A biochip for simultaneous detection of multiple abundance markers and its application

Abstract

A biochip for simultaneous detection of multiple abundance markers and its application, belonging to the field of immunoassay technology. The biochip includes a PDMS layer and a glass substrate layer, a detection channel formed between the PDMS layer and the glass substrate layer, and a chip inlet and a chip outlet connected to the detection channel; the detection channel includes a CA19-9 detection channel, a CA125 detection channel and a LIF detection channel; the CA19-9 detection channel and the CA125 detection channel are filled with capture antibody-modified immunomagnetic beads and immunofluorescence-modified detection antibodies; the LIF detection channel is filled with immunofluorescence-modified detection antibodies, and the surface of the glass substrate layer in the LIF detection channel is modified with Anti-LIF capture antibodies. The present invention discloses a biochip for simultaneous detection of multiple abundance markers with high capture concentration and its application.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, and in particular to a biochip for simultaneously detecting multiple abundance markers and applications thereof. Background Art

[0002] In the process of disease diagnosis and treatment, single biomarkers often have problems with insufficient specificity and sensitivity. By simultaneously detecting multiple markers with different abundances, the disease state can be evaluated from multiple perspectives, and the comprehensive analysis of the results of these markers can greatly improve the accuracy of diagnosis. In the early diagnosis of certain cancers, a single marker may not increase in some patients, while the combination of multiple markers can more comprehensively reflect the occurrence and development of the tumor, reducing the probability of missed diagnosis and misdiagnosis. In addition, the occurrence and development of the disease is a dynamic process, and different combinations of markers may change at different stages. The multi-abundance marker simultaneous detection chip can monitor the dynamic changes of multiple markers in real time, providing more accurate information for the progression of the disease and the evaluation of treatment effects. For complex multifactorial diseases, it is difficult to accurately screen with a single marker. The multi-abundance marker chip can cover multiple biological pathways and molecular mechanisms related to the disease, has good compatibility with biomarkers of different contents, and improves the screening ability for complex diseases. Considering the importance of detecting markers of different abundance in early diagnosis and prognosis monitoring, there is still a need to develop low-cost, fast, efficient, and multi-marker compatible biodetection chips to further reduce detection costs and improve detection efficiency to meet the current clinical medical needs for rapid and convenient detection.

[0003] Single molecule detection can accurately detect samples with very low concentrations, and can detect substances with concentrations of femtograms (fg / ml) or even lower. This is of great significance for the early diagnosis of diseases and the discovery of biomarkers. In the detection of neurological diseases, tumors, infectious diseases, etc., single molecule detection technology can fully detect low-abundance biomarkers in blood samples, providing strong support for the early diagnosis and monitoring of diseases. Due to the ultra-high sensitivity of single molecule detection technology, it has obvious advantages in the detection of extremely low-content biomarkers, but this is also accompanied by the problem of a low detection range that is difficult to cover the conventional content of different types of markers. Therefore, a method is needed for the simultaneous detection of extremely low-abundance markers and conventional concentration markers. Summary of the invention

[0004] The purpose of the present invention is to provide a biochip for simultaneously detecting multiple abundance markers and its application, which can not only realize the simultaneous detection of multiple abundance markers, but also improve the ability to capture markers.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] A biochip for simultaneous detection of multiple abundance markers comprises a PDMS layer and a glass substrate layer, a detection channel formed between the PDMS layer and the glass substrate layer, and a chip inlet and a chip outlet connected to the detection channel; the detection channel comprises three parallel branch channels: a CA19-9 detection channel, a CA125 detection channel and a LIF detection channel; both the CA19-9 detection channel and the CA125 detection channel are filled with capture antibody modified immunomagnetic beads and immunofluorescence modified detection antibodies; the LIF detection channel is filled with immunofluorescence modified detection antibodies, and the surface of the glass substrate layer in the LIF detection channel is modified with Anti-LIF capture antibodies; the capture antibody modified immunomagnetic beads are prepared by connecting magnetic beads and capture antibodies, and the magnetic beads comprise carboxyl magnetic beads, or modified carboxyl magnetic beads, and the modified carboxyl magnetic beads have fluorine elements and siloxy groups.

[0007] The present invention performs functional carboxyl modification on the surface of magnetic beads to prepare modified carboxyl magnetic beads, and connects the modified carboxyl magnetic beads with capture antibodies, which are used to capture high-abundance target markers in biochips to improve the capture concentration of high-abundance target markers; at the same time, the present invention uses capture antibodies to modify the glass substrate layer, which is used to capture low-abundance target markers in biochips to improve the capture performance of low-abundance target markers. The present invention is a biochip with good capture performance and simultaneous detection of multiple abundance markers.

[0008] Preferably, the diameter of the chip inlet is 0.5-2 mm, and the diameter of the chip outlet is 0.5-2 mm.

[0009] Preferably, in the preparation of modified carboxyl magnetic beads, the ferroferric oxide nanoreagent is first reacted with ethyl silicate, and then reacted with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and sodium pentafluoropropionic acid salt to obtain modified carboxyl magnetic beads. The modified carboxyl magnetic beads of the present invention include ferroferric oxide magnetic particles and a surface modified shell layer, and the surface modified shell layer has a siloxy group and a carboxyl group, which helps to improve the binding ability of the modified carboxyl magnetic beads with the organic interface, thereby improving the capture performance of high-abundance target markers.

[0010] Preferably, the preparation of carboxyl magnetic beads is specifically as follows:

[0011] Weigh the ferroferric oxide nanoreagent, add anhydrous ethanol and deionized water, ultrasonically disperse for 20-40 minutes, slowly add ammonia water, stir at a rate of 300-700 r / min for 5-20 minutes, add ethyl silicate dropwise, heat in a water bath at 25-35°C for 6-18 hours, then add SDS sodium dodecyl sulfate and sodium bicarbonate, ultrasonically disperse for 20-40 minutes, stir at a rate of 100-500 r / min for 5-20 minutes in a water bath at 60-90°C, add styrene, acrylic acid and potassium persulfate solution to react for 8-24 hours, separate by magnet after the reaction, wash with ethanol 2-5 times, dry, add isopropanol to a final concentration of 0.1-10 mg / mL, and obtain carboxyl magnetic beads.

[0012] More preferably, the ferrosoferric oxide nanoreagent comprises ferrosoferric oxide nanoparticles and pure water, and the dosage ratio of the ferrosoferric oxide nanoparticles to the pure water is 1 mg: 0.1-10 mL.

[0013] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to anhydrous ethanol is 1:5-20.

[0014] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to deionized water is 1:0.1-1.0.

[0015] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to the ammonia water is 1:0.2-1.

[0016] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to ethyl silicate is 1:0.1-1.

[0017] More preferably, the usage ratio of the ferrosoferric oxide nanoreagent to SDS sodium dodecyl sulfate is 1 mL: 0.01-0.1 g.

[0018] More preferably, the mass ratio of SDS sodium dodecyl sulfate to sodium bicarbonate is 1:0.1-1.

