CN108896754A - A kind of sliding micro-fluidic chip of quick detection biomarker - Google Patents
A kind of sliding micro-fluidic chip of quick detection biomarker Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
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- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a kind of sliding micro-fluidic chips of quickly detection biomarker, micro-fluidic chip will be slided, nanotechnology and biotechnology three combine, utilize the specific recognition principle of antigen-antibody, fluorescent nano particles and detection antibody are assembled into combined probe, by sliding chip, the oxygen that platinum nano-solution and hydrogen peroxide mixing generate pushes the reaction of sample and combined probe, in conjunction with fluorescence counting technology, additional power device and complicated artificial cleaning procedure are not needed in detection process.Sample and reagent consumption are few, it is easy to operate, production, testing cost are low, can Reusability, measurement accuracy is high, and test scope is big, analysis time is short, the rapid quantitative detection that more biomarkers can also be generalized to by replacing different antibodies, will not influence the specificity of this method in different systems, detects immediately for biomarker in disease prevention, medical diagnosis on disease, personalized medicine and provides a kind of new analysis means.
Description
Technical Field
The invention relates to a sliding microfluidic chip, in particular to a sliding microfluidic chip for rapidly detecting biomarkers.
Background
The current detection methods of hCG include radioimmunoassay, enzyme-linked immunosorbent assay, and colloidal gold labeled test strip; the CEA detection method comprises chemiluminescence immunoassay, radioimmunoassay, and enzyme-linked immunosorbent assay. The colloidal gold labeled test strip method is simple to operate, but has low sensitivity; several other methods are highly sensitive, but the analysis method is time-consuming and the sample size is large; some methods require expensive chemicals such as radioisotopes, enzymes, or fluorescently labeled antibodies/antigens.
The microfluidic technology has the characteristics of low sample consumption, high integration level, good portability, low cost and the like, and the enzyme-linked immunosorbent assay plays an important role in detection and treatment of biomarkers and diseases when introduced into a microfluidic platform. However, conventional microfluidic technology introduces fluidic accessories and pump control systems, increasing device space and cost.
The slide chip does not require an additional accessory, and can operate the liquid only by sliding. However, when performing enzyme-linked immunoassay, it usually requires multi-step sliding or manual operation to perform the washing procedure, which increases the analysis time, and also complicates the design of the chip and the actual operation steps, which is not suitable for popularization.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a sliding microfluidic chip and a method for rapidly detecting a biomarker (ELISA) based on the chip without an additional power device and a complicated manual cleaning procedure.
In order to achieve the above object, the present invention adopts the following technical solutions:
the slide micro-control flow chip comprises a slide base material and a slide control flow chip, wherein the slide base material comprises a first slide and a second slide which are matched, and the first slide and the second slide are rectangular and have the same size;
the binding surface of the slide is carved with a plurality of channels, the channels comprise a reagent tank, a gas tank, an antibody tank, a sample tank and a detection tank which are sequentially connected in series through a main channel, and the front end and the rear end of the antibody tank are respectively connected in series with a buffer tank;
the reagent tank comprises a main reagent tank and a secondary reagent tank;
the main reagent groove, the gas groove, the antibody groove, the sample groove, the detection groove and the buffer groove are engraved on the first slide glass,
the auxiliary reagent groove and the main flow channel are carved on the second slide;
the buffer tank is provided with a plurality of groups of interval runners, consists of parallel main runners and auxiliary runners and is engraved on the second glass slide.
The main reagent tank, the auxiliary reagent tank, the buffer tank, the antibody tank and the sample tank are respectively provided with a matched injection hole and a matched outflow hole which are communicated through an injection flow channel and an outflow flow channel;
the injection runner and the outflow runner of the secondary reagent groove are carved on the first glass slide, and the injection hole and the outflow hole are carved on the second glass slide;
the main reagent groove, the buffer groove, the antibody groove, the injection hole and the outflow hole of the sample groove, the injection flow channel and the outflow flow channel are engraved on the second glass slide;
and when the auxiliary flow channel is communicated: the main reagent tank, the buffer tank, the antibody tank and the sample tank are respectively communicated with the injection hole and the outflow hole.
