Microfluidic immune quantitative analysis system and application thereof

Abstract

The invention discloses a micro-fluidic immune quantitative analysis system, which comprises a micro-fluidic chip and immune colloidal gold, and a preparation method of the micro-fluidic chip comprises the following steps: carrying out corona activation on a polydimethylsiloxane chip with a channel and a glass slide together for 30-60 s, attaching the polydimethylsiloxane chip and the glass slide, and sequentially carrying out amination treatment and formylation treatment on the channel; adding a cTN I monoclonal capture antibody into the treated chip for modification; carrying out vacuum degassing and sealing on the modified chip; the immune colloidal gold is obtained by marking a cTNI detection antibody with colloidal gold. The microfluidic immune quantitative analysis system disclosed by the invention can realize rapid and quantitative detection of cTNI, can quantitatively detect 0.5 ng / ml-60ng / ml of cTNI through strip length without depending on instruments and equipment, and is good in specificity.

Description

Technical Field

[0001] The invention belongs to the field of immune detection, and in particular relates to a microfluidic immune quantitative analysis system and an application thereof. Background Art

[0002] Cardiovascular disease is the leading cause of death worldwide, accounting for nearly half of the population's deaths. Among them, acute myocardial infarction (AMI) is the main cause of death. AMI is often caused by coronary atherosclerosis, which is related to genetic and environmental factors and is extremely lethal and disabling.

[0003] In recent years, point-of-care testing (POCT) has been in a rapid growth stage. The lateral flow immunoassay (LFIA) occupies the largest share of the home self-testing market. Most of the LFIA products used in clinical practice are colloidal gold immunochromatographic test strips, which use colloidal gold as a marker for determination. The steps are simple, the test is fast, and it is easy to carry. In addition, there is no need to use a readout instrument, and it will not cause radioactive pollution to the environment. However, this method can only perform qualitative or semi-quantitative analysis. In addition, the accuracy and repeatability of colloidal gold test strips are also relatively poor, and the coefficient of variation (CV) can only be controlled between 15% and 20%. This is because the test strips are sampled by capillary action, and the fluid cannot be controlled; the pore sizes of the chromatographic membranes are different and irregularly distributed.

[0004] Microfluidic chips are a technology platform that integrates routine operations of chemical and biological experiments such as sample pretreatment, reaction, separation, and detection on a chip of a few square centimeters, enabling low-cost, fast, and efficient analysis. Figure 1 As shown in the figure, the chip consists of a glass slide, a polydimethylsiloxane (PDMS) chip with four immune reaction channels and a scale, and a piece of tape. The bottom of the immune reaction channel is made of glass, and the injection port and the waste liquid pool are located at both ends of the immune channel. The bottom layer is a glass slide, the middle layer is a PDMS chip, and the upper layer is a tape. The tape is attached before the test after vacuuming. The immunofluorescence method on the chip uses a microfluidic chip to replace the chromatographic membrane, which improves the precision and sensitivity, but it also requires the use of detection instruments such as fluorescence microscopes, and the versatility of signal reading is poor. Another limitation is that the use of bulky external fluid-driven equipment such as syringe pumps and pressure pumps is usually required for detection using microfluidics and immunological methods. This is not convenient and simple for home devices. Summary of the invention

[0005] The present invention aims to combine microfluidics technology with colloidal gold immunoassay technology to develop a convenient, fast and easy-to-operate immunoassay system for quantitative detection of the cardiac biomarker cTN I.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A microfluidic chip comprises an immune response channel, the bottom of the immune response channel is made of glass, the glass surface is treated with amino and aldehyde groups, and a capture antibody is fixed to the front end of the immune response channel through chemical bonds.

[0008] In a preferred embodiment, the capture antibody is a cTN I monoclonal capture antibody.

[0009] A microfluidic immune quantitative analysis system comprises the microfluidic chip and immune colloidal gold; the sample to be tested and the immune colloidal gold are sequentially added to the injection port of the microfluidic chip, the sample injection is driven by PDMS degassing, the sample to be tested and the immune colloidal gold are captured by the capture antibody, the immune colloidal gold is used as a color developer to form a red strip, and quantitative analysis is performed according to the linear relationship with the strip length;

[0010] The immune colloidal gold is obtained by labeling detection antibodies with colloidal gold.

[0011] In a preferred embodiment, the capture antibody is a cTNI monoclonal capture antibody, and the detection antibody is a cTNI detection antibody.

[0012] The sample being tested is captured by the cTNI capture antibody in decreasing concentrations.

[0013] In one preferred embodiment, the preparation method of the microfluidic chip includes: corona activating a PDMS chip with a channel and a glass slide for 30 to 60 seconds and then bonding them together, and then performing amino treatment and aldehyde treatment on the channel in sequence; adding capture antibodies to the treated chip for modification; and vacuum degassing and sealing the modified chip.

[0014] In one preferred embodiment, the method for preparing colloidal gold comprises: reacting a chloroauric acid solution and a trisodium citrate solution under boiling conditions to obtain colloidal gold.

[0015] In one preferred embodiment, the mass ratio of chloroauric acid to citric acid is 1:(1-2).

[0016] In a preferred embodiment, the mass ratio of chloroauric acid to citric acid is 1:1.5.

[0017] Colloidal gold is prepared using trisodium citrate reduction method, and the amount of trisodium citrate used is closely related to the size of colloidal gold particles. Light scattering is affected by the size of gold particles, and the particle size changes, and the color of the colloidal gold solution will also change visibly to the naked eye. Keeping other conditions unchanged, the ratio of the two can prepare colloidal gold solutions of different particle sizes. Within a certain range, the more trisodium citrate is added, the smaller the average particle size of the colloidal gold, and the smaller the wavelength of its ultraviolet maximum absorption peak. This is because the concentration of trisodium citrate increases within a certain range, which can accelerate the nucleation rate of gold, with more crystal nuclei and smaller particle size. When labeling IgG, 40nm colloidal gold particles provide maximum visibility and minimum steric hindrance. Therefore, it is preferred that the mass ratio of chloroauric acid to citric acid is 1:1.5.

[0018] In one preferred embodiment, during the process of using colloidal gold to label the detection antibody, the pH value of the system is adjusted by adding 0-10 μL of 0.2 mol / mL K2CO3 to 1 mL of colloidal gold.

[0019] In one preferred embodiment, the amount of 0.2 mol / mL K2CO3 added is 4 μL.

[0020] Colloidal gold labeling of proteins is often considered to be completed through electrostatic adsorption of negatively charged colloidal gold and positively charged proteins. Therefore, the pH value and ionic strength of the environment during labeling are the main factors affecting the labeling process. When less than 4μL 0.2mol / mL K2CO3 is added to 1mL colloidal gold, the absorbance value of colloidal gold decreases and presents different degrees of gray-blue. This is because the environmental pH is lower than the isoelectric point of the protein at this time, and the antibody carries multiple positively charged groups, which attract multiple gold particles at the same time, resulting in the phenomenon of cross-linked dead gold. When 4μL K2CO3 is added to 1mL colloidal gold, the pH is close to the isoelectric point, the protein is in a weak hydration state, and it is easy to adsorb on the gold surface, and the formation of a protein protective layer prevents the phenomenon of dead gold caused by the aggregation of gold particles. In addition, labeling should not be carried out in an environment with too high pH, ​​because at this time the protein is negatively charged, and there is negative electrical repulsion between it and the colloidal gold, the two become difficult to contact, the labeling efficiency will decrease, and unnecessary waste of reagents will occur. Therefore, in order to ensure the stability and high efficiency of colloidal gold-labeled antibodies, the optimal labeling pH was selected to be 4 μL of 0.2 mol / mL K2CO3 added to 1 mL of colloidal gold.

[0021] In one preferred embodiment, the added amount of the detection antibody is: 1-40 μg of detection antibody is added to 1 mL of colloidal gold.

[0022] In one preferred embodiment, the added amount of the detection antibody is: 24 μg of detection antibody is added to 1 mL of colloidal gold.

[0023] When no protein is added or the amount of protein added is insufficient, colloidal gold cannot be stabilized, so the color of colloidal gold shows a coagulation phenomenon from red to blue; when the amount of protein added is sufficient, the quality of colloidal gold is stable, the color does not change, and the maximum absorption wavelength remains unchanged. When the amount of antibody added in 1mL colloidal gold is 20μg, the colloidal gold-antibody complex tends to be stable, is not affected by NaCl, and no coagulation of dead gold occurs. Therefore, 20μg is the minimum amount of 1mL colloidal gold labeled detection antibody. In actual work, the amount is increased by 10-20%, so the optimal amount of labeled antibody for 1mL colloidal gold with a maximum absorption wavelength of 527nm is 24μg.

[0024] In a preferred embodiment, the time for labeling the detection antibody with colloidal gold is 1 to 10 minutes.

[0025] In order to ensure that the color is obvious and the reading is clear while further shortening the entire detection process, the reaction time of the colloidal gold labeled antibody needs to be optimized. When the colloidal gold labeled antibody reaches saturation after 3 minutes of reaction, even if the reaction time continues to increase, a stronger signal cannot be obtained; and when the reaction time is less than 3 minutes, the color of the colloidal gold is obviously lighter, and the color also changes significantly between 1 minute and 2 minutes of reaction. Therefore, in order to ensure the detection sensitivity and shorten the detection time, 3 minutes is preferred.

[0026] In one preferred embodiment, the amination treatment includes: injecting 10-20% wt aminopropyltriethoxysilane (APTES) ethanol solution into the channel, reacting in the dark at room temperature for 1-60 minutes, repeatedly rinsing with ethanol solution for 1-10 minutes, and fixing at 110-120°C to complete the amination treatment.

[0027] The silanization time has a significant effect on the coating of the antibody. The modification of the glass surface at the bottom of the immune reaction channel is completed by injecting APTES solution into the immune reaction channel. The same situation will occur if the reaction time is too long. Even if the humidity can be increased and new solution can be injected into the channel frequently and timely, the complicated operation greatly reduces the efficiency of the reaction. At room temperature, the glass at the bottom of the channel reacts with the APTES solution for 1min, 5min, 10min, 20min and 1h respectively. The color of the reaction for more than 5min is obviously darker than the color of the reaction for 1min, and there is no obvious difference when it is more than five minutes. Therefore, it is preferred to use 10-20%wt APTES ethanol solution for 5min.