[0019] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to styrene is 1:0.2-1.

[0020] More preferably, the volume ratio of the ferrosoferric oxide nano-agent to acrylic acid is 1:0.02-0.1.

[0021] More preferably, the concentration of the potassium persulfate solution is 0.01-0.1 g / mL, and the volume ratio of the ferrosoferric oxide nanoreagent to the potassium persulfate solution is 1:0.1-1.

[0022] Preferably, the preparation of modified carboxyl magnetic beads is specifically as follows:

[0023] Weigh the ferroferric oxide nanoreagent, add anhydrous ethanol and deionized water, ultrasonically disperse for 20-40 minutes, slowly add ammonia water, stir at a rate of 300-700r / min for 5-20 minutes, add ethyl silicate dropwise, heat in a water bath at 25-35°C for 6-18 hours, add N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and ultrapure water at 2-10°C, mix and stir for 0.5-2 hours, add sodium pentafluoropropionic acid salt, ultrasonically disperse for 5-20 minutes, react at 70-100°C for 1-3 hours, cool to room temperature after the reaction, magnetically separate, wash alternately with ethanol and ultrapure water for 2-5 times, add isopropanol to a final concentration of 0.1-10 mg / mL, and obtain modified carboxyl magnetic beads.

[0024] More preferably, the ferrosoferric oxide nanoreagent comprises ferrosoferric oxide nanoparticles and pure water, and the dosage ratio of the ferrosoferric oxide nanoparticles to the pure water is 1 mg: 0.1-10 mL.

[0025] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to anhydrous ethanol is 1:5-20.

[0026] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to deionized water is 1:0.1-1.0.

[0027] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to the ammonia water is 1:0.2-1.

[0028] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to ethyl silicate is 1:0.1-1.

[0029] More preferably, the usage ratio of the ferrosoferric oxide nanoreagent and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane is 1 mL: 2-10 g.

[0030] More preferably, the usage ratio of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane to ultrapure water is 1 g:50-150 mL.

[0031] More preferably, the dosage ratio of the ferrosoferric oxide nanoreagent to the sodium pentafluoropropionic acid salt is 1 mL: 4-8 g.

[0032] Preferably, the preparation of modified carboxyl magnetic beads is also modified using 3-(vinylsulfonyl) propionamide and allylmalonic acid, specifically,

[0033] Weigh the ferroferric oxide nanoreagent, add anhydrous ethanol and deionized water, ultrasonically disperse for 20-40 minutes, slowly add ammonia water, stir at a rate of 300-700r / min for 5-20 minutes, drop ethyl silicate, heat in a water bath at 25-35℃ for 6-18 hours, add N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and ultrapure water at 2-10℃, mix and stir for 0.5-2 hours, add sodium pentafluoropropionic acid, ultrasonically disperse for 5-20 minutes, and heat at 70-1 00℃ for 1-3h, after the reaction is completed, cool to room temperature, add 3-(vinylsulfonyl) propionamide and allylmalonic acid, stir at a rate of 100-500r / min for 10-20min, add potassium persulfate and methylenebisacrylamide after nitrogen is introduced for 20-40min, react for 6-12h in a water bath at 60-90℃, separate magnetically, wash alternately with ethanol and ultrapure water for 2-5 times, add isopropanol to a final concentration of 0.1-10mg / mL, and obtain modified carboxyl magnetic beads. The present invention further uses 3-(vinylsulfonyl) propionamide and allylmalonic acid to coat the surface of the magnetic beads with a carboxyl-rich hydrogel shell layer, further improving the binding ability of the magnetic beads to the organic interface, thereby improving the capture concentration of the target marker.

[0034] More preferably, the ferrosoferric oxide nanoreagent comprises ferrosoferric oxide nanoparticles and pure water, and the dosage ratio of the ferrosoferric oxide nanoparticles to the pure water is 1 mg: 0.1-10 mL.

[0035] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to anhydrous ethanol is 1:5-20.

[0036] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to deionized water is 1:0.1-1.0.

[0037] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to the ammonia water is 1:0.2-1.

[0038] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to ethyl silicate is 1:0.1-1.

[0039] More preferably, the usage ratio of the ferrosoferric oxide nanoreagent and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane is 1 mL: 2-10 g.

[0040] More preferably, the usage ratio of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane to ultrapure water is 1 g:50-150 mL.

[0041] More preferably, the dosage ratio of the ferrosoferric oxide nanoreagent to the sodium pentafluoropropionic acid salt is 1 mL: 4-8 g.

[0042] More preferably, the usage ratio of the ferrosoferric oxide nanoreagent to 3-(vinylsulfonyl)propionamide is 1 mL: 0.5-1.0 g.

[0043] More preferably, the volume ratio of the ferrosoferric oxide nanoreagent to allylmalonic acid is 1:0.4-0.85.

[0044] More preferably, the dosage ratio of the ferrosoferric oxide nanoreagent to potassium persulfate is 1 mL: 0.5-1.0 g.

[0045] More preferably, the dosage ratio of the ferrosoferric oxide nanoreagent to methylenebisacrylamide is 1 mL: 1-2 g.

[0046] Preferably, the mass concentration of the capture antibody is 0.1-10 mg / mL, and the volume ratio of the magnetic beads to the capture antibody is 1:1-2.

[0047] Preferably, the volume ratio of the capture antibody-modified immunomagnetic beads to the immunofluorescence-modified detection antibody is 1:1-2.

[0048] Preferably, the preparation of capture antibody modified immunomagnetic beads comprises:

[0049] Weigh carboxyl magnetic beads, add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to activate for 20-40 minutes, then add capture antibody to react for 2-10 hours to obtain capture antibody modified immunomagnetic beads

[0050] More preferably, the capture antibody is an Anti-CA19-9 capture antibody, or an Anti-CA125 capture antibody, or an Anti-LIF capture antibody.

[0051] More preferably, the concentration of the N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 0.1-10 mg / mL, and the volume ratio of the carboxyl magnetic beads to the N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:5-100.

[0052] More preferably, the concentration of the N-hydroxysuccinimide solution is 0.1-10 mg / mL, and the volume ratio of the carboxyl magnetic beads to N-hydroxysuccinimide is 1:0.5-10.

[0053] More preferably, the mass concentration of the capture antibody is 0.1-10 mg / mL, and the volume ratio of the magnetic beads to the capture antibody is 1:1-2.

[0054] Preferably, in the preparation of the immunofluorescence modified detection antibody, the detection antibody is mixed with Atto 565 NHS ester fluorescent dye to obtain the immunofluorescence modified detection antibody.

[0055] More preferably, the concentration of the detection antibody is 0.1-100 μg / mL, and the molar ratio of the detection antibody to the Atto 565 NHS ester fluorescent dye is 1:5-100.

[0056] Preferably, the preparation of immunofluorescence modified detection antibodies is specifically as follows:

[0057] The detection antibody was mixed with Atto 565 NHS ester fluorescent dye and reacted for 2-10 hours to obtain the immunofluorescence modified detection antibody.