The detection groove is connected with the indicating channel and comprises a second secondary reagent groove, an indicating gas groove, an indicator groove and a scale groove matched with the scale mark, which are sequentially connected in series through a secondary flow channel;
the second secondary reagent groove and the indicator groove are respectively provided with a filling hole, a flow-out hole, a filling flow channel and a flow-out flow channel which are matched with each other;
the secondary flow channel, the indicating gas groove and the injection flow channel, the outflow flow channel and the scale groove of the indicator groove are engraved on the first glass slide;
the indicator groove and the injection hole and the outflow hole thereof, the second auxiliary reagent groove and the injection hole, the outflow hole and the scale mark thereof are marked on the second slide glass;
and when the main runner is connected: the indicator groove and the second sub-reagent groove are respectively communicated with the injection hole and the outflow hole. The preparation method of the sliding micro-control flow chip comprises the following steps:
a1, designing a chip mask, and etching a glass slide by adopting standard photoetching and/or wet method;
and A2, perforating the etched glass slide, modifying the surface of the glass slide into hydrophobicity, and then forming the chip.
The method of claim 4, wherein the standard photolithography in step A1 uses SPR220-7 as a photoresist, AZ400K as a developer, 1, 1, 1, 3, 3, 3-hexamethyldisilazane as a hydrophilic agent; the wet etching adopts HF and NH as etching liquid4F and HNO3Mixing the solution, wherein the molar ratio is preferably 1:0.5: 0.75;
the hydrophobizing agent adopted in the hydrophobizing treatment in the step A2 is perfluorooctyl trichlorosilane; the composition agent is dimethyl silicone oil.
The method for rapidly detecting the biomarker by the sliding micro-control flow chip comprises the following steps:
s1, covalently fixing the capture antibody by reacting with an epoxy group in the detection groove;
s2, adding the standard solution of the biological marker into the sample groove,
the buffer solution is added into the buffer tank,
adding the composite probe solution into an antibody groove,
adding a hydrogen peroxide solution and a platinum nanoparticle solution into a main reagent tank and an auxiliary reagent tank respectively;
s3, connecting the slide glass with the main flow channel, and pushing the liquid to pass through the detection groove by the gas generated in the reagent groove to finish the ELISA reaction of the antigen and the antibody;
s4, acquiring a fluorescence picture during detection in the detection tank by using a full-automatic fluorescence microscope, and analyzing by combining Image J to obtain the number of the fluorescent nanoparticles in unit area;
s5, adding the biomarker standard solutions with different concentrations into the sample tank, repeating the steps S2-S4, and drawing a standard curve chart;
and S6, adding the biomarker sample into the sample tank, repeating the steps S2-S4, and analyzing by combining the standard curve chart of the step S5 to obtain the concentration of the antibody sample to be detected.
The step of immobilizing the capture antibody in step S1 is:
b1, incubating the detection tank with the piranha solution for a period of time, and then cleaning;
b2, incubating the detection tank with a mixed solution of 10% 3- (2, 3-epoxypropoxy) propyl trimethoxy silane (GPS) and toluene for a period of time, washing and drying;
b3, incubating the capture antibody in the detection groove for a period of time, covalently fixing, and washing.
Further, the method comprisesThe piranha solution obtained in the step B1 is H2SO4:H2O2The incubation time is 1-2h, and the cleaning solution is deionized water;
the incubation time in the step B2 is 1-2h, the cleaning solution is toluene, and the drying temperature is 100-120 ℃;
the incubation temperature in the step B3 is 3-5 ℃, the cleaning solution is BSA solution with the mass content of 4-6%, and the BSA can effectively avoid non-specific adsorption after cleaning the detection area for many times;
further, the preparation method of the platinum nanoparticles in the step S2 includes: reducing chloroplatinic acid by ascorbic acid to prepare nano platinum particles; the reaction temperature is preferably 80 ℃ and the reaction time is preferably 30 min.
Further, the method for preparing the composite probe in step S2 includes:
c1, preparing carboxylated fluorescent nano-silica particles: synthesizing fluorescent nano-silica particles by using a reverse microemulsion method through a fluorescent substance bipyridyl ruthenium and nano-silica, and then carrying out covalent coupling with a silane carboxylation reagent to prepare carboxylated fluorescent nano-silica particles;
c2, preparing a composite probe: and (3) carrying out covalent coupling on the detection antibody and the carboxylated fluorescent nano-silica particles by adopting a carbodiimide method to prepare the composite probe.
