8) is 0.1-0.3 mm.

4. The on-chip PCR-based multi-channel surface plasmon resonance image sensor of claim 1, wherein: the filter is used for improving the monochromaticity of incident light, and the peak wavelength of the filter is 632.8 nm.

5. A detection method of a multi-channel surface plasma resonance image sensor based on-chip PCR is characterized by comprising the following steps:

1) firstly, simultaneously introducing a mixture phosphate PBS buffer solution of biotinylated thiol derivatives and OH radical thiol diluent with the molar ratio of 1: 9 into A, B, C three channels, reacting for 16-30 hours for a long time, generating a self-assembled monomolecular film SAM tightly covered on the surface of each channel, and then washing with the PBS buffer solution;

2) then sequentially introducing phosphate PBS buffer solution and Streptavidin with high enough concentration into each channel, reacting for 5-20 minutes, and simultaneously detecting the resonance angle of A, B channel in real time;

3) calculating the difference value of resonance angles before and after reaction, and calculating the surface sensitivity;

4) washing the three channels by phosphate PBS buffer solution, and then introducing the phosphate PBS buffer solution containing the rat liver f protein gene probe into the C channel to complete the fixation of the probe molecules;

5) washing C channel with PBS, washing three channels with purified water, and washing three channels simultaneouslyPurified water and ethylene glycol are sequentially introduced into the channels, the difference value of the resonance angle of each channel when the purified water and the ethylene glycol are introduced is calculated, and the body sensitivity and the K are calculated_C；

6) The reactants are injected into a PCR amplification reaction pool by using a peristaltic pump, the reaction volume is about 20 mu l, and the PCR amplification sample is rat liver f protein gene and has the concentration of 1.3 mu g/mu l. The reaction buffer solution contains 20pmol each of the upstream and downstream primers, 1.25u of Taq enzyme and 0.2mmol each of dNTP;

7) stabilizing the temperature of reactants at 95 ℃, 55 ℃ and 72 ℃ in sequence, and performing 30 temperature cycles to achieve the purpose of DNA amplification;

8) before a sample solution to be detected is introduced into the resonance SPR sample pool for detection, the resonance PBS is introduced to clean each channel of the SPR sample pool;

9) injecting the amplified product in the PCR reaction pool into a resonance SPR sample pool by using a peristaltic pump, controlling the hybridization temperature, and detecting the resonance angle of each channel in real time, wherein the result is shown in figure 5;

10) and (3) obtaining the response of each channel at any time t by taking the resonance angle of the PBS as a reference: delta theta_A(t)、Δθ_B(t)、Δθ_C(t), the specific dynamic response curve of the C channel can be obtained from the obtained parameters.

Images