CN104849254B - A kind of micro-fluidic detection chip based on Surface enhanced Raman scattering - Google Patents

Abstract

The invention discloses a microfluidic detection chip based on surface-enhanced Raman scattering. A microfluidic detection chip disclosed in the present invention is made based on the principle of surface-enhanced Raman scattering. Said sequence is successively overlapped and fixed on the carrier. The advantages of the present invention are: (1) the active substrate of the microtip array is conducive to uniform sputtering or deposition of metal layers without leaving any dead space; (2) the introduction of a highly sensitive surface on the basis of inheriting the advantages of traditional chromatography test strips Enhanced Raman scattering technology can not only effectively improve the sensitivity, but also carry out quantitative analysis; (3) The use of capillary force self-driven mode reduces the difficulty of design and processing of supporting detection equipment, reduces the weight of the equipment, and enhances the portability of the equipment , while also reducing costs.

Description

A microfluidic detection chip based on surface-enhanced Raman scattering

technical field

The invention relates to a microfluidic detection chip based on surface-enhanced Raman scattering.

Background technique

When a beam of monochromatic light is incident on an inhomogeneous medium, it will be scattered by the medium molecules. For example, when the frequency of the incident light, the energy of the photon and the direction of the photon are all changed, we call this inelastic scattering Raman scattering. Raman scattering spectroscopy can be used to characterize the vibrational energy levels of molecules, and has important applications in chemistry, physics, biomedicine and other fields. There is no special requirement for the state of the sample to be tested when Raman scattering spectroscopy is used for detection. Liquid phase, solid phase, and gas phase samples can be measured, and it has the advantages of non-contact and non-destructive to the sample. Therefore, it is used in biological sample analysis and medical testing. , environmental monitoring and other aspects have great application potential.

However, before the advent of laser light sources, the sensitivity of Raman scattering spectroscopy was too low, which once hindered the application of this analysis method. Since Fleischman (Fleischman M, et al, Chem. Phys. Lett. 1974, 26, 163-166) and other researchers first observed the enhanced Raman scattering signal of pyridine molecules on the rough silver electrode surface in 1974, this kind of The new surface photochemical effect - Surface Enhanced Raman Scattering (Surface Enhanced Raman Scattering, SERS) has played an important role in promoting the application of Raman scattering spectroscopy in the field of analysis, especially in ultrasensitive biomedical detection. Surface-enhanced Raman scattering refers to the Raman scattering spectral signal that can be significantly enhanced when the rough noble metal (such as gold, silver, copper, etc.) or its nano-sol is used as the substrate surface to analyze the molecules to be tested. The order of magnitude of enhancement of surface-enhanced Raman scattering is extremely high. In our laboratory, hollow porous silver microspheres synthesized by bacteria as a template were used as a substrate to detect mercaptopyridine. The minimum detection limit can reach 10 ^-15 M, and the enhancement factor can reach 10 ¹¹ (Dapeng Yang, et al, Green Chem. 2010, 12, 2038-2042).

Immunochromatography detection technology is a biological analysis method developed in the 1980s. Colloidal gold, colloidal selenium, colored latex microspheres, etc. are used as tracer probes to label antibodies or antigens. Driven to pass through the sample pad, the tracer probe pad, the nitrocellulose membrane immobilized with the target ligand, and the water-absorbing pad, the corresponding target molecules are captured and colored on the nitrocellulose membrane. This technology has the advantages of extremely low preparation cost, simple operation (only one operation step of adding samples), flexible and fast detection throughput (results can be obtained within 15 minutes), and thus has been widely used in the field of biomedical detection. However, due to the above-mentioned common labeling materials and the inherent limitations of the nitrocellulose membrane-based detection scheme, the detection sensitivity of this technique is low, and the precise quantitative analysis of target molecules cannot be performed.

So far, a variety of microfluidic chip solutions based on surface-enhanced Raman scattering spectroscopy have been used in the field of analysis, such as the patent application number 201110131032.1 "Microfluidic surface-enhanced Raman scattering detection device and its preparation Methods and Applications", using a microfluidic chip prepared based on a nano-hole surface roughened active substrate; patent application number 201010117672.2 "A microfluidic detection device based on a surface-enhanced Raman scattering active substrate", using Fabricate nanocolumns or nanofiber upright structures on the substrate and sputter metal nanoparticle layers to obtain surface-enhanced Raman scattering active substrates to construct microfluidic detection devices; another example is the patent application number 201110040128.7 "A Array Micro Fluidic Surface-Enhanced Raman Scattering Special Detection Chip Analysis System", which uses multi-lattice active reaction areas to solidify different tumor markers, and uses the Raman fingerprint library of various tumor markers to perform multi-marker unlabeled tumor Marker analysis; another example is the patent application number 200610008767.4 "Microfluidic chip with surface-enhanced Raman scattering spectrum active substrate and its preparation method", using physical evaporation, sputtering or chemical deposition methods combined with mask technology to prepare coin families Metal thin film layers to prepare microfluidic chips with surface-enhanced Raman scattering spectroscopy active substrates, etc. Using exogenous driving force to drive the injection of the sample to be tested, the design scheme of this type of patent must be equipped with a sample drive module in the matching detector, which complicates the design of the detector and also increases the preparation cost of the detector . From the perspective of practical application, the increase of functional modules will also increase the weight of the device and reduce the portability of the device. In addition, vertical pore-like structures with high aspect ratios or vertical columnar structures with aspect ratios are not conducive to sputtering or depositing metal layers during processing and preparation, which increases the complexity of preparation and processing.

Contents of the invention

The purpose of the present invention is to provide a microfluidic detection chip based on surface-enhanced Raman scattering.