Still further, the inverse microemulsion system in step C1 above: 1.77g Triton X-100, 7.5 mL cyclohexane, 1.6mL hexanol, 400 μ L deionized water, 80 μ L0.1M ruthenium bipyridine, 100 μ L ethyl orthosilicate (TEOS), 65 μ L ammonia, 100 μ L L N-trimethoxysilylpropylethylenediamine triacetate; stirring the fluorescent nano silicon dioxide particles at room temperature for reacting for 18 hours; carboxylated coupling was reacted for 4h in PBS (10 mmol/mL, pH = 7-8) buffer at room temperature.
Further, the biomarker includes hCG and/or CEA.
The invention has the advantages that:
the invention combines a sliding micro-fluidic chip, a nanotechnology and a biotechnology, utilizes the principle of antigen-antibody specificity recognition, assembles fluorescent nanoparticles and a detection antibody into a composite probe in the presence of a specific antigen, pushes the reaction of a sample and the composite probe by oxygen generated by mixing a sliding chip, a platinum nanometer solution and hydrogen peroxide, and combines a fluorescence counting technology to obtain a result. The detection process does not need an additional power device, and the sample amount only needs one drop of blood, so the method is a detection method with low cost, short analysis time, high precision and high sensitivity.
In the ELISA reaction, the fluorescent nano-silica particles are introduced to mark the analyte, the quantitative detection can be realized by a fluorescence counting technology without complex data processing process, and the detection limit is 100mIU ml compared with the detection limit of the radio immunoassay hCG−1The detection limit of enzyme-linked immunosorbent assay (ELISA) hCG is 17 mIUml−1(ii) a The detection limit of the colloidal gold labeled test strip method hCG is 50 mIUml−1The analysis time of the traditional microchip for detecting CEA is 4 h; the detection limits of hCG and CEA are 2mIU and 1ng/mL respectively, and the analysis time is 10 min.
The self-driven sliding microfluidic chip disclosed by the invention can be used for quantitative detection and is suitable for analysis of various biomarkers. By replacing other detection antibodies which have specific response to the sample, the detection of various markers can be realized only by replacing the detection antibodies of the composite probes with corresponding markers.
The sliding microfluidic chip is prepared from two common glass sheets, the consumption of samples and reagents is less, the operation is simple, the manufacturing and detection cost is low, the sliding microfluidic chip can be repeatedly used, the measurement precision is high, the test range is wide, the sliding microfluidic chip can be popularized to quantitative detection of more biomarkers by replacing different antibodies, the specificity of the method cannot be influenced in different systems, and a new analysis means is provided for instant detection of the biomarkers in disease prevention, disease diagnosis and personalized medical treatment. The portable fluorescence detection device is combined, the portable fluorescence detection device is more suitable for on-site and in-situ detection, and further combined with a mobile phone intelligent terminal, analysis results are uploaded to a cloud medicine system at the first time, personalized diagnosis can be realized, and the portable fluorescence detection device has strong practicability and wide applicability.
Drawings
FIG. 1 is a schematic view of the structure of a first slide of the present invention;
FIG. 2 is a schematic view of the structure of a second slide of the present invention;
FIG. 3 is a schematic structural view of the auxiliary flow channel of the present invention in the on state;
FIG. 4 is a schematic structural diagram of a main flow channel in a closed state according to the present invention;
FIG. 5 is a schematic view of the structure of the secondary flow path in the on state of the present invention;
figure 6 is a standard graph of hCG according to the invention.
FIG. 7 is a standard curve chart of CEA of the present invention.
The designations in the drawings have the following meanings:
a. an injection hole, b, an injection flow channel, c, an outflow hole, d, an outflow flow channel;
1. a main reagent tank, 2, a gas tank, 3, a buffer tank, 4, an antibody tank, 5, a sample tank, 6, a detection tank, 7, a scale tank, 8, an indication gas tank, 9, a secondary flow channel, 10, a correction tank, 11, a secondary reagent tank, 12, a main flow channel, 13, an auxiliary flow channel, 14, a secondary reagent tank, 15, an indicator tank, 16 and a scale mark.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Structure of sliding micro-control flow chip
The slide micro-flow control chip is characterized in that the base material of the slide micro-flow control chip is a glass slide, preferably a first glass slide and a second glass slide which are rectangular and have the same size; the binding face of the slide is carved with a plurality of channels, including a reagent tank, a gas tank 2, a buffer tank 3, an antibody tank 4, a buffer tank 3, a sample tank 5 and a detection tank 6 which are connected in series in sequence through a main channel 12.