[0028] In one preferred embodiment, the aldehyde treatment comprises: injecting 2.5% to 5% glutaraldehyde PBS solution into the channel, reacting at room temperature in the dark for 10 to 120 minutes, and then repeatedly washing with PBS solution to complete the aldehyde treatment.

[0029] Aldehydeation is another step for treating the glass surface, which also has a significant effect on the fixation of the capture antibody. If the aldehyde reaction time is not long enough, the number of capture antibodies on the glass surface cannot reach saturation. When the antibody is saturated, continuing to increase the aldehyde reaction time cannot obtain better results. In the present invention, when the reaction time is less than 60 minutes, the color of the colloidal gold deepens as the aldehyde time increases. When the reaction time reaches 60 minutes, the color no longer changes significantly. Therefore, it is preferred to use 2.5% to 5% glutaraldehyde PBS solution for aldehydeation for 60 minutes.

[0030] In one preferred embodiment, the method of driving sample injection by PDMS degassing is vacuum degassing, including: placing the modified chip in a vacuum drying oven, evacuating the air at a pressure of 10 to 50 kPa; after taking the chip out of the vacuum drying oven, covering the top of the microfluidic chip with tape for sealing.

[0031] In one preferred embodiment, the flow rate of the sample added to the chip is adjusted by adjusting the vacuum degassing time, and the flow rate of the sample is no greater than 0.4 μL / min.

[0032] In one preferred embodiment, the vacuum degassing time is 10 to 120 minutes.

[0033] According to the basic principle of the present invention, the sample flows into the immune response channel and is preferentially captured by the capture antibody at the front end of the immune response channel. The concentration of the sample to be tested that flows to the rear end of the immune response channel decreases. After it decreases to a certain extent, it cannot be combined due to the low concentration. Therefore, a red stripe of a certain length is observed to be formed by the naked eye, and the sample to be tested can be quantified by the length of the red stripe. Therefore, it is very important to control the flow rate of the sample flow through the channel. When the sample flows into the reaction channel, it will be captured by the specific antibody modified at the bottom. If the antigen injection flow rate is too fast, some antigens will not have time to bind to the specific antibody modified on the glass substrate, resulting in a test result lower than the true value; if the antigen injection flow rate is too slow, the required time will increase significantly, and the bedside detection requirements cannot be met.

[0034] In the present invention, the flow rate is adjusted by the PDMS vacuum time, so the vacuum time needs to be optimized. The approximate range of the flow rate is first measured using a syringe pump. When the flow rate is greater than 0.4 μL / min, the antigen binding is insufficient, and the strip color is obviously lighter than other flow rate strips. When the flow rate is reduced to 0.4 μL / min, the color is not significantly different. Therefore, the injection flow rate should not be greater than 0.4 μL / min.

[0035] When the vacuum was drawn for 10 min, the reaction time required 30 min, which was significantly longer than that of the other groups. When the vacuum was drawn for 30, 60, and 120 min, the reaction times were similar and the flow rate did not change much. Therefore, the preferred vacuum time was 30 min.

[0036] In one preferred embodiment, the method for preparing the PDMS chip with channels comprises: preparing a photosensitive resin mold by dry film photolithography, pouring a PDMS prepolymer and a curing agent into the photosensitive resin mold by casting, and peeling off after drying to prepare a PDMS substrate.

[0037] In one preferred embodiment, the glass slide is cleaned and then bonded to the polydimethylsiloxane chip.

[0038] In one preferred embodiment, the slide glass cleaning step comprises: washing the slide glass with a detergent, tap water, ultrapure water, and piranha solution in sequence, and then blowing it dry.

[0039] In a preferred embodiment, the treated chip is modified with cTN I monoclonal capture antibody using glutaraldehyde method or EDC / NHS method.

[0040] After the glass surface is silanized, glutaraldehyde can be added to aldehyde the glass surface. The antibody is fixed on the glass surface by condensation of the aldehyde group on the glass surface and the amino group of the antibody to form an imine. The carboxyl group of the antibody is also activated by the EDC / NHS method, and then the complex condenses with the amino group on the glass surface to form an amide. Both methods are covalent binding methods, which can bind antibodies more stably and make the antibody more active to a certain extent.

[0041] In one preferred embodiment, the glutaraldehyde method includes: using 2.5% to 3% glutaraldehyde solution to formaldehyde the bottom glass of the chip channel, adding capture antibodies to each channel after the reaction is completed, flushing the channel after the reaction is completed, and then sealing the channel.

[0042] In one preferred embodiment, the EDC / NHS method includes: adding EDC and NHS to the capture antibody, mixing evenly and reacting, and adding PBS to terminate the reaction after the reaction is completed; then, injecting the reaction solution into the channel to react for 12 to 14 hours; after the reaction is completed, flushing the channel and then sealing the channel.

[0043] In a preferred embodiment, the captured antibody is modified by glutaraldehyde method on the treated chip.

[0044] We used two methods to fix the antibodies on the glass surface at the bottom of the channel in the chip, and compared the effect of modifying the capture antibodies by the ability to capture antigens, that is, the color depth of colloidal gold. The results showed that the background signals of the GA (glutaraldehyde) method and the EDC / NHS method were very weak, but the GA method was obviously darker than the EDC / NHS method, and the effect was better. This may be due to the fact that EDC / NHS is more active and is easily inactivated or dissociated in the reverse equilibrium during the coupling process.

[0045] In one preferred embodiment, the capture antibody concentration is 0.5-100 μg / mL.

[0046] The glass at the bottom of the channel is chemically modified to covalently bind to the capture antibody, and the efficiency of binding the antibody is limited, and only a certain number of antibodies can be bound. Since the surface area of ​​the chip channel does not change, it is necessary to select a suitable antibody concentration. If the concentration is too low, the antibody is too little, the amount of bound antibody is too little, and the sensitivity decreases; if the antibody concentration is too high, the antibody amount is excessive, which will increase the detection cost and cause unnecessary waste. In the present invention, when the capture antibody concentration is 0-50μg / mL, the color of the channel reacted with the same antigen concentration gradually deepens, which is more obvious in the quantified bar chart. When the antibody concentration reaches 75μg / mL, the channel color no longer deepens, indicating that the glass-bound antibody under this surface area has reached saturation. It is preferably modified with a capture antibody at a concentration of 75μg / mL.

[0047] The present invention also claims to protect the use of the microfluidic chip in preparing a reagent for quantitatively detecting cardiac troponin.

[0048] In one of the preferred embodiments, the application includes: adding the test sample and immune colloidal gold to the injection port of the microfluidic chip in sequence, using PDMS degassing to drive sample injection, the test sample and immune colloidal gold are captured by capture antibodies, using immune colloidal gold as a color developer to form a red strip, and performing quantitative analysis based on the linear relationship with the strip length; the immune colloidal gold is obtained by using colloidal gold to label the detection antibody.

[0049] The present invention also claims to protect the use of the microfluidic immunoquantitative analysis system in the preparation of a reagent for quantitatively detecting cardiac troponin.

[0050] In one preferred embodiment, the application includes: opening the sealed package of the microfluidic chip, dripping the sample and immunocolloidal gold into the injection port for reaction, and detecting the content of cardiac troponin by observing the length of the strip.

[0051] The present invention also claims a method for quantitatively detecting antigens, comprising: dripping a sample and immunocolloidal gold into a sample inlet of a microfluidic chip for reaction, wherein the sample to be tested and the immunocolloidal gold are captured by a capture antibody fixed on an immune reaction channel, using the immunocolloidal gold as a color developer to form a red strip, and detecting the content of the antigen according to a linear relationship with the strip length;

[0052] The microfluidic chip comprises an immune response channel, the bottom of which is made of glass, and the glass surface is treated with amino and aldehyde groups, and the capture antibody is fixed to the front end of the immune response channel through chemical bonds;

[0053] The immune colloidal gold is obtained by labeling detection antibodies with colloidal gold.

[0054] The present invention is further explained below:

[0055] The bulky injection pump not only increases the cost of detection but also reduces the convenience of detection; in the capillary action method, when the narrow immune response channel is filled with liquid, the pumping will basically stop, and it is also unable to meet the detection requirements. The detection principle of the present invention is that at a certain flow rate, the sample is slowly injected, and as it binds to the specific monoclonal antibody at the front end of the channel, the concentration of the target antigen in the sample solution decreases. When the solution flows to the back end of the channel, the concentration decreases, and the number of bound antigens decreases, resulting in too few sandwich immune complexes at the back end to develop color, thereby instantly quantifying the concentration of the target antigen in the sample according to the length of the strip. Therefore, the pumping method without complex external pumping instruments, the flow rate in the channel, and the stability of the pumping are the key to achieving detection. The feasibility of the degassing drive technology was determined by examining the flow rate and repeatability of the PDMS drive.

