A microfluidic sepsis marker detection chip, preparation method and detection method thereof

Abstract

The present invention belongs to the technical field of detecting sepsis markers using biosensor technology, and specifically relates to a microfluidic sepsis marker detection chip, a preparation method and a detection method thereof. The present invention provides a microfluidic sepsis marker detection chip, comprising a substrate and a reaction layer located thereon; the substrate has a layer of titanium carbide (Ti3C2T x ) Mxene nanomaterial-based glass slide substrate, the titanium carbide (Ti3C2T x ) The Mxene nanomaterial substrate is printed with N parallel S-shaped capture antibody barcodes; the reaction layer is provided with a plurality of vertically connected reaction chambers, and the bottom of each reaction chamber is covered with N parallel capture antibody barcodes. The detection chip has high sensitivity and a large detection range, and can realize rapid detection of multiple markers and multiple samples, thereby improving detection efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of detecting sepsis markers using biosensor technology, and specifically relates to a microfluidic sepsis marker detection chip, a preparation method and a detection method thereof. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Sepsis is mostly caused by various types of trauma and infection, and is common in pneumonia, digestive system infection, peritonitis, cholangitis, urinary system infection, cellulitis, bacteremia, multiple injuries and other diseases. It poses a serious threat to human health, with high mortality and morbidity, especially in young children or the elderly. After the body is infected, bacteria and toxins enter the body, activate the body's inflammatory response cells, produce and release a large number of inflammatory mediators (such as TNF-α, IL-4, IL-6, etc.), and cause local or systemic inflammatory reactions. Due to nonspecific clinical manifestations, the rapid clinical diagnosis of sepsis remains a major challenge. At present, C-reactive protein (CRP) and procalcitonin (PCT) are used as specific markers for sepsis, and each of them plays an irreplaceable role in the diagnosis of infectious diseases. Studies have shown that the simultaneous detection of multiple markers (CRP+PCT+SAA+IL-6+TNF-α+IL-4) can help quickly identify clinical patients, provide accurate treatment options and improve the success rate of treatment. However, the concentrations of these markers in human serum samples range over six orders of magnitude, from pg mL -1 to μg mL -1 This requires the construction of a highly sensitive biosensor that can detect multiple targets simultaneously within a dynamic detection range in the same real sample.

[0004] At present, the commonly used methods for detecting inflammatory mediators mainly include: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoblotting (Western Blot), flow cytometry (Flow-Cytometry), etc. Among them, although radioimmunoassay has high sensitivity, it is rarely used now because of radioactive contamination; immunoblotting can determine the size of molecules and has no non-specific reaction, but the operation is cumbersome, the sensitivity is low, and it can only detect a single indicator, which is not suitable for the detection of inflammatory factors; flow cytometry can detect the level of inflammatory molecules at the cellular level, but it has the disadvantages of low sensitivity, low throughput, and high cost; enzyme-linked immunosorbent assay is the most commonly used method, with the advantages of high sensitivity, good specificity, and simple operation, but it can only detect a single indicator in one test, with low throughput and high cost. In the detection of inflammatory factors with multi-indicator characteristics, this method has obvious shortcomings.

[0005] Compared with conventional methods, microfluidic chip technology can be used in highly integrated and automated clinical diagnosis, providing a unique platform for multiple target measurements. At the same time, microfluidic chip analysis requires less reagents and time, making it suitable for point-of-care testing (POCT). This type of biosensor usually requires the use of materials that can immobilize a large number of biomacromolecules on the surface. Currently commonly used 2D nanomaterials, such as graphene, MoS2, and boron nitride nanosheets, are difficult to immobilize biomacromolecules on the surface of the material due to the difficulty of surface functionalization, resulting in problems including insufficient sensitivity and narrow detection range. Summary of the invention

[0006] In view of the problem that it is difficult to fix biological molecules on the surface of 2D nanomaterials in microfluidic chips in the prior art, the present invention provides a microfluidic sepsis marker detection chip to improve the solidification strength of biological molecules and the detection sensitivity; at the same time, a new chip structure is provided to improve the detection efficiency.

[0007] In addition, the present invention also provides a preparation method of the above-mentioned microfluidic sepsis marker detection chip and a detection method thereof.

[0008] To achieve the above purpose, the present invention adopts the following technical solution.