The reagent groove consists of a main reagent groove 1 and an auxiliary reagent groove 11, the main reagent groove 1, a gas groove 2, an antibody groove 4, a sample groove 5, a detection groove 6 and a buffer groove 3 are engraved on a first glass slide, and the auxiliary reagent groove 11 and a main flow passage 12 are engraved on a second glass slide;
the buffer grooves 3 are respectively provided with 2 groups of interval flow channels consisting of parallel main flow channels 12 and auxiliary flow channels 13 which are carved on the second slide glass.
The main reagent tank 1, the auxiliary reagent tank 11, the buffer tank 3, the antibody tank 4 and the sample tank 5 are respectively provided with a matched injection hole a and a matched outflow hole c which are communicated through an injection flow channel b and an outflow flow channel d; the injection flow channel b and the outflow flow channel d of the secondary reagent groove 11 are engraved on the first slide; an injection hole a and a discharge hole c of the sub reagent well 11 engraved on the second slide glass; a main reagent groove 1, a buffer groove 3, an antibody groove 4, an injection hole a and an outflow hole c of a sample groove 5, an injection flow channel b and an outflow flow channel d are engraved on the second slide glass; and when the auxiliary flow channel 13 is connected: the main reagent tank 1, buffer tank 3, antibody tank 4, and sample tank 5 are connected to the injection hole a and the outflow hole c, respectively.
In addition, the detection groove 6 is connected with an indication channel and comprises a second secondary reagent groove 14, an indication gas groove 8, an indicator groove 15 and a scale groove 7 matched with scale marks 16 which are sequentially connected in series through a secondary flow channel 9; the second secondary reagent groove 14 and the indicator groove 15 are respectively provided with a matched injection hole a, a matched outflow hole c, an injection flow channel b and a matched outflow flow channel d; the injection flow channel b, the outflow flow channel d and the scale groove 7 of the secondary flow channel 9, the indicating gas groove 8 and the indicator groove 15 are engraved on the first glass slide; the indicator groove 15 and the injection hole a and the outflow hole c thereof, the second sub-reagent groove 14 and the injection hole a and the outflow hole c thereof, and the scale 16 are engraved on the second slide; when main flow passage 12 is on: the indicator groove 15 and the second sub-reagent groove 14 are respectively communicated with the injection hole a and the outflow hole c.
Preferably, the first and second slides are provided with matching correction slots 10 at the corner ends for slide position correction when the main channel 12 is switched on.
Preparation method of sliding micro-control flow chip
The method comprises the following steps:
(1) respectively designing and drawing masks of the upper layer and the lower layer of the glass slide by using AutoCAD;
(2) the size of a single glass chip is 75mm multiplied by 50mm multiplied by 1mm, the glass chip is cleaned by using isopropanol and deionized water, and the glass chip is dried by using nitrogen;
(3) treating the surface of the glass sheet for 3min by oxygen plasma;
(4) placing the glass sheet into a glass storage box, placing 1mL of 1, 1, 1, 3, 3, 3-hexamethyldisilazane into a small beaker, placing the glass sheet and the glass sheet into a closed box, standing for 30min, and bonding a volatile reagent to the surface of the glass sheet;
(5) placing the modified glass slide on a spin coater, and spin-coating photoresist SPR220-7 under a dark condition, rotating at a low speed of 300 rpm/min for 5 s; rotating at high speed of 1000 rpm/min for 40 s;
(6) placing the glass slide coated with the photoresist on a heating table, drying the photoresist for 3min at 70 ℃, then raising the temperature to 100 ℃, continuing to dry for 8min, and naturally cooling to room temperature;
(7) covering a mask on the glass slide after the glue is dried, fixing four corners of the glass slide by using adhesive tape, and removing the mask after exposing the glass slide on a photoetching machine for 100s by using an ultraviolet lamp;
(8) pouring the AZ400K developing solution into a crystallizing dish, putting the exposed glass slide into the developing solution, continuously shaking the developing solution, and gradually developing the exposed area of the chip;
(9) wrapping the edge and the back of the glass slide with a transparent adhesive tape, and placing the glass slide on HF and NH at a molar ratio of 1:0.5:0.754F and HNO3Hydrophobization of the mixed solutionThe adopted hydrophobic reagent is perfluorooctyl trichlorosilane, and the etching is carried out for 40min at room temperature;
(10) after etching, cleaning the residual etching solution on the surface by using deionized water, sequentially washing the residual photoresist by using acetone, isopropanol and deionized water, and drying by using nitrogen;
(11) the slide glass is fixed to the slide glass lining with a glue, and holes are punched in the positions of the injection hole a and the discharge hole c of the slide glass by a small engraving and milling machine. After punching, adding acetone for ultrasonic treatment, washing off the glue, and taking out the punched slide;
(12) cleaning the perforated glass slide with oxygen plasma for 3min, adding 3 muL microliter of perfluorooctyl trichlorosilane reagent on the chip, sliding the first glass slide and the second glass slide, uniformly smearing the reagent, and standing in a vacuum box for 12 h;
(13) and cleaning the silane reagents on the surfaces of the first glass slide and the second glass slide by using acetone, isopropanol and deionized water, and drying by using nitrogen. And 3 muL of dimethyl silicone oil is taken to attach and assemble the first glass slide and the second glass slide, and the silicone oil can increase the lubrication of the chip and reduce the oxygen leakage. A 1% sodium hydroxide solution was added to the sample and reagent wells using a pipette gun to increase the hydrophilicity of this region.
