WO2007060989A1

WO2007060989A1 - Method and apparatus for detecting trace substances by surface-enhanced raman scattering (sers), and microchannel chip for detection of microanalyte

Info

Publication number: WO2007060989A1
Application number: PCT/JP2006/323314
Authority: WO
Inventors: Tomohiro Marui; Akito Sekiguchi
Original assignee: Intellectual Property Bank Corp.
Priority date: 2005-11-22
Filing date: 2006-11-22
Publication date: 2007-05-31

Abstract

This invention provides a method and apparatus for detecting trace substances with a SERS substrate that are highly practical and can realize rapid observation. In the method, a microanalyte is discriminated and detected using surface-enhanced Raman scattered light obtained by applying a laser beam to a site at which an analyte is close to metallic fine particles. The method comprises the steps of mixing an analyte with a previously provided metallic fine particle colloid, supplying the mixture of the analyte with the metallic fine particle colloid to a solid plane, on which a plurality of juxtaposed grooves or a plurality of juxtaposed recesses are arranged, along the direction of the arrangement of the grooves or recesses, drying the supplied mixture on the solid plane, and applying a laser beam onto the dried mixture in the grooves or recesses on the solid plane to observe Raman scattered light. The Raman scattered light is observed by applying a laser beam to the dried product formed in the plurality of juxtaposed grooves or the plurality of juxtaposed recesses. The solid surface may also be a metallic surface having a plasmon mirror effect.

Description

Specification

Detection method and apparatus for trace substances by surface enhanced Raman scattering (SERS), microchannel chip for trace analyte detection

Technical field

[0001] The present invention relates to a trace substance detection method and apparatus by surface enhanced Raman scattering (SERS). In the present invention, the Raman scattering enhancement effect in the vicinity of the metal fine particles in the dried body obtained by drying the colloid in which the metal fine particles are suspended is used. The present invention also utilizes a relatively clean metal surface that induces surface plasmon polaritons with irradiated light energy. Such a metal surface that has the effect of further enhancing surface enhanced Raman scattering (SERS) in the vicinity of the metal surface is tentatively referred to as a “metal surface having a plasmon mirror effect”, and the metal surface is used. Naturally, the metal surface having the plasmon mirror effect is preferably a gold clean surface or a silver clean surface. Background art

[0002] It is well known to detect and detect trace substances by Raman analysis of analytes by observing surface enhanced Raman scattering (SERS) due to plasmon effect of noble metal nanoparticles such as gold and silver (See Non-Patent Document 1 and Non-Patent Document 2). In the absence of nanoparticles, the Raman signal is enhanced by the plasmon effect and can be discriminated even with a very small amount of material with a weak Raman signal. The mechanism of SERS plasmon effect expression has not been fully elucidated, but research and development is being conducted with the aim of applying it to harmful substance sensors. Here, the substance for discrimination detection is also called an analyte.

[0003] A method for obtaining a solid substrate having SERS activity by dropping a SERS base material containing noble metal nanoparticles such as gold and silver and drying it is known (see FIG. 2). For example, Patent Document 1 discloses a simple method for producing a substrate exhibiting high SERS activity by dispersing a SERS base material in a liquid and fixing it on a solid surface.

[0004] A device for obtaining strong SERS activity by self-assembling nanoparticle groups has also been studied. For example, a SERS substrate in which metal fine particles are lined up by scraping a group of metal fine particles that have been self-assembled at the interface according to the LB film (Langmuir-Blodgett film) manufacturing method. This method is also known (see FIG. 3 and Patent Document 3). Patent Document 2 is also a method of forming a SERS substrate in which particles are periodically aligned and deposited by repeatedly pulling up a flat plate immersed in a solution containing metal fine particles at a predetermined speed.

[0005] Besides the contrivance of nanoparticle alignment using LB films, many SERS substrate manufacturing methods have been proposed. For example, there is a method for producing a SERS substrate using a surface having affinity for water (hydrophilicity / water repellency) (see Patent Document 5).

[0006] A colloid in which noble metal nanoparticles suspended in a solid-state SERS active substrate as described above is suspended as a SERS substrate (it is not called a substrate because it is liquid), and this is a liquid phase mixture of analyte. It is also known to detect analytes by observing surface-enhanced Raman scattering.

(See FIG. 4 and Patent Document 4).

[0007] A colloidal SERS substrate is easier to produce than a solid-phase SERS substrate, but has the disadvantage that the SERS activity disappears when it is separated and precipitated or immediately after separation and precipitation. For this reason, a method of stabilizing a SERS substrate for a long period of time with a clay-like substance such as smectite (see Patent Document 4) has been studied.

[0008] It is also known to use a capillary phenomenon (capillary) for analysis of an extremely small amount of analyte (see Patent Document 6). It is also known that analytes and colloidal SERS substrates are mixed in a capillary phenomenon (capillary), and SERS Raman signals are analyzed to detect trace substances.