A microfluidic detection chip provided by the present invention is made based on the principle of surface-enhanced Raman scattering. The sequence is sequentially overlapped and fixed on the carrier;

The carrier can be any carrier used in the preparation of test strips or chips, specifically, it can be a plastic casing;

The conjugate pad is a polyester film or glass fiber film cured with exogenous Raman probes;

The method of curing is non-chemical bonding;

The exogenous Raman probe is a targeting probe composed of noble metal nanoparticles coated with a ligand capable of interacting with the substance to be tested and an exogenous molecule with Raman activity;

The targeting refers to specificity for the substance to be tested;

The active substrate is obtained by setting a test area and a quality control area on the surface of the substrate used in surface-enhanced Raman scattering analysis;

The test area is connected with the ligand that can interact with the substance to be tested, and the quality control area is connected with a substance that cannot interact with the substance to be tested but can interact with the substance to be tested. ;

The test area is on the side of the conjugate pad, and the quality control area is on the side of the material that can generate capillary driving force;

The ligand that can interact with the substance to be tested on the test area of the active substrate can be combined with the substance to be tested, and the substance to be tested can be combined with the ligand that can interact with the substance to be tested on the exogenous Raman probe. Body binding, the exogenous Raman probe is immobilized on the detection area by the double-antibody sandwich method, and the intensity of the characteristic Raman scattering peak of the exogenous molecule on the exogenous Raman probe is used to reflect the concentration of the substance to be tested. content;

The substance on the quality control area of the active substrate that cannot interact with the substance to be tested but can interact with the substance to be tested can interact with the substance on the exogenous Raman probe. Ligand binding, the exogenous Raman probe can be fixed in the quality control area without passing through the substance to be tested, and the characteristic Raman scattering peak intensity of the exogenous molecule on the exogenous Raman probe can be detected Quality control of the microfluidic chip.

In the above chip, the material capable of generating capillary driving force is filter paper, glass fiber membrane or polymer water-absorbing material;

The sample pad is a glass fiber membrane;

The noble metal nanoparticles are gold nanoparticles or gold nanorods;

The ligand capable of interacting with the substance to be tested is connected to the nano-gold particle through an action including a gold-sulfur bond;

The exogenous molecule is linked to the nano-gold particle through an action including a gold-sulfur bond;

The substrate used in the surface-enhanced Raman scattering analysis is a polymer thick film or plate with an upwardly protruding pointed cone structure array covered with a thin layer of noble metal;

The polymer is a polymer that can be solidified and formed under certain conditions;

The top of the cone hole of the pointed cone is any closed geometric figure.

The closed geometric figure is specifically a triangle, trapezoid, ellipse or circle;

The array of conical structures is an array of micro-conical structures;

The height of the cones in the microtip structure array is 20-500 microns, and the area of the cone bottom is 225-250000 square microns;

The thickness of the noble metal thin layer is 30-300nm;

The thickness of the polymer thick film or plate is 100-3000um;

The polymer that can be cured and shaped under certain conditions is polyimide or polydimethylsiloxane;

The ligand capable of interacting with the substance to be tested is an antibody or an antigen.

In any of the chips described above, the exogenous molecule with Raman activity is 5,5' dithiobis(2-nitrobenzoic acid);

The substance to be tested is ricin;

The antibody is an anti-ricin polyclonal antibody;

The ligand-binding substance that cannot interact with the substance to be tested but can interact with the substance to be tested is a goat anti-rabbit polyclonal antibody;

The polymer thick film or plate with an upwardly protruding pointed cone structure array covered with a thin layer of noble metal is a polyethylene glycol functionalized polymer thick film or plate that has been double-functionalized with mercapto carboxyl groups;

The mercapto carboxyl bifunctional polyethylene glycol is connected to the polymer thick film or plate with an upwardly protruding pointed pyramid structure array covered with a thin layer of noble metal through a gold-sulfur bond;

The anti-ricin polyclonal antibody and the goat anti-rabbit polyclonal antibody are connected to the mercapto carboxyl bifunctional polyethylene glycol through an amide bond, and then fixed on the surface covered with a thin layer of precious metal with an upwardly protruding tip. Polymer thick film or plate with pyramid structure array;

The polymer is polydimethylsiloxane;

The polymer thick film or plate has a thickness of 5000 μm;

The precious metal is gold;

The thickness of the noble metal thin layer is 200nm;

The closed geometric figure is a circle;

The pattern diameter is 50 μm, and the distance between circles is 30 μm.

The conjugate pad is 3mm long and 2mm wide;

The sample pad is 3mm long and 2mm wide;

The polymer water-absorbing material has a length of 5mm and a width of 2mm;

The active substrate is 4 mm long and 2 mm wide.

In any of the chips described above, the preparation method of the exogenous Raman probe is as follows: adding the ligand capable of interacting with the substance to be tested into the noble metal nanoparticle solution, incubating, and centrifuging The supernatant was precipitated; the precipitate was resuspended with 0.1M PBS containing BSA at pH 7.4, the exogenous molecule with Raman activity was added, reacted, and centrifuged to obtain a precipitate; it was resuspended in a solution containing BSA, PEG20000 , Na ₃ N, sucrose, trehalose and Tween-20 in the PBS buffer solution of 10mM pH7.4 to obtain the exogenous Raman probe solution;

The conjugate pad is prepared as follows: immerse the polyester film or glass fiber membrane in the exogenous Raman probe solution and dry overnight;

The function of the BSA is to block the ligands capable of interacting with the substance to be tested and the remaining active sites after the exogenous molecules coat the noble metal nanoparticles;

The active substrate is prepared according to the following method: soak the substrate used in surface-enhanced Raman scattering analysis with Piranha solution, wash it, and then immerse it in an aqueous polyethylene glycol solution with mercapto carboxyl bifunctionalization to react to obtain functionalized After the substrate; use 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide to couple the ligands that can interact with the substance to be tested on the functional A test area is made on a functionalized substrate, and a substance that cannot interact with the substance to be tested but can be coupled with a ligand that can interact with the substance to be tested is coupled to a functionalized substrate to make a quality control area.

