CN113311160A - Micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody - Google Patents
Micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody Download PDFInfo
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Abstract
The invention discloses a micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody and a preparation method of the chip, the biochip is composed of GOQDs functional substrate with a detection bar code array, a capture antibody/antigen array micro-printing PDMS layer and a precise quantitative sample detection PDMS layer, and is used for quantitatively detecting SARS-CoV-2 antigen protein and IgG/IgM antibody in serum with high flux, rapidness, sensitivity and simultaneously, the application of the chip not only is helpful for rapid diagnosis of patients with COVID-19, but also provides a valuable screening approach for infectors, drug therapy and vaccinees.
Description
Technical Field
The invention relates to a micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody, belonging to the field of biotechnology.
Background
COVID-19 still poses serious threat to global health at present, the current commonly used method for diagnosing SARS-CoV-2 infection is to detect the virus RNA in a nasal swab sample by RT-PCR, the method is simple and reliable, but the sample collection process needs operation details, takes more than hours, and can not track the immune response of virus infection.
Recent studies on SARS-CoV-2 have shown that SARS-specific antibodies 8-9, such as IgG, IgM and IgA, appear in the serum after viral infection. Research shows that the dynamic levels of neutralizing antibodies of different populations at different time points show different clinical characteristics, antibodies (IgG and IgM) appear from day 4 after COVID-19 infection, and the seropositive rate reaches 100.0% within 20 days after 16 outbreaks and still reaches 100.0% within 41-53 days. Antibody levels in patients over the age of 31 are higher than in patients 16-30 years of age, while female patients tend to produce more IgG antibody in early disease and in severe disease than male patients, and female patients tend to have higher average IgG antibody levels than male patients. Therefore, the virus-specific antibody in the serum can be used as an effective diagnostic biomarker for screening virus infection and evaluating adaptive immune response of patients after discharge and injection of vaccines. In addition, the viral antigens (spike (S) protein 18-20 and nucleocapsid (N) protein 21-23) in circulating blood can also be used for prognostic studies of COVID-19-associated viremia.
The existing detection methods of SARS-CoV-2 specific antibody and virus antigen mainly include electronic transistor biosensor method, gold nanoparticle lateral flow method and enzyme linked immunosorbent method. The electronic transistor biosensor has higher sensitivity in SARS-CoV-2 antigen protein detection, but the practical application is limited by very complicated manufacturing process; the gold nanoparticle lateral flow method is used for rapidly detecting SARS-CoV-2IgG/IgM antibody, but has limited sensitivity and can carry out quantitative detection; enzyme-linked immunosorbent assay (ELISA) can provide quantitative detection, but the operation process is complex and the detection time is long.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody, which consists of a GOQDs functional substrate with a detection bar code array, a capture antibody/antigen array micro-printing PDMS layer and a precise quantitative sample detection PDMS layer and is used for quantitatively detecting SARS-CoV-2 antigen protein and IgG/IgM antibody in serum at the same time with high flux and rapidness and sensitivity.
The micro flow control biochip for fast detecting SARS-CoV-2 antigen and IgG/IgM antibody and its making process includes the following steps:
(1) preparing GOQDs base layer with detection bar code array;
(2) manufacturing a substrate mould by utilizing a substrate (such as a silicon wafer) and an ICP (inductively coupled plasma) etching process;
(3) preparing a PDMS layer by using the substrate mould obtained in the step (2), punching holes on the PDMS layer to generate an inlet and an outlet of an antibody/antigen array, combining the punched holes with the GOQDs base layer obtained in the step (1) to form a space coding microfluidic biochip, loading a capture antibody/antigen specific probe into an inlet of the obtained space coding microfluidic biochip, which is connected with a microchannel on the chip, and micro-printing the antibody/antigen probe array on the base layer in the microchannel to obtain the space coding microfluidic biochip loaded with the capture antibody/antigen;
(4) then preparing a PDMS layer with the thickness of 0.5-1mm and a PDMS layer with the thickness of 2-3mm, aligning the two PDMS layers, and then baking at 75-80 ℃ for 50-60min to form a PDMS layer for accurate quantitative sample detection;
(5) combining the interval coding micro-fluidic biochip loaded with capture antibody/antigen obtained in step (3) with the PDMS layer for accurate quantitative sample detection obtained in step (4), so as to form the micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody.