[0009] Similarly, it is also known that a microliter level trace amount of analyte and a colloidal SERS substrate are flow-mixed in a known microchannel, and the SERS Raman signal is analyzed to detect a trace amount substance. (See Patent Document 7 and Non-Patent Document 3). Non-Patent Document 4 and Non-Patent Document 5 are cited in the best mode for carrying out the invention.

[0010] On the other hand, Patent Document 8 discloses a trace substance detection method and apparatus using the "Blasmon mirror effect". Here, the plasmon mirror effect means that the nanoparticles are self-assembled (self-aligned) in the vicinity of a relatively clean metal surface that induces surface plasmon polaritons with the same light energy, and a stronger SERS activity is obtained. is there. The conditions and mechanism for obtaining the plasmon mirror effect are not clear, but it is considered important to self-assemble the nanoparticles in the vicinity of a relatively clean metal surface. (See Patent Document 8).

[0011] On the other hand, a group of nanoparticles is self-assembled on the metal surface, and metal deposition is further performed on the upper surface thereof, or nanoparticles are stacked in two steps on the metal surface to form a sandwich. ! "Sandwich Substrates J" or "Sandwich architecturej is also known. In some cases, a strong sandwich configuration can provide stronger SERS activity than a single layer (see Patent Document 9, Non-Patent Document 6, and Non-Patent Document 7).

Patent Document 1: Japanese Patent Laid-Open No. 2005-077362 “Method for creating surface-enhanced Raman scattering active substrate” (Keio University)

Patent Document 2: Japanese Patent Application Laid-Open No. 2004-170334 “Raman scattering measurement sensor and manufacturing method thereof” (Japan Science and Technology Agency, etc.)

Patent Document 3: Japanese Laid-Open Patent Publication No. 2005-233637 “Raman Spectroscopic Analysis with Gold Nanorod Thin Film” (Japan Science and Technology Agency)

Patent Document 4: Japanese Patent Application Laid-Open Publication No. 2004-205435 “Analytical method of binding analysis target substance that does not require labeling dye and analysis kit used therefor” (Fukuo Takao)

Patent Document 5: Japanese Laid-Open Patent Publication No. 2005-219184 “Metal Nano-Triangular Column Structure for Single-Molecular Raman Spectroscopy: Array Substrate Formation Method and Single-Molecule Analysis Method Using the Same” (National Institute of Advanced Industrial Science and Technology) No. "Capillary electrophoresis detector" (Nippon Telegraph and Telephone Corporation)

Patent Document 7: Special Table 2005-507500 Publication “Detection of microfluids by surface-enhanced resonance Raman scattering” (University of Strathclyde et al.)

Patent Document 8: International Publication WO2005 114298 Publication `` OPTICAL SENSOR WITH LAYERED P LASMON STRUCTURE FOR ENHANCED DETECTION OF CHEMICAL GROUPS BY SERS J VP HODLINGS.LLC

Patent Document 9: US Patent US6149868 `` Surface enhanced raman scattering from meta

1 nanoparticle-analyte-no Die metal substrate sandwiches ”(Michael J. Natan) Non-patent document 1: Kotaro Kajikawa (Tokyo Tech), Yuta Mitsui“ Bio-sensing using localized plasmon resonance ”Applied Physics, No. 72, No. 12, p.1541-1544 (2003)

Non-Patent Document 2: Takayuki Okamoto (RIKEN) “Metal Nanoparticle Interaction and Biosen Survey Research on Sir ”2002 Grant-in-Aid for Scientific Research (Basic Research C)“ Survey Research on Localization of Surface Brasmon and Its Application ”Research Results Report Individual Report

Non-Patent Document 3: Keir R, Igata E, Arundell M, Smith WE, Graham D, McHugh C, Cooper JM.TSERRS.In situ substrate formation and improved detection using microfluidic sJ Anal Chem. 2002 Apr 1; 74 (7) : 1503-8.

Non-Patent Document 4: Jaeyoun Kim, Gang L. Liu, and Luke P. Lee, `` Lens- scanning Raman microspectroscopy system using compact disc optical pickup technology '', 13 June 2 005 Vol. 13, No. 12 OPTICS EXPRESS 4780

Non-Patent Document 5: Cameron L. Jones ^ `` Cryptograpmc Hash Functions and CD-Based Optical Biosensors '', Problems of Nonlinear Analysis in Engineering Systems.No.2 (23), v.ll, 2005, pp.17-36.

Non-Patent Document 6: Jacquitta K. Daniels and George and humanov (Clemson University) “Na noparticle— Mirror Sandwich Substrates for Surface-Enhanced Raman Scattering” J. Phys. Chem. B, 109 (38), 17936 -17942, 2005.

Non-Patent Document 7: Orendorff, CJ Gole, A. Sau, TK Murphy, CJ (University of South Carolina) `` Surface- enhanced Raman spectroscopy of Self-assembled monolayers: S andwich architecture and nanoparticle shape dependencej Anal Chem. 2005 May 15 ; 77 (10): 3261-6.