In any of the chips described above, in the preparation method of the exogenous Raman probe, the temperature of the incubation is 4°C; The step of resuspending the precipitate in PBS buffer and standing at 25°C, centrifuging at 4°C to discard the supernatant to obtain the precipitate; the condition for adding exogenous molecules with Raman activity to react is to continuously stir at 4°C for 1 hour ; said resuspending it in the PBS buffer of 10mM pH7.4 containing BSA, PEG20000, Na ₃ N, sucrose, trehalose and Tween-20 also included washing two by centrifugation with 0.1M PBS of pH7.4. The second precipitation step;

In the preparation method of the active substrate, the temperature of the reaction is 25° C., and the reaction time is 4 hours.

A method for preparing any of the chips described above also belongs to the protection scope of the present invention, including a method for preparing the conjugate pad and a method for preparing the active substrate;

The method for preparing the conjugate pad comprises the following steps: immersing the glass fiber membrane in an exogenous Raman probe solution and drying it overnight;

The drying temperature is 36°C;

The exogenous Raman probe solution is prepared according to the following method: incubate the ligand that can interact with the substance to be tested in any of the above-mentioned chips with the noble metal nano solution at 4°C, and centrifuge at 4°C to obtain a precipitate , Dissolve the precipitate in 0.1M PBS with pH 7.4 containing BSA, add exogenous molecules with Raman activity, stir at 4°C, and centrifuge at 4°C to obtain a ligand that can interact with the substance to be tested Precious metal nanoparticles coated with exogenous molecules with Raman activity are finally obtained by adding 10mM PBS buffer at pH 7.4 containing BSA, PEG20000, Na ₃ N, sucrose, trehalose and Tween-20;

The concentration of the BSA in the exogenous Raman probe solution is 1g/100ml, the concentration of the PEG20000 in the exogenous Raman probe solution is 0.2g/100ml, and the Na ₃ N The concentration in the exogenous Raman probe solution is 0.02g/100ml, the concentration of the sucrose in the exogenous Raman probe solution is 10g/100ml, and the trehalose in the exogenous The concentration in the exogenous Raman probe solution is 2.5g/100ml, and the concentration of Tween-20 in the exogenous Raman probe solution is 0.1g/100ml;

The noble metal nanometer is specifically gold nanometer;

The ligand capable of interacting with the substance to be tested is combined with the gold nanometer through an action including a gold-sulfur bond, and the exogenous molecule is combined with the nano-gold through an action including a gold-sulfur bond, The BSA seals the ligands that can interact with the substance to be tested and the remaining sites after the exogenous molecules coat the gold nanoparticles, and finally obtains the exogenous Raman probes;

The method for preparing the active substrate comprises the steps of: functionalizing the substrate used in surface-enhanced Raman scattering analysis to obtain a functionalized substrate; setting a test area and a quality control area on the functionalized substrate, and Ligands that can interact with the substance to be tested are connected to the surface of the functionalized substrate to make a test area, and substances that cannot interact with the substance to be tested but can interact with the ligand to be tested are connected to the functionalized substrate. The surface of the substrate is made into a quality control area;

The substrate used in the surface-enhanced Raman scattering analysis is specifically a polymer thick film or a polymer plate with an upwardly protruding pointed cone structure array covered with a thin layer of noble metal;

The functionalization is to soak the substrate used in the surface-enhanced Raman scattering analysis with Piranha solution, wash it with water, and then immerse it in an aqueous solution of polyethylene glycol with mercaptocarboxy bifunctionalization at 25°C for reaction;

Described Piranha solution is that the aqueous solution of the concentrated sulfuric acid and the hydrogen peroxide of volume percentage content 30% is mixed according to the volume ratio 3:1 to obtain;

The soaking time is 10-15 minutes;

The polymer thick film or polymer plate with upwardly protruding tapered structure arrays covered with a thin layer of noble metal is sputtering noble metal on the polymer thick film or polymer plate with upwardly protruding tapered structure arrays made;

The polymer thick film with upwardly protruding pointed cone structure array is poured on the female mold after uniformly mixing the polymer that can be cured and shaped under certain conditions with the corresponding curing agent, and peeled off from the female mold after curing Obtain the solidified polymer thick film obtained;

The curing agent is a substance that can promote or control the curing reaction, specifically such as hydroxysilane;

or,

The polymer plate with an upwardly protruding tapered structure array is obtained by bonding the cured polymer plate to the silicon female mold by thermocompression, and then peeling off the cured polymer plate from the female mold;

The female mold is a female mold with an array of conical holes on the surface of the hard substrate;

The hard substrate is a silicon wafer or ceramics;

The negative mold is a silicon negative mold;

The silicon negative mold is obtained by corroding a silicon wafer having an etched array of silicon cone holes in a silicon etching solution;

The silicon corrosion solution is obtained by mixing potassium hydroxide: isopropanol: water according to the mass ratio of 1:2:2;

The silicon wafer with the etched array of silicon cones and sharp holes is obtained by oxidizing the surface of the silicon wafer to obtain an oxide layer, polishing the oxide layer while throwing a layer of positive photoresist to develop the silicon dioxide pattern to be etched, Etch the silicon wafer with _SiO2 etching solution to expose the silicon dioxide layer, remove the positive photoresist, and perform wet etching;

The _SiO2 corrosion solution is obtained by mixing HF, NH4F and water according to the ratio of 84ml, _339g , and 510ml;

The negative mold can also be processed into a negative mold with an array of cone holes by using microelectronics and micromechanics (MEMS) and precision machinery technology;

The mass ratio of the polymer to the curing agent is specifically 10:1;

The thickness of the oxide layer is specifically 0.1-2 μm;

The thickness of the positive photoresist is specifically 5-50 μm.