[0009] A microfluidic sepsis marker detection chip, characterized by comprising a substrate and a reaction layer located thereon;

[0010] The substrate has a layer of titanium carbide (Ti3C2T x ) Mxene nanomaterial-based glass slide substrate, the titanium carbide (Ti3C2T x) The Mxene nanomaterial substrate is printed with N parallel S-shaped capture antibody barcodes;

[0011] The reaction layer is provided with a plurality of reaction chambers which are connected vertically, and the bottom of each reaction chamber is covered with N parallel capture antibody barcodes;

[0012] The N is a natural number greater than 1.

[0013] The above-mentioned microfluidic sepsis marker detection chip is preferably:

[0014] The substrate is made by the following method: a glass slide is roughened by ultraviolet laser, titanium carbide (Ti3C2T x ) MXene nanomaterials are bound to the treated glass slide;

[0015] The capture antibody barcode is made by the following method: first, a transfer layer is prepared using polydimethylsiloxane, and the bottom of the transfer layer is provided with N uniformly arranged S-shaped microfluidic grooves, which are arranged in parallel and spaced apart; the transfer layer is also provided with N inflow holes and N outflow holes that are connected from top to bottom, and the two ends of each microfluidic groove are respectively connected to the corresponding inflow hole and outflow hole; secondly, the transfer layer is aligned with the glass slide substrate and attached together by self-weight; the titanium carbide (Ti3C2T x ) The substrate of the Mxene nanomaterial is placed opposite to the groove of the microchannel; thirdly, a monoclonal capture antibody is added to each flow channel from the inflow hole, and the monoclonal capture antibody is evenly spread over the S-shaped array flow channel through negative pressure and adsorbed on the titanium carbide (Ti3C2T x ) on the Mxene nanomaterial substrate to form a capture antibody barcode; fourth, the transfer layer is removed from the substrate.

[0016] The above-mentioned microfluidic-based sepsis marker detection chip is preferably: the transfer layer is printed with positioning holes for identifying positions, and the reaction layer is provided with positioning chambers corresponding to the positioning holes on the transfer layer.

[0017] The above-mentioned microfluidic sepsis marker detection chip is preferably: the N is 3-9; the number of reaction chambers is 20-100; and the depth of the microchannel groove is 20-30 um.

[0018] In the above-mentioned microfluidic sepsis marker detection chip, preferably: the monoclonal capture antibody is one or more of CRP, PCT, SAA, TNF-α, IL-4 and IL-6.

[0019] The method for preparing the above-mentioned sepsis marker detection chip comprises the following steps:

[0020] A. The glass slide was roughened by ultraviolet laser and titanium carbide (Ti3C2T x ) MXene nanomaterials are bound to the treated glass slide;

[0021] B. First, a transfer layer is prepared using polydimethylsiloxane, wherein the bottom of the transfer layer is provided with N uniformly arranged S-shaped microfluidic grooves, which are arranged in parallel and spaced apart; the transfer layer is also provided with N inflow holes and N outflow holes which are connected from top to bottom, and the two ends of each microfluidic groove are respectively connected with the corresponding inflow hole and outflow hole; secondly, the transfer layer is aligned with the glass slide substrate and attached together by self-weight; the titanium carbide (Ti3C2T x ) The substrate of the Mxene nanomaterial is opposite to the groove of the microchannel; thirdly, a monoclonal capture antibody is added into each channel from the inflow hole, and the monoclonal capture antibody is evenly spread over the S-shaped array channel through negative pressure and adsorbed on the titanium carbide (Ti3C2T x ) on the Mxene nanomaterial substrate, forming a capture antibody barcode; fourth, removing the transfer layer (2) from the substrate; forming an S-shaped antibody barcode, and obtaining a substrate;

[0022] C. Use polydimethylsiloxane to prepare the reaction layer; after the reaction layer and the substrate are aligned with the positioning hole and the positioning chamber, they are attached together by their own weight to obtain the sepsis marker detection chip.

[0023] In the above preparation method, preferably: the coupling agent is 3-aminopropyltriethoxysilane, the coupling agent and titanium carbide (Ti3C2T x ) The mass ratio of Mxene is 1:1.

[0024] A sepsis marker detection kit using the above-mentioned sepsis marker detection chip includes a detection chip with one or more monoclonal capture antibodies fixed in an integrated order, one or more corresponding recombinant proteins, and corresponding monoclonal detection antibodies with fluorescent groups; a treatment solution and a stop solution.