Disclosure of the invention

Problems to be solved by the invention

[0013] An object of the present invention is to propose a method and a device that are more practical than conventional methods and devices for detecting trace substances using a SERS substrate or a SERS substrate. The present invention proposes a method and apparatus that uses a colloidal SERS substrate without using a substrate in which metal fine particles are aligned in a solid phase, but does not cause a problem of separation and deposition of the substrate.

Means for solving the problem

[0014] The present invention (Claim 1) is a method for discriminating and detecting a trace amount of analyte with surface-enhanced Raman scattering light obtained by irradiating a laser beam to a site where the analyte and metal fine particles are close to each other. Mixing the analyte with the colloidal metal particle colloid prepared in advance, Supplying a mixture of the colloidal particles and the metal fine particle colloid to a solid plane provided with a plurality of parallel grooves or a plurality of parallel recesses along the alignment direction of the grooves or the recesses, and supplying the supplied mixture to the solid This is a method for detecting trace substances by surface-enhanced Raman scattering, which has a step of drying on a flat surface and a step of observing Raman scattered light by irradiating a laser beam onto a dried product of grooves or recesses on a solid surface. (See Figure 1 and Figure 6.) The step of supplying the mixture of the analyte and the metal fine particle colloid to the solid flat surface in which the plurality of grooves or the plurality of parallel recesses are arranged along the direction in which the grooves or the recesses are arranged is the same as that of electric energy. It may be a process of spraying the mixture onto a stationary solid surface by the pressure due to the volume change of the changing piezoelectric element.

[0015] In the mixture supplying step of the present invention (Claim 2), after bringing the mixture into contact with the stationary solid plane, the mixture or the solid plane is moved in the direction in which the grooves or recesses are arranged. Is preferably supplied to the solid plane along the direction of the grooves or recesses. If supplied in this way, the mixture is smoothly extended. If the supply is not done in this way, it may not be spread and become a lump.

[0016] Here, the colloidally prepared metal fine particle colloid is a clay-like material such as smectite, and it is not necessary to stabilize the SERS substrate for a long period of time. For example, known metal fine particle colloids obtained by reduction of metal salts such as citrate reduction of silver salts such as silver nitrate and reduction of silver salts such as silver nitrate with borohydride (borane). As the borohydride reagent, sodium borohydride is often used. The solid to be dried is preferably glass or polycarbonate. Conventionally, it has been difficult to dry and observe a liquid phase mixture of metal fine particle colloid and analyte on a solid surface. In this plan, it is possible to prepare only ordinary metal fine particle colloids, mix them with an analyte, dry it, and observe the dried body without any particular elaboration of the stability.

The conventional method is shown in FIGS. 2 to 4, and the method of the present invention contrasted with this is shown in FIG. In these figures, SS is a known SERS substrate, SB is a bottle containing a known SERS substrate (SERS vial), and SX is a solid plane of the present plan for supplying a mixture of metal fine particle colloid and analyte. Or a plurality of parallel recesses are provided. Ev indicates a part of a solid plane provided with a plurality of parallel grooves or a plurality of parallel recesses. A dried body of a dried analyte and metal colloid mixture is shown. P is a liquid supply means such as a micropipette. Fig. 6 shows a flowchart of the method for detecting trace substances according to the present invention.

[0018] Fig. 5 shows a plurality of parallel grooves arranged on the solid plane SX of the present invention, and Fig. 9 shows a plurality of concave portions arranged in parallel. G in the figure is the interval between the grooves of 5 m or less arranged in parallel or the interval of the arrangement of the recesses of 5 μm or less. The mixture of metal fine particle colloid and analyte supplied to the site with multiple parallel grooves or multiple parallel recesses becomes a thin and large surface area extended at the groove or recess site, and the drying phenomenon proceeds quickly. To do. In addition, an aggregate of metal fine particles and analyte suitable for obtaining the plasmon effect is easily formed. Supplying along the direction in which the grooves or recesses are arranged is because the mixture is thin and suitable for extending the specific surface area.

[0019] The reason why the dried body formed by the grooves or the recesses is advantageous for the formation of the aggregate of the metal fine particles and the analyte is not clear, but the metal fine particles and the analyte in the dispersed state before drying are dried. Since there is a tendency to form an aggregate near the surface, it is estimated that a thin and large specific surface area is advantageous for the formation of aggregates of fine metal particles and analyte.

[0020] If the groove or the concave portion is made to have a hydrophilic surface or a mixture of a hydrophilic surface and a hydrophobic surface, it may be effective for extending the mixture. Therefore, a hydrophilic / hydrophobic surface treatment may be appropriately performed. That is, (Claim 6), there is a difference between the water affinity of the plurality of groove recess surface materials arranged in parallel and the water affinity of the groove protrusion surface material, or the water affinity of the plurality of recess surface materials arranged in parallel There should be a difference in the water affinity of the surface material other than the recess.