A method for assisting in the qualitative determination of whether a sample to be tested contains a target substance also belongs to the protection scope of the present invention, comprising the following steps: adding a sample that does not contain the target substance to the sample pad of any of the above-mentioned chips, and waiting to contain no target substance. After the sample is completely absorbed by the polymer water-absorbing material on the chip, the chip is placed on a Raman spectrometer to detect the exogenous The characteristic Raman peak intensity of the characteristic molecule, the value of taking the average value plus twice the standard deviation is recorded as the Cut-off value; according to the same method, the sample to be tested is detected to obtain the characteristic Raman peak intensity of the sample to be tested, If the characteristic Raman peak intensity of the sample to be tested is higher than or equal to the Cut-off value, the sample to be tested is judged as a sample containing the target substance; if the characteristic Raman peak intensity of the sample to be tested is lower than the Cut-off value, the The test sample is judged to be a sample that does not contain the target substance.

A method for quantitatively detecting the target substance in a sample also belongs to the protection scope of the present invention, comprising the following steps: adding standard substances of different concentrations of the target substance to the sample pad of any of the above-mentioned chips, and waiting for the target substance to be completely absorbed by the After the polymer water-absorbing material on the chip has been absorbed, the chip is placed on a Raman spectrometer to detect the characteristic Raman peak of the exogenous molecule on the wavenumber of the Raman scattering peak region of the exogenous molecule Intensity, with the lg value of the target substance concentration as the abscissa, and the characteristic Raman peak intensity as the ordinate, a standard curve is made to obtain the standard curve formula; according to the same method, the sample to be tested is detected to obtain the characteristic of the sample to be tested. Raman peak intensity, the characteristic Raman peak intensity of the sample to be tested is brought into the standard curve formula to obtain the content of the target substance in the sample to be tested;

The exogenous molecule is specifically 5,5' dithiobis(2-nitrobenzoic acid);

The wavenumber of the Raman scattering peak region of the exogenous molecule is specifically 1330cm ^-1 ;

The specific unit of the target substance concentration is pg/mL;

The target substance is specifically ricin;

The standard curve formula is specifically y=1395.7x-2077.5.

The application of any of the chips described above in the preparation of products for qualitative or quantitative detection of target substances in samples also falls within the protection scope of the present invention.

In the above application, the target substance is ricin.

The advantages of the present invention are: (1) the active substrate of the microtip array is conducive to uniform sputtering or deposition of metal layers without leaving any dead space; (2) the introduction of a highly sensitive surface on the basis of inheriting the advantages of traditional chromatography test strips Enhanced Raman scattering technology can not only effectively improve the sensitivity, but also carry out quantitative analysis; (3) The use of capillary force self-driven mode reduces the difficulty of design and processing of supporting detection equipment, reduces the weight of the equipment, and enhances the portability of the equipment , while also reducing costs.

Description of drawings

Figure 1 is the standard curve for the detection of ricin by the microfluidic detection chip.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Castor (Ricinus communis) seeds were purchased from Beijing Xiuhe Seed Co., Ltd., and the product catalog number is Castor No. 1.

The balance solution is 0.01M PBS buffer, pH7.2.

Agarose affinity chromatography column was purchased from Amersham Company, the product catalog number is 17-5080-01.

New Zealand purebred white rabbits were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army.

The preparation method of anti-ricin polyclonal antibody in the following examples is as follows:

(1) Preparation of detoxified ricin

Crude extraction: remove the castor seeds and weigh 100g, soak them in PBS (0.01M, pH7.2) for 24 hours until the seeds swell; remove the shells and wash them, and place the washed seeds in 500mL PBS (0.01M, pH7.2) buffer solution, homogenate in a homogenizer, and leached at 4°C for 24 hours overnight (placed for 24 hours) to obtain the extract; the next day, filter the extract with degreasing gauze to remove the residue , Centrifuge at 12000rpm for 20min at 4°C, take the supernatant, filter it with a 0.45μm filter membrane, adjust the supernatant to pH7.2; slowly add saturated ammonium sulfate solution (pH7.4) to the supernatant until the saturation is 30% , stirred magnetically at 4°C for 1 hour, then stood still for 1 hour, centrifuged at 12,000 rpm for 20 minutes, discarded the precipitate to obtain the supernatant; continued to add saturated ammonium sulfate liquid to the supernatant to 90% saturation, stirred magnetically at 4°C for 1 hour, and stood still overnight for 24 hours to obtain a mixture The next day, the mixture was centrifuged at 12000rpm at 4°C for 20min, and the precipitate was white; the precipitate was dissolved in 50mL of PB (10mM, pH7.4), and then dialyzed against PB for 48h at 4°C (change every 8h After dialysis, it was centrifuged at 1200g at 4°C for 20min, and the supernatant was taken, which was the crude extract of ricin, for protein quantification, 12% denatured and non-denatured SDS electrophoresis analysis; it was stored at -20°C.

Affinity chromatography purification: the affinity chromatography medium is aminophenyl-β-D-galactoside agarose affinity medium (purchased from sigma company, product catalog number is A-0414). After vacuum degassing 5mL of affinity medium, slowly load it into the chromatography column, pay attention to avoid bubbles and faults, and wash and balance with PBS at least 10 times the volume of the column bed. After the crude ricin extract was filtered through a 0.45 μm filter membrane, it was diluted with an equilibrium solution 10 times in volume, and added to the chromatography column at a flow rate of 1 mL/min, so that the ricin and lectin were fully adsorbed on the column. After the ricin and lectin are fully adsorbed, use the balance solution to elute the impurity protein, and the flow rate is controlled at about 1mL/min. After the impurity protein is completely eluted, use PBS buffer containing 0.1M galactose to elute the target protein in one step. , collect elution peaks, 12% denaturing, non-denaturing SDS electrophoresis analysis.