[0056] The present invention combines microfluidics technology with colloidal gold immunoassay technology to develop a convenient, fast and easy-to-operate immunoassay system for quantitative detection of cardiac biomarker cTN I. An exogenous micropump based on PDMS with high gas solubility characteristics is used to replace bulky external pumping equipment to develop a biomarker instant detection device, which makes up for the shortcomings of poor reproducibility and only qualitative or semi-quantitative detection of existing colloidal gold immunochromatographic test strips on the market, as well as high detection costs and complexity of other quantitative detection equipment, and provides a promising family self-service quantitative detection approach for early diagnosis of AMI.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The present invention covalently fixes the monoclonal specific capture antibody in the immune reaction channel by surface chemical modification of the bottom glass of the immune reaction channel of the glass PDMS microfluidic chip, combines the microfluidic chip with immune colloidal gold, and uses PDMS degassing to drive sample injection, and successfully constructs a microfluidic quantitative detection system based on immune colloidal gold. Antibodies are micro-detected with colloidal gold-labeled antibodies, and the sample flows into the immune reaction channel, and is preferentially captured by the capture antibody at the front end of the immune reaction channel. The cTN I antigen concentration flowing to the rear end of the immune reaction channel is reduced, forming a red strip visible to the naked eye of a certain length, and cTN I is quickly quantified by the length of the red strip. The present invention optimizes the antibody fixation conditions such as the glass surface silanization modification time and temperature, the aldehyde-forming time, the capture antibody concentration, and the related experimental conditions such as the injection flow rate and the vacuum pumping time, and the detection antibody reaction time, and investigates the methodology-related content. The results showed that in the range of 0.5-60 ng / mL, the length of cTN I band was linearly related to the concentration of cTN I, and the linear equation was: Y = 0.442X + 0.6246 (R 2 =0.9942), and has good specificity and high precision and accuracy, RSD is less than 11%, and the recovery rate is between 99% and 119%. In addition, the present invention does not require any signal reading equipment, and only 4 μL sample solution is needed to achieve the determination within 30 minutes. In the detection of actual samples, the cTNI concentration of serum samples of patients with myocardial infarction measured by the immune microfluidic chip is close to the data measured by Xiangya Hospital, which preliminarily proves that the immune microfluidic chip is accurate and effective in detecting actual cTNI samples. In short, the present invention has successfully developed a microfluidic detection system that can realize rapid quantitative detection of cTNI, which is independent of instruments and equipment, and can quantitatively detect 0.5ng / ml-60ng / ml of cTNI by strip length, with good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a schematic diagram of the microfluidic chip structure; 1. Inlet; 2. Waste liquid pool; 3. Immune reaction channel; 4. Scale; 5. Glass slide; 6. PDMS chip; 7. Tape;

[0060] Figure 2 It is the immune response channel mask and photosensitive resin mold;

[0061] Figure 3 is the repeatability of the degassing drive pump;

[0062] Figure 4 a) Colloidal gold structure; 4b) Principle of colloidal gold binding to antibodies;

[0063] Figure 5 is the effect of the amount of trisodium citrate added on the maximum wavelength;

[0064] Figure 6 It is a homemade colloidal gold characterization;

[0065] Figure 7 It is the UV absorbance value and physical picture of colloidal gold at different pH at 520nm wavelength;

[0066] Figure 8 It is the UV absorbance value and physical picture of colloidal gold-antibody complex at 520nm wavelength after adding different amounts of antibody;

[0067] Fig. 9 This is a comparison of the UV absorption curves of colloidal gold (purple) and colloidal gold-antibody complex (red);

[0068] Fig.10 This is a schematic diagram of the quantitative detection principle of immunocolloidal gold on a microfluidic chip; (a) the antibody is covalently bound to the glass slide at the bottom of the chip channel; (b) the PDMS degassing drives the antigen flow injection to be fixed by the captured antibody in a concentration-reducing manner; (c) the colloidal gold-labeled antibody binds to the antigen-antibody complex to show a red strip visible to the naked eye;

[0069] Fig.11 a is a graph of band lengths at different antigen concentrations; 11b is a histogram of band lengths at different antigen concentrations;

[0070] Fig.12 It is a comparison of the ability to capture cTn I antigen by fixing the antibody to the bottom of the channel using different methods;

[0071] Fig.13 It is the change of contact ratio during the GA method glass surface modification process;

[0072] Fig.14 It is the optimization of the silanization time and temperature of the glass at the bottom of the channel;

[0073] Fig.15 It is the optimization of the glutaraldehyde alkylation time of the bottom glass of the channel;

[0074] Fig.16 It is the optimization of the amount of capture antibody used on the glass surface at the bottom of the channel;

[0075] Fig.17 It is the selection of the flow rate range for antigen injection;

[0076] Fig.18 It is the choice of vacuuming time;

[0077] Fig.19 It is an option to test the duration of antibody reaction;

[0078] Fig. 20 It is specific to the immunoassay system;

[0079] Fig.21 This is the standard curve of the quantitative microfluidic chip based on immunocolloidal gold. DETAILED DESCRIPTION

[0080] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0081] Example 1

[0082] Fabrication of degassing-driven glass-PDMS microfluidic chip

[0083] Solution preparation

[0084] (1) Piranha solution: Measure 70 mL of concentrated sulfuric acid into a glass beaker, slowly add 30 mL of H2O2 and stir continuously with a glass rod. Wait until the solution is completely cooled before use. Prepare and use immediately.

[0085] (2) 0.85% (w / v) Na2CO3 developer: Weigh 8.5 g of Na2CO3 solid and place it in a beaker. Add about 100 mL of ultrapure water and stir to dissolve. Continue to add water to dilute to 1000 mL to obtain 0.85% Na2CO3 solution. Transfer it to a reagent bottle and store at room temperature for later use.

[0086] (3) PDMS: Use an electronic balance to weigh the silicone prepolymer and curing agent to a mass ratio of 10:1. Use a dropper to mix the two in the same direction in a disposable plastic cup to obtain a PDMS solution. Prepare and use immediately. If there is any residue, store it in a refrigerator at 4°C and use it as soon as possible.

[0087] 1. Design of Glass-PDMS Chip

[0088] Microfluidic chips were designed using Adobe Illustrator. Figure 1 As shown, the chip consists of a piece of glass, a PDMS chip with four vertical channels and a scale, and a piece of tape. The bottom layer is a glass slide, the middle layer is a PDMS chip, and the upper layer is a tape. The tape is attached after vacuuming and before detection. The entire chip system consists of three areas: ① The injection port 1 and the waste liquid pool 2 are located at both ends of the chip. The diameter of the injection port is equal to the channel width of 600μm, and the diameter of the waste liquid pool is 2.5mm; ② Immune reaction channel 3: Including the injection port, the channel is 3cm long, 600μm wide, and 75μm deep; the bottom glass is modified with specific monoclonal capture antibodies; ③ Scale 4: The scale is 3cm long and the graduation value is 0.2mm.

[0089] 2. Fabrication of Microfluidic Chip Immunoreaction Channel Mask and Photosensitive Resin Mold

[0090] Adobe Illustrator software was used to draw the immune response channel pattern and scale of the glass-PDMS composite chip, and then a high-definition printer was used to transfer the pattern to a transparent film to make a photolithography mask containing microfluidic channels and scales. The printing was observed under a microscope, and the following was obtained: Figure 2 The photolithography mask shown in A, where the film background is black, and the channels and scale are transparent white. The process of making the photosensitive resin mold of the PDMS chip is as follows Figure 2 As shown in B: Take out the DuPont dry film and saturated polyester (Polyethylene terephthalate, PET) substrate in a light-proof environment, cut them into appropriate sizes with scissors, remove the protective layer of the PET substrate, and laminate the DuPont dry film to the PET substrate. Pay attention to avoid bubbles and creases in the process; after the laminator is fully preheated, the laminated composite film is hot-pressed for multiple times to make the DuPont dry film and the PET substrate more firmly bonded; then irradiate with ultraviolet light in a UV exposure box to copy the pattern on the mask to the DuPont dry film, where the mask faces down, and the irradiation time is 120s; soak and dissolve the unexposed DuPont dry film in the developer prepared in advance, and stir the developer with a cotton swab to accelerate the dissolution speed, then rinse it with ultrapure water, dry it with a hair dryer, and place it in an electric hot air drying oven for 1h to make the dry film more firmly bonded while removing residual moisture; fix the dried composite film with channels and scales in a plastic culture dish to obtain a reusable PDMS chip mold.

[0091] 3. Fabrication of Microfluidic Chip

[0092] 3.1 PDMS chip fabrication

[0093] Use an electronic balance to weigh appropriate amounts of PDMS prepolymer and curing agent in a ratio of 10:1, stir and mix them evenly in the same direction, then slowly and evenly spread them on a culture dish with a channel and a scale pattern, cover it with a lid to prevent dust from falling, and place it at room temperature until the bubbles disappear; after debubbling, place the mold containing the PDMS solution horizontally in an electric blast drying oven and heat it at 60°C for 2h. During this process, the mixture changes from liquid to solid. Touch the edge of the PDMS with your finger. If there is no sticky feeling, it means that it has been completely cured and can be taken out of the drying oven; after cooling at room temperature, use a scalpel to cut and peel off the solid PDMS with channels and scales from the mold, and quickly clean the surface with cleaning tape to obtain a PDMS chip; finally, use punches with diameters of 0.6mm and 2.5mm, respectively, to punch holes in the injection port and waste liquid pool.

[0094] 3.2 Treatment of glass bottom plate

[0095] Take out a new glass slide, cut it into a suitable size with a glass cutter, soak it in detergent for 10 minutes and then wash it, then rinse it with tap water until there are no bubbles, and then rinse it thoroughly with ultrapure water to remove organic dirt; put the dried glass slide into a 500mL beaker containing piranha solution, soak it at room temperature for 1 hour, take it out and rinse it thoroughly with ultrapure water, and use it after drying with cold air from a hair dryer.

[0096] 3.3 Fabrication of glass-PDMS composite chip

[0097] Use ultrapure water and ethanol to clean the punched PDMS chip and the glass slide treated with piranha solution to wash away surface contaminants such as punching debris. After drying, observe the immune response channel and the injection port under a microscope. The qualified PDMS chip and the glass slide are placed horizontally on an insulating writing board and activated with a corona treatment device to fully oxidize the PDMS surface and increase its hydrophilicity. Then, the PDMS is quickly bonded to the glass slide to avoid bubbles during the bonding process. Finally, it is placed in an electric blast drying oven and heated at 60°C for 2 hours to obtain an irreversibly bonded glass-PDMS composite chip.

[0098] 4. Construction of Degassing-driven Glass-PDMS Microfluidic System

[0099] 4.1 PDMS degassing driven flow rate measurement

[0100] Take out four prepared glass-PDMS composite chips, number them AD, and place them in a vacuum drying oven for evacuation for 10, 30, 60 and 120 min respectively; after taking them out of the vacuum drying oven, stick transparent tape on the top of the PDMS to expose the injection port, drop 4 μL of red ink into the injection port, time the time required for the fluid to flow through the channel, and use formula 1 to calculate the flow rate of the fluid in the reaction channel.

[0101]

[0102] Formula 1

[0103] Note: In the above formula, T is the time required for 4 μL of fluid to flow through the chip channel.