Further, the method for preparing GOQDs functionalized substrates with detection barcode arrays as described in step (1) comprises the following steps:
s1, cleaning the glass substrate, treating the glass substrate by using a 3-aminopropyl trimethoxy broccoli (APTES) solution, and cleaning to obtain the glass substrate treated by the APTES;
s2, placing the glass substrate processed by the APTES obtained in the step S1 into GOQDs solution for self-assembly of GOQDs, washing to remove redundant GOQDs, and micro-printing an antibody/antigen detection probe bar code array on the glass substrate with the GOQDs to obtain a GOQDs base layer with a detection bar code array.
Further, the concentration of the 3-aminopropyltrimethoxy-broccoli (APTES) solution is 1-5%, and the concentration of the GOQDs solution is 1-5 mg/mL.
Further, the substrate mold in the step (2) is a silicon wafer mold having 20 to 30 parallel stripe-shaped projections with a width of 5 to 50 μm.
Further, the method for preparing the PDMS chip layer described in the step (3) includes the steps of:
and (3) soaking the silicon wafer mold obtained in the step (2) in Trimethylchlorosilane (TMCS) for 15-20min, mixing Sylgard 184A and Sylgard 184B according to the weight ratio of 10:1, loading the mixture on the silicon wafer mold treated by TMCS, vacuumizing bubbles, curing the mixture at 70-80 ℃ for 60min, and stripping the PDMS layer from the silicon wafer mold to obtain the PDMS layer.
The application can also obtain a micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody, which is prepared according to the above preparation method.
Furthermore, the micro-fluidic biochip is applied to the rapid detection of SARS-CoV-2 antigen and IgG/IgM antibody.
Further, the application method of the application comprises the following steps:
introducing a sample to be detected into a microcavity of a microfluidic biochip, incubating at room temperature for 3-20 min, stripping a precise quantitative sample detection PDMS layer from the interval coding microfluidic biochip by using 1% BSA, washing the interval coding microfluidic biochip by using 1% BSA solution, loading and dispersing 300 mu L of a fluorescent conjugate detection antibody compound onto the surface of the whole interval coding microfluidic biochip, sequentially washing by using 1% BSA, 1 XPBS, 0.5 XPBS and deionized water, drying, and scanning the interval coding microfluidic biochip by using a fluorescence scanner (such as GenePix-4400) to obtain and analyze the concentration of a target antibody/antigen.
Further, the concentration of the fluorescence conjugated detection antibody complex is 5-20 μ g/ml.
Has the advantages that:
the microfluidic biochip prepared by the application integrates the advantages of the nano material and the microfluidic chip, can simultaneously detect various SARS-CoV-2 antigens or IgG/IgM antibodies of 60 samples, has the sample volume of each patient of only 2 mu L, the ultralow detection limit of about 0.3pg/mL and the qualitative detection time of less than 10min, and realizes the high-throughput, rapid, sensitive and quantitative detection of SARS-CoV-2 antigen protein and IgG/IgM antibodies in serum. The application of the chip not only facilitates the rapid diagnosis of COVID-19 patients, but also provides a valuable screening way for infectors, drug therapy and vaccinees.
Drawings
FIG. 1 is a schematic diagram of SARS-CoV-2 virus and microfluidic biochips.
FIG. 2 is a schematic view of a microfluidic biochip.
Fig. 3 is a chip manufacturing process diagram.
FIG. 4 shows the construction and detection mechanism verification of the sensing biochip.
FIG. 5 is a schematic diagram of the detection scheme of anti-Covid-19 antigen and antibody IgM/IgG on the biosensing chip.