Gel filtration chromatography: the gel filtration column is a SephacrylTMS-100G75 gel prepacked column (purchased from Amersham). The sample concentrated by affinity chromatography was filtered through a 0.22 μm filter membrane and loaded with a sample volume of 5 mL and a concentration of 2 mg/mL; PBS was used as the eluent for elution at a flow rate of 0.5 mL/min. The components of the second absorption peak were combined and analyzed by 12% denatured and non-denatured SDS electrophoresis as ricin protein; the obtained ricin protein was dialyzed, concentrated and stored at -20°C.

Detoxification: Dilute the ricin protein after gel filtration with PBS (0.1M, pH7.4) to 0.2-0.3mg/ml, place it in PBS buffer (0.1M, pH8.1), and dialyze at 35°C for 7 days , and then dialyzed in PBS (0.01M, pH7.4) for 48h, centrifuged at 12000rpm for 10min, the supernatant was taken and stored at -20°C.

The amino acid sequence of ricin is shown in SEQ ID No.1.

(2) Animal immunity

Using detoxified ricin protein as the immunogen, take 200 μg and mix well with an equal volume of Freund's complete adjuvant to immunize New Zealand purebred white rabbits. Two weeks later, boost immunization once with 0.5 mg detoxified ricin protein and an equal volume of Freund's incomplete adjuvant; after three weeks, use 1 mg detoxified ricin protein and an equal volume Freund's incomplete adjuvant Immunization was boosted once; thereafter, blood was collected from the ear vein every week, and the titer was determined by indirect ELISA. The titer of the serum on the 7th day after the booster immunization was the highest, 10 ⁶ , and the serum was taken for polyantibody purification.

(3) Antibody purification

Polyclonal antibodies were purified using agarose affinity chromatography columns.

The specific steps are as follows: equilibrate the affinity column with equilibration buffer (1L equilibration buffer is 1000ml 20mM PB buffer containing 300mmol NaCl, pH7.8), after the column is equilibrated, filter the serum obtained in step (2) through 0.45μm After membrane filtration, put it on the column at a flow rate of 1mL/min, use the equilibrium buffer to elute the impurity protein at a flow rate of 1mL/min, monitor, and change the eluent (0.1M citric acid buffer, pH4.0) after the impurity protein is completely eluted Wash down the antibody. Collect the washed antibody and add an appropriate amount of 1mol/L, pH9.0 Tris-HCl to the collection tube to restore the pH to about 7.4, so as not to lose activity. The purified antibody was dialyzed against PBS (5mM, pH7.4) working solution at 4°C for 24h, the solution was changed every 8h, and then centrifuged at 10000r/min for 30min, the supernatant was collected and stored at -70°C for later use.

Goat anti-rabbit polyclonal antibody was purchased from Aldrich-Sigma, USA, the product catalog number is SAB3700883.

SiO ₂ corrosive solution: Mix HF 84ml, NH ₄ F 339g, and water 510ml.

Silicon etching solution: Mix potassium hydroxide: isopropanol: water according to the mass ratio of 1:2:2.

Mercapto carboxyl bifunctional polyethylene glycol (PEG), namely (HS-C ₂ H ₄ -CONH-PEG-C ₃ H ₆ -COOH), was purchased from Rapp Polymere, Germany.

Embodiment 1, preparation of microfluidic detection chip

1. Preparation of exogenous Raman probes and preparation of glass fiber membranes cured with Raman probes

(1) Preparation of gold nanoparticles

The preparation scheme of nano gold is sodium citrate reduction method. Specific steps are as follows:

Take 99mL of ultrapure water and heat it in a firing vessel. After the solution boils, add 1mL of 0.1g/100ml aqueous solution of chloroauric acid and keep boiling for more than 2min. Add 1mL of 1g/100ml trisodium citrate aqueous solution preheated to 80°C, keep boiling and heat and stir quickly evenly, when the reaction solution suddenly changes from black purple to wine red, the reaction is over, reduce the speed, and continue boiling and heating for 10min Stop heating, stir slowly until it cools down naturally, and then set the volume to 100mL for later use. This solution is the nano-gold solution.

(2) Slowly add 1ml of 80μg/ml anti-ricin polyclonal antibody PBS solution into 1ml of nano-gold solution while stirring, incubate at 4°C for 1h, then centrifuge at 4°C at 8000rpm for 45min, and discard the supernatant overnight to retain the precipitate. Resuspend the pellet with an equal volume of pH7.40.1M phosphate buffered saline (PBS) and let stand at 25°C for 30min, then centrifuge at 8000rpm for 45min at 4°C, discard the supernatant to retain the pellet. Then resuspend the pellet with 0.1 M PBS at pH 7.4 containing 1 g/100 ml of BSA, add 20 μl of 1 mM 5,5' dithiobis(2-nitrobenzoic acid) (DTNB) dissolved in PBS at pH 8.5 In the buffer solution, stir continuously at 4°C for 1 hour, centrifuge at 8000 rpm for 45 minutes, wash twice with 0.1M PBS with pH 7.4, and resuspend in 1ml containing 1g/100ml BSA, 0.2g/100ml PEG20000, 0.02g/100mlNa ₃ N, 10g/100ml sucrose, 2.5g/100ml trehalose, 0.1g/100ml Tween-20, 10mM pH7.4 PBS buffer solution to obtain exogenous Raman probe solution.

(3) Immerse the glass fiber membrane in the exogenous Raman probe solution prepared in step (2) to obtain the glass fiber membrane with the exogenous Raman probe cured, dry it at 36°C overnight and cut it into 3×2mm size spare.