[0104] Since the vacuuming time affects the amount of air degassed from the solid PDMS, thereby affecting the pressure difference between PDMS and the atmosphere, the time for vacuuming the microfluidic chip affects the flow rate driven by degassing. As shown in Table 1, the average flow rate calculated by injecting 4 μL is 0.13 μL / min for group A, 0.19 μL / min for group B, 0.20 μL / min for group C, and 0.23 μL / min for group D. When the vacuuming time is 120 min, the flow rate is slightly higher than that of the groups with 60 min and 30 min, while the flow rates of the vacuuming time of 60 min and 30 min are almost the same, and are also significantly higher than that of degassing for 10 min. The average flow rate of the four groups within 30 min of vacuuming time is between 0.1 and 0.3 μL / min. As the vacuuming time shortens, less air is extracted from the solid PDMS, resulting in a small pressure difference, which slows down the flow rate of the fluid entering the channel. Therefore, we also speculate that when the air in PDMS is completely evacuated, the maximum flow rate is reached, which is also the maximum flow rate driven by degassing of the microfluidic chip. We need to further select the degassing time based on subsequent experiments.

[0105] Table 1 PDMS degassing drive flow rate determination

[0106]

[0107] 4.2 PDMS degassing drive repeatability determination

[0108] A single PDMS device with different vacuum times was tested four times to investigate the repeatability of the PDMS degassing-driven flow process. Between each test, the device was cleaned with ethanol and deionized water, dried in an electric blast drying oven, and then the PDMS was degassed for 10, 30, 60, and 120 min, respectively, numbered as groups AD. Subsequently, transparent tape was attached to the top of the PDMS, 4 μL of red ink was dropped into the inlet, and the time required for the fluid to flow through the channel was timed. The reliability and practicality of the process were tested based on the coefficient of variation (CV) of the four experiments.

[0109] like Figure 3 As shown, the CV values ​​of the flow rate calculated by repeating 4 times of 4 μL injection were 2% for group A, 1.6% for group B, 3.5% for group C, and 2.1% for group D, all less than 5%, proving that PDMS degassing-driven injection can repeatedly load samples at a consistent flow rate, which meets the requirements of subsequent experiments.

[0110] The present invention uses a simple and convenient dry film photolithography method to prepare a photoresist mold with good quality, then pours PDMS liquid into the photosensitive resin mold through a casting method to prepare a PDMS substrate, and then performs corona activation after scalpel peeling and punching, and bonds with a glass slide treated with a piranha solution to obtain a glass-PDMS chip. The composite chip has a smooth surface, is transparent and free of impurities, has uniform thickness, fits tightly without bubbles, and has a clearly visible scale, which provides a good basic preparation for the subsequent construction of an immune chip. In addition, flow rate measurement and repeatability investigation are carried out to conduct a preliminary evaluation of the feasibility of the PDMS degassing drive technology. The flow rate of the degassing drive can be adjusted within a certain range by adjusting the time of vacuuming to degas the PDMS, and the maximum flow rate is achieved when the air in the PDMS is completely extracted. In addition, the chip has good repeatability in driving the sample, and the flow rate CV values ​​under different vacuuming times are all less than 5%, which lays a solid foundation for subsequent experiments.

[0111] Example 2

[0112] Trisodium citrate was used as a reducing agent to prepare colloidal gold solution. The required 40nm colloidal gold was prepared by optimizing the amount of reducing agent added, and it was used to label human cTN I antibody, providing a solid foundation for subsequent experiments. The basic principle is to add a certain amount of sodium citrate to the chloroauric acid solution to reduce the chloroaurate ions to gold atoms to form a gold particle suspension. In this process, trisodium citrate can form a stable charge layer on the gold surface to prevent gold nanoparticles from precipitating in the solution. Colloidal gold particles such as Figure 4 -a is composed of a gold core and a double ion layer surrounding it. The negatively charged gold particles can bind to the positively charged antibodies (lysine residues) through electrostatic adsorption, as shown in Figure 4 -b as shown.

[0113] 1. Solution Preparation

[0114] (1) Potassium dichromate washing solution: Weigh 40 g of potassium dichromate crystals using an electronic balance, place in a glass beaker, add 80 mL of water, and heat to dissolve; then measure 720 mL of concentrated sulfuric acid and slowly add it to the potassium dichromate solution while stirring continuously with a glass rod. After the mixture is prepared, store in a wide-mouth bottle with a lid.

[0115] (2) 1% chloroauric acid solution: Take out the chloroauric acid reagent, remove the outer packaging, clean the inner ampoule with ultrapure water, open the ampoule, add 3-5 mL of ultrapure water to dissolve the chloroauric acid, pour into a 100 mL volumetric flask, and then rinse the ampoule repeatedly with 3-5 mL of ultrapure water. Add all the rinse solution into the volumetric flask, add ultrapure water to the scale line, mix well, and you will get 1% chloroauric acid solution. After preparation, store in a brown reagent bottle in a dark place at 4°C refrigerator for future use, and filter with a 0.22 μm filter before use.

[0116] (3) 1% trisodium citrate solution: Weigh 1.14 g of trisodium citrate solid into a glass beaker, add a small amount of ultrapure water and stir to dissolve, then continue to add water to dilute to 100 mL, stir evenly, and transfer to a reagent bottle to obtain a 1% trisodium citrate solution; store in a refrigerator at 4°C for future use, and filter with a 0.22 μm filter before use.

[0117] (4) 0.2 mol / L K2CO3 solution: Use an electronic analytical balance to accurately weigh 27.6 mg of K2CO3 solid particles into a 1.5 mL centrifuge tube, add 1 mL of ultrapure water, and vortex on a vortex machine to dissolve. After mixing, 0.2 mol / L K2CO3 solution is obtained and stored in a refrigerator at 4°C for later use.

[0118] (5) 10% NaCl solution: weigh 100 mg of NaCl solid into a 1.5 mL centrifuge tube, add 1 mL of ultrapure water to dissolve, vortex mix on a vortex machine to obtain a 10% NaCl solution, and store in a refrigerator at 4°C until use.

[0119] (6) PBS buffer: Take a bag of commercially available PBS dry powder and place it in a 1-liter glass beaker. Take a small amount of ultrapure water and stir to dissolve it. Then continue to add water to dilute to 1 liter and stir evenly to obtain a 0.01 mol / L, pH=7.4 PBS buffer solution. Store it in a refrigerator at 4°C for later use.

[0120] (7) 10% BSA stock solution: Use an electronic analytical balance to weigh 100 mg of BSA powder into a 1.5 mL centrifuge tube, add 1 mL of the prepared PBS solution, vortex to mix and dissolve to obtain a 10% BSA solution, and place in a refrigerator at 4°C for later use.

[0121] (8) 5% PEG MW20000: Use an electronic analytical balance to weigh 50 mg of PEG20000 solid into a 1.5 mL centrifuge tube, add 1 mL of ultrapure water, vortex to mix and dissolve to obtain a 5% (w / v) PEG20000 solution, and place in a refrigerator at 4°C for later use.

[0122] (9) Reconstitution solution: Accurately weigh 50 mg of sucrose and 10 mg of BSA and add them to a 1.5 mL centrifuge tube. Dissolve them in 1 mL of the prepared PBS solution and vortex to mix evenly. The colloidal gold reconstitution solution is obtained and stored in a refrigerator at 4°C until use.

[0123] 2. Preparation of Colloidal Gold

[0124] 2.1 Effect of the ratio of chloroauric acid to trisodium citrate on particle size

[0125] (1) Cleaning: prepare a 100 mL round-bottom flask and 7 reagent bottles, wash away dust with running tap water, add the prepared potassium dichromate cleaning solution and soak for 24 hours, wash the cleaning solution with a large amount of tap water, wash with detergent, rinse with tap water, and then wash with ultrapure water for 4-6 times, dry in an electric blast drying oven for 2 hours and set aside;

[0126] (2) Preparation: Measure 1 mL of 1% chloroauric acid solution and add 49 mL of sterile ultrapure water to dilute it so that the final concentration of the chloroauric acid solution is 0.02%; add 50 mL of 0.02% chloroauric acid solution to a cleaned 100 mL round-bottom flask, mark the liquid surface, then put in a magnetic stirrer, heat to 100° C. in an oil bath and stir; after the chloroauric acid solution boils, use a dropper to quickly add 1% trisodium citrate solution at one time and continue stirring to react; during the process, obvious color changes can be observed, first from light yellow to gray-black, and after the color of the solution changes from gray-black to translucent red, continue to boil for 10 minutes and stop heating; remove from the oil bath, continue to stir at a low speed to cool to room temperature, and add water to the marked position; repeat the preparation 7 times, and the amounts of trisodium citrate are: 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2 mL respectively;

[0127] (3) Determination: The ultraviolet absorption curve and particle size were measured using an ultraviolet spectrophotometer and a particle size analyzer, respectively.

[0128] The results show that the amount of trisodium citrate used is closely related to the size of colloidal gold particles. Light scattering is affected by the size of gold particles. When the particle size changes, the color of the colloidal gold solution will also change visibly. As shown in Table 2, keeping other conditions unchanged and only changing the amount of trisodium citrate used, colloidal gold solutions of different particle sizes can be prepared. From the table below and Figure 5 It can be seen that within a certain range, the more trisodium citrate is added, the smaller the average particle size of colloidal gold is, and the smaller the wavelength of its maximum ultraviolet absorption peak is. This is because within a certain range, the increase in the concentration of trisodium citrate can accelerate the nucleation rate of gold, resulting in more crystal nuclei and smaller particle size.

[0129] Table 2 Relationship between the amount of trisodium citrate added and the particle size of colloidal gold

[0130]

[0131] 2.2 Preparation of colloidal gold

[0132] Clean all glassware; measure 2mL of 1% chloroauric acid solution, add 98mL of sterile ultrapure water to dilute, so that the final concentration of chloroauric acid solution is 0.02%; add 100mL of 0.02% chloroauric acid solution to the cleaned 250mL round-bottom flask, mark the liquid surface, then put in a magnetic stirrer, use an oil bath to heat and stir; after the chloroauric acid solution initially boils, use a dropper to quickly add 3mL of 1% trisodium citrate solution at one time and continue stirring to react; stop heating after the color of the solution turns to clear red; remove from the oil bath, continue stirring to cool to room temperature, add water to the mark, and store in a wide-mouth brown reagent bottle in a 4°C refrigerator away from light.