[0021] Silver having a particle size of 20 to 30 nanometers produced by reducing citrate with silver nitrate by arranging grooves with a groove interval of about 2 μm and a depth of about 0.5 μm on a flat polycarbonate surface. A mixture of particulate colloid and analyte oligonucleotide was supplied. Fig. 8 shows an optical micrograph of the condition in which the dried body was formed in the groove after standing for 5 minutes. Oligonucleotide has approximately 1000 bases. Similarly, the polycarbonate is provided with recesses of approximately 2 μm in length, approximately 0.5 m deep, approximately 0. Fig. 10 shows an optical micrograph of the condition in which the dried body was formed in the recess after standing for a minute. F represents a dried product. FIG. 7 shows an example of Raman spectrum data obtained by the trace substance detection method of the present invention. FF is a position that shows the Raman spectrum observed when a laser beam is irradiated after moving through a part of F that is dried by Ev. Ν is the position of the Raman spectrum that is observed when the part is moved linearly and irradiated with laser. (Ev indicates a part of a solid plane in which a plurality of parallel grooves or a plurality of parallel recesses are arranged)

The invention's effect

[0023] Although the present invention uses a colloidal SERS substrate, separation and precipitation of the substrate is not a problem. Therefore, it is practical because it suffices to prepare only ordinary colloidal metal fine particles that do not devise any means to stabilize the substrate. In addition, since it is possible to obtain a large Raman signal enhanced by the grooves or recesses formed in the peripheral edge of the dry body, it is necessary to obtain the Raman signal only by the grooves or recesses when observing the Raman spectrum. Oh ,. In the detection of trace substances, it may take time to identify the observation site. However, in this proposal, the position of the groove or recess is known, so it can be observed quickly.

Brief Description of Drawings

[0024] [Fig. 1] Schematic diagram showing the work flow of the method for detecting a trace substance of the present invention.

[Fig.2] Schematic diagram of the first method for producing a known SERS substrate (drop-and-dry method) and the method for detecting minute substances using it

[Fig. 3] Schematic diagram of the second method for producing a known SERS substrate and the method for detecting trace substances using it. The metal particles formed on the interface in the form of an LB film (Langmuir-Blodgett film) are trapped! SERS substrate manufacturing method

[Fig. 4] Schematic diagram of a known SERS substrate (not a substrate because it is liquid) that has been designed to stabilize metal colloids for a long period of time and a method for detecting trace substances using it.

FIG. 5 is an explanatory diagram of a solid plane provided with parallel grooves. The distance G between the parallel grooves is 5 microns or less.

[Fig. 6] Flow chart of trace substance detection method of the present invention

FIG. 7 shows an example of Raman spectrum data obtained by the method for detecting a trace substance of the present invention. FF is a position that shows the Raman spectrum observed when the laser beam is irradiated after moving in a straight line through a part of F that is dried by Ev. N is F, and the laser beam is observed after moving the part linearly. The position showing the man spectrum.

FIG. 8 is an optical micrograph of a surface obtained by drying a mixture of silver nanoparticle colloid and a nucleic acid (analyte) analyte on a solid surface provided with parallel grooves of about 2 microns. The part written with a brush is the dry mixture.

FIG. 9 is an explanatory diagram of a solid plane provided with a plurality of parallel recesses. The gap G between the multiple concave groups in parallel is 5 microns or less.

[FIG. 10] An optical microscope photograph of a surface obtained by drying a mixture of silver nanoparticle colloid and nucleotide (nucleic acid) analyte on a solid surface having approximately 2 micrometer concave portions arranged in parallel. A site like a deposit is a dry mixture.

圆 11] Microchannel chip explanatory diagram

[Fig. 12] Schematic diagram showing the work flow of the trace amount detection method of this proposal using multiple 8-strip multipipettes (A) is in standby state, (B) is the first 8-strip multipipette with metal particulate colloid The liquid phase mixture of id and 8 analytes is in contact with the solid plane at the same time, (C) moves the solid plane and supplies Ev to the colloid 'analyte mixture in the first 8 multipipette. It is in a state.

[Fig. 13] Schematic diagrams ( _a , b, c, d) showing the work flow of the trace substance detection method of this proposal using multiple 8-series multipipettes are 8 analysts for MP1, MP2, MP3 and MP4, respectively. (E, Al, A2, A3, A4, respectively), the liquid phase mixture is simultaneously brought into contact with the solid plane, the solid plane is moved, and the colloid-analyte mixture is supplied to Ev. , F) Move Rp to the position of A1 supplied by MP1 and dried, move Rh traverse on Rp, irradiate the laser for Raman analysis, receive Raman scattered light and collect data. Similarly, h) collects Raman scattered light data of A2 supplied with MP2 and dried. Similarly, (i, j) collects Raman scattered light data of A3 supplied and dried by MP3. Similarly, (k, 1) collects Raman scattered light data of A3 supplied by MP3 and dried.

[Fig.14] An example of the spreading means for extending the mixture on the solid plane to the solid plane. Z has a plane that is roughly parallel to the solid plane, and the mixture is thinly stretched on the plane to increase the surface area and dry. To promote.

15] An explanatory view of a metal surface having a plasmon mirror effect in which parallel grooves are arranged. In parallel The distance G between the grooves is 5 microns or less.