2. Preparation of microtip array active substrate

(1) After the silicon wafer is cleaned, it is first oxidized with wet oxygen at 1130°C for 5 hours, and then oxidized with dry oxygen for 2 hours (high-temperature water vapor oxidation at 1130°C with wet oxygen, and the water flow is 800ml/min; dry oxygen oxidation is carried out in air. Pure oxygen oxidation, oxygen flow 500ml/min), a uniform and dense oxide layer with a thickness of about 2 μm is oxidized on the surface, and this oxide layer is used as a mask layer for etching silicon (the barrier layer in the process of etching silicon, that is, Is there a silicon dioxide layer in the place that needs to be etched, and there is a silicon dioxide layer to cover the place that does not need to be etched). Polish one side of the oxidized silicon wafer and throw a layer of 5-50μm thick positive photoresist. After photolithography, develop the silicon dioxide pattern to be etched in 0.5% developer solution (after the positive photoresist, The pattern area is exposed to ultraviolet light by a photolithography machine, and then developed in a developer, the positive photoresist in the exposed area is removed, and the positive photoresist in other places is retained). The pattern is a circle with a diameter of 50 μm, and the distance between circles is 30 μm. After rising from room temperature to 50°C, keep warm for 60 minutes, rise to 90°C within 30 minutes, keep warm for 120 minutes, and complete film hardening with furnace cooling.

(2) Etch the silicon wafer with SiO ₂ etching solution for about 10 minutes to expose the silicon dioxide layer. Directly soak in acetone and alcohol sequentially or directly wipe to remove the positive photoresist. Control the wet etching time according to the size of the silicon pattern to be etched (the pattern after development remains on the silicon wafer). The etching parameters are: temperature: 45°C, pH value 3, etching rate 400nm/min. Until the etching stops, the etched silicon cone hole array is left on the silicon wafer. Put the silicon wafer etched in the predetermined area into the prepared silicon etching solution, and etch it at 78°C. Due to the anisotropy of silicon etching, the required circular cone hole can be obtained after a certain etching time. In addition, in order to prevent "islands" from appearing at the bottom of the cone hole, a certain amount of stirring is required during the etching process to obtain a silicon negative mold. Mix PDMS (polydimethylsiloxane) and curing agent hydroxysilane uniformly at a mass ratio of 10:1 and pour it on the silicon negative mold. Remove the air bubbles in the mixture under vacuum and negative pressure, and cure at 75°C. , the cured PDMS is peeled off from the silicon wafer at room temperature to leave an array of cone tips on the PDMS surface in contact with the silicon negative mold surface.

(3) The PDMS prepared in step (2) with the cone-tip array was cut into strips of 4mm and 2mm in width, and then a 200nm gold film was sputtered on it. Using LEYBOLD-HERAEUS Z550 sputtering machine, the basic parameters of the sputtering seed layer process: the power is 600W, the flow rate of high-purity argon gas is 4.5sccm, the background vacuum is 3.0× ^10-6 mbar, and the working pressure is 5.0×10-3mbar , the sputtering time was 6min, and finally a PDMS strip covered with gold film was obtained.

(4) Soak the PDMS strip covered with gold film in Piranha solution (concentrated sulfuric acid: 30% by volume hydrogen peroxide solution mixed according to the volume ratio of 3:1) for 10-15 minutes, wash with pure water and then The PDMS strips covered with gold film were immersed in 1mM mercapto carboxyl bifunctional polyethylene glycol (PEG) aqueous solution at 25°C for 4 hours, washed with pure water and then washed with conventional 1-ethyl-3- (3-Dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) reagents were used to couple anti-ricin polyclonal antibody and goat anti-rabbit polyclonal antibody to micro The test area and the quality control area on the active substrate of the cone array.

3. Assembly of microfluidic detection chip

Cut the glass fiber membrane, the glass fiber membrane cured with Raman probes prepared in step 1, and the polymer water-absorbing material into 3×2mm, 3×2mm and 5×2mm sizes respectively, and then combine with the anti- The microtip array active substrates of ricin polyclonal antibody and goat anti-rabbit polyclonal antibody are in the order of glass fiber membrane, glass fiber membrane cured with Raman probes, microtip array active substrate and polymer water-absorbing material The microfluidic detection chip is formed by overlapping and pasting on the plastic shell. Among them, the goat anti-rabbit polyclonal antibody coupling area on the active substrate of the microtip array is in the direction of the polymer water-absorbing material, and the anti-ricin polyclonal antibody coupling area is in the direction of the glass fiber membrane cured with Raman probes.

4. Detection of microfluidic detection chip

Take 10 parts of PBS solution not containing ricin (100ul each) and add it to the glass fiber membrane side of the microfluidic chip prepared in step 3. After the sample is completely absorbed by the polymer water-absorbing material, place it in the Raman spectrometer i-Raman (B&WTekInc, Newark, DE) detects the intensity of the characteristic Raman peak of DTNB with a wavenumber of 1330cm ^-1 in the corresponding area, and takes the mean value plus twice the standard deviation as the cut-off value (Cut-off value) for the judgment result. In the detection of unknown samples, those higher than and equal to this value are judged as positive, and those lower than this value are judged as negative.

During quantitative detection, prepare the ricin standard substance of different concentrations, obtain the DTNB characteristic Raman peak intensity of ^1330cm by the above-mentioned method, take the lg value of ricin concentration as abscissa, and Raman characteristic peak intensity as vertical coordinates, make a standard curve, and obtain the standard curve formula, as shown in Figure 1. Quantitative analysis of ricin content in unknown samples can be carried out by substituting the characteristic Raman peak intensity of 1330cm ^-1 of DTNB in samples containing ricin in unknown concentration into the standard curve formula.