[0133] Characterization of colloidal gold

[0134] The prepared 40nm colloidal gold was subjected to dynamic light scattering particle size measurement and UV scanning to determine its particle size and maximum absorption wavelength.

[0135] like Figure 6 -a, the prepared colloidal gold is a bright, uniform wine red solution. The prepared wine red colloidal gold solution is scanned by an ultraviolet spectrophotometer in the range of 350-800nm, with ultrapure water as a reference. Figure 6 -b shows that the maximum absorption peak wavelength appears at 527nm, OD 527 =1.098. The particle size of colloidal gold was measured by a particle size analyzer. Figure 6 -c, the colloidal gold particle size distribution is around 40 nm, and the PDI is 0.183.

[0136] 3. Preparation of Immunocolloidal Gold

[0137] 3.1 Optimal pH for colloidal gold-labeled cTNI detection antibody

[0138] Take 6 1.5mL centrifuge tubes, add 1mL colloidal gold to each, then add 0, 2, 4, 6, 8 and 10μL of K2CO3 solution with a concentration of 0.2mol / L respectively, and mix thoroughly immediately after adding to prevent K2CO3 from settling to the bottom naturally, causing excessive local ionic strength, and even causing the gold at the bottom to die directly and turn black, resulting in obvious stratification; then add 10μg of the labeled antibody to each centrifuge tube, mix well, react for 30 minutes, and observe the color changes of each tube; finally, add 100μL of 10% BSA solution, vortex mix well, and react for 30 minutes. Observe the color changes of each tube, and use a UV absorption photometer to measure the UV absorption light intensity at a wavelength of 520nm.

[0139] like Figure 7As shown in the figure, when less than 4μL 0.2mol / mL K2CO3 is added to 1mL colloidal gold, the absorbance value of colloidal gold decreases and presents gray-blue to varying degrees. This is because the environmental pH is lower than the isoelectric point of the protein at this time, and the antibody carries multiple positively charged groups, which attract multiple gold particles at the same time, resulting in the phenomenon of cross-linked dead gold. When 4μL K2CO3 is added to 1mL colloidal gold, the pH is close to the isoelectric point, the protein is in a weak hydration state, and it is easy to adsorb on the gold surface, and the formation of a protein protective layer prevents the dead gold phenomenon caused by the aggregation of gold particles. In addition, labeling cannot be carried out in an environment with too high pH, ​​because at this time the protein is negatively charged, and there is a negative electrical repulsion between the protein and the colloidal gold, the contact between the two becomes difficult, the labeling efficiency will decrease, and unnecessary waste of reagents will be caused. Therefore, in order to ensure the stability and high efficiency of colloidal gold labeled antibodies, we choose to add 4μL 0.2mol / mL K2CO3 to 1mL colloidal gold as the optimal labeling pH.

[0140] 3.2 Optimal amount of colloidal gold labeled cTN I antibody for detection

[0141] Take 8 1.5mL centrifuge tubes, add 1mL colloidal gold to each, then add 4μL of K2CO3 solution with a concentration of 0.2mol / L, and vortex mix; then, dilute the cTN I detection antibody to be labeled with 0.01mol / L PBS with a pH of 7.4 to 0, 10, 15, 20, 25, 30, 35 and 40μg / mL, take 1mL and add it to the colloidal gold solution in each tube, vortex mix; after reacting for 5min, add 0.1mL 10% NaCl solution, mix and stand for 2h to observe the color change, and use a UV absorption photometer to measure the ultraviolet absorption light intensity at a wavelength of 520nm. When no protein is added or the amount of protein added is insufficient, the colloidal gold cannot be stabilized, so the color of the colloidal gold shows a coagulation phenomenon from red to blue; when the amount of protein added is sufficient, the quality of the colloidal gold is stable, the color does not change, and the maximum absorption wavelength remains unchanged. The minimum amount of protein that can keep the quality of colloidal gold unchanged is the necessary amount of protein to stabilize 1 mL of colloidal gold. In practical applications, adding 10 to 20% is the optimal amount of protein added.

[0142] like Figure 8 As shown in the figure, when too little protein is added, the colloidal gold sol state is destroyed by NaCl, the colloidal gold precipitates and turns purple or blue, and the absorbance value decreases. When the amount of antibody added to 1mL colloidal gold is 20μg, the colloidal gold-antibody complex tends to be stable, is not affected by NaCl, and does not precipitate. Therefore, 20μg is the minimum amount of 1mL colloidal gold labeled cTN I. In actual work, the amount is increased by 10-20%, so the optimal amount of antibody labeled with 1mL colloidal gold with a maximum absorption wavelength of 527nm is 24μg.

[0143] 3.3 Colloidal gold labeled cTN I detection antibody

[0144] Take 1mL of colloidal gold in a 1.5mL centrifuge tube, add 4μL of 0.2mol / L K2CO3 to adjust the pH of the colloidal gold; add 24μL of 1mg / mL antibody stock solution dropwise, vortex mix and react at room temperature for 60min; add 10μL of 10% BSA and 10μL of 5% PEG20000, vortex mix thoroughly, and continue to react for 60min; centrifuge at 1200r / min for 15min to remove the precipitate, then centrifuge at 12000r / min for 25min, remove the supernatant, and re-dissolve the precipitate with 100μL of PBS buffer solution containing 5% sucrose, 1% BSA, and 0.02% NaN3, and store in a refrigerator at 4℃ for later use.

[0145] 3.4 Characterization of immunocolloidal gold

[0146] The prepared colloidal gold and colloidal gold-antibody complex were scanned by ultraviolet wavelength, and the peak shape and maximum absorption peak wavelength were compared to evaluate whether the antibody was successfully connected to the colloidal gold.

[0147] like Fig. 9 As shown, the purple curve is the UV absorption curve of colloidal gold, and the red curve is the UV absorption curve of colloidal gold-antibody complex. Compared with the purple curve, the maximum absorption peak of the red curve increases, the peak shape becomes wider, and the red shift occurs. The colloidal gold λmax=527nm, while the gold-labeled complex is λmax=530nm. This is because colloidal gold interacts with the surrounding medium, and the colloidal gold-antibody has been purified and concentrated by centrifugation. The change in the UV absorption peak proves that the antibody has been connected to the colloidal gold.

[0148] Example 3

[0149] Solution preparation

[0150] (1) 10% APTES ethanol solution: Take 200 μL of APTES solution in a 2 mL centrifuge tube, add ethanol to dilute to a total volume of 2 mL, vortex and mix thoroughly to obtain a 10% APTES ethanol solution, wrap it in tin foil and protect it from light before use.

[0151] (2) 2.5% GA solution: Take 100 μL of 25% GA solution in a 1.5 mL centrifuge tube, then add PBS to dilute to a total volume of 1 mL. Vortex thoroughly to mix, wrap in tin foil and protect from light before use.

[0152] (3) Standard NaOH solution: Weigh 400 mg of NaOH solid into a glass beaker, add a small amount of ultrapure water and stir to dissolve, then continue to add water to dilute to 10 mL, stir evenly to obtain the standard NaOH solution, and store in a refrigerator at 4°C for later use.

[0153] (4) 50 mmol / L MES buffer solution: Use an electronic balance to weigh 533 mg of MES powder and place it in a 50 mL glass beaker. Add a small amount of ultrapure water and stir to dissolve. Continue to add ultrapure water to dilute to a total volume of 50 mL. Stir and mix thoroughly to obtain a 50 mmol / L MES solution. Subsequently, adjust the pH to 5.0 with a standard NaOH solution to obtain a MES buffer solution.

[0154] (5) PBST solution: Use a measuring cylinder to take 50 mL of the prepared PBS solution into a 50 mL centrifuge tube, add 5 μL of Tween 20 solution, vortex to mix, and then obtain the PBST buffer solution. Store in a refrigerator at 4°C until use.

[0155] (6) EDC solution: Accurately weigh 10.0 mg of EDC powder into a 1.5 mL centrifuge tube, add 1 mL of MES solution and vortex to mix and dissolve to obtain a 10.0 mg / mL EDC solution. Prepare and use immediately to avoid hydrolysis.

[0156] (7) NHS solution: Accurately weigh 10.0 mg of NHS into a 1.5 mL centrifuge tube. Add 1 mL of MES solution and vortex to mix and dissolve to obtain a 10.0 mg / mL NHS solution. Prepare and use immediately to avoid hydrolysis.

[0157] (8) cTN I antigen solutions of different concentrations: Use a pipette to take 1 μL of 1.1 mg / mL cTN I stock solution into a 2 mL centrifuge tube, add 1099 μL of PBS buffer solution to dilute, vortex to mix, obtain a cTN I antigen solution with a solubility of 1 μg / mL, divide into 11 portions, 100 μL each, and store in a -20°C refrigerator. Then dilute stepwise to 100 ng / mL in proportion, and use the 100 ng / mL antigen solution to prepare 7 different antigen concentration solutions of 60, 40, 30, 20, 10, 5, and 0.5 ng / mL, and store in a 4°C refrigerator for later use.

[0158] (9) 1% (w / v) BSA blocking solution: Weigh 10 mg of BSA powder into a 1.5 mL centrifuge tube, add PBS solution, vortex and mix to dissolve, and obtain BSA solution.

[0159] (10) Other antigen solutions: Take 1 μL of 1 mg / mL AFP, CEA, or human IgG antigen solution in a 2 mL centrifuge tube, add 999 μL PBS buffer solution to dilute to 1 μg / mL, and store in a refrigerator at 4°C until use.