[0058] More preferably, the concentration of the detection antibody is 0.1-100 μg / mL, and the molar ratio of the detection antibody to the Atto 565 NHS ester fluorescent dye is 1:5-100.

[0059] More preferably, the detection antibody is an Anti-CA19-9 detection antibody, or an Anti-CA125 detection antibody, or an Anti-LIF detection antibody.

[0060] Preferably, in the method of modifying the surface of the glass substrate layer in the LIF detection channel with the Anti-LIF capture antibody, 3-aminopropyltriethoxysilane is first added to the LIF detection channel for amino treatment, and then streptavidin is used to connect the glass to the biotinylated capture antibody.

[0061] More preferably, the biotinylated capture antibody is obtained by a mixed reaction of NHS-Biotin and Anti-LIF capture antibody, and the molar ratio of NHS-Biotin to Anti-LIF capture antibody is 1:5-100.

[0062] Preferably, the method for modifying the surface of the glass substrate layer in the LIF detection channel with an Anti-LIF capture antibody is specifically as follows:

[0063] Add 3-aminopropyltriethoxysilane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, anhydrous ethanol and acetic acid into the LIF detection channel, shake to mix evenly and spread the reagents on the surface of the glass substrate layer, react in the dark for 20-40 minutes, then add the functional polymer biomaterial, shake to mix evenly and spread the solvent on the surface of the glass substrate layer, react in a humid environment for 6-12 hours, then add streptavidin, shake to mix evenly and spread the solvent on the surface of the glass substrate layer, react for 20-40 minutes, then add NHS-biotin-Anti-LIF capture antibody, shake to mix evenly and spread the solvent on the surface of the glass substrate layer, react for 1-2 hours, discard the reaction reagents after the reaction, wash the LIF detection channel with ethanol 2-5 times, and dry to obtain a glass substrate modified by the Anti-LIF capture antibody in the LIF detection channel.

[0064] More preferably, the mass concentration of the 3-aminopropyltriethoxysilane solution is 1-5%, and the volume ratio of the 3-aminopropyltriethoxysilane solution to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:1-2.

[0065] More preferably, the volume ratio of 3-aminopropyltriethoxysilane to anhydrous ethanol is 1:10-30.

[0066] More preferably, the volume ratio of 3-aminopropyltriethoxysilane to acetic acid is 1:1-5.

[0067] More preferably, the volume ratio of 3-aminopropyltriethoxysilane to the functional polymer biomaterial is 1:0.2-1.

[0068] More preferably, the functional polymer biomaterial includes Biotin-PEG-SVA, mPEG-SVA and a sodium bicarbonate solution with a concentration of 0.1-1 mol / L, the mass ratio of Biotin-PEG-SVA to mPEG-SVA is 1:20-60, and the amount ratio of mPEG-SVA to the sodium bicarbonate solution is 1 g:5-10 mL.

[0069] More preferably, the concentration of streptavidin is 1-2 mg / mL, and the volume ratio of 3-aminopropyltriethoxysilane to streptavidin is 1:0.2-1.

[0070] More preferably, the volume ratio of 3-aminopropyltriethoxysilane to NHS-biotin-Anti-LIF capture antibody is 1:0.2-1.

[0071] More preferably, the concentration of NHS-biotin-Anti-LIF capture antibody is 0.1-10 μg / mL, the NHS-biotin-Anti-LIF capture antibody is obtained by mixing NHS-biotin and Anti-LIF capture antibody for 2-10 hours, and the molar ratio of NHS-biotin to Anti-LIF capture antibody is 1:5-100.

[0072] The invention also discloses the application of the biochip to simultaneously detect multiple abundance markers in serum.

[0073] The present invention adopts modified carboxyl magnetic beads to prepare capture antibody modified immunomagnetic beads, and then uses the capture antibody modified immunomagnetic beads to detect high abundance markers in a biochip; and uses the capture antibody to modify a glass substrate to detect low abundance markers in a biochip, so it has the following beneficial effects: the present invention uses silicon dioxide, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, 3-(vinylsulfonyl) propionamide and allylmalonic acid to functionalize the surface of magnetic beads with carboxyls, thereby improving the dispersibility and biocompatibility of magnetic beads, and improving the capture performance of magnetic beads for high abundance markers, and the capture concentration of CA19-9 markers in serum reaches 538.32-621.73U / mL, and the capture concentration of CA125 markers reaches 270.70-321.62U / mL; the present invention uses the capture antibody to modify the glass substrate, which can improve the capture performance of low abundance markers. Therefore, the present invention improves a biochip for simultaneous detection of multiple abundance markers with high capture concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is a structural diagram of the biochip obtained in Example 1 of the present invention.

[0075] Figure 2 This is a fluorescence signal diagram of the biochip capturing the LIF marker in Example 1 of the present invention.

[0076] Figure 3 This is a fluorescence signal diagram of the CA19-9 marker captured by the biochip obtained in Example 1 of the present invention.

[0077] Figure 4 This is a fluorescence signal diagram of the CA125 marker captured by the biochip obtained in Example 1 of the present invention.

[0078] Figure 5 This is the standard curve of the LIF marker captured by the biochip obtained in Example 1 of the present invention.

[0079] Figure 6 This is the standard curve of the CA19-9 marker captured by the biochip obtained in Example 1 of the present invention.

[0080] Figure 7 This is the standard curve of the CA125 marker captured by the biochip obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0081] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0082] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0083] Embodiment 1:

[0084] S1. Preparation of carboxyl magnetic beads, including:

[0085] Weigh the ferroferric oxide nanoreagent, add anhydrous ethanol and deionized water, ultrasonically disperse for 30 minutes, slowly add ammonia water, stir at a rate of 500 r / min for 10 minutes, drop ethyl silicate, heat in a water bath at 30°C for 10 hours, then add SDS sodium dodecyl sulfate and sodium bicarbonate, ultrasonically disperse for 30 minutes, stir at a rate of 300 r / min for 10 minutes in a water bath at 70°C, add styrene, acrylic acid and potassium persulfate solution to react for 12 hours, separate by magnet after the reaction, wash with ethanol 3 times, dry, add isopropanol to a final concentration of 10 mg / mL, and obtain carboxyl magnetic beads;

[0086] The ferrosoferric oxide nanoreagent includes ferrosoferric oxide nanoparticles and pure water, and the dosage ratio of the ferrosoferric oxide nanoparticles to pure water is 1 mg:0.1 mL; the volume ratio of the ferrosoferric oxide nanoreagent to anhydrous ethanol is 1:10; the volume ratio of the ferrosoferric oxide nanoreagent to deionized water is 1:0.2; the volume ratio of the ferrosoferric oxide nanoreagent to ammonia water is 1:0.3; the volume ratio of the ferrosoferric oxide nanoreagent to ethyl silicate is 1:0.1; the dosage ratio of the ferrosoferric oxide nanoreagent to SDS sodium dodecyl sulfate is 1 mL:0.016 g; the mass ratio of SDS sodium dodecyl sulfate to sodium bicarbonate is 1:0.1; the volume ratio of the ferrosoferric oxide nanoreagent to styrene is 1:0.4; the volume ratio of the ferrosoferric oxide nanoreagent to acrylic acid is 1:0.02; the concentration of the potassium persulfate solution is 0.03 g / mL, and the volume ratio of the ferrosoferric oxide nanoreagent to the potassium persulfate solution is 1:0.1.