Claims (10)

1. it is the micro-fluidic detection chip based on made of the principle of Surface enhanced Raman scattering a kind of micro-fluidic detection chip Overlapped and be fixed on successively in the order described above by material, active substrate, conjugate pad and the sample pad that can produce REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power Formed on carrier；

The material for producing REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power is filter paper, glass film or polymer absorbent material；

The conjugate pad has the polyester film or glass film of exogenous Raman microprobe to cure；

The sample pad is glass film；

The exogenous Raman microprobe is can be with the ligand of matter interaction to be checked and the exogenous molecule with Raman active The targeting probe that the noble metal nano particles coated jointly are formed；

The noble metal nano particles are nano Au particle or nanometer gold bar；

The targeting refers to that specificity is directed to material to be checked；

The active substrate is to set test section and quality control region on the surface of substrate used when Surface enhanced Raman scattering is analyzed Obtained from；

The substrate used when Surface enhanced Raman scattering is analyzed is covered with having for noble metal thin layer for surface and projects upwards The polymer thick film or plate of cone-shaped array of structures；

The thickness of the noble metal thin layer is 30-300nm；

The thickness of the polymer thick film or plate is 100-3000um；

Be connected with the test section it is described can with the ligand of matter interaction to be checked, be connected with quality control region cannot with it is to be checked Matter interaction but can be with the material of the ligand binding of the matter interaction to be checked；

Described can be antibody or antigen with the ligand of matter interaction to be checked；

Side is padded in the test section in the conjugate, and quality control region is in the material side for producing REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power.

2. chip according to claim 1, it is characterised in that：

The polymer for can curing molding under certain condition polymer；

It is any enclosed geometric figure at the top of the cone hole of the pointed cone；

It is described can the polymer of curing molding under certain condition be polyimides or dimethyl silicone polymer.

3. chip according to claim 2, it is characterised in that：

The exogenous molecule with Raman active is 5,5 ' two thiobis (2- nitrobenzoic acids)；

The material to be checked is ricin (WA)；

The antibody is antiricin polyclonal antibody；

It is described cannot be with matter interaction to be checked but can be goat-anti rabbit with the material of the ligand binding of matter interaction to be checked Polyclonal antibody；

What the surface was covered with noble metal thin layer is first to pass through with cone-shaped the array of structures polymer thick film or plate projected upwards Cross the polymer thick film or plate of the polyethylene glycol functionalization of mercapto carboxy difunctionalization；

The polyethylene glycol of mercapto carboxy difunctionalization is connected to the tool that the surface is covered with noble metal thin layer by golden sulfide linkage Have on cone-shaped the array of structures polymer thick film or plate projected upwards；

It is difunctional that the antiricin polyclonal antibody and goat-anti rabbit polyclonal antibody by amido link are connected to mercapto carboxy On the polyethylene glycol of change, so be fixed on the surface be covered with noble metal thin layer with the cone-shaped array of structures projected upwards On polymer thick film or plate；

The polymer is dimethyl silicone polymer；

The noble metal is gold；

The thickness of the noble metal thin layer is 200nm；

The closure geometric figure is circle；

The pattern diameter is 50 μm, and the distance between circular and circle is 30 μm.

4. chip according to claim 1 or 2, it is characterised in that：The preparation method of the exogenous Raman microprobe is as follows： It can add in the noble metal nano particles solution, incubate, supernatant is abandoned in centrifugation with the ligand of matter interaction to be checked by described It must precipitate；Sediment is resuspended with the PBS of the 0.1M of the pH 7.4 containing BSA, adds exogenous point with Raman active Son, reaction, centrifuging to precipitate；It is resuspended in containing BSA, PEG20000, Na₃N, sucrose, trehalose and Tween-20 In the PBS buffer of 10mM pH7.4, the exogenous Raman microprobe solution is obtained；

The conjugate pad is prepared as follows：The polyester film or glass film are immersed into the exogenous Raman microprobe In solution, it is dried overnight to obtain the final product；

The active substrate is prepared as follows：Analyzed with the immersion of Piranha solution is described in Surface enhanced Raman scattering Shi Suoyong substrates, are cleaned, then are dipped in the Aqueous Solutions of Polyethylene Glycol of mercapto carboxy difunctionalization and are reacted, after obtaining functionalization Substrate；Can be with material to be checked with 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides and n-hydroxysuccinimide Test section is made in the ligand coupling of interaction in the substrate of functionalization, equally cannot with matter interaction to be checked but It can be coupled at the material of the ligand binding of matter interaction to be checked in the substrate of functionalization and quality control region is made.

5. chip according to claim 4, it is characterised in that：It is described in the preparation method of the exogenous Raman microprobe The temperature of incubation is 4 DEG C；The centrifugation abandons after supernatant must precipitate to further include is hanged again with the PBS buffer of 7.4 0.1M of pH Drift along and form sediment and stood at 25 DEG C, the step of supernatant obtains sediment is abandoned in 4 DEG C of centrifugations；Described add has the exogenous of Raman active The condition that molecule is reacted be continuously stirred at 4 DEG C 1 it is small when；It is described to be resuspended in containing BSA, PEG20000, Na₃N, sugarcane The PBS centrifugations with the 0.1M of pH 7.4 are further included before in the PBS buffer of the 10mM pH7.4 of sugar, trehalose and Tween-20 Wash twice the step of must precipitating；

In the preparation method of the active substrate, the temperature of the reaction is 25 DEG C, when the time is 4 small.