[0160] 1 Feasibility analysis of detecting cTN I

[0161] Take a prepared glass-PDMS composite chip, and number the channels 1 to 4. Use a 1mL disposable syringe to inject a 10% (v / v) APTES anhydrous ethanol solution into the channel at the bottom of the channel, and react at room temperature in the dark for 2 hours. Since anhydrous ethanol is volatile, in order to prevent the solvent from volatilizing and causing APTES to precipitate and block the channel, the liquid is replenished once every 10 minutes to ensure that the channel reacts fully. After the reaction, the channel is flushed with anhydrous ethanol for 3 minutes. After rinsing, place it in a 110℃ electric blast drying oven for heating and drying for 1 hour, then take it out and cool it to room temperature. Use a 1mL disposable syringe to cover the channel with 2.5% glutaraldehyde solution for aldehyde modification, and react at room temperature in the dark for 2 hours. After the reaction is completed, use PBS solution to flow wash the channel for 3 minutes. Add a 1mg / mL cTN I monoclonal capture antibody to each channel and react in a 4℃ refrigerator for 12 hours. After the antibody modification is completed, use PBST to rinse the channel for 3 minutes. Subsequently, 1% BSA was used to react at room temperature for 1 hour to block the channel. After the reaction was completed, the channel was rinsed with PBST for 3 minutes. The modified chip was taken out and placed in a vacuum drying oven for vacuum degassing for 2 hours. After degassing, the waste liquid pool was sealed by sticking transparent tape on the top of PDMS, and 4 μL of cTN I antigen solution with concentrations of 0, 5, 50, and 500 ng / mL was dropped into the injection ports of channels 1 to 4 respectively. After the reaction was completed, it was rinsed with PBST for 1 minute. The detection antibody labeled with colloidal gold was added to react for 10 minutes, and the channel color was observed.

[0162] The principle of quantitative detection of immunocolloidal gold on microfluidic chip is as follows Fig.10 As shown: First, the antibody is covalently fixed to the glass slide at the bottom of the microfluidic chip channel by the glutaraldehyde method; then the cTN I antigen is driven to flow and sample by the degassing self-driving method based on the high gas solubility of PDMS. Since the cTN I antigen is slowly flowed in the channel, it is fixed on the channel surface by the capture antibody on the channel with a gradually decreasing concentration; then, the gold-labeled antibody is directly added to form a double antibody sandwich structure. Since the colloidal gold is aggregated in large quantities and presents a visible red color, the red strip can be directly observed in the immune response channel. The cTN I can be quantified based on the relationship between the strip length and the cTN I antigen concentration.

[0163] According to the basic principle, since the amount of cTN I in the sample is certain, the sample flows into the immune response channel and is preferentially captured by the capture antibody at the front end of the immune response channel. The concentration of cTN I flowing to the back end of the immune response channel decreases. After it decreases to a certain level, it cannot bind because the concentration is too low. Therefore, a red strip of a certain length is observed by the naked eye, and cTN I is quantified by the length of the red strip. Therefore, the feasibility of this method can be verified based on the change in the length of the red strip. Fig.11 As shown in the figure, the red bands of the three groups with different concentrations of antigen (500 / 50 / 5ng / mL) are longer than those without antigen, indicating that the signal transduction method is feasible. In addition, it can be seen from the three groups with the addition of antigen that the length of the band increases with the increase of antigen concentration, indicating that this method has the potential for quantitative detection.

[0164] 2. Optimization of modification conditions

[0165] 2.1 Comparison of different modification methods on the bottom glass surface of the channel

[0166] Prepare two composite chips, numbered A and B.

[0167] (1) Silanization modification: Use a 1mL disposable syringe to inject a 10% (v / v) APTES anhydrous ethanol solution into the channel and react for 2 hours at room temperature in the dark. Since anhydrous ethanol is volatile, in order to prevent the solvent from volatilizing and APTES from precipitating and blocking the channel, the solution is replenished every 10 minutes to ensure that the channel reacts fully. After the reaction, flush the channel with anhydrous ethanol for 3 minutes. After rinsing, place it in an electric blast drying oven at 110°C to heat and dry it for 1 hour, then take it out and cool it to room temperature;

[0168] (2) Capture antibody modification: Chip A uses the glutaraldehyde method to modify the capture antibody at the bottom of the channel, and chip B uses the EDC / NHS method to modify the capture antibody;

[0169] A. Glutaraldehyde method: Use 2.5% glutaraldehyde solution to formaldehyde the bottom glass of the chip channel, and react at room temperature in the dark for 2 hours. After the reaction is completed, use the pre-prepared PBS to flow and rinse the reaction channel for 3 minutes. Add 2μL of cTN I monoclonal capture antibody with a concentration of 1mg / mL to each channel, react in a 4℃ refrigerator for 12 hours, and rinse the channel with PBST for 3 minutes after the reaction is completed. Then use 1% BSA to block the channel at room temperature for 1 hour, then rinse the channel with PBST for 3 minutes, and store it in a 4℃ refrigerator for later use.

[0170] B. EDC / NHS method: Take 5μL of 2mg / mL capture antibody and add it to a 1.5mL centrifuge tube, add 10mg / mL EDC solution and 4μL and 6μL of NHS solution respectively, vortex mix evenly and react at 37℃ for 30min to activate the antibody carboxyl group. After the reaction is completed, add 60μL PBS to terminate the esterification reaction. Subsequently, inject the reaction solution into the channel and react in a 4℃ refrigerator for 12h. After the reaction is completed, rinse the channel with PBST solution for 3min. Use 1% BSA to block the channel at room temperature for 1h. After the reaction is completed, rinse the channel with PBST for 3min and store it in a 4℃ refrigerator for later use.

[0171] (3) Detection: Take out the modified chip and place it in a vacuum drying oven for 2 hours to evacuate and degas. After degassing, take out the waste liquid pool and stick it on the top of PDMS with transparent tape to seal it. Add 4 μL of 1 μg / mL cTN I antigen to the injection ports of channels 1-3 respectively, and add 4 μL PBS to channel 4 as a blank control. After the reaction is completed, rinse with PBST for 1 minute. Add colloidal gold-labeled detection antibody to react for 10 minutes, and observe the channel color.

[0172] Two methods were used to fix the antibodies on the glass surface at the bottom of the channel in the chip, and the effects of the two methods on modifying cTN I antibodies were compared by their ability to capture antigens, i.e., the color depth of colloidal gold. Fig.12 It can be seen that the background signals of the GA (glutaraldehyde) method and the EDC / NHS method are very weak, but the GA method is obviously darker and more effective than the EDC / NHS method. This may be because EDC / NHS is more active and is easily inactivated or dissociated in the reverse equilibrium during the coupling process. Therefore, we chose the GA method to fix the antibody in subsequent experiments.

[0173] In addition, the glass surface modification effects at each stage of the GA method were briefly investigated by measuring the glass surface contact angle. Fig.13 As shown in the figure, the new clean glass slide has strong hydrophilicity, and the water contact angle (θ) is about 19.7°C; after piranha cleaning, the hydroxyl groups on the glass slide surface are fully exposed, the hydrophilicity is enhanced, and θ is 13.6°C; after APTES treatment, the hydroxyl groups are replaced by amino groups, the hydrophilicity is reduced, and θ increases to 66.9°C; after GA treatment, the amino groups are replaced by aldehyde groups, the hydrophilicity continues to decrease, and θ increases to 78.3°C. The change in the contact angle of the glass surface further shows that the glass slide has been successfully modified.

[0174] 2.2 Selection of silanization time and temperature

[0175] Take 6 chips and divide them into two groups, three chips in each group; Group A is the room temperature group and Group B is the heating group. According to the silanization modification conditions in "2.1", the channels of Group A and Group B are treated with 10% APTES anhydrous ethanol for 1min, 5min, 10min, 20min and 1h respectively, and two groups are set up in parallel at each time point. Modify the capture antibody in each channel according to the glutaraldehyde method and perform detection.

[0176] Silanization time and temperature have a significant effect on antibody coating. The modification of the glass surface at the bottom of the immune reaction channel is completed by injecting APTES solution into the immune reaction channel. At high temperatures, the ethanol solvent is easy to volatilize, and APTES precipitates white solids, which not only fails to complete the modification of the glass surface, but also causes channel blockage. In a similar situation, the same situation will occur if the reaction time is too long. Even if the humidity can be increased and new solutions can be injected into the channel frequently and timely, the complicated operation greatly reduces the efficiency of the reaction. Therefore, the feasibility of silanization reaction with APTES in a short time and at room temperature was studied. The silanization modification effect of the slide was examined by its ability to capture antigens, that is, the color depth of colloidal gold. Fig.14 a is a chip channel image of the reaction of the bottom glass of the channel with the APTES solution at room temperature for 1min, 5min, 10min, 20min and 1h, respectively. It can be seen that the color of the reaction for more than 5min is obviously darker than that of the reaction for 1min, while no obvious difference is seen when the reaction is longer than 5 minutes. Fig.14 As can be seen in b, the same trend is also seen in the high temperature group. However, there is no obvious difference between the same reaction time at room temperature and high temperature. Fig.14 It is easier to observe in b.

[0177] 2.3 Optimization of aldehyde conditions

[0178] Take 4 chips and divide them into three parallel groups, with channels in each group numbered 1 to 5. Change the treatment time of 10% APTES ethanol solution to 5 minutes, and treat the chip channels according to the silanization modification in "2.1". Modify the capture antibody in each channel according to the glutaraldehyde method, and the glutaraldehyde reaction time in channels 1 to 5 is 0, 10, 30, 60, and 90 minutes respectively. Then perform the test.

[0179] Aldehydation is another step for treating the glass surface, which also has a significant effect on the fixation of the capture antibody. If the aldehydation reaction time is not long enough, the number of capture antibodies on the glass surface cannot reach saturation. When the antibody is saturated, further increasing the aldehydation reaction time will not produce better results. The aldehydation modification effect of the slide is examined by comparing the ability of the immune response channel to capture antigens under different aldehydation reaction times, that is, the color depth of colloidal gold. Fig.15As shown in the figure, when the reaction time is less than 60 min, the color of colloidal gold deepens with the increase of the formaldehyde reaction time. When the reaction time reaches 60 min, the color no longer changes significantly. Therefore, 60 min was selected as the optimal reaction time for formaldehyde reaction in subsequent experiments.