[Fig. 16] Flow chart of the method for detecting a trace substance of the present invention (in the case of a metal surface having a plasmon mirror effect)

FIG. 17 is an explanatory view of a metal surface having a plasmon mirror effect in which a plurality of parallel recesses are arranged. The distance G between the plurality of concave groups arranged in parallel is 5 microns or less.

[Fig.18] Example of plasmon mirror effect in which mixing of two analytes (analyte A = adenosine, analyte B = guanosine) is detected

Explanation of symbols

A1 First analyte group mixed with metal colloid and dried by Ev

Α2 Second analyte group mixed with metal colloid and dried by Ev

A3 Third analyte group mixed with metal colloid and dried by Ev

A4 4th analyte group mixed with metal colloid and dried by Ev

Ca The first low-pressure cavity (low-pressure chamber) to dry the analyte and metal colloid mixture, and to seal the area where Va becomes low-pressure

Cb In the second low-pressure cavity (low-pressure chamber), Vb is used to suck and move the analyte and metal colloid mixture.

Ev Solid part with multiple parallel grooves or multiple parallel recesses

Dry body of analyte and metal colloid mixture dried by F Ev

A position showing the Raman spectrum observed when a laser beam is radiated from a part of F dried by FF Ev.

MP1 1st 8 multipipette

MP2 second 8 multipipette

MP3 3rd 8 multipipette

MP4 4th 8 multipipette

In the case of N F, the position showing the Raman spectrum observed by moving the part linearly and irradiating the laser

G Spacing between parallel grooves of 5 μm or less or spacing between multiple recesses of 5 μm or less P1 Flow path for transporting mixed metal salt and metal salt reducing agent (first flow path) P2 Analyte channel (second channel)

P3 Channel part where metal colloid and analyte are mixed by reduced metal salt

P4 Channel for transporting mixed metal colloid and analyte from reduced metal salt P5 Channel for drying mixed metal colloid and analyte from reduced metal salt (third channel)

Rh Laser irradiation head for Raman analysis, Raman scattering light receiving head

Pickup with Rp Rh

SB A bottle containing a known SERS substrate (SERS vial)

SS Known SERS substrate (SERS = surface enhanced Raman scattering)

SX Ev is placed on the solid surface of the present plan, on which a mixture of metal fine particle colloid and analyte is supplied

Va first low pressure suction source

Vb Second low pressure suction source

W1 Metal salt solution introduction tool (hole)

W2 Metal salt reducing agent solution introduction well (hole)

W3 Analyst introduction well (hole)

W4 Flow outlet side hole

Z Extension means for extending the mixture on the solid plane to the solid plane

Zp Parts of metal surface with plasmon mirror effect in parallel grooves or recesses (indicated by diagonal lines)

Zx Parts other than metal surfaces with plasmon mirror effect in multiple parallel grooves or multiple parallel recesses

BEST MODE FOR CARRYING OUT THE INVENTION

[0026] In the present invention (Claim 3), the interval between the plurality of parallel grooves or the interval between the plurality of parallel recesses is 5 micrometers or less, and the depth of the grooves or recesses is 1 micrometer or less. Preferably, a mixture of 1-10 microliters of analyte and 1-10 microliters of metal particulate colloid is supplied to the solid surface.

[0027] The dried product according to the present invention (Claim 8) is obtained by mixing an analyte and a metal fine particle colloid and drying. A dried body for detecting an analyte discrimination, wherein a plurality of grooves arranged in parallel at intervals of 5 μm or less or a plurality of grooves arranged in parallel at intervals of 5 μm or less are disposed on the solid flat grooves or the recesses. 1 to 10 microliters of metal along the direction of the alignment Fine particle colloid and 1 to: Drying by supplying a mixture of LO microliters of analyte is a dried body of width 5 microns or less and length 10 microns or more . The reason why a dry body formed by grooves or recesses is advantageous for the formation of aggregates of fine metal particles and analyte, and it is not suitable for obtaining a plasmon effect, is not clear. This method is effective in discriminating and detecting trace analytes using surface-enhanced Raman scattering light obtained by irradiating a laser beam on the surface.

[0028] In the present invention, the analyte and the colloid may be caused to flow in the flow path. Ie

(Claim 4), a step of fluidly mixing an analyte and a metal colloid prepared in advance in a liquid phase, and a plurality of parallel grooves or a plurality of parallel recesses provided in the fluid mixing step. A flow transfer process on the solid plane, a process of static drying on the solid plane provided with a plurality of parallel grooves or a plurality of parallel recesses, and a static drying process. This is a method for detecting trace substances by surface-enhanced Raman scattering, which has a step of observing Raman scattered light by irradiating the dried mixture obtained in step 1 with laser light.

[0029] Even when the analyte and the colloid are caused to flow in the flow path (Claim 5), the interval between the plurality of parallel grooves arranged in the flow portion or the interval between the plurality of parallel recesses is 5 microns. It is preferable that the depth of the groove or the recess is 1 micrometer or less. Further, (Claim 6), there is a difference between the water affinity of the plurality of groove recess surface materials arranged in parallel and the water affinity of the groove projection surface material, or the water affinity of the plurality of recess surface materials arranged in parallel There is a difference in the water affinity of the surface material other than the recesses.