Prepare the method for any chip in claim 1-5 6. a kind of, including prepares the method for the conjugate pad with The method for preparing the active substrate；

The method for preparing the conjugate pad, includes the following steps：Glass film is immersed in exogenous Raman microprobe solution, It is dried overnight to obtain the final product；

The exogenous Raman microprobe solution is prepared as follows：By institute in any chip in claim 1-5 Stating can be with 4 DEG C of incubation of ligand and noble metal nano solution of matter interaction to be checked, and 4 DEG C centrifuge and must precipitate, and precipitation is dissolved in The PBS of the 0.1M of pH 7.4 containing BSA, adds the exogenous molecule with Raman active, 4 DEG C of stirrings, 4 DEG C centrifuge and must sink Form sediment, the noble metal for obtaining to coat jointly with the ligand of matter interaction to be checked and the exogenous molecule with Raman active is received Rice corpuscles, is eventually adding containing BSA, PEG20000, Na₃N, the PBS of the 10mM pH7.4 of sucrose, trehalose and Tween-20 delay Fliud flushing to obtain the final product；

Concentration of the BSA in the exogenous Raman microprobe solution is 1g/100ml, and the PEG20000 is in the external source Concentration in property Raman microprobe solution is 0.2g/100ml, the Na₃Concentration of the N in the exogenous Raman microprobe solution is 0.02g/100ml, concentration of the sucrose in the exogenous Raman microprobe solution is 10g/100ml, and the trehalose exists Concentration in the exogenous Raman microprobe solution is 2.5g/100ml, and the Tween-20 is molten in the exogenous Raman microprobe Concentration in liquid is 0.1g/100ml；

The noble metal nano is specially nanogold；

The method for preparing the active substrate, includes the following steps：Will when Surface enhanced Raman scattering is analyzed base used Bottom carries out functionalization, obtains the substrate of functionalization；Test section and quality control region are set in the substrate of functionalization, can be with thing to be checked The ligand of matter interaction is connected to the substrate surface of functionalization and test section is made, it is impossible to matter interaction to be checked but Quality control region is made in the substrate surface that functionalization can be connected to the material of the ligand binding of matter interaction to be checked；

The substrate used when Surface enhanced Raman scattering is analyzed is specially that surface is covered with dashing forward with upward for noble metal thin layer Cone-shaped the array of structures polymer thick film or polymer sheet gone out；

The functionalization is by the substrate Piranha solution immersion used when Surface enhanced Raman scattering is analyzed, and is eluted with water It is dipped in 25 DEG C of reactions in the Aqueous Solutions of Polyethylene Glycol of mercapto carboxy difunctionalization again afterwards；

The Piranha solution is the aqueous solution of the hydrogen peroxide of the concentrated sulfuric acid and volumn concentration 30% according to volume ratio 3:1 It is uniformly mixed so as to obtain；

The surface be covered with noble metal thin layer with cone-shaped the array of structures polymer thick film or polymer sheet projected upwards It is to be formed described with cone-shaped the array of structures polymer thick film or polymer sheet that project upwards sputtering noble metal；

It is described with the cone-shaped array of structures polymer thick film projected upwards be by can curing molding under certain condition it is poly- Compound uniformly mixes after-pouring on former with corresponding curing agent, is peeled off after curing from former and obtains the polymerization of curing molding Thing thick film obtains；

The curing agent is the material that can promote or control curing reaction, specific such as silicol；

Or,

Described with the cone-shaped array of structures polymer sheet projected upwards is by cured polymer sheet and silicon former hot pressing key After conjunction, cured polymer sheet is peeled off from former and is obtained；

The former is the former for having cone hole array on hard substrate surface；

The former is silicon former；

The silicon former is that the silicon chip with the silicon cone hole array being etched is put into corrosion in silicon etch solution to obtain；

The silicon etch solution is by potassium hydroxide：Isopropanol：Water is according to quality 1：2：2 ratio mixes to obtain the final product；

The silicon chip with the silicon cone hole array being etched is to aoxidize silicon chip surface to obtain oxide layer, in oxide layer upthrow Light one side gets rid of one layer of positive photoetching rubber, the silicon dioxide pattern of the needs that develop etching, by SiO₂Corrosive liquid corrosion of silicon exposure two Silicon oxide layer, removes positive photoetching rubber, wet etching to obtain the final product；

The SiO₂Corrosive liquid is by HF, NH₄F and water are mixed according to the ratio of 84ml, 339g, 510ml to obtain the final product.

7. in a kind of qualitative measuring samples of auxiliary whether the method containing desired substance, include the following steps：It will be free of purposeful The sample of material is added in claim 1-5 in the sample pad of any chip, and the sample of desired substance not contained is complete After the material absorption that REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power can be produced described on the chip entirely, the chip is placed in Raman spectrum inspection Survey on instrument, detect the characteristic Raman peak intensity of the exogenous molecule in the Raman scattering peak region wave number of exogenous molecule Degree, is averaged plus the value of twice of standard deviation is denoted as Cut-off values；After the same method, measuring samples are detected, obtained To the characteristic Raman peak intensity of measuring samples, if the characteristic Raman peak intensity of measuring samples is greater than or equal to Cut-off Then measuring samples are determined as the sample containing desired substance to value, if the characteristic Raman peak intensity of measuring samples is less than Cut- Then measuring samples are judged to not containing the sample of desired substance to off values.

8. a kind of method for quantitatively detecting desired substance in sample, includes the following steps：By the mark of the desired substance of various concentrations In quasi- product addition claim 1-5 in the sample pad of any chip, treat desired substance completely by the institute on the chip After stating the material absorption that can produce REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power, chip is placed on Raman spectrum detector, is detected at exogenous point The characteristic Raman peak intensity of the exogenous molecule in the Raman scattering peak region wave number of son, with the lg of desired substance concentration It is ordinate to be worth for abscissa, characteristic Raman peak intensity, makes standard curve, obtains calibration curve formula；According to same Method, is detected measuring samples, obtains the characteristic Raman peak intensity of measuring samples, by the characteristic Raman of measuring samples Peak intensity substitutes into calibration curve formula, obtains the content of desired substance in measuring samples.

9. any chips of claim 1-5 are in the product of the desired substance in preparing qualitative or quantitative detection sample Using.

10. application according to claim 9, it is characterised in that：The desired substance is ricin (WA).