[0180] 2.4 Optimization of capture antibody concentration

[0181] Take 6 chips and divide the chip channels into 3 groups in parallel, with each group of channels numbered 1 to 7. Change the treatment time of 10% APTES ethanol solution to 5 minutes, and treat the chip channels according to the silanization modification conditions in "2.1". Change the glutaraldehyde reaction time to 1 hour, and modify the monoclonal capture antibody at the bottom of each channel according to the glutaraldehyde method. The capture antibody concentrations used in channels 1 to 7 are 100, 75, 50, 25, 5, 0.5, and 0 μg / mL, respectively. Then perform the test.

[0182] The glass at the bottom of the channel is chemically modified and covalently bound to the capture antibody. The efficiency of binding the antibody is limited and it can only bind a certain number of antibodies. Since the surface area of ​​the chip channel does not change, it is necessary to select an appropriate antibody concentration. If the concentration is too low, there will be too few antibodies, and the amount of bound antibodies will be too small, resulting in reduced sensitivity. If the antibody concentration is too high, there will be an excess of antibodies, which will increase the detection cost and cause unnecessary waste. We select the optimal capture antibody concentration by examining the ability of antibodies on the channel surface to capture antigens. Fig.16 As shown in the figure, when the capture antibody concentration is 0-50μg / mL, the channel color of the same antigen concentration reaction gradually deepens, which is more obvious in the quantified bar graph. When the antibody concentration reaches 75μg / mL, the channel color no longer deepens, indicating that the glass-bound antibody under this surface area has reached saturation. Therefore, the capture antibody with a concentration of 75μg / mL was selected for subsequent experiments.

[0183] 2.5 Vacuuming time selection

[0184] The vacuum time affects the fluid flow rate, so the vacuum time is optimized. Take a chip, change the treatment time of 10% APTES ethanol solution to 5min, treat the chip channel according to the silanization modification in "2.1", change the glutaraldehyde reaction time to 1h, change the capture antibody concentration to 75μg / mL, and modify the capture antibody in each channel according to the glutaraldehyde method in "2.1". Take out the modified chip and vacuum degas for 10, 30, 60, and 120min respectively. After degassing, take out and stick it on the top of PDMS with transparent tape to close the waste liquid pool, and add 4μL of cTN I antigen solution with a concentration of 60ng / mL to the channel inlet. After the reaction is completed, rinse with PBST for 1min. Add colloidal gold-labeled detection antibody to react for 10min and observe the channel color.

[0185] According to the basic principle, the sample flows into the immune response channel and is preferentially captured by the capture antibody at the front end of the immune response channel. The concentration of cTNI flowing to the back end of the immune response channel decreases. After it decreases to a certain level, it cannot bind due to the low concentration. Therefore, a red strip of a certain length is observed by the naked eye, and the cTNI can be quantified by the length of the red strip. Therefore, it is very important to control the flow rate of the sample flowing through the channel. When the sample flows into the reaction channel, it will be captured by the specific antibody modified at the bottom. If the antigen injection flow rate is too fast, some antigens will not have time to bind to the specific antibody modified on the glass substrate, resulting in the test result being lower than the true value; if the antigen injection flow rate is too slow, the time required will increase significantly, and the bedside detection requirements cannot be met. In the present invention, the flow rate is adjusted by the PDMS vacuum time, so the vacuum time also needs to be optimized. First, the approximate range of the flow rate is determined using a syringe pump, such as Fig.17 As shown in the figure, when the flow rate is greater than 0.4μL / min, the antigen binding is insufficient and the color of the strip is obviously lighter than that of the strips at other flow rates. When the flow rate is reduced to 0.4μL / min, the color is not significantly different. Therefore, it is preliminarily determined that the injection flow rate should not be greater than 0.4μL / min. From the above flow rate measurement, it can be seen that the PDMS degassing driven injection flow rate is less than 0.4μL / min, which meets the requirements. Fig.18 It can also be seen from a that the length of the bands of the same concentration at different vacuum times is close. Fig.18 b It can be seen that when the vacuum is pumped for 10 minutes, the reaction time takes half an hour, which is significantly longer than that of other groups; while when the vacuum is pumped for 30, 60 and 120 minutes, the reaction time is similar and the flow rate does not change much. Therefore, the vacuum time of 30 minutes is selected as the subsequent reaction condition.

[0186] 2.6 Optimization of antibody detection reaction time

[0187] Take 4 chips and divide the channels into 3 groups in parallel, with the channels in each group numbered 1 to 5. Change the treatment time of 10% APTES ethanol solution to 5 minutes, treat the chip channels according to the silanization modification in "2.1", change the glutaraldehyde reaction time to 1 hour, change the capture antibody concentration to 75μg / mL, and modify the capture antibody in each channel according to the glutaraldehyde method. Finally, change the vacuum time to 30 minutes for detection.

[0188] In order to ensure clear color and clear reading while further shortening the entire detection process, the reaction time of colloidal gold labeled antibody and antigen-capture antibody complex was optimized. Fig.19As shown in the figure, the colloidal gold labeled antibody reached saturation after 3 minutes of reaction, and no stronger signal could be obtained even if the reaction time continued to increase; when the reaction time was less than 3 minutes, the color of the colloidal gold was obviously lighter, and the color also changed significantly between 1 minute and 2 minutes of reaction. Therefore, in order to ensure the detection sensitivity and shorten the detection time, 3 minutes was selected as the subsequent reaction time.

[0189] 2.7 Methodological considerations

[0190] 1 Exclusiveness

[0191] Specificity analysis is used to illustrate the anti-interference ability of the analytical method to other nonspecific targeted antigens. In order to examine the specificity of the immunomicrofluidic chip to the target cTN I, the 0ng / mL control group, 60ng / mL cTN I group, and 6000ng / mL CEA and AFP, human immunoglobulin G (IgG) were measured respectively. Under the optimal experimental conditions, the length of the colloidal gold stripe of each group was simultaneously detected by the microfluidic immune chip, and the specificity of the immunoassay method was examined according to the length of the stripe.

[0192] The results are as follows Fig. 20 As shown in the figure, the length of the cTN I band measured by the chip at 60 ng / mL is significantly longer than the length of the CEA, AFP and human IgG bands at 6000 ng / mL. This shows that the immunoassay system can specifically detect cTN I, has good specificity, and is expected to be used for quantitative determination of actual samples.

[0193] 2 Standard curve

[0194] Using this system, cTN I antigen solutions with concentrations of 0, 0.5, 5, 10, 20, 30, 40, and 60 ng / mL were measured simultaneously, and the length of the colloidal gold band in each group was observed. A linear curve was prepared based on the relationship between the band intensity and the cTN I concentration.

[0195] The standard curve was drawn with cTN I concentration as the horizontal axis and band length as the vertical axis. Fig.21 As shown in the figure, within the concentration range of 0.5-60 ng / mL, the measured band length increases with the increase of cTN I concentration, and the linear relationship is shown. The linear regression equation is: Y = 0.442*X + 0.6246 (R 2 =0.9942), the linearity is good.

[0196] 3. Precision

[0197] Precision analysis is a prerequisite for measuring reproducibility and ensuring accuracy. The intra-chip precision and inter-chip precision were investigated respectively.

[0198] In-chip precision: Three chips were taken and divided into three groups: low concentration (0.5 ng / mL), medium concentration (30 ng / mL), and high concentration (60 ng / mL). According to the optimal experimental conditions, four parallel experiments were conducted on each group using the immunomicrofluidic system. The relative standard deviation was used to evaluate the length of the colloidal gold strip on the same chip.

[0199] Inter-chip precision: Four chips were taken, and three concentrations of low (0.5 ng / mL), medium (30 ng / mL), and high (60 ng / mL) were set for each chip. Each concentration was measured four times in parallel. The immunomicrofluidic system was used for measurement under the optimal experimental conditions, and the relative standard deviation was used to evaluate the detection results of the same concentration on different chips.

[0200] As shown in Table 3, the RSD of the antigen band lengths at various concentrations in the same chip was <9.1%, the RSD of the detection signals of different immune chips was <10.9%, and the RSD values ​​of high concentrations were generally smaller than those of low concentrations. Therefore, the microfluidic system had good precision in quantitative detection of cTn I antigen.

[0201] Table 3 Intra-chip and inter-chip precision investigation

[0202]

[0203] 4. Accuracy

[0204] Accuracy refers to the degree of conformity between the measured value and the true value. Under the best experimental conditions, the accuracy of the system is examined.

[0205] Six chips were taken and divided into three groups: low concentration (0.5 ng / mL), medium concentration (30 ng / mL), and high concentration (60 ng / mL). Each concentration was measured four times in parallel. According to the optimal experimental conditions, the immune microfluidic system was used to perform experiments on each group in parallel. The concentration of cTN I antigen was calculated according to the standard curve, and the recovery rate was calculated.

[0206]

[0207] The results showed that the lengths of the red bands at 0.5, 30 and 60 ng / mL were 0.9, 14.5 and 27.1 mm, respectively. The calculated concentrations were 0.6, 31.4 and 59.8 ng / mL, respectively. The recoveries were 118.9%, 99.6% and 104.6%, respectively, indicating that the system had good accuracy.

[0208] 5 Actual sample testing

[0209] Accurate detection of cTN I content in actual clinical samples is a very important evaluation indicator for this immunocolloidal gold-based microfluidic quantitative detection system.

[0210] The glass-PDMS microfluidic chip was prepared in advance, and the bottom of the glass was chemically modified to fix the capture antibody. The serum samples of myocardial infarction patients provided by the Department of Immunology of Xiangya Hospital in Changsha, Hunan were tested in time. The serum was tested in time according to the optimal detection conditions. The optimal detection conditions are: degas the glass-PDMS composite chip in a vacuum oven for 30 minutes, and after taking it out, a transparent tape was attached to the top of the PDMS except for the injection port. The PDMS degassing was driven to inject 4μL cTN I antigen solution, and the average injection speed was 0.2μL / min; the channel was rinsed with PBST buffer solution for 3 minutes; the gold-labeled antibody with an OD value of 0.5 was injected into each immune chip microchannel, reacted at room temperature for 3 minutes, and then the immune chip microchannel was rinsed with PBST for 1 minute, the strip length was immediately observed, and a CCD camera was used to take pictures and record.

[0211] The serum samples were operated in parallel in three different immune response channels of the same chip, and the length of the red strip was observed. The measured length of the red strip was used as the y value, substituted into the linear regression equation, and the concentration value was calculated. The RSD analysis of the three groups of concentrations was performed, and the measured concentration values ​​were compared with the hospital data to test the reliability and practicality of the immune detection system.