[0025] The above-mentioned sepsis marker detection kit is preferably: the monoclonal capture antibody is one or more of CRP, PCT, SAA, TNF-α, IL-4 and IL-6 monoclonal capture antibodies; the recombinant protein is one or more of CRP, PCT, SAA, TNF-α, IL-4 and IL-6 recombinant proteins; the monoclonal detection antibody with a fluorescent group is one or more of CRP, PCT, SAA, TNF-α, IL-4 and IL-6 monoclonal detection antibodies; the fluorescent group is AlexaFluor 647.

[0026] The method for detecting sepsis markers using the sepsis marker detection kit comprises the following steps:

[0027] A. Based on microfluidic integrated titanium carbide (Ti3C2T x ) Add the sample to be tested into the reaction chamber of the sepsis marker detection chip based on the Mxene nanomaterial, so that the sample covers the entire reaction chamber, so that the antigen in the sample to be tested fully combines with the monoclonal capture antibody on the substrate, and after the excess liquid is drained by a negative pressure pump, the sample is washed with a treatment solution to clean the antigen to be tested that is not adsorbed on the substrate;

[0028] B. Add the mixed fluorescent monoclonal detection antibodies into the reaction chamber to cover the sample wells, so that the fluorescent monoclonal detection antibodies specifically bind to the antigens in the sample to be tested on the binding substrate, use a negative pressure pump to drain the excess liquid, and then rinse with a treatment solution to clean the fluorescent monoclonal detection antibodies that are not adsorbed on the substrate;

[0029] C. peeling off the reaction layer (3) in the treatment solution, then washing the substrate with the stop solution, using a microarray scanner to detect the intensity of the optical signal on the substrate, and outputting a fluorescent strip with a positioning point after scanning. The data processing software can accurately find the fluorescent strip of the reaction detection result by identifying the positioning point, thereby reading the fluorescence intensity of the fluorescent strip of the fluorescent monoclonal detection antibody;

[0030] D. According to the relationship between the fluorescence value and the marker concentration obtained from the standard curve, the measured fluorescence intensity is substituted into the equation to calculate the content of the monoclonal capture antibody in the sample.

[0031] MXenes are a new type of 2D nanomaterials, and have advantages in hydrophilicity, conductivity, and biocompatibility for sensor design. Most importantly, due to the presence of surface functional groups, they are easy to functionalize and can stably fix proteins, which has obvious advantages in detection without the need for complex multiple amplification methods to improve detection sensitivity. x )Mxene nanomaterials are bound to the glass slide through a coupling agent, and then bound to the protein through a π-π bond. The monoclonal capture antibody is fixed on the glass slide. After adding the sample to be tested, the detection is achieved through antibody-antigen specific binding. The basic principle of the detection is as follows Figure 1 shown.

[0032] The present invention has the following advantages:

[0033] (1) The present invention uses titanium carbide (Ti3C2T x) After the glass slide is treated with Mxene nanomaterials, due to the surface functional groups such as hydroxyl and oxygen, it can non-specifically bind to the protein and fully fix the target protein. The monoclonal antibody corresponding to the marker to be detected is fixed to the glass slide by designing the transfer layer of the site, realizing the directional transfer of the capture antibody. At the same time, the corresponding reaction layer is designed according to the transfer layer, and the design of the positioning point and the corresponding positioning reaction chamber enables the data processing software to accurately find the fluorescent strip of the reaction detection result by identifying the positioning point, thereby realizing data reading and outputting the detection result.

[0034] (2) The detection chip has high sensitivity and a wide detection range, and can achieve measurements at nanogram or even picogram levels. The multi-channel capture antibody array design that does not interfere with each other can achieve rapid detection of multiple markers and multiple samples, thereby improving detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The present invention is a microfluidic integrated titanium carbide (Ti3C2T x ) Schematic diagram of the experimental chip for detecting sepsis markers based on Mxene nanomaterials;

[0036] Figure 2 It is a schematic diagram of the substrate structure of the present invention;

[0037] Figure 3 It is a schematic diagram of the transfer layer structure of the present invention;

[0038] Figure 4 It is a schematic diagram of the reaction layer structure of the present invention;

[0039] Figure 5 This is a schematic diagram of the structure of the sepsis marker detection chip based on the microfluidic substrate of the present invention;