FIG. 6 micro-fluidic biochip for detecting specificity and sensitivity of SARS-CoV-2 antibody/antigen.
FIG. 7 is a graph showing the effect of detection time on the detection limit and quantitative detection amount of a microfluidic biochip.
FIG. 8 bar graphs of fluorescence intensities observed at (a)5min, (b)10min, (c)20min and (d)40min at antigen concentrations of 5 to 500 ng/mL.
FIG. 9 is a bar graph showing fluorescence intensities observed at (a)5min, (b)10min, (c)20min and (d)40min at S-IgG concentrations of 5 to 500 ng/mL.
FIG. 10 is a bar graph showing fluorescence intensities observed at (a)5min, (b)10min, (c)20min and (d)40min at S-IgM concentrations of 5 to 500 ng/mL.
FIG. 11 shows reproducibility tests of (a) S-antigen, (b) N-antigen, (c) S-IgG, (d) N-IgG, (e) S-IgM, and (f) N-IgM.
FIG. 12 shows the fluorescence intensity of (a) S-antigen, (b) N-antigen, (c) S-IgG, (d) N-IgG, (e) S-IgM and (f) N-IgM repeated experiments.
FIG. 131 shows the results of measurement of a sample mixture of bovine serum albumin and healthy serum.
Figure 141% biomarker assay concentration in BSA deviation from biomarker assay concentration in healthy serum.
FIG. 15 detection of SARS-CoV-2S antigen, S-IgG and S-IgM in serum of healthy persons.
FIG. 16 is a graph showing the result of the fluorescence intensity distribution of SARS-CoV-2s antigen.
FIG. 17 is a graph showing the result of the fluorescence intensity distribution of SARS-CoV-2 s-IgG.
FIG. 18 is a graph showing the result of the distribution of fluorescence intensity of SARS-CoV-2 s-IgM.
FIG. 19 is a schematic representation of an enzyme-linked immunosorbent assay method.
FIG. 20 ELISA detection of SARS-CoV-2 antigen/antibody.
FIG. 21 is a graph comparing the results of SARS-CoV-2S antigen and S-IgG detection using microfluidic chip and ELISA method.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, which are only a part of examples of the present application, but not all examples, and the present invention is not limited by the following examples.
EXAMPLE 1 preparation of microfluidic biochip for Rapid high-throughput detection of SARS-CoV-2 antigen and IgG/IgM antibody
Micro-fluidic biochip for preparing rapid high-throughput detection SARS-CoV-2 antigen and IgG/IgM antibody
(1) Cleaning a glass substrate, treating the glass substrate by using a 3-aminopropyl trimethoxy broccoli (APTES) solution, and cleaning to obtain the APTES-treated glass substrate; placing the obtained glass substrate treated by APTES into GOQDs solution for self-assembly of GOQDs, washing to remove redundant GOQDs, and micro-printing an antibody/antigen detection bar code array on the glass substrate with the GOQDs to prepare a GOQDs base layer with the detection bar code array, as shown in (a) in FIG. 2.
(2) The silicon chip die is manufactured by utilizing a silicon chip and an ICP (inductively coupled plasma) etching process, and the die is a silicon chip die with 20 parallel strip-shaped bulges with the width of 20 mu m.
(3) Soaking the silicon wafer mold obtained in the step (2) in Trimethylchlorosilane (TMCS) for 15-20min, mixing Sylgard 184A and Sylgard 184B according to the weight ratio of 10:1, loading the mixture onto the silicon wafer mold treated by TMCS, vacuumizing bubbles, curing the mixture at 70-80 ℃ for 60min, peeling the PDMS layer from the silicon wafer mold to obtain a PDMS layer, micro-printing an antibody/antigen array on the PDMS layer (as shown in (B) in fig. 2), combining the PDMS layer with GOQDs obtained in the base layer in the step (1) to form a space coding microfluidic biochip (as shown in fig. 3), and loading captured antibodies/antigens into an inlet of the space coding microfluidic biochip obtained in the step (3) and connected with a microchannel on the chip.