[0030] An apparatus for carrying out the method of the present invention (Claim 10) comprises a solid plane provided with a plurality of grooves arranged in parallel at intervals of 5 microns or less or a plurality of recesses arranged in parallel at intervals of 5 microns or less. A solid surface provided with a mixing means for mixing an analyte and a metal colloid prepared in advance and a mixture obtained by the mixing means in which a plurality of parallel grooves or a plurality of parallel recesses are arranged. Supply along the direction in which the grooves or the recesses are arranged Supply means, a drying means for drying the mixture supplied on the solid plane provided with a plurality of grooves or a plurality of parallel recesses on the supply means, and drying the mixture obtained by the drying means It is a trace substance detection device that has means for irradiating a body with laser light and observing Raman scattered light. The means for supplying the mixture obtained by the mixing means to a solid flat surface provided with a plurality of parallel grooves or a plurality of parallel recesses along the direction in which the grooves or the recesses are arranged is a piezoelectric element whose volume is changed by electric energy. It may be a means for spraying the mixture onto a stationary solid surface by the pressure due to the volume change.

[0031] Another apparatus for carrying out the method of the present invention by flowing an analyte and a colloid in a flow path (Claim 12) is a plurality of grooves arranged in parallel at intervals of 5 microns or less, or 5 microns or less. An apparatus having a flow path having a solid plane as an inner surface with a plurality of recesses arranged in parallel at intervals, and a fluid mixing means for fluidly mixing an analyte and a metal particulate colloid prepared in advance in a liquid phase; A flow transfer means for flow-transferring the mixture obtained by the flow mixing means to a flow path having a solid plane provided with a plurality of parallel grooves or a plurality of parallel recesses as an inner surface; and a plurality of grooves paralleled by the flow transfer means or Drying means for standing and drying the mixture transferred to a solid plane provided with a plurality of parallel recesses, and observing Raman scattered light by irradiating the dried mixture obtained by the drying means with laser light Means to This is a detection device for trace substances.

[0032] The drying means for drying (Claim 13) of the apparatus of the present invention may also serve as a pressure reducing means for lowering the solid plane below atmospheric pressure. It may be combined with a V-type evaporator (constant pressure dryer). The drying means (Claim 14) may also have an extending means for extending the mixture on the solid plane to the solid plane. The extending means has, for example, a surface substantially parallel to the solid surface disposed on the solid surface, and the mixture is thinly stretched on the surface to increase the surface area to promote drying (see FIG. 14).

[0033] In addition, in order to quickly detect and discriminate a plurality of analytes, a configuration in which a plurality of analytes can be supplied simultaneously may be adopted. For example, as shown in Fig. 12, a mixture of analyte and metal fine particle colloid is dispensed to each of a plurality of 8-multipipette MP1, MP2, MP3, and MP4 pipettes using a known automatic dispensing means. In this form, a plurality of grooves arranged in parallel with a circumferential track on a solid plane or a plurality of parallel recesses arranged in a circle are prepared. Circle While rotating the disk-shaped solid plane around the target axis, MP1, MP2, MP3, and MP4 sequentially supply the mixture along the circumferential direction of the grooves or recesses (see Fig. 13).

[0034] After supply, the pickup Rp with the laser irradiation head Rh for Raman analysis is moved to the mixture supply position, and the surface is enhanced by irradiating the laser beam while traversing Rp on the Rh (lateral movement). By collecting Raman scattered light, a large number of analytes can be distinguished and detected efficiently and quickly.

[0035] Non-Patent Document 4 discloses a Raman device and an applied technology using an optical device portion of a known CD (Compact Disk) player. Non-Patent Document 5 discloses a technique for analyzing a biological substance using a known CD (Compact Disc). By combining these with the present invention, it is possible to easily realize a compact trace substance discrimination detection device that is small and portable.

[0036] More specifically, the apparatus using flow may be implemented by being incorporated in the microchannel described in Patent Document 7 and Non-Patent Document 3. In other words, the micro-channel for detecting a small amount of analyte by a method for discriminating and detecting a small amount of analyte with surface-enhanced Raman scattering light obtained by irradiating a laser beam to a site where the analyte and metal fine particles are close to each other. A first channel for flowing and mixing a metal salt and a reducing agent for the metal salt to reduce the metal salt during the flow, and a second channel for flowing the analyte that merges with the first channel. And a micro-channel chip having a third channel having a surface on which a plurality of grooves continuous in parallel with the flow direction or a plurality of recesses parallel to the flow direction are arranged at the confluence of the first second flow channel Please implement as.