Method for rapidly detecting biomarkers based on sliding micro-control flow chip
S1, immobilization of capture antibody in detection well 6:
b1, 5 mu L piranha solution (H) for detection tank 62SO4:H2O2=7: 3), washing for 1h, washing with deionized water and drying with nitrogen,
b2, then incubating 10% 3- (2, 3-epoxypropoxy) propyl trimethoxy silane (GPS)/toluene for 1h by using a straw, adding the incubated solution into a detection tank 6, preserving for 30min, washing by using fresh toluene, and drying at 120 ℃ to remove redundant GPS;
b3, addition of the corresponding capture antibody by reaction with epoxy groups on the glass surface, covalent immobilization in ELISA channels, incubation overnight at 4 ℃ and then washing multiple times with 5% bovine serum albumin (BSA, Sigma) to avoid non-specific binding.
S2, adding the biomarker standard solution to the sample tank 5, adding the PBS (PH = 7.4) buffer solution to the buffer tank 3, adding the composite probe solution to the antibody tank 4, and adding the platinum nanoparticle solution and the hydrogen peroxide solution to the main reagent tank 1 and the auxiliary reagent tank 11, respectively.
Wherein,
(1) the biomarkers selected in this example were human chorionic gonadotropin (hCG) and carcinoembryonic antigen (CEA).
The concentrations of the hCG standard solution were: 0.2, 20, 50, 150, 250, 750, and 1250 mIU;
the concentration of the CEA standard solution is as follows: 1. 1.5, 2.5, 5, 10, 50, 100, 200 and 500 ng/mL.
(2) The preparation method of the composite probe comprises the following steps:
c1, synthesizing carboxylated fluorescent nano-silica particles:
(C11) weighing 1.77g of Triton X-100, adding into a conical flask, and mixing and stirring 7.5 mL of cyclohexane and 1.6mL of n-hexanol for 700 r/min; (a2) preparing 0.1M bipyridyl ruthenium solution by 400 muL deionized water, adding 80uL bipyridyl ruthenium solution and 400 muL deionized water into a conical flask, adding 100 muL ethyl orthosilicate (TEOS), and stirring for 30 min;
(C12) adding 65 mu L of ammonia water into a conical flask, and reacting for 10 hours;
(C13) stirring 100 mu of L N-trimethoxysilylpropyl ethylenediamine triacetate and fluorescent nano-silica particles at room temperature and reacting for 4 hours in PBS (10 mmol/m1, PH = 7.4) buffer solution at room temperature;
(C14) centrifuging at 12000r/min for 10min after the reaction is finished, washing with ethanol for three times, and washing with deionized water for three times to prepare the carboxylated fluorescent nano-silica particles.
C2, preparing a composite probe:
(C21) preparing 1mL of 1mg/mL carboxylated fluorescent nano-silica particles, dispersing the particles into 1.0mL of PBS (10 mmol/m1, pH7.4) buffer solution, mixing the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) 10 mg of N-hydroxysuccinimide (NHS) 2 mg of PBS buffer solution with 1.0mL of water, continuously mixing the mixture at room temperature for reaction for 15 min, centrifuging the mixture for three times at 12000r/min 10min after the reaction is finished, and washing the mixture for three times with PBS (10 mmol/m1, pH = 7.4) buffer solution for later use;
(C22) 200 mu L of 250ug/mL hCG detection antibody or 200 mu L of 10 mu g/mL CEA detection antibody is added into the sample to be used, the mixture is fixed and reacted at room temperature for 4h, and the mixture is centrifuged for a plurality of times by PBS (10 mmol/m1, pH7.4) buffer solution, is centrifuged at 12000 rpm for 10min, is placed in PBS (10 mmol/m1, pH = 7.4) buffer solution and is kept at 4 ℃, and the hCG or CEA composite probe is prepared.