[0087] S2. Preparation of capture antibody modified immunomagnetic beads, comprising:

[0088] Weigh the carboxyl magnetic beads prepared in step S1 of this embodiment, add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to activate for 30 minutes, then add capture antibody to react for 4 hours to obtain capture antibody modified immunomagnetic beads. When Anti-CA19-9 capture antibody is used, Anti-CA19-9 capture antibody modified immunomagnetic beads are prepared, when Anti-CA125 capture antibody is used, Anti-CA125 capture antibody modified immunomagnetic beads are prepared, and when Anti-LIF capture antibody is used, Anti-LIF capture antibody modified immunomagnetic beads are prepared;

[0089] The concentration of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride was 1:5; the concentration of N-hydroxysuccinimide solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-hydroxysuccinimide was 1:0.5; the mass concentration of the capture antibody was 1 mg / mL, and the volume ratio of carboxyl magnetic beads to the capture antibody was 1:1.

[0090] S3. Preparation of immunofluorescence modified detection antibodies, including:

[0091] The detection antibody was mixed with Atto 565 NHS ester fluorescent dye and reacted for 4 hours to obtain an immunofluorescence modified detection antibody. When using Anti-CA19-9 detection antibody, an immunofluorescence modified Anti-CA19-9 detection antibody was prepared, when using Anti-CA125 detection antibody, an immunofluorescence modified Anti-CA125 detection antibody was prepared, and when using Anti-LIF detection antibody, an immunofluorescence modified Anti-LIF detection antibody was prepared. The concentration of the detection antibody was 10 μg / mL, and the molar ratio of the detection antibody to Atto565 NHS ester fluorescent dye was 1:50.

[0092] S4. A biochip for simultaneous detection of multiple abundance markers, comprising a PDMS layer and a glass substrate layer, a detection channel formed between the PDMS layer and the glass substrate layer, and a chip inlet and a chip outlet connected to the detection channel; the detection channel comprises three parallel detection channels: a CA19-9 detection channel, a CA125 detection channel and a LIF detection channel; the CA19-9 detection channel is filled with the Anti-CA19-9 capture antibody modified immunomagnetic beads prepared in step S2 of this embodiment and the immunofluorescence modified Anti-CA19-9 detection antibody prepared in step S3 of this embodiment, and the Anti-CA19-9 capture antibody is prepared in step S4 of this embodiment. The volume ratio of the obtained antibody-modified immunomagnetic beads to the immunofluorescence-modified Anti-CA19-9 detection antibody is 1:1; the CA125 detection channel is filled with the Anti-CA125 capture antibody-modified immunomagnetic beads prepared in step S2 of this embodiment and the immunofluorescence-modified Anti-CA125 detection antibody prepared in step S3 of this embodiment, and the volume ratio of the Anti-CA125 capture antibody-modified immunomagnetic beads to the immunofluorescence-modified Anti-CA125 detection antibody is 1:1; the surface of the glass substrate layer facing the detection channel in the LIF detection channel is modified with the Anti-LIF capture antibody, and the modification steps are as follows:

[0093] Add 3-aminopropyltriethoxysilane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, anhydrous ethanol and acetic acid into the LIF detection channel, shake to mix evenly and spread the reagents on the surface of the glass substrate layer, react in the dark for 20 minutes, then add the functional polymer biomaterial, shake to mix evenly and spread the solvent on the surface of the glass substrate layer, react in a humid environment for 8 hours, then add streptavidin, shake to mix evenly and spread the solvent on the surface of the glass substrate layer, react for 20 minutes, then add NHS-biotin-Anti-LIF capture antibody, shake to mix evenly and spread the solvent on the surface of the glass substrate layer, react for 1 hour, discard the reaction reagent after the reaction, wash the LIF detection channel with ethanol for 4 times, and dry it to obtain a glass substrate modified by the Anti-LIF capture antibody in the LIF detection channel;

[0094] The mass concentration of 3-aminopropyltriethoxysilane solution was 2%, the volume ratio of 3-aminopropyltriethoxysilane solution to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was 1:1.2; the volume ratio of 3-aminopropyltriethoxysilane to anhydrous ethanol was 1:20; the volume ratio of 3-aminopropyltriethoxysilane to acetic acid was 1:2; the volume ratio of 3-aminopropyltriethoxysilane to functional polymer biomaterials was 1:0.6, and the functional polymer biomaterials included Biotin-PEG-SVA, mPEG-SVA and sodium bicarbonate solution with a concentration of 0.1 mol / L, the mass ratio of Biotin-PEG-SVA to mPEG-SVA was 1:40, m The dosage ratio of PEG-SVA and sodium bicarbonate solution is 1g:7.5mL; the concentration of streptavidin is 1mg / mL, the volume ratio of 3-aminopropyltriethoxysilane and streptavidin is 1:0.4; the volume ratio of 3-aminopropyltriethoxysilane and NHS-biotin-Anti-LIF capture antibody is 1:0.4, the concentration of NHS-biotin-Anti-LIF capture antibody is 10ug / mL, and the NHS-biotin-Anti-LIF capture antibody is obtained by mixing NHS-biotin and Anti-LIF capture antibody for 4h, and the molar ratio of NHS-biotin and Anti-LIF capture antibody is 1:50.

[0095] Embodiment 2:

[0096] S1. Preparation of modified carboxyl magnetic beads, comprising:

[0097] Weigh the ferroferric oxide nanoreagent, add anhydrous ethanol and deionized water, ultrasonically disperse for 30 minutes, slowly add ammonia water, stir at a rate of 500r / min for 10 minutes, add ethyl silicate dropwise, heat in a water bath at 30°C for 10 hours, add N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and ultrapure water at 4°C, mix and stir for 1 hour, add sodium pentafluoropropionic acid salt, ultrasonically disperse for 10 minutes, react at 95°C for 2 hours, cool to room temperature after the reaction, magnetically separate, wash alternately with ethanol and ultrapure water for 4 times, add isopropanol to a final concentration of 10 mg / mL, and obtain modified carboxyl magnetic beads;

[0098] The ferrosoferric oxide nanoreagent includes ferrosoferric oxide nanoparticles and pure water, the dosage ratio of the ferrosoferric oxide nanoparticles to pure water is 1 mg:0.1 mL; the volume ratio of the ferrosoferric oxide nanoreagent to anhydrous ethanol is 1:10; the volume ratio of the ferrosoferric oxide nanoreagent to deionized water is 1:0.2; the volume ratio of the ferrosoferric oxide nanoreagent to ammonia water is 1:0.3; the volume ratio of the ferrosoferric oxide nanoreagent to ethyl silicate is 1:0.1; the dosage ratio of the ferrosoferric oxide nanoreagent to N-(2-aminoethyl)-3-aminopropyltrimethoxysilane is 1 mL:5 g; the dosage ratio of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane to ultrapure water is 1 g:100 mL; the dosage ratio of the ferrosoferric oxide nanoreagent to sodium pentafluoropropionic acid salt is 1 mL:8 g.