[0212] The results showed that the cTNI concentration in the serum of myocardial infarction patients provided by Changsha Xiangya Hospital was 0.712 ng / mL. The cTNI concentrations in serum samples measured by the quantitative microfluidic chip based on immunocolloidal gold were 0.6, 0.8, and 0.8 ng / mL, respectively, with an average measurement value of 0.7 ng / mL. The measured cTNI concentration results were very close, indicating that the developed immunomicrofluidic chip is feasible for the detection of actual cTNI samples.

[0213] Comparative Example 1

[0214] For comparison, a total of 10 unprocessed microfluidic chips having fan-shaped depressions connected in series via tubular cavities were prepared according to the etching method of the prior art CN 109655611 A, and each chip was designed with 6 sample loading holes.

[0215] Dopamine treatment: The microfluidic chip body was immersed in 12 mM Tris-HCl dopamine solution for 8 h. Air was introduced into the system during the immersion process. After immersion, the chip was washed with water for 3 times and then dried naturally.

[0216] Glutaraldehyde treatment: The microfluidic chip body treated with dopamine was soaked in anhydrous ethanol solution of glutaraldehyde with a volume fraction of 10% at a temperature of 25° C. for 60 minutes, then taken out and washed with water for 3 times, and then naturally air-dried to obtain a microfluidic chip substrate.

[0217] Coating antigen: Use a micropipette to apply the antigen to the fan-shaped depression of the microfluidic substrate, keep it at 25℃ for 120min, wash it 3 times, then block it with 0.5% skim milk for 12h, repeat the washing 3 times, and dry it at 25℃.

[0218] Take the prepared microfluidic chip, and add 200 μl of serum sample from a myocardial infarction patient, 800 μl of PBST cleaning solution, and 200 μl of 2000-fold diluted antibody into different sample wells.

[0219] Comparative Example 2

[0220] For comparison, a microfluidic chip having a Zn-based material layer was prepared according to the prior art CN 110320355 A, as follows:

[0221] Zn-based compound nanorods bonded to the substrate

[0222] A reaction solution of ZnO was prepared, and a certain amount of zinc nitrate, ethanolamine and ammonia water were mixed. The reaction solution was injected into the quartz capillary (the inner diameter of the tube was 100 μm and the outer diameter was 200 μm) substrate of the microfluidic channel using a constant flow injection pump, and reacted at 75°C for 1 hour. After that, it was washed with deionized water at 25 μL / min to remove the residual reaction substances; then, a sodium polystyrene sulfonate solution was injected into the microchannel, and after incubation for 1 hour, deionized water was passed for washing to complete the binding of Zn-based compound nanorods to the substrate of the microfluidic channel.

[0223] Capture antibodies loaded on Zn-based compound nanorods

[0224] After the Zn-based compound nanorods are bound to the substrate, an EDC / NHS solution containing capture antibodies is introduced at an injection rate of 25 μL / min, and after being filled, it is left for 2 hours, and then PBS buffer is introduced for washing to complete the binding of the capture antibodies to the surface of the Zn-based compound nanorods.

[0225] Detection

[0226] 4 μL cTN I antigen solution was injected through PDMS degassing drive with an average injection speed of 0.2 μL / min; the channel was rinsed with PBST buffer solution for 3 min; gold-labeled antibody with an OD value of 0.5 was injected into each immune chip microchannel, reacted at room temperature for 3 min, and then the immune chip microchannel was continued to be rinsed with PBST for 1 min, the strip length was immediately observed, and photographed and recorded using a CCD camera.

[0227] Test example

[0228] Table 1 Test results of Example 3 and Comparative Examples 1-2

[0229]

[0230] By comparing the test results of Example 3 and Comparative Example 1, it can be seen that under the same concentration test, the standard deviation and coefficient of variation of Comparative Example 1 are larger than those of Example 3. It can be seen that the microfluidic quantitative detection system using the immunocolloidal gold in Example 3 improves the quantitative detection sensitivity and accuracy of the microfluidic chip compared with Comparative Example 1.

[0231] By comparing the test results of Example 3 and Comparative Example 2, it can be seen that under the same concentration test, the standard deviation and coefficient of variation of Comparative Example 2 are larger than those of Example 3. It can be seen that the microfluidic quantitative detection system using the immune colloidal gold in Example 3 improves the quantitative detection sensitivity and accuracy of the microfluidic chip compared with Comparative Example 2.

[0232] In summary, the present invention covalently fixes the monoclonal specific capture antibody in the immune reaction channel by surface chemical modification of the bottom glass of the immune reaction channel of the glass-PDMS microfluidic chip, combines the microfluidic chip with immune colloidal gold, and uses PDMS degassing to drive sample injection, and successfully constructs a microfluidic quantitative detection system based on immune colloidal gold. Antibodies are micro-detected with colloidal gold labeled antibodies, and the sample flows into the immune reaction channel, and is preferentially captured by the capture antibody at the front end of the immune reaction channel. The concentration of cTN I antigen flowing to the rear end of the immune reaction channel is reduced, forming a red strip visible to the naked eye of a certain length, and cTN I is quickly quantified by the length of the red strip. The present invention optimizes the antibody fixation conditions such as the glass surface silanization modification time and temperature, the aldehyde modification time, the capture antibody concentration, and the related experimental conditions such as the injection flow rate and the vacuum pumping time, and the detection antibody reaction time, and investigates the methodology-related content. The results showed that in the range of 0.5-60 ng / mL, the length of cTN I band was linearly related to the concentration of cTN I, and the linear equation was: Y = 0.442X + 0.6246 (R 2 =0.9942), and has good specificity and high precision and accuracy, RSD is less than 11%, and the recovery rate is between 99% and 119%. In addition, the present invention does not require any signal reading equipment, and only 4 μL sample solution is needed to achieve the determination within 30 minutes. In the detection of actual samples, the cTN I concentration of serum samples of myocardial infarction patients measured by the immune microfluidic chip is close to the data measured by Xiangya Hospital, which preliminarily proves that the immune microfluidic chip is accurate and effective in detecting actual samples of cTN I. In short, the present invention has successfully developed a microfluidic detection system that can realize rapid quantitative detection of cTN I.

[0233] The contents explained in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the embodiments made by those skilled in the art fall within the scope defined by the claims attached to the present invention.

Claims

1. A microfluidic chip, characterized in that: The microfluidic chip comprises an immune response channel, the bottom of which is made of glass, and after the glass surface is treated with amino and aldehyde groups, the capture antibody is fixed to the front end of the immune response channel through chemical bonds.

2. The microfluidic chip according to claim 1, characterized in that: The amination treatment comprises: injecting a 10-20%wt APTES ethanol solution into the channel, reacting in the dark at room temperature for 1-60 minutes, repeatedly washing with the ethanol solution for 1-10 minutes, and fixing at 110-120°C to complete the amination treatment; preferably, treating with a 10-20%wt APTES ethanol solution for 5 minutes.

3. The microfluidic chip according to claim 1, characterized in that: The formalization treatment includes: injecting 2.5%-5% glutaraldehyde PBS solution into the channel, reacting in the dark at room temperature for 10-120 minutes, and then repeatedly washing with PBS solution to complete the formalization treatment; preferably, formalization with 2.5%-5% glutaraldehyde PBS solution for 60 minutes.

4. A microfluidic immunoquantitative analysis system, characterized in that: The method comprises the microfluidic chip and immune colloidal gold as claimed in claim 1; the sample to be tested and the immune colloidal gold are sequentially added to the injection port of the microfluidic chip, the sample to be tested and the immune colloidal gold are driven to be injected by the PDMS degassing, the sample to be tested and the immune colloidal gold are captured by the capture antibody, the immune colloidal gold is used as a color developer to form a red stripe, and quantitative analysis is performed according to the linear relationship with the stripe length; The immune colloidal gold is obtained by labeling detection antibodies with colloidal gold.

5. The microfluidic immunoquantitative analysis system according to claim 4, characterized in that: The preparation method of the microfluidic chip comprises: corona activating a polydimethylsiloxane chip with a channel and a glass slide for 30-60 seconds and then laminating them, and then sequentially performing amino treatment and aldehyde treatment on the channel; adding capture antibody to the treated chip for modification; and vacuum degassing and sealing the modified chip to obtain the microfluidic chip.

6. The microfluidic immunoquantitative analysis system according to claim 5, characterized in that: The vacuum degassing comprises: placing the modified chip in a vacuum drying oven and evacuating the air at a pressure of 10 to 50 kPa; preferably, adjusting the flow rate of the sample added to the chip by adjusting the vacuuming time, and the flow rate of the sample is not greater than 0.4 μL / min.

7. The microfluidic immunoquantitative analysis system according to claim 4, characterized in that: In the process of using colloidal gold to label the detection antibody, the pH value is adjusted by adding 0 to 10 μL of 0.2 mol / mL K2CO3 to 1 mL of colloidal gold; preferably, the amount of 0.2 mol / mL K2CO3 added is 4 μL.

8. The microfluidic immunoquantitative analysis system according to any one of claims 4 to 7, characterized in that: The capture antibody is a cTN I monoclonal capture antibody, and the detection antibody is a cTN I detection antibody.

9. Use of the microfluidic chip according to any one of claims 1 to 3 and the microfluidic immunoquantitative analysis system according to any one of claims 4 to 7 in preparing a reagent for quantitative detection of cardiac troponin.

10. A method for non-diagnostic quantitative detection of antigens, characterized in that: include: The sample and immunocolloidal gold are dripped into the injection port of the microfluidic chip for reaction. The sample and immunocolloidal gold are captured by the capture antibody fixed on the immune reaction channel. The immunocolloidal gold is used as a color developer to form a red strip. The content of the antigen is detected based on the linear relationship with the strip length. The microfluidic chip comprises an immune response channel, the bottom of which is made of glass, and the glass surface is treated with amino and aldehyde groups, and the capture antibody is fixed to the front end of the immune response channel through chemical bonds; The immune colloidal gold is obtained by labeling detection antibodies with colloidal gold.

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