(3) The preparation method of the platinum nano-particles comprises the following steps:
(D1) taking 500 mu L of 100mM chloroplatinic acid solution, adding 45mL ultrapure water, heating at 80 ℃ for 40min, quickly adding 9mL 0.2M ascorbic acid, and continuing heating at 85 ℃ for 30 min;
(D2) and centrifugally cleaning with ultrapure water for several times at 5000rpm for 5min to obtain 1mg/mL platinum nanoparticle aqueous solution, and storing at 4 ℃ in a dark place.
And S3, sliding the glass slides to enable the two glass slides to be overlapped, switching on the main flow channel 12, pushing the liquid in the channel to sequentially pass through the detection tank 6 by the gas generated in the reagent tank within 5min, and finishing the ELISA reaction of the antigen and the antibody.
S4, acquiring a fluorescence picture during detection in the detection tank 6 by using a full-automatic fluorescence microscope, and analyzing by combining Image J to obtain the number of the fluorescent nanoparticles in unit area; the exposure time was set to 500ms and the exposure intensity was 13.
S5, adding the biomarker standard solutions with different concentrations into the sample tank 5, repeating the steps S2-S4, and drawing a standard curve chart;
s6, adding the to-be-detected biomarker sample into the sample tank 5, repeating the steps S2-S4, and analyzing by combining the standard curve chart of the step S5 to obtain the concentration of the to-be-detected antibody sample.
Further, as shown in fig. 5:
s7, when the main channel 12 is closed after the first slide of the slide glass, H is injected into each of the second sub-reagent reservoir 14 and the indicator reservoir 152O2Solutions and red inks;
triggering an ELISA reaction when H2O2Solution and platinum nano-solution to produce O2When the reagents are sequentially pushed to the detection groove 6 as a driving force, the slide sliding is carried out for the second time, and the secondary flow channel 9 is connected; the Pt modified detection antibody and the capture antibody form a sandwich structure, and simultaneously can be added with H for the second time2O2Solution reaction to produce O2Promote the red ink column and remove thereby produce the reading, realize visual detection, whether can reach the degree of disease according to the concentration reading of the biomarker that detects from scale mark 16.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (10)
1. The sliding micro-flow control chip is characterized in that the base material is a glass slide and comprises a first glass slide and a second glass slide which are matched;
the binding surface of the slide is carved with a plurality of channels, the channels comprise a reagent tank, a gas tank, an antibody tank, a sample tank and a detection tank which are sequentially connected in series through a main channel, and the front end and the rear end of the antibody tank are respectively connected in series with a buffer tank;
the reagent tank comprises a main reagent tank and a secondary reagent tank;
the main reagent groove, the gas groove, the antibody groove, the sample groove, the detection groove and the buffer groove are engraved on the first slide glass,
the auxiliary reagent groove and the main flow channel are carved on the second slide;
the buffer tank is provided with a plurality of groups of interval runners, consists of parallel main runners and auxiliary runners and is engraved on the second glass slide.
2. The sliding micro-control flow chip according to claim 1, wherein the main reagent tank, the sub-reagent tank, the buffer tank, the antibody tank and the sample tank are respectively provided with a matched injection hole and a matched outflow hole which are communicated through an injection flow channel and an outflow flow channel;
the injection runner and the outflow runner of the secondary reagent groove are carved on the first glass slide, and the injection hole and the outflow hole are carved on the second glass slide;
the main reagent groove, the buffer groove, the antibody groove, the injection hole and the outflow hole of the sample groove, the injection flow channel and the outflow flow channel are engraved on the second glass slide;
and when the auxiliary flow channel is communicated: the main reagent tank, the buffer tank, the antibody tank and the sample tank are respectively communicated with the injection hole and the outflow hole.