FIG. 11 is an explanatory diagram of the microchannel chip of the present invention. W1 is a metal salt solution introduction hole (hole), W2 is a metal salt reducing agent solution introduction well (hole), and W3 is an analyte introduction well (hole). P1 is a channel that transports metal salt and metal salt reducing agent mixed (first channel), P2 is a channel that transports analyte (second channel), and P3 is a metal core made of a reduced metal salt. The flow path part where Lloyd and the analyte are mixed, the flow path where the metal colloid and the analyte are mixed with the P4 reduced metal salt and the analyte is transferred, and the metal colloid and the analyte are mixed with the P5 reduced metal salt. This is a flow path (third flow path) for drying the mixture. Here, as described in Patent Document 7 and Non-Patent Document 3, P3 and P4 generate metal particulate colloids during flow. Therefore, a metal salt solution and a reducing agent solution as raw materials may be prepared. [0038] In the first low-pressure cavity (low-pressure chamber) arranged in the well (hole) W4 on the flow outlet side, in order to move the analyte and the metal colloid mixture by suction, at the sealed part Cb where the pressure is reduced by Vb, Cb is fluidized by connecting to the second low-pressure suction source Vb and suctioning. On the other hand, it may be pressurized at the inlet side Wl, W2, W3, but it sucks with a laminar flow that does not disturb the flow when sucked. There is a part of Ev provided with a plurality of grooves parallel to the third flow path P5 or a plurality of concave parts arranged in parallel, and the mixture is fluidly transferred to the part of Ev.

[0039] To promote drying, a first low-pressure cavity (low-pressure chamber) Ca that is connected to the first low-pressure suction source Va and seals the third flow path P5 may be provided. In that case, Ca should be made of a material with small attenuation of the laser and Raman scattered light for Raman observation, or a certain type of material should be made to exclude Ca and be excluded after drying to observe Raman. Anyway.

[0040] (Claims 7, 9, 15, 17) In the method, dry body, and apparatus of the present invention, the solid surface may be a metal surface having a plasmon mirror effect (see FIGS. 15 to 18). .

[0041] The plasmon mirror effect will be supplemented. At present, the plasmon mirror effect is not academically recognized. When the surface on which the metal colloid is dropped is called the `` lower layer '', and when the lower layer is made of glass resin and the lower layer is made of metal, it is a fact that the latter gives a larger Raman signal. And reproducible (see Figure 18). However, whether this is due to the electromagnetic enhancement field called “gap mode” described in Patent Document 8 between the lower layer metal and the upper layer metal fine particles is unknown and has not been acknowledged or verified scientifically.

A supplementary explanation will be given for the formation of a metal surface having a plasmon mirror effect. As an experimental fact, when compared to the case where the lower layer is a glass resin surface and the lower layer is a metal deposition surface that is simply formed on an arbitrary component layer, the latter gives a larger Raman signal. That is, the effect of the present invention can be obtained even by using a simple method called metal vapor deposition. More preferably, a clean and flat metal surface described as “mirror surface” in Patent Document 8 is formed by the method described in Patent Document 8 or cited in Patent Document 8! If you do, you can get a much larger Raman signal.

[0043] In this specification, the analyte is dropped on the microscopic Raman observation stage and dried. It is assumed that the measurement is performed on the observation stage of the microscopic Raman, but a configuration in which the analyte is supplied in a direction not necessarily downward such as an inverted microscope or a side surface or an upper surface of the microchannel is also conceivable. Therefore, the dropping step and the dropping means in this specification are a supply process and a supplying means in a broader sense, and it is also effective to use the spraying means as in claim 11 as one supply means. In this way, the analyte can be supplied in a direction that is not necessarily downward, and the accuracy of the analyte supply amount is increased due to the property of deriving a quantitative pressure by the electric energy of the piezoelectric element, so that drying can be performed. This is more preferable because the prediction accuracy of the time required is improved. The spraying means may be a known piezoelectric element that is practically used in ink jet printers!

Claims

The scope of the claims

[1] Obtained by irradiating laser light to the area where the analyte and metal fine particles are close to each other

A method for discriminating and detecting a trace amount of analyte with surface-enhanced Raman scattered light,

The process of mixing the analyte with the metal particulate colloid prepared in advance, and the mixture of the analyte and the metal particulate colloid

On a solid plane with multiple parallel grooves or multiple parallel recesses

Supplying along the direction in which the grooves or the recesses are arranged;

Drying the supplied mixture on the solid plane;

Irradiate laser beam to dry mixture of groove or recess on solid surface

A step of observing Raman scattered light

A method for detecting trace substances by surface-enhanced Raman scattering.

[2] The step of supplying the mixture of the analyte and the metal fine particle colloid to the solid plane

After contacting the mixture with a stationary solid plane,

By moving the mixture or solid plane in the direction of the grooves or recesses

Supply the mixture to the solid plane along the direction of the grooves or recesses

The method for detecting a trace substance according to claim 1.

[3] The distance between the parallel grooves or the distance between the parallel recesses

5 micrometer or less, groove or recess depth of 1 micrometer or less, 1-10 microliters of analyte and 1-10 microliters of metal particulate colloid mixture

The method for detecting a minute amount of analyte according to claim 1, wherein the method is supplied to a solid plane.

