WO2023236519A1 - 一种光子晶体生物芯片及其蛋白检测方法 - Google Patents

一种光子晶体生物芯片及其蛋白检测方法 Download PDF

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WO2023236519A1
WO2023236519A1 PCT/CN2022/142780 CN2022142780W WO2023236519A1 WO 2023236519 A1 WO2023236519 A1 WO 2023236519A1 CN 2022142780 W CN2022142780 W CN 2022142780W WO 2023236519 A1 WO2023236519 A1 WO 2023236519A1
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photonic crystal
photocrystal
detection
antibody
biochip
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PCT/CN2022/142780
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English (en)
French (fr)
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杜杰
王媛
王雪
薛冰洁
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北京市心肺血管疾病研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention belongs to the field of testing or analyzing materials by means of chemical or physical properties of the test materials, and specifically relates to a photonic crystal biochip and a protein detection method thereof.
  • ST2L human growth stimulating expressed gene 2
  • ST2L is The functional receptor of IL-33, the IL-33/ST2L signaling pathway, has been shown to have a cardioprotective effect, and the binding of sST2 to IL-33 will lead to the downregulation of the IL-33/ST2L signaling pathway.
  • sST2 is mainly produced by vascular endothelial cells at all levels under stress or injury. Its concentration has been confirmed to be increased in patients with acute myocardial infarction and acute heart failure, and significantly increased in patients with aortic dissection.
  • the sensitivity of the detection method can be effectively improved by expanding and enhancing the detectable signal.
  • photonic crystals are widely used. Based on the photonic crystal fluorescence enhancement effect, the photonic crystal fluorescence sensor introduces the photonic crystal structure into the traditional fluorescence detection system to enhance the fluorescence signal intensity, improve detection sensitivity, and reduce detection limits. Photonic crystals have a periodic structure. When fluorescent molecules enter the local photon position, the interaction between the electromagnetic field excitation light and the fluorescent molecules is enhanced, and the fluorescence signal of spontaneous emission is enhanced, making it easy to detect.
  • Patent CN102161284A discloses a method for preparing responsive and patterned colloidal photonic crystal films using inkjet printing technology.
  • Patent 200510011219.2 discloses a method for preparing polymethyl methacrylate-acrylic acid-styrene latex balls.
  • Patent CN113912729A discloses a method for preparing Nanobodies.
  • the technical problem to be solved by the present invention is how to obtain a photonic crystal biochip that can detect proteins quickly, easily and/or sensitively.
  • the invention provides a photonic crystal biochip, which includes a reaction base layer. At least one stable capture area and at least one unstable detection area are provided on the reaction base layer.
  • the stable capture area includes a photonic crystal array and at least one capture area.
  • Antibody; the unstable detection area includes a photonic crystal array and at least one detection antibody, and the detection antibody is labeled with a fluorescent group.
  • the fluorescent group is selected from at least one of FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED460, LC RED705, Quasar705 or Texas Red.
  • the capture antibody is an antibody that can capture the protein to be tested
  • the detection antibody is an antibody that can bind to the protein to be tested and is labeled with a fluorescent group
  • the fluorescent signal can be a Cy5 group.
  • the reaction base layer is a polymer sheet or polymer film.
  • the reactive base layer is made of polyester, polystyrene, polymethacrylic acid, polypropylene or polyvinyl chloride.
  • the photonic crystal is a polymethyl methacrylate-acrylic acid-styrene latex ball.
  • the particle size (diameter) of the polymethylmethacrylate-acrylic-styrene latex sphere is 300 nm.
  • the chip is a photonic crystal biochip for detecting soluble ST2 protein
  • the capture antibody is a soluble ST2 protein capture antibody
  • the detection antibody is a soluble ST2 protein detection antibody.
  • the soluble ST2 protein capture antibody was named capture antibody NbA, and the capture