[0099] S2. Preparation of capture antibody-modified immune-modified magnetic beads, comprising:

[0100] Weigh the modified carboxyl magnetic beads prepared in step S1 of this embodiment, add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to activate for 30 minutes, then add capture antibody to react for 4 hours to obtain capture antibody modified immune-modified magnetic beads; when Anti-CA19-9 capture antibody is used, Anti-CA19-9 capture antibody modified immune-modified magnetic beads are prepared; when Anti-CA125 capture antibody is used, Anti-CA125 capture antibody modified immune-modified magnetic beads are prepared; when Anti-LIF capture antibody is used, Anti-LIF capture antibody modified immune-modified magnetic beads are prepared;

[0101] The concentration of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride was 1:5; the concentration of N-hydroxysuccinimide solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-hydroxysuccinimide was 1:0.5; the mass concentration of the capture antibody was 1 mg / mL, and the volume ratio of carboxyl magnetic beads to the capture antibody was 1:1.

[0102] S3. Preparation of immunofluorescence modified detection antibody, same as Example 1.

[0103] S4. A biochip for simultaneous detection of multiple abundance markers, comprising a PDMS layer and a glass substrate layer, a detection channel formed between the PDMS layer and the glass substrate layer, and a chip inlet and a chip outlet connected to the detection channel; the detection channel comprises three parallel detection channels: a CA19-9 detection channel, a CA125 detection channel and a LIF detection channel; the CA19-9 detection channel is filled with the Anti-CA19-9 capture antibody-modified immune-modified magnetic beads prepared in step S2 of this embodiment and the immunofluorescence-modified Anti-CA19-9 detection antibody prepared in step S3 of this embodiment; the CA125 detection channel is filled with the Anti-CA125 capture antibody-modified immune-modified magnetic beads prepared in step S2 of this embodiment and the immunofluorescence-modified Anti-CA125 detection antibody prepared in step S3 of this embodiment; the surface of the glass substrate layer in the LIF detection channel facing the detection channel is modified with the Anti-LIF capture antibody, and the modification steps are the same as those in Example 1.

[0104] Embodiment 3:

[0105] S1. Preparation of modified carboxyl magnetic beads. Except that the ratio of ferrosoferric oxide nanoreagent and sodium pentafluoropropionic acid salt is changed to 1 mL:4 g, other conditions are the same as those in Example 2.

[0106] S2. Preparation of capture antibody-modified immune-modified magnetic beads, comprising:

[0107] Weigh the modified carboxyl magnetic beads prepared in step S1 of this embodiment, add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to activate for 30 minutes, then add capture antibody to react for 4 hours to obtain capture antibody modified immune-modified magnetic beads; when Anti-CA19-9 capture antibody is used, Anti-CA19-9 capture antibody modified immune-modified magnetic beads are prepared; when Anti-CA125 capture antibody is used, Anti-CA125 capture antibody modified immune-modified magnetic beads are prepared; when Anti-LIF capture antibody is used, Anti-LIF capture antibody modified immune-modified magnetic beads are prepared;

[0108] The concentration of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride was 1:5; the concentration of N-hydroxysuccinimide solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-hydroxysuccinimide was 1:0.5; the mass concentration of the capture antibody was 1 mg / mL, and the volume ratio of carboxyl magnetic beads to the capture antibody was 1:1.

[0109] S3. Preparation of immunofluorescence modified detection antibody, same as Example 1.

[0110] S4. A biochip for simultaneous detection of multiple abundance markers, same as in Example 2.

[0111] Embodiment 4:

[0112] S1. Preparation of modified carboxyl magnetic beads, comprising:

[0113] Weigh the ferroferric oxide nanoreagent, add anhydrous ethanol and deionized water, ultrasonically disperse for 30 minutes, slowly add ammonia water, stir at 500r / min for 10 minutes, add ethyl silicate dropwise, heat in a water bath at 30°C for 10 hours, add N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and ultrapure water at 4°C, mix and stir for 1 hour, add sodium pentafluoropropionic acid salt, ultrasonically disperse for 10 minutes, react at 95°C for 2 hours, cool to room temperature after the reaction, add 3-(vinylsulfonyl)propionamide and allylmalonic acid, stir at 300r / min for 15 minutes, add potassium persulfate and methylenebisacrylamide after nitrogen is introduced for 30 minutes, react in a water bath at 70°C for 8 hours, separate magnetically, wash alternately with ethanol and ultrapure water 4 times, add isopropanol to a final concentration of 10 mg / mL, and obtain modified carboxyl magnetic beads;

[0114] The ferroferric oxide nanoreagent includes ferroferric oxide nanoparticles and pure water, wherein the dosage ratio of the ferroferric oxide nanoparticles to the pure water is 1 mg:0.1 mL; the volume ratio of the ferroferric oxide nanoreagent to anhydrous ethanol is 1:10; the volume ratio of the ferroferric oxide nanoreagent to deionized water is 1:0.2; the volume ratio of the ferroferric oxide nanoreagent to ammonia water is 1:0.3; the volume ratio of the ferroferric oxide nanoreagent to ethyl silicate is 1:0.1; the dosage ratio of the ferroferric oxide nanoreagent to N-(2-aminoethyl)-3-aminopropyltrimethoxysilane is 1 mL:5 g; The dosage ratio of -(2-aminoethyl)-3-aminopropyltrimethoxysilane and ultrapure water is 1g:100mL; the dosage ratio of ferroferric oxide nanoreagent and sodium pentafluoropropionic acid salt is 1mL:8g; the dosage ratio of ferroferric oxide nanoreagent and 3-(vinylsulfonyl)propionamide is 1mL:0.75g; the volume ratio of ferroferric oxide nanoreagent and allylmalonic acid is 1:0.85; the dosage ratio of ferroferric oxide nanoreagent and potassium persulfate is 1mL:0.65g; the dosage ratio of ferroferric oxide nanoreagent and methylenebisacrylamide is 1mL:1.6g.

[0115] S2. Preparation of capture antibody-modified immune-modified magnetic beads, comprising:

[0116] Weigh the modified carboxyl magnetic beads prepared in step S1 of this embodiment, add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to activate for 30 minutes, then add capture antibody to react for 4 hours to obtain capture antibody modified immune-modified magnetic beads; when Anti-CA19-9 capture antibody is used, Anti-CA19-9 capture antibody modified immune-modified magnetic beads are prepared; when Anti-CA125 capture antibody is used, Anti-CA125 capture antibody modified immune-modified magnetic beads are prepared; when Anti-LIF capture antibody is used, Anti-LIF capture antibody modified immune-modified magnetic beads are prepared;

[0117] The concentration of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride was 1:5; the concentration of N-hydroxysuccinimide solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-hydroxysuccinimide was 1:0.5; the mass concentration of the capture antibody was 1 mg / mL, and the volume ratio of carboxyl magnetic beads to the capture antibody was 1:1.