3. The sliding micro-flow control chip according to claim 1, wherein the detection groove is connected with the indication channel, and comprises a second secondary reagent groove, an indication gas groove, an indicator groove and a scale groove matched with scale marks which are connected in series in sequence through the secondary flow channel;
the second secondary reagent groove and the indicator groove are respectively provided with a filling hole, a flow-out hole, a filling flow channel and a flow-out flow channel which are matched with each other;
the secondary flow channel, the indicating gas groove and the injection flow channel, the outflow flow channel and the scale groove of the indicator groove are engraved on the first glass slide;
the indicator groove and the injection hole and the outflow hole thereof, the second auxiliary reagent groove and the injection hole, the outflow hole and the scale mark thereof are marked on the second slide glass;
and when the main runner is connected: the indicator groove and the second sub-reagent groove are respectively communicated with the injection hole and the outflow hole.
4. The method of making a sliding microfluidic chip according to claim 1, comprising the steps of:
a1, designing a chip mask, and etching a glass slide by adopting standard photoetching and/or wet method;
and A2, perforating the etched glass slide, modifying the surface of the glass slide into hydrophobicity by using a hydrophobic reagent, and forming the chip.
5. The method for rapidly detecting biomarkers based on the sliding micro-control flow chip of any one of claims 1 to 4, which comprises the following steps:
s1, covalently fixing the capture antibody by reacting with an epoxy group in the detection groove;
s2, adding the standard solution of the biological marker into the sample groove,
the buffer solution is added into the buffer tank,
adding the composite probe solution into an antibody groove,
adding a hydrogen peroxide solution and a platinum nanoparticle solution into a main reagent tank and an auxiliary reagent tank respectively;
s3, connecting the slide glass with the main flow channel, and pushing the liquid to pass through the detection groove by the gas generated in the reagent groove to finish the ELISA reaction of the antigen and the antibody;
s4, acquiring a fluorescence picture during detection in the detection tank by using a full-automatic fluorescence microscope, and analyzing by combining Image J to obtain the number of the fluorescent nanoparticles in unit area;
s5, adding the biomarker standard solutions with different concentrations into the sample tank, repeating the steps S2-S4, and drawing a standard curve chart;
and S6, adding the biomarker sample into the sample tank, repeating the steps S2-S4, and analyzing by combining the standard curve chart of the step S5 to obtain the concentration of the antibody sample to be detected.
6. The method of claim 5, wherein the step of immobilizing the capture antibody in step S1 is:
b1, incubating the detection tank with the piranha solution for a period of time, and then cleaning;
b2, incubating the detection tank with a mixed solution of 10% 3- (2, 3-epoxypropoxy) propyl trimethoxy silane (GPS) and toluene for a period of time, washing and drying;
b3, incubating the capture antibody in the detection groove for a period of time, covalently fixing, and washing.
7. The method of claim 6, wherein the piranha solution in step B1 is H2SO4:H2O2The incubation time is 1-2h, and the cleaning solution is deionized water;
the incubation time in the step B2 is 1-2h, the cleaning solution is toluene, and the drying temperature is 100-120 ℃;
the incubation temperature in the step B3 is 3-5 ℃, and the cleaning solution is BSA solution with the mass content of 4-6%.
8. The method according to claim 5, wherein the composite probe in step S2 is prepared by:
c1, preparing carboxylated fluorescent nano-silica particles: synthesizing fluorescent nano-silica particles from bipyridine ruthenium and nano-silica by adopting a reverse microemulsion method, and then covalently coupling the fluorescent nano-silica particles with a silane carboxylation reagent to prepare carboxylated fluorescent nano-silica particles;
c2, preparing a composite probe: and (3) carrying out covalent coupling on the detection antibody and the carboxylated fluorescent nano-silica particles by adopting a carbodiimide method to prepare the composite probe.
9. The method according to claim 8, wherein the inverse microemulsion system in step C1: 1.77g Triton X-100, 7.5 mL cyclohexane, 1.6mL hexanol, 400 μ L deionized water, 80 μ L0.1M ruthenium bipyridine, 100 μ L ethyl orthosilicate (TEOS), 65 μ L ammonia, 100 μ L L N-trimethoxysilylpropylethylenediamine triacetate; stirring the fluorescent nano silicon dioxide particles at room temperature for reacting for 18 hours; carboxylated coupling was reacted for 4h in PBS (10 mmol/mL, pH = 7-8) buffer at room temperature.
10. The method of claim 5, wherein the biomarker comprises hCG and/or CEA.
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