The concentration of 3-aminopropyl trimethoxy broccoli (APTES) solution is 1-5%, and the concentration of the GOQDs solution is 1-5 mg/mL.
(4) And then manufacturing a PDMS layer with the thickness of 0.5-1mm and a PDMS layer with the thickness of 2-3mm (the manufacturing method is the same as that of the PDMS layer in the step (3)), aligning the two PDMS layers, and then baking the two PDMS layers at the temperature of 75-80 ℃ for 50-60min to form a PDMS layer for accurately detecting the quantitative sample (shown in (c) in figure 2).
(5) Combining the space coding microfluidic biochip loaded with the capture antibody/antigen obtained in the step (3) with the PDMS layer for accurate quantitative sample detection obtained in the step (4), so as to form the microfluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody.
SARS-CoV-2 is a novel particle consisting of antigenic proteins (surface spinous process (S), membrane (M) and nucleocapsid (N) proteins) and RNA viral genome, which invades human by interaction of S protein with human cells and replicates itself in human cells by RNA viral genome, as shown in a in fig. 1. Once infected with SARS-CoV-2, the adaptive immune system will produce SARS-CoV-2 antibodies (IgG/IgM) to prevent invasion of SARS-CoV-2. The schematic diagram of the designed microfluidic biochip for detecting SARS-CoV-2 antigen protein and IgG/IgM antibody is shown in b in FIG. 1, and has the capability of detecting 60 samples simultaneously, a small sample volume of 2 μ L, a detection time of less than 10 minutes, and a lower detection limit of about 0.3 pg/ml.
For SARS-CoV-2 antigen detection, the target antigen is bracketed by the capture and detection antibodies, as shown in c in FIG. 1. To perform the anti-SARS-CoV-2 IgG/IgM detection, a specific antigen is micro-imprinted on a functionalized substrate, the target antibody is first captured, a fluorescently labeled goat anti-human secondary antibody IgG and IgM is used to specifically bind to IgG/IgM, and a fluorescent signal is given as indicated by d in FIG. 1.
Secondly, detecting the manufacturing condition of the microfluidic biochip
To confirm successful engagement at each step of the fabrication and detection process, AFM and raman (fig. 4) characterization was performed on the microfluidic biochips. FIG. 4 (a) is an AFM image of a slide glass, FIG. 4 (b) is an AFM image of a GOQDs assembled glass, (c) is an AFM image of a capture antibody immobilized on a GOQDs substrate, and FIG. 4 (d) is an AFM image of an antigen captured by an antibody immobilized on a GOQDs substrate. FIG. 4 (e) to FIG. 4 (h) are the corresponding Raman spectra detected from the slide glass, the assembled glass of GOQDs, the capture antibody immobilized on the substrate of GOQDs, and the antigen captured by the antibody immobilized on the substrate of GOQDs, respectively.
The glass substrate has a relatively small roughness, which is reduced even by the deposition of the molecular thin GOQDs layer, as shown in fig. 4 (a) and 4 (b). But after immobilizing the capture antibody and the antigen, it becomes rougher as shown in (c) in fig. 4 and (d) in fig. 4. In order to confirm successful bonding at each step, raman spectroscopy was performed on these substrates as shown in fig. 4 (e) to fig. 4 (h). It exhibited representative peaks at 576 and 1092cm-1 on bare slides, and peaks at 1400 and 1641cm-1 after immobilization of GOQDs. Raman peaks at 1400 and 1641cm-1 indicate successful assembly of GOQDs on slides. Finally, after immobilization of the capture antibody, representative peaks of the capture antibody appeared in the Raman spectrum at 1096cm-1 and 1407cm-1, indicating successful immobilization of the capture antibody. Representative peaks of antigen at 1196 and 1513cm-1 indicate successful capture of antigen
The result shows that the microfluidic biochip for detecting SARS-CoV-2 igg/IgM/antigen is successfully prepared and the working sensing mechanism is established.