[0118] S3. Preparation of immunofluorescence modified detection antibody, same as Example 1.

[0119] S4. A biochip for simultaneous detection of multiple abundance markers, comprising a PDMS layer and a glass substrate layer, a detection channel formed between the PDMS layer and the glass substrate layer, and a chip inlet and a chip outlet connected to the detection channel; the detection channel comprises three parallel detection channels: a CA19-9 detection channel, a CA125 detection channel and a LIF detection channel; the CA19-9 detection channel is filled with the Anti-CA19-9 capture antibody-modified immune-modified magnetic beads prepared in step S2 of this embodiment and the immunofluorescence-modified Anti-CA19-9 detection antibody prepared in step S3 of this embodiment; the CA125 detection channel is filled with the Anti-CA125 capture antibody-modified immune-modified magnetic beads prepared in step S2 of this embodiment and the immunofluorescence-modified Anti-CA125 detection antibody prepared in step S3 of this embodiment; the surface of the glass substrate layer in the LIF detection channel facing the detection channel is modified with the Anti-LIF capture antibody, and the modification steps are the same as those in Example 1.

[0120] Embodiment 5:

[0121] S1. Preparation of modified carboxyl magnetic beads, except that the volume ratio of ferrosoferric oxide nanoreagent and allylmalonic acid is changed to 1:0.4, other conditions are the same as those in Example 4.

[0122] S2. Preparation of capture antibody-modified immune-modified magnetic beads, comprising:

[0123] Weigh the modified carboxyl magnetic beads prepared in step S1 of this embodiment, add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to activate for 30 minutes, then add capture antibody to react for 4 hours to obtain capture antibody modified immune-modified magnetic beads; when Anti-CA19-9 capture antibody is used, Anti-CA19-9 capture antibody modified immune-modified magnetic beads are prepared; when Anti-CA125 capture antibody is used, Anti-CA125 capture antibody modified immune-modified magnetic beads are prepared; when Anti-LIF capture antibody is used, Anti-LIF capture antibody modified immune-modified magnetic beads are prepared;

[0124] The concentration of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride was 1:5; the concentration of N-hydroxysuccinimide solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-hydroxysuccinimide was 1:0.5; the mass concentration of the capture antibody was 1 mg / mL, and the volume ratio of carboxyl magnetic beads to the capture antibody was 1:1.

[0125] S3. Preparation of immunofluorescence modified detection antibody, same as Example 1.

[0126] S4. A biochip for simultaneous detection of multiple abundance markers, comprising a PDMS layer and a glass substrate layer, a detection channel formed between the PDMS layer and the glass substrate layer, and a chip inlet and a chip outlet connected to the detection channel; the detection channel comprises three parallel detection channels: a CA19-9 detection channel, a CA125 detection channel and a LIF detection channel; the CA19-9 detection channel is filled with the Anti-CA19-9 capture antibody-modified immune-modified magnetic beads prepared in step S2 of this embodiment and the immunofluorescence-modified Anti-CA19-9 detection antibody prepared in step S3 of this embodiment; the CA125 detection channel is filled with the Anti-CA125 capture antibody-modified immune-modified magnetic beads prepared in step S2 of this embodiment and the immunofluorescence-modified Anti-CA125 detection antibody prepared in step S3 of this embodiment; the surface of the glass substrate layer in the LIF detection channel facing the detection channel is modified with the Anti-LIF capture antibody, and the modification steps are the same as those in Example 1.

[0127] Comparative Example 1:

[0128] S1. Preparation of modified carboxyl magnetic beads, comprising:

[0129] Weigh the ferroferric oxide nanoreagent, add anhydrous ethanol and deionized water, ultrasonically disperse for 30 minutes, slowly add ammonia water, stir at a rate of 500 r / min for 10 minutes, add ethyl silicate dropwise, heat in a water bath at 30°C for 10 hours, then add SDS sodium dodecyl sulfate and sodium bicarbonate, ultrasonically disperse for 30 minutes, stir at a rate of 300 r / min for 10 minutes in a water bath at 70°C, add styrene, acrylic acid and potassium persulfate solution to react for 12 hours, add 3-(vinylsulfonyl) propionamide and allylmalonic acid, stir at a rate of 300 r / min for 15 minutes, add potassium persulfate and methylenebisacrylamide after nitrogen is introduced for 30 minutes, react in a water bath at 70°C for 8 hours, separate magnetically, wash alternately with ethanol and ultrapure water 4 times, add isopropanol to a final concentration of 10 mg / mL, and obtain modified carboxyl magnetic beads;

[0130] The ferroferric oxide nanoreagent includes ferroferric oxide nanoparticles and pure water, the dosage ratio of ferroferric oxide nanoparticles to pure water is 1 mg:0.1 mL; the volume ratio of ferroferric oxide nanoreagent to anhydrous ethanol is 1:10; the volume ratio of ferroferric oxide nanoreagent to deionized water is 1:0.2; the volume ratio of ferroferric oxide nanoreagent to ammonia water is 1:0.3; the volume ratio of ferroferric oxide nanoreagent to ethyl silicate is 1:0.1; the dosage ratio of ferroferric oxide nanoreagent to SDS sodium dodecyl sulfate is 1 mL:0.016 g; the mass ratio of SDS sodium dodecyl sulfate to sodium bicarbonate is 1:0.1; ferroferric oxide The volume ratio of the nanoreagent and styrene is 1:0.4; the volume ratio of the ferroferric oxide nanoreagent and acrylic acid is 1:0.02; the concentration of the potassium persulfate solution is 0.03g / mL, and the volume ratio of the ferroferric oxide nanoreagent and potassium persulfate solution is 1:0.1; the dosage ratio of the ferroferric oxide nanoreagent and 3-(vinylsulfonyl)propionamide is 1mL:0.75g; the volume ratio of the ferroferric oxide nanoreagent and allylmalonic acid is 1:0.85; the dosage ratio of the ferroferric oxide nanoreagent and potassium persulfate is 1mL:0.65g; the dosage ratio of the ferroferric oxide nanoreagent and methylenebisacrylamide is 1mL:1.6g.