[0040] Figure 6 This is a specific bar graph of the test kit provided by the present invention; in the figure, the ordinate is the concentration and the abscissa is the fluorescence value;

[0041] Figure 7The standard curve diagram for the detection of the kit provided by the present invention; in the figure, (a) is a schematic diagram of the standard curve fitted by the fluorescence values ​​obtained by detecting the optical signal of CRP with different concentration gradients through a microarray scanner; (b) is a schematic diagram of the standard curve fitted by detecting the optical signal of PCT with different concentration gradients through a microarray scanner; (c) is a schematic diagram of the standard curve fitted by detecting the optical signal of SAA with different concentration gradients through a microarray scanner; (d) is a schematic diagram of the standard curve fitted by detecting the optical signal of TNF-α with different concentration gradients through a microarray scanner; (e) is a schematic diagram of the standard curve fitted by detecting the optical signal of IL-4 with different concentration gradients; (f) is a schematic diagram of the standard curve fitted by detecting the optical signal of IL-6 with different concentration gradients;

[0042] Figure 8 The present invention provides a test result diagram of detecting CRP, PCT and IL-6 in serum using the kit provided by the present invention; in the figure, (a) is a comparison diagram of the test results of 20 samples of IL-6 using the Roche test kit and the test kit of the present invention; (b) is a comparison diagram of the test results of 20 samples of CRP using the Roche test kit and the test kit of the present invention; (c) is a comparison diagram of the test results of 20 samples of PCT using the Roche test kit and the test kit of the present invention; (d) is a statistical diagram of the test results of 20 samples of IL-6 using the two test kits; (e) is a statistical diagram of the test results of 20 samples of CRP using the two test kits; (f) is a statistical diagram of the test results of 20 samples of PCT using the two test kits;

[0043] Among them, 1 is a substrate, 2 is a transfer layer, 3 is a reaction layer, 4 is an inlet hole, 5 is a positioning hole, 6 is a positioning chamber, 7 is a reaction chamber, 8 is an outlet hole, and 9 is a microfluidic groove. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0045] Example 1

[0046] The present invention provides a titanium carbide Ti3C2T based on microfluidic integration x ) Mxene nanomaterial-based sepsis marker detection chip, such as Figure 5 As shown, it includes a substrate 1 and a reaction layer 3 located thereon; Figure 2 As shown, the substrate 1 has a layer of titanium carbide (Ti3C2T x ) Glass slide substrate based on Mxene nanomaterials, titanium carbide (Ti3C2T x) The Mxene nanomaterial substrate is printed with an S-shaped capture antibody barcode; Figure 3 As shown, the bottom of the transfer layer 2 is provided with three S-shaped microchannel grooves 9 arranged evenly and arranged in parallel and spaced apart, which are used to print the monoclonal capture antibody onto the titanium carbide (Ti3C2T x ) On the Mxene nanomaterial substrate, the depth of the microchannel groove 9 is between 20-30um. The transfer layer 2 is provided with a positioning hole 5, and the transfer layer 2 is also provided with an inflow hole 4 and an outflow hole 8 that pass through from top to bottom. The two ends of the microchannel groove 9 are connected to the inflow hole and the outflow hole respectively; Figure 4 As shown, the reaction layer 3 is provided with a positioning chamber 6 corresponding to the positioning hole on the transfer layer 2 and a reaction chamber 7 corresponding to the S-shaped capture antibody barcode.

[0047] Example 2

[0048] The sepsis marker detection chip of Example 1 was prepared by the following method.

[0049] The glass slide substrate was roughened by ultraviolet laser at room temperature for 60 seconds to bind oxygen ions to the glass slide, and then immersed in a coupling agent. The coupling agent and the roughened glass slide underwent a cross-linking reaction, and then immersed in titanium carbide (Ti3C2T x ) In the Mxene nanomaterial solution, the coupling agent and titanium carbide (Ti3C2T x ) Mxene nanomaterials react to achieve large-area, highly uniform nanomaterial self-assembly, making titanium carbide Ti3C2T x ) MXene nanomaterials are fully bonded to the glass slide substrate.

[0050] The glass slide substrate is made of calcium silicate, sodium silicate or silicon dioxide, the coupling agent is 3-aminopropyltriethoxysilane (APTES), titanium carbide (Ti3C2T x ) Mxene was purchased from Xianfeng Nanomaterial Technology Co., Ltd.