EXAMPLE 2 microfluidic biochip detection of SARS-CoV-2 igg/IgM/antigen
The target antibody/antigen sample is introduced into the microcavity of the microfluidic biochip. And after incubation for 3-20 min at room temperature, stripping an accurate quantitative sample detection PDMS layer from the microfluidic biochip interval coding substrate in 1% BSA, and flushing the substrate with 1% BSA. For antigen detection, 300. mu.L of fluorescent conjugated detection antibody complex (concentration 10. mu.g/ml) was loaded onto the substrate and dispersed throughout the substrate area. The reaction time of the detection antibody and the antigen is 2-20 min. The antibody detection adopts 300 mu L of fluorescence labeled goat anti-human IgG secondary antibody with the concentration of 10 mu g/ml, and the goat anti-human IgG secondary antibody is incubated for 2 to 20 minutes at room temperature. Finally, the substrate with the barcode array for detection of fluorescent signals was washed with 1% BSA, 1 XPBS, 0.5 XPBS and deionized water, and then span dried. The target antibody/antigen concentration was analyzed by scanning the substrate with the detectable fluorescent signal using GenePix-4400. A schematic detection process of SARS-CoV-2 IgM/IgG/antigen on a biosensor chip is shown in FIG. 5.
Example 3 specificity and sensitivity and detection
2. mu.L of different concentrations (1-10)5pg/ml) of recombinant S antigen and N antigen are loaded into a microfluidic biochip, and then a fluorescently labeled detection antibody is added to bind to the captured target antigen. As shown in fig. 6 (a), both S antigen and N antigen detection showed good specificity. The dependence of fluorescence intensity on the concentration of the target antigen, and their fitting equation are given in (b) and (c) of fig. 6, which indicates that the S antigen and the N antigen have similar binding affinity. The result shows that the micro-fluidic chip has wide detection range for SARS-CoV-2s antigen and N antigen, good specificity and high hypersensitivityUp to 0.3 pg/ml.
Human S-IgM, S-IgG, N-IgM and N-IgG were loaded in each sample chamber at concentrations between 1-105pg/ml, respectively, and the complete capture antigen array was placed on the microfluidic biochip to perform its specificity and sensitivity. In fact, the S antigen is used to capture S-IgM and S-IgG and the N antigen is used to capture N-IgM and N-IgG, but their signals are separated by fluorescently labeled secondary IgM and IgG. Fig. 6 (d) shows that they both specifically react with the corresponding capture antigen and secondary antibody, because the paired capture antigen-antibody exhibits a higher fluorescence signal than the unmatched capture antigen-antibody. The dependence of fluorescence intensity on antibody concentration is shown in fig. 6 (e) - (h), and further analysis shows that for all four antibodies, the fluorescence intensity has a similar linear relationship on a logarithmic scale with the target antibody concentration. The detection limits for two target antigens and four target antibodies were calculated according to the representative equation LOD ═ 3S _ b 1/S29, 30 and are listed in table 1, with detection limits for S antigen, N antigen, S-IgM, S-IgG, N-IgM, and N-IgG of 0.23pg/mL, 0.35pg/mL, 0.21pg/mL, 0.32pg/mL, 0.31pg/mL, and 0.29pg/mL, respectively. The result shows that the developed microfluidic biochip has good specificity and sensitivity to SARS-CoV-2 antibody/antigen detection.
TABLE 1 calculation of detection limits
EXAMPLE 4 SARS-CoV-2 antibody/antigen detection Limit and detection time
The time of SARS-CoV-2 antibody/antigen detection is an important factor affecting influenza pandemic outbreaks. At the same time, a critical detection limit is required to distinguish between healthy and infected patients. To determine the effective detection time of SARS-CoV-2 antibody/antigen, we have similar specificity and sensitivity to the developed microfluidic biochip and similar specificity and sensitivity to the SARS-CoV-2 antibody/antigen detection, so we performed S antigen, S-IgG and S-IgM detections at different detection times. The results of fluorescence intensity detected in serum at 5 to 500ng/mL S antigen, S-IgG and S-IgM samples over 5min, 10min, 20min and 40min are shown in fig. 8, which is used to determine the limit of detection according to the representative formula LOD ═ 3S U b 1/S29, 30. The detection limits of S antigen and S-IgG/IgM increased from-0.3 pg/ml to-4 ng/ml, and the detection time decreased from 40min to 5min, as shown in FIGS. 7 (a) - (c), which is caused by partial reaction of SARS-CoV-2 antibody/antigen with the substrate and the fluorescently labeled detection antibody. In order to further verify the shortest effective detection time and reasonable sensitivity, the relatively low concentrations of S antigen, S-IgG and S-IgM samples are selected to be 5ng/ml, and the detection is carried out by using a microfluidic biochip of 5-40 min. The results (d) - (f) in FIG. 7 show that SARS-CoV-2 antibody/antigen 5ng/ml can be identified quantitatively within 5min and quantitatively within 10-40 min.
Example 5 reproducibility of microfluidic biochips
The same sample of SARS-CoV-2 antibody/antigen (100ng/ml) in 1% BSA was subjected to 5 replicates and the fluorescence intensity of the sample detected was shown in SI FIG. 12. The detected concentration variation was small and the Relative Standard Deviation (RSD) of all six biomarkers was less than 3%, as shown in (a) - (f) of fig. 11, indicating that the proposed SARS-CoV-2 antibody/antigen detection microfluidic biochip has good reproducibility.
Example 6
In practical applications, all biomarkers are mixed with large amounts of biological substances in serum or other body fluids, and high performance detection platforms with good specifications and sensitivity are required. It is necessary to test the platform with a mix of target samples before performing the serum sample analysis. First, the S antigen protein and the N antigen protein were mixed into 1% BSA or healthy serum to form 3 samples of different concentrations, and 4 kinds of target IgG/IgM antibodies were mixed into 1% BSA or healthy serum to form 3 samples, as shown in table 2.
TABLE 2 Mixed concentration of recombinant SARS-CoV-2 antigen/antibody in each sample
The quantitative fluorescence analysis of the target antigen was measured, as shown in FIGS. 13(a) - (c). The fluorometric assay of target antibodies in pooled BSA and healthy serum is shown in FIGS. 13(d) - (f). The test concentrations obtained from the relational expression in FIG. 6 are shown in FIGS. 13(g) to (l), and the recovery rates were 90.36% to 109.98%. Furthermore, the biomarker concentrations detected in the spiked 1% BSA were consistent with the biomarker concentrations in the spiked healthy sera and showed a slight deviation of less than 5%, as shown in fig. 14. These results provide the basis for the practical application of microfluidic biochips.
Example 7
To demonstrate the high throughput and statistical sensitivity of microfluidic biochips, we performed multiple assays for different concentrations of SARS-CoV-2 antigen/antibody. SARS-CoV-2S antigen, S-IgG and S-IgM were detected in a sample volume of 2. mu.L for 5 min. A total of 60 samples of each antigen/antibody were tested on each biochip, 12 negative controls (0ng/mL) and 16 positive mock samples, three different concentrations of 5, 50 and 100ng/mL, respectively.
FIG. 15 shows the relationship between the fluorescence intensity and the concentration of SARS-CoV-2S antigen, S-IgG and S-IgM (a) S antigen, (b) S-IgG, and (c) S-IgM). (d) -f-statistical comparison between negative and positive samples. p <0.05, the difference between the two groups was statistically significant. The results of the experiments are shown in FIGS. 15(a) - (c), which show clear fluorescence signals from different samples, consistent with the concentrations of the prepared mock samples. The quantitative fluorescence intensities of S antigen, S-IgG and S-IgM are shown in FIGS. 16 to 18, respectively. FIGS. 15(d) - (f) show statistical analysis of the detected fluorescence intensities. The negative control and the positive simulation sample have obvious difference, and the positive control group of the t test has obvious difference compared with the positive control group (p < 0.0001). The simulated S antigen, the S-IgG and the S-IgM are in the range of 0.001-100 ng/mL, and the recovery rate is 90.36-109.98%. Therefore, the developed microfluidic biochip provides a new high-throughput and rapid simultaneous detection and analysis method for the detection and analysis of SARS-CoV-2 antigen/antibody.
Example 8 comparison with commercial enzyme-linked immunosorbent assay (ELISA)
1. ELISA detection of SARS-CoV-2 igg/IgM/antigen.
SARS-CoV-2 antibody or antigen was immobilized on each well of the ELISA plate. Then 100 μ L of target antigen/antibody sample was introduced into each well of the ELISA plate. After incubation at room temperature for 2 hours, samples were removed from each well and the substrate was rinsed 5 times with 1% BSA. Then 100 μ L of the corresponding enzyme-linked antibody/antigen was added to bind to the antigen/antibody attached to the solid support. After 2 hours of incubation, the corresponding enzyme-linked antibody/antigen was removed from each well and the substrate was washed 5 times with 1% BSA. Finally, a substrate solution is added, catalytically causing a color change, which can be measured. (ELISA comparison was chosen because this method is also the method of using protein biomarker analysis.)
2. Micro-fluidic biochip for detecting SARS-CoV-2 igg/IgM/antigen
Detection was carried out as in example 2.
3. Results
ELISA detection procedure for SARS-CoV-2S antigen and S-IgG is shown in FIG. 19, which takes about 4.5 hours. As shown in FIG. 20, the results of ELISA detection of SARS-CoV-2S antigen and S-IgG are shown, and a calibration curve and a negative control line were generated using SARS-CoV-2S antigen and S-IgG (S antigen in FIG. 20 (a) and S-IgG in FIG. 20 (b)). Error bars are generated by repeated measurements. Chemiluminescent signals from 96-well plates were used to detect various concentrations of analyte (S-antigen in c and S-IgG in d)).
The ELISA detection limits for healthy serum and 1% BSA samples were 1ng/ml, with a linear range of 1-100 ng/ml. In the ELISA detection range, the comparison of the detection results of our biochip and the commercial ELISA is shown in FIG. 21, the specific analysis data is shown in Table 3, and the recovery rate is 97.19-103.01%.
TABLE 3 results of the detection of spiked samples in serum of healthy persons using microfluidic biochips and ELISA method
EXAMPLE 9 comparison of different detection methods for SARS-CoV-2 antibody
TABLE 4 comparison of different detection methods for SARS-CoV-2 antibody
Comparisons were made for the different detection methods and are summarized in table 4. Most detection modes detect a single biomarker, the detection range is narrow, the detection limit is high, and the qualitative detection time is longer than 15 min. The provided microfluidic biochip platform has a relatively large detection range of 5 orders of magnitude, can simultaneously detect 4 or more (at most 20 in the design of the biochip of the invention) biomarkers, each sample is 2 muL, the high flux (60 samples per biochip), the qualitative detection is less than 10min, the quantitative detection is less than 40min, the ultra-low detection limit is about 0.3pg/mL, and the detection cost, the time consumption and the propagation probability of SARS-CoV-2 antigen/antibody are fundamentally reduced. Therefore, compared with the reported SARS-CoV-2 antigen/antibody detection method, the proposed microfluidic biochip strategy is more suitable for the current SARS-CoV-2 pandemic needs.
The invention develops a microfluidic immunoassay method, which can simultaneously carry out rapid (less than 10 minutes), high-flux (60 samples per chip), reliable and ultrasensitive detection on various SARS-CoV-2 antigens and antibodies. The platform integrates the advantages of nano materials, combines high-density biomolecules with microfluid, and realizes a high-efficiency, low-volume and high-integration biosensing platform. The micro-fluidic biochip not only has great potential value for rapidly screening SARS-CoV-2 infection, but also provides a valuable tool for effective vaccine screening after vaccination.