[0131] S2. Preparation of capture antibody-modified immune-modified magnetic beads, comprising:

[0132] Weigh the modified carboxyl magnetic beads prepared in step S1 of this embodiment, add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution to activate for 30 minutes, then add capture antibody to react for 4 hours to obtain capture antibody modified immune-modified magnetic beads; when Anti-CA19-9 capture antibody is used, Anti-CA19-9 capture antibody modified immune-modified magnetic beads are prepared; when Anti-CA125 capture antibody is used, Anti-CA125 capture antibody modified immune-modified magnetic beads are prepared; when Anti-LIF capture antibody is used, Anti-LIF capture antibody modified immune-modified magnetic beads are prepared;

[0133] The concentration of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride was 1:5; the concentration of N-hydroxysuccinimide solution was 10 mg / mL, and the volume ratio of carboxyl magnetic beads to N-hydroxysuccinimide was 1:0.5; the mass concentration of the capture antibody was 1 mg / mL, and the volume ratio of carboxyl magnetic beads to the capture antibody was 1:1.

[0134] S3. Preparation of immunofluorescence modified detection antibody, same as Example 1.

[0135] S4. A biochip for simultaneous detection of multiple abundance markers, comprising a PDMS layer and a glass substrate layer, a detection channel formed between the PDMS layer and the glass substrate layer, and a chip inlet and a chip outlet connected to the detection channel; the detection channel comprises three parallel detection channels: a CA19-9 detection channel, a CA125 detection channel and a LIF detection channel; the CA19-9 detection channel is filled with the Anti-CA19-9 capture antibody-modified immune-modified magnetic beads prepared in step S2 of this embodiment and the immunofluorescence-modified Anti-CA19-9 detection antibody prepared in step S3 of this embodiment; the CA125 detection channel is filled with the Anti-CA125 capture antibody-modified immune-modified magnetic beads prepared in step S2 of this embodiment and the immunofluorescence-modified Anti-CA125 detection antibody prepared in step S3 of this embodiment; the surface of the glass substrate layer in the LIF detection channel facing the detection channel is modified with the Anti-LIF capture antibody, and the modification steps are the same as those in Example 1.

[0136] Experimental example:

[0137] 1. Detection of markers

[0138] Serum containing leukemia inhibitory factor (LIF), cancer antigen 19-9 (CA19-9) and cancer antigen 125 (CA125) is added to the chip inlet of the biochip obtained in Example 1 of the present invention, the serum flows through the biochip and is incubated, fluorescent magnetic beads capturing CA19-9 markers are separated in the CA19-9 detection channel, fluorescent magnetic beads capturing CA125 markers are separated in the CA125 detection channel, and a glass substrate capturing LIF markers is separated in the LIF detection channel, and the fluorescent signals of the fluorescent magnetic beads capturing CA19-9 markers, the fluorescent magnetic beads capturing CA125 markers and the glass substrate capturing LIF markers are detected.

[0139] Figure 1 This is a structural diagram of a biochip obtained in Example 1 of the present invention; Figure 2 This is a fluorescence signal diagram of the captured LIF marker obtained in Example 1 of the present invention, where the red fluorescence is the captured LIF marker; Figure 3 This is a fluorescence signal diagram of the biochip capturing the CA19-9 marker obtained in Example 1 of the present invention, and the red fluorescence is the captured CA19-9 marker; Figure 4 This is a fluorescence signal diagram of the biochip of Example 1 of the present invention capturing the CA125 marker, and the red fluorescence is the captured CA125 marker. This shows that the biochip of Example 1 of the present invention can simultaneously capture the markers LIF, CA19-9 and CA125 in serum.

[0140] 2. Standard curve of the marker to be tested

[0141] Serum containing different concentrations of leukemia inhibitory factor (LIF), cancer antigen 19-9 (CA19-9) and cancer antigen 125 (CA125) was added to the chip inlet of the biochip obtained in Example 1 of the present invention, the serum flowed through the biochip and was incubated, fluorescent magnetic beads capturing CA19-9 markers were separated in the CA19-9 detection channel, fluorescent magnetic beads capturing CA125 markers were separated in the CA125 detection channel, and a glass substrate capturing LIF markers was separated in the LIF detection channel, and a standard curve was drawn according to the fluorescent signals of the captured CA19-9, CA125 and LIF markers.

[0142] Figure 5 The standard curve of the biochip capturing the LIF marker obtained in Example 1 of the present invention, the linear regression equation is y=0.273x+201.19, x is the detection concentration (pg / mL), y is the number of fluorescence events, and the linear correlation coefficient is 0.995; Figure 6 The standard curve of the biochip capturing the CA19-9 marker obtained in Example 1 of the present invention, the linear regression equation is y=0.26x+52.38, x is the detection concentration (U / mL), y is the fluorescence intensity (au), and the linear correlation coefficient is 0.998; Figure 7 The standard curve of the biochip capturing CA125 marker obtained in Example 1 of the present invention has a linear regression equation of y=0.41x+50.96, where x is the detection concentration (U / mL), y is the fluorescence intensity (au), and the linear correlation coefficient is 0.995.

[0143] 3. Capture and determination of CA19-9 marker concentration

[0144] Serum containing cancer antigen 19-9 (CA19-9) was added to the chip inlet of the biochip obtained in Examples 1-5 of the present invention and Comparative Example 1, the serum flowed through the biochip and incubated, the fluorescent magnetic beads capturing the CA19-9 marker were separated, the fluorescence signal intensity of the fluorescent magnetic beads capturing the CA19-9 marker was detected, and the capture concentration (U / mL) of CA19-9 was calculated according to the standard curve equation for capturing the CA19-9 marker. The measurement results are shown in Table 1.

[0145] Table 1 Capture concentration of CA19-9 marker (U / mL)

[0146]

[0147] As shown in Table 1, the capture concentration of CA19-9 marker obtained in Example 2-3 of the present invention is higher than that in Example 1. This is because in Example 2-3, ethyl silicate is first used to coat the surface of the ferroferric oxide nanoreagent with a silica layer, and then N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and sodium pentafluoropropionic acid are used to functionalize the surface carboxyl groups to prepare modified carboxyl magnetic beads, and the modified carboxyl magnetic beads are used to capture the CA19-9 marker; while in Example 1, ethyl silicate is first used to coat the surface of the ferroferric oxide nanoreagent with a silica layer, and then SDS sodium dodecyl sulfate and sodium bicarbonate are used to functionalize the surface carboxyl groups to prepare modified carboxyl magnetic beads. Carboxyl functional modification is performed to prepare carboxyl magnetic beads, and the carboxyl magnetic beads are used to capture the CA19-9 marker; the capture concentration of the CA19-9 marker obtained in Example 2 of the present invention is higher than that in Example 3, because the amount of sodium pentafluoropropionic acid salt used in the preparation of the modified carboxyl magnetic beads is different; this shows that Example 2-3 of the present invention uses N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and sodium pentafluoropropionic acid salt to perform carboxyl functional modification on the surface of magnetic beads, which can improve the capture ability of the capture antibody-modified immunomagnetic beads for the CA19-9 marker, thereby increasing the capture concentration of the CA19-9 marker.