[4] Obtained by irradiating the area where the analyte and metal fine particles are close to each other with laser light

The process of fluidly mixing the analyte and the prepared metal fine particle colloid in the liquid phase and the mixture obtained by the fluidized mixing process

Flow-transferring on a solid plane provided with a plurality of parallel grooves or a plurality of parallel recesses; and The mixture transferred in the fluid transfer process

A process of standing and drying on a solid plane provided with a plurality of parallel grooves or a plurality of parallel recesses;

It has a step of observing Raman scattered light by irradiating laser light to the dried mixture obtained in the stationary drying step

A method for detecting trace substances by surface-enhanced Raman scattering.

[5] The distance between the parallel grooves or the distance between the parallel recesses is

5. The method for detecting a minute amount of analyte according to claim 4, wherein the depth is 5 μm or less and the depth of the groove or the recess is 1 μm or less.

[6] There is a difference between the water affinity of the plurality of groove concave surface materials arranged in parallel and the water affinity of the groove convex surface material, or

There is a difference between the water affinity of a plurality of concave surface materials arranged in parallel and the water affinity of surface materials other than the concave portions,

The method for detecting a trace substance by surface-enhanced Raman scattering according to claim 1 or 4.

[7] Solid plane is a metal surface with plasmon mirror effect

The method for detecting a trace substance according to any one of claims 1 to 6.

[8] Obtained by irradiating the area where the analyte and metal fine particles are close to each other with laser light

Used in the method of discriminating and detecting trace analytes with surface enhanced Raman scattered light

A dried substance for detecting an analyte discrimination, which is obtained by mixing an analyte and a metal fine particle colloid and drying it.

Multiple grooves in parallel with an interval of 5 microns or less or

A plurality of recesses arranged in parallel at intervals of 5 microns or less

Along the direction of the alignment of the grooves or recesses on a solid plane

1-10 microliters of fine metal particle colloid and 1-10 microliters of analyte mixture

Supply and dry a dried body with a width of 5 microns or less and a length of 10 microns or more.

[9] Solid plane is a metal surface with plasmon mirror effect

The dried body according to claim 8.

[10] For carrying out the method for detecting a trace substance according to claim 1,

Multiple grooves in parallel with an interval of 5 microns or less or

A device having a solid plane provided with a plurality of recesses arranged in parallel at intervals of 5 microns or less,

A mixing means for mixing the analyte and the metal fine particle colloid prepared in advance, and a mixture obtained by the mixing means

On a solid plane with multiple parallel grooves or multiple parallel recesses

Supply means for supplying along the direction in which the grooves or the recesses are arranged;

The mixture supplied on the solid plane provided with a plurality of grooves or a plurality of parallel recesses by the supply means

Drying means for drying on the solid plane;

A trace substance detection device having means for observing Raman scattered light by irradiating a dry mixture obtained by a drying means with laser light.

11. The apparatus for detecting a trace substance according to claim 10, wherein the supply means sprays the mixture onto a stationary plane that is stationary by a pressure generated by the volume change of the piezoelectric element that changes in volume by electric energy.

[12] For carrying out the method for detecting trace substances according to claim 4,

Multiple grooves in parallel with an interval of 5 microns or less or

An apparatus having a flow path with a solid plane as an inner surface, in which a plurality of recesses arranged in parallel at intervals of 5 microns or less are arranged,

A fluid mixing means for fluidly mixing an analyte and a metal colloid prepared in advance in a liquid phase;

The mixture obtained by fluid mixing means

Fluid transfer means for fluidly transferring to a flow path having a solid plane provided with a plurality of parallel grooves or a plurality of parallel recesses as an inner surface;

A mixture transferred to a solid plane provided with a plurality of parallel grooves or a plurality of parallel recesses by a fluid transfer means.

Drying means for standing and drying on the solid plane; A trace substance detection device having means for observing Raman scattered light by irradiating a dry mixture obtained by a drying means with laser light.

[13] The drying means for drying the mixture on the solid plane reduces the solid plane below atmospheric pressure.

13. The trace substance detection device according to claim 10 or claim 12, which also comprises:

[14] A spreading means in which the drying means extends the mixture on the solid plane to the solid plane

[15] Solid plane is a metal surface with plasmon mirror effect

15. The trace substance detection device according to any one of claims 10 to 14.

[16] Obtained by irradiating the area where the analyte and metal fine particles are close to each other with laser light

A micro-channel chip for detecting a small amount of analyte by a method of discriminating and detecting a small amount of analyte with surface-enhanced Raman scattered light,

A first channel for fluidly mixing a metal salt and a reducing agent for the metal salt to reduce the metal salt during the flow;

A second flow path for flowing the analyte that merges with the first flow path;

A microchannel chip comprising a third channel having a fixed plane in which a plurality of grooves continuous in parallel with the flow direction or a plurality of recesses arranged in parallel with the flow direction are arranged at a confluence of the first second channel.

[17] Solid plane is a metal surface with plasmon mirror effect

17. The microchannel chip according to claim 16.