Claims (10)
1. The method for manufacturing the micro-fluidic biochip for rapidly detecting the SARS-CoV-2 antigen and the IgG/IgM antibody is characterized by comprising the following steps:
(1) preparing GOQDs base layer with detection bar code array;
(2) manufacturing a substrate mould by utilizing a substrate and an ICP (inductively coupled plasma) etching process;
(3) preparing a PDMS layer by using the substrate mould obtained in the step (2), punching holes on the PDMS layer to generate an inlet and an outlet of an antibody/antigen array, combining the punched holes with the GOQDs base layer obtained in the step (1) to form a space coding microfluidic biochip, loading a capture antibody/antigen specific probe into an inlet of the obtained space coding microfluidic biochip, which is connected with a microchannel on the chip, and micro-printing the antibody/antigen probe array on the base layer in the microchannel to obtain the space coding microfluidic biochip loaded with the capture antibody/antigen;
(4) then manufacturing two PDMS layers, aligning the two PDMS layers, and baking to form a precise quantitative sample detection PDMS layer;
(5) combining the interval coding micro-fluidic biochip loaded with capture antibody/antigen obtained in step (3) with the PDMS layer for accurate quantitative sample detection obtained in step (4), so as to form the micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody.
2. The method for preparing microfluidic biochips according to claim 1, wherein the method for preparing GOQDs functionalized substrates with detection barcode arrays described in step (1) comprises the following steps:
s1, cleaning the glass substrate, treating the glass substrate by using an APTES solution, and cleaning to obtain the glass substrate treated by the APTES;
s2, placing the glass substrate processed by the APTES obtained in the step S1 into GOQDs solution for self-assembly of GOQDs, washing to remove redundant GOQDs, and micro-printing an antibody/antigen detection probe bar code array on the glass substrate with the GOQDs to obtain a GOQDs base layer with a detection bar code array.
3. The method for preparing a microfluidic biochip according to claim 2, wherein the concentration of the APTES solution is 1-5% and the concentration of the GOQDs solution is 1-5 mg/mL.
4. The method of claim 1, wherein the substrate mold in the step (2) is a silicon wafer mold having 20 to 30 parallel stripe-shaped protrusions with a width of 5 to 50 μm.
5. The method for fabricating a microfluidic biochip according to claim 1, wherein the method for preparing the PDMS layer in step (3) comprises the steps of:
and (3) soaking the silicon wafer mold obtained in the step (2) in TMCS for 15-20min, mixing Sylgard 184A and Sylgard 184B according to the weight ratio of 10:1, loading the mixture on the silicon wafer mold treated by TMCS, vacuumizing bubbles, curing the mixture at 70-80 ℃ for 60min, and stripping the PDMS layer from the silicon wafer mold to obtain the PDMS layer.
6. The method of claim 1, wherein the two PDMS layers in step (4) are a layer of 0.5-1mm thick PDMS and a layer of 2-3mm thick PDMS.
7. A microfluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody prepared according to the method of claim 1.
8. The use of the microfluidic biochip of claim 7 for rapid detection of SARS-CoV-2 antigen and IgG/IgM antibody.
9. The method for applying an application as claimed in claim 8, characterized by comprising the steps of:
introducing a sample to be detected into a microcavity of a microfluidic biochip, incubating at room temperature for 3-20 min, stripping a precise quantitative sample detection PDMS layer from the interval coding microfluidic biochip by using 1% BSA, washing the interval coding microfluidic biochip by using 1% BSA solution, loading and dispersing 300 mu L of a fluorescence conjugate detection antibody compound onto the surface of the whole interval coding microfluidic biochip, sequentially washing by using 1% BSA, 1 XPBS, 0.5 XPBS and deionized water, drying, and scanning the interval coding microfluidic biochip by using a fluorescence scanner to obtain and analyze the concentration of a target antibody/antigen.
10. The use of claim 9, wherein the concentration of the fluorescent conjugated detection antibody complex is 5-20 μ g/ml.
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