[0148] The capture concentration of CA19-9 marker obtained in Example 4-5 of the present invention is higher than that in Example 2 because Example 4-5 further uses 3-(vinylsulfonyl) propionamide and allyl malonic acid to modify the modified carboxyl magnetic beads prepared in Example 2, and the modified carboxyl magnetic beads are used to capture the CA19-9 marker; the capture concentration of CA19-9 marker obtained in Example 4-5 is higher than that in Comparative Example 1 because in the preparation of the modified carboxyl magnetic beads, Comparative Example 1 does not use N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and sodium pentafluoropropionic acid for modification; the capture concentration of CA19-9 marker obtained in Example 4 is higher than that in Example 5 because the amount of allyl malonic acid used in the preparation of the capture antibody modified immunomagnetic beads is different; this shows that the present invention uses 3-(vinylsulfonyl) propionamide and allyl malonic acid to further modify the carboxyl magnetic beads, which can further improve the capture ability of the capture antibody modified immunomagnetic beads for CA19-9 markers, thereby improving the capture concentration of CA19-9 markers.

[0149] 4. Capture and determination of CA19-9 marker concentration

[0150] Serum containing cancer antigen 125 (CA125) was added to the chip inlet of the biochip obtained in Examples 1-5 of the present invention and Comparative Example 1, the serum flowed through the biochip and was incubated, the fluorescent magnetic beads capturing the CA125 marker were separated, the fluorescence signal intensity of the fluorescent magnetic beads capturing the CA125 marker was detected, and the capture concentration (U / mL) of CA125 was calculated according to the standard curve equation for capturing the CA125 marker. The measurement results are shown in Table 2.

[0151] Table 2 Concentration of captured CA125 marker (U / mL)

[0152]

[0153] As can be seen from Table 2, the capture concentration of CA125 marker obtained in Example 2-3 of the present invention is higher than that in Example 1. This is because in Example 2-3, ethyl silicate is first used to coat the surface of the ferroferric oxide nanoreagent with a silica layer, and then N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and sodium pentafluoropropionic acid are used to functionalize the surface carboxyl groups to prepare modified carboxyl magnetic beads, and the modified carboxyl magnetic beads are used to capture the CA125 marker; while in Example 1, ethyl silicate is first used to coat the surface of the ferroferric oxide nanoreagent with a silica layer, and then SDS sodium dodecyl sulfate and sodium bicarbonate are used to functionalize the surface carboxyl groups. Carboxyl functional modification is performed to prepare carboxyl magnetic beads, and the carboxyl magnetic beads are used to capture the CA125 marker; the capture concentration of the CA125 marker obtained in Example 2 of the present invention is higher than that in Example 3, because the amount of sodium pentafluoropropionic acid salt used in the preparation of the modified carboxyl magnetic beads is different; this shows that Example 2-3 of the present invention uses N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and sodium pentafluoropropionic acid salt to perform carboxyl functional modification on the surface of magnetic beads, which can improve the capture ability of the capture antibody-modified immunomagnetic beads for the CA125 marker, thereby increasing the capture concentration of the CA125 marker.

[0154] The capture concentration of CA125 marker obtained in Examples 4-5 of the present invention is higher than that in Example 2 because, on the basis of the modified carboxyl magnetic beads prepared in Example 2, Examples 4-5 further use 3-(vinylsulfonyl) propionamide and allylmalonic acid for modification, and the modified carboxyl magnetic beads are used to capture the CA125 marker; the capture concentration of CA125 marker obtained in Examples 4-5 is higher than that in Comparative Example 1 because, in the preparation of the modified carboxyl magnetic beads, Comparative Example 1 does not use N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and sodium pentafluoropropionic acid for modification; the capture concentration of CA125 marker obtained in Example 4 is higher than that in Example 5 because the amount of allylmalonic acid used in the preparation of the capture antibody modified immunomagnetic beads is different; this shows that the present invention uses 3-(vinylsulfonyl) propionamide and allylmalonic acid to further modify the carboxyl magnetic beads, which can further improve the capture ability of the capture antibody modified immunomagnetic beads for CA125 markers, thereby improving the capture concentration of CA125 markers.

[0155] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0156] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A biochip for simultaneous detection of multiple abundance markers, comprising a PDMS layer and a glass substrate layer, a detection channel formed between the PDMS layer and the glass substrate layer, and a chip inlet and a chip outlet connected to the detection channel; the detection channel comprises three parallel branch channels: a CA19-9 detection channel, a CA125 detection channel and a LIF detection channel; the CA19-9 detection channel and the CA125 detection channel are both filled with capture antibody modified immunomagnetic beads and immunofluorescence modified detection antibodies; the LIF detection channel is filled with immunofluorescence modified detection antibodies; Fluorescence modified detection antibody, the surface of the glass substrate layer in the LIF detection channel is modified with Anti-LIF capture antibody; the capture antibody modified immunomagnetic beads are prepared by connecting magnetic beads and capture antibodies, and the magnetic beads include carboxyl magnetic beads or modified carboxyl magnetic beads; in the preparation of the modified carboxyl magnetic beads, ferrosilicate nanoreagents are first reacted with ethyl silicate, and then reacted with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and sodium pentafluoropropionic acid to obtain modified carboxyl magnetic beads; the volume ratio of the modified carboxyl magnetic beads to the capture antibody is 1:1-2.

2. A biochip for simultaneous detection of multiple abundance markers according to claim 1, characterized in that: The diameter of the chip inlet is 0.5-2 mm, and the diameter of the chip outlet is 0.5-2 mm.

3. A biochip for simultaneous detection of multiple abundance markers according to claim 1, characterized in that: The mass concentration of the capture antibody is 0.1-10 mg / mL, and the volume ratio of the magnetic beads to the capture antibody is 1:1-2.

4. A biochip for simultaneous detection of multiple abundance markers according to claim 1, characterized in that: The volume ratio of the capture antibody modified immunomagnetic beads to the immunofluorescence modified detection antibody is 1:1-2.

5. The biochip for simultaneous detection of multiple abundance markers according to claim 1, characterized in that: In the preparation of the immunofluorescence modified detection antibody, the detection antibody is mixed with Atto 565 NHS ester fluorescent dye to react and obtain the immunofluorescence modified detection antibody.

6. The biochip for simultaneous detection of multiple abundant markers according to claim 5, characterized in that: The concentration of the detection antibody is 0.1-100 μg / mL, and the molar ratio of the detection antibody to the Atto 565 NHS ester fluorescent dye is 1:5-100.

7. A biochip for simultaneous detection of multiple abundance markers according to claim 1, characterized in that: In the method for modifying the surface of the glass substrate layer in the LIF detection channel with the Anti-LIF capture antibody, 3-aminopropyltriethoxysilane is first added to the LIF detection channel for amino treatment, and then streptavidin is used to connect the glass with the biotinylated capture antibody.

8. A biochip for simultaneous detection of multiple abundance markers according to claim 7, characterized in that: The biotinylated capture antibody is obtained by a mixed reaction of NHS-Biotin and Anti-LIF capture antibody, and the molar ratio of NHS-Biotin to Anti-LIF capture antibody is 1:5-100.

9. Use of the biochip according to claim 1 in preparing a detection kit for CA19-9, CA125 and LIF markers.

Images