[0051] The coupling agent used is similar to titanium carbide (Ti3C2T x ) The mass ratio of Mxene is 1:1.

[0052] The transfer layer 2 and the reaction layer 3 are made by mixing polydimethylsiloxane (PDMS) and a curing agent (Sylgard silicone elastomer Kit) in a certain ratio (ratio of 10:1) and then processed by injection molding.

[0053] The transfer layer 2 is combined with titanium carbide (Ti3C2T x) After the glass slide substrate of the Mxene nanomaterial substrate is aligned through the positioning points, it is attached together by its own weight, and a monoclonal capture antibody is added to each flow channel from the inflow hole. The monoclonal capture antibody can be selected from one or more of CRP, PCT, SAA, TNF-α, IL-4, and IL-6.

[0054] CRP, PCT, and SAA were purchased from Nanjing Baikang Biotechnology Co., Ltd., and TNF-α, IL-4, and IL-6 were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0055] A negative pressure of about -80 kPa was applied to the outflow hole at the other end to make the monoclonal capture antibody evenly spread over the S-shaped microchannel groove. The titanium carbide (Ti3C2T x ) The Mxene nanomaterial substrate is combined with the monoclonal capture antibody through a π-π bond, and the monoclonal capture antibody is fixed on a glass slide to form an S-shaped monoclonal capture antibody barcode to obtain a substrate 1;

[0056] After the reaction layer 3 and the substrate 1 are aligned with the positioning chamber through the positioning hole, they are attached together by their own weight. The bottom of each reaction chamber 7 is covered with three parallel capture antibody barcodes, and a sepsis marker detection chip can be obtained. Figure 5 shown.

[0057] One detection chip can detect 60 samples from different sources, and each sample can detect 3 antibodies at the same time, thus greatly improving the efficiency of detection.

[0058] Example 3

[0059] In this embodiment 3, the microfluidic integrated titanium carbide (Ti3C2T x ) Mxene nanomaterial-based sepsis marker detection chip, preparing a sepsis marker detection kit, including the microfluidic integrated titanium carbide (Ti3C2T x ) Mxene nanomaterial-based sepsis marker detection chip; purified recombinant proteins CRP, PCT, SAA, TNF-α, IL-4, IL-6; CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibodies with fluorescent groups; treatment solution and stop solution.

[0060] The concentration of the monoclonal detection antibody is greater than 100 μg / mL.

[0061] The fluorescent group is Alexa Fluor 647.

[0062] The working concentration dilution multiple of the monoclonal detection antibody is 1:100-1:500, preferably 1:200.

[0063] The diluent used was DPBS solution.

[0064] The treatment solution used is a DPBS solution containing 1% bovine serum albumin (BSA); the DPBS solution is a Dulbecco's phosphate buffer solution with a pH of 7.2-7.4 and a concentration of 0.01 mol / L.

[0065] The stop solution is DPBS solution, 50% DPBS solution prepared with ultrapure water as solvent, and ultrapure water.

[0066] This kit should be stored at -20℃ for long-term storage and 2-8℃ for short-term storage.

[0067] Example 4

[0068] The kit of the above embodiment was used to test the specificity of the kit of the present invention. The recombinant proteins CRP, PCT, SAA, TNF-α, IL-4, IL-6, IL-17, IFN-γ, and IL-10 were diluted to 27.7ug / ml, 3750pg / ml, 10ug / ml, 2350pg / ml, 2000pg / ml, 5000pg / ml, 20ng / ml, 1.5ug / ml, and 20ng / ml respectively as standard products. x ) Add the sample to be tested to the reaction chamber of the sepsis marker detection chip based on the Mxene nanomaterial to cover the reaction chamber, incubate for 10 minutes, allow the antigen in the sample to be tested to fully bind with the CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal capture antibodies on the substrate 1, drain the excess liquid with a negative pressure pump, and wash with a treatment liquid to clean the antigen to be tested that is not adsorbed on the substrate. At this time, the antigen to be tested is specifically bound to the CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal capture antibodies and is fixed on the substrate 1.

[0069] Add mixed fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibodies to the reaction chamber, so that they cover the sample wells, incubate for 10 minutes, so that the fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibodies specifically bind to the antigens in the sample to be tested on the binding substrate, use a negative pressure pump to drain excess liquid and then rinse with a treatment liquid to clean the fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibodies that are not adsorbed on the substrate 1. At this time, the fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibodies specifically bind to the antigens in the sample to be tested and are fixed on the substrate 1.

[0070] (3) Remove the reaction layer 3 in the treatment solution, then wash the substrate 1 with the stop solution, use a microarray scanner to detect the intensity of the optical signal on the substrate 1 (the detection wavelength is 635nm), the scanning time is 10min, and the fluorescent strips with positioning points are output after scanning. The data processing software can accurately find the fluorescent strips of the reaction detection results by identifying the positioning points, thereby reading the fluorescence intensity of the fluorescent CRP, PCT, SAA, TNF-α, IL-4, and IL-6 monoclonal detection antibody fluorescent strips. Each standard product is measured three times using three microfluidic chip systems.

[0071] The results are as follows Figure 6 As shown in the results, it can be seen that the kit has good specificity, and the marker only has detection results for the corresponding protein, and no other non-specific results are detected.

[0072] Example 5

[0073] In this example, the kit of Example 3 was used to prepare the standard curve.

[0074] The standard curve of the sepsis marker detection kit was drawn, and the recombinant proteins CRP, PCT, SAA, TNF-α, IL-4, and IL-6 were gradiently diluted as standards. The concentration gradient of CRP was 277ug / ml, 27.7ug / ml, 2.77ug / ml, 138.5ng / ml, and 13.85ng / ml, the concentration gradient of PCT was 8800pg / ml, 3570pg / ml, 1890pg / ml, 506pg / ml, and 205pg / ml, and the concentration gradient of SAA was 100ug / ml, 1 0ug / ml, 1ug / ml, 10ng / ml, 1ng / ml, TNF-α concentration gradient is 2350pg / ml, 235pg / ml, 23.5pg / ml, 7.8pg / ml, 2.6pg / ml, IL-4 concentration gradient is 2000pg / ml, 1000pg / ml, 500pg / ml, 250pg / ml, 125pg / ml, IL-6 concentration gradient is 5000pg / ml, 1000png / ml, 100pg / ml, 10pg / ml, 1pg / ml. Take 2μL of each concentration of recombinant protein and add it to different reaction chambers of the detection chip.

[0075] The antigen in the sample to be tested is fully combined with the CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal capture antibody on the substrate, incubated for 10 minutes, and the excess liquid is drained with a negative pressure pump and rinsed with a treatment liquid to clean the antigen to be tested that is not adsorbed on the substrate. At this time, the antigen to be tested is specifically bound to the CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal capture antibody and is fixed on the substrate; then the mixed fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibody is added to the reaction chamber to cover the reaction chamber, incubated for 10 minutes, so that the fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibody is specifically bound to the antigen in the sample to be tested on the substrate, and the excess liquid is drained with a negative pressure pump and rinsed with a treatment liquid to clean the fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibody that is not adsorbed on the substrate.

[0076] The reaction layer is removed in the treatment solution, and the substrate is then washed with the stop solution to achieve fluorescence detection of CRP, PCT, SAA, TNF-α, IL-4, and IL-6. The intensity of the optical signal is detected by a microarray scanner (with a detection wavelength of 635 nm) and the test results are output.

[0077] Each standard product was tested three times using three kits, and the average of the results was taken to perform linear fitting and draw a standard curve. Figure 7 shown.

[0078] Example 6

[0079] The following tests were performed using the kit of the above embodiment.

[0080] Human serum sample collection: With the consent of the subjects, a certain amount of blood was drawn using a non-anticoagulant tube, placed at room temperature, and allowed to stand for about 4 hours or centrifuged at 3500rpm for 10 minutes, and the top layer of light yellow liquid was taken.

[0081] The present invention provides a method for detecting a sepsis marker, comprising the following steps:

[0082] (1) Based on microfluidic integrated titanium carbide (Ti3C2T x ) Add the sample to be tested to the reaction chamber of the sepsis marker detection chip based on the Mxene nanomaterial to cover the reaction chamber, incubate for 10 minutes, allow the antigen in the sample to be tested to fully bind with the CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal capture antibodies on the substrate 1, drain the excess liquid with a negative pressure pump, and wash with a treatment liquid to clean the antigen to be tested that is not adsorbed on the substrate. At this time, the antigen to be tested is specifically bound to the CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal capture antibodies and is fixed on the substrate 1.

[0083] (2) Add mixed fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibodies to the reaction chamber, so that they cover the sample wells, and incubate for 10 minutes to allow the fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibodies to specifically bind to the antigens in the sample to be tested on the binding substrate. Use a negative pressure pump to drain the excess liquid and then rinse with a treatment solution to clean the fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibodies that are not adsorbed on the substrate 1. At this time, the fluorescent CRP, PCT, SAA, TNF-α, IL-4, IL-6 monoclonal detection antibodies specifically bind to the antigens in the sample to be tested and are fixed on the substrate 1.

[0084] (3) The reaction layer 3 is peeled off in the treatment solution, and the substrate 1 is then washed with the stop solution. The intensity of the optical signal on the substrate 1 is detected using a microarray scanner (the detection wavelength is 635 nm). The scanning time is 10 min. After scanning, a fluorescent strip with a positioning point is output. The data processing software can accurately find the fluorescent strip of the reaction detection result by identifying the positioning point, thereby reading the fluorescence intensity of the fluorescent CRP, PCT, SAA, TNF-α, IL-4, and IL-6 monoclonal detection antibody fluorescent strip.

[0085] (4) According to the relationship between the fluorescence value and the marker concentration obtained from the standard curve, the measured fluorescence intensity was substituted to obtain the content of CRP, PCT, SAA, TNF-α, IL-4, and IL-6 in the sample. 15 patients with sepsis were used as the sepsis disease group, and 5 healthy controls were used as the non-sepsis disease group. The results of CRP, PCT, and IL-6 were shown in Figure 8 .

[0086] The results show that the minimum detection limit of the sepsis marker concentration detected by this kit is 1.1pg / mL, and the detection range is 1.1pg / mL-100ug / mL. By comparing the test results with the test results of the existing commercial detection method (electrochemical method), it is found that the test results obtained using the kit of the present invention and the test results of the existing commercial detection method (electrochemical method) are highly consistent, indicating that the test results obtained using the kit of the present invention are accurate. At the same time, by measuring the same serum sample multiple times (10 times in this embodiment), the coefficient of variation between the test results obtained is less than 10%, indicating that this detection kit has good repeatability and can be further used as a reference for medical diagnosis of blood system diseases.

[0087] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A microfluidic sepsis marker detection chip, characterized in that: It comprises a substrate (1) and a reaction layer (3) located thereon; The substrate (1) is a glass slide substrate having a layer of titanium carbide Mxene nanomaterial substrate on the surface, and the titanium carbide Mxene nanomaterial substrate is printed with N parallelly arranged S-shaped capture antibody barcodes; The reaction layer (3) is provided with a plurality of reaction chambers (7) that are connected vertically, and the bottom of each reaction chamber (7) is covered with N parallel capture antibody barcodes; The N is a natural number greater than 1; The substrate (1) is manufactured by the following method: a glass slide is roughened by ultraviolet laser, and a coupling agent is used to bond the titanium carbide Mxene nanomaterial to the treated glass slide; The capture antibody barcode is prepared by the following method: first, a transfer layer (2) is prepared using polydimethylsiloxane, and the bottom of the transfer layer (2) is provided with N uniformly arranged S-shaped microfluidic grooves (9) and arranged in parallel and spaced apart; the transfer layer (2) is also provided with N inflow holes (4) and N outflow holes (8) that are connected from top to bottom, and the two ends of each microfluidic groove are respectively connected to the corresponding inflow hole (4) and outflow hole (8); second, the transfer layer (2) is aligned with the glass slide substrate and bonded together by self-weight; the substrate combined with titanium carbide Mxene nanomaterial is placed opposite to the microfluidic groove; third, a monoclonal capture antibody is added to each flow channel from the inflow hole (4), and the monoclonal capture antibody is evenly spread over the S-shaped array flow channel by negative pressure, and adsorbed on the titanium carbide Mxene nanomaterial substrate to form a capture antibody barcode; fourth, the transfer layer (2) is removed from the substrate; The transfer layer (2) is printed with a positioning hole (5) for identifying a position, and the reaction layer (3) is provided with a positioning chamber (6) corresponding to the positioning hole on the transfer layer (2).

2. The microfluidic sepsis marker detection chip according to claim 1, characterized in that: The N is 3-9; the number of reaction chambers (7) is 20-100; and the depth of the microchannel groove (9) is 20-30 um.

3. The microfluidic sepsis marker detection chip according to claim 1, characterized in that: The monoclonal capture antibody is one or more of CRP, PCT, SAA, TNF-α, IL-4 and IL-6.

4. A method for preparing a sepsis marker detection chip as claimed in claim 1, characterized in that: The following steps are involved: A. The glass slide is roughened by UV laser, and the titanium carbide Mxene nanomaterial is bonded to the treated glass slide using a coupling agent; B. First, a transfer layer (2) is prepared using polydimethylsiloxane, wherein the bottom of the transfer layer (2) is provided with N uniformly arranged S-shaped microchannel grooves, which are arranged in parallel and spaced apart; the transfer layer (2) is also provided with N inflow holes (4) and N outflow holes (8) which are connected from top to bottom, and the two ends of each microchannel groove are respectively connected to the corresponding inflow hole (4) and outflow hole (8); second, the transfer layer (2) is aligned with the glass slide substrate and bonded together by self-weight; the substrate combined with titanium carbide Mxene nanomaterial is opposite to the microchannel groove; third, a monoclonal capture antibody is added into each flow channel from the inflow hole (4), and the monoclonal capture antibody is uniformly spread over the S-shaped array flow channel by negative pressure, and adsorbed on the titanium carbide Mxene nanomaterial substrate to form a capture antibody barcode; fourth, the transfer layer (2) is removed from the substrate; an S-shaped antibody barcode is formed, and the substrate (1) is obtained; C. The reaction layer (3) is prepared using polydimethylsiloxane; the reaction layer (3) and the substrate (1) are aligned with the positioning hole and the positioning chamber, and then bonded together by their own weight, thereby obtaining a sepsis marker detection chip.

5. The preparation method according to claim 4, characterized in that: The coupling agent is 3-aminopropyltriethoxysilane, and the mass ratio of the coupling agent to titanium carbide Mxene is 1:

1.

6. A sepsis marker detection kit comprising the sepsis marker detection chip according to claim 1, characterized in that: It includes a detection chip on which one or more monoclonal capture antibodies are fixed in an integrated sequence, one or more corresponding recombinant proteins, and corresponding monoclonal detection antibodies with fluorescent groups; a treatment solution and a stop solution.

7. The sepsis marker detection kit according to claim 6, characterized in that: The monoclonal capture antibody is one or more of CRP, PCT, SAA, TNF-α, IL-4 and IL-6 monoclonal capture antibodies; the recombinant protein is one or more of CRP, PCT, SAA, TNF-α, IL-4 and IL-6 recombinant proteins; the monoclonal detection antibody with a fluorescent group is one or more of CRP, PCT, SAA, TNF-α, IL-4 and IL-6 monoclonal detection antibodies; the fluorescent group is Alexa Fluor 647.

8. A method for detecting sepsis markers using the sepsis marker detection kit according to claim 7, characterized in that: The following steps are involved: A. adding a sample to be tested into the reaction chamber (7) of a sepsis marker detection chip based on a microfluidic integrated titanium carbide Mxene nanomaterial substrate, so that the sample covers the entire reaction chamber, allowing the antigen in the sample to be tested to fully bind to the monoclonal capture antibody on the substrate (1), draining excess liquid with a negative pressure pump, and washing with a treatment solution to clean the antigen that is not adsorbed on the substrate; B. adding mixed fluorescent monoclonal detection antibodies to the reaction chamber (7) so that they cover the sample wells, allowing the fluorescent monoclonal detection antibodies to specifically bind to the antigens in the sample to be tested on the binding substrate, draining excess liquid with a negative pressure pump, and then rinsing with a treatment solution to clean the fluorescent monoclonal detection antibodies that are not adsorbed on the substrate (1); C. peeling off the reaction layer (3) in the treatment solution, then washing the substrate (1) with the stop solution, using a microarray scanner to detect the intensity of the optical signal on the substrate (1), and outputting a fluorescent strip with a positioning point after scanning. The data processing software can accurately find the fluorescent strip of the reaction detection result by identifying the positioning point, thereby reading the fluorescence intensity of the fluorescent strip of the fluorescent monoclonal detection antibody; D. According to the relationship between the fluorescence value and the marker concentration obtained from the standard curve, the measured fluorescence intensity is substituted into the equation to calculate the content of the monoclonal capture antibody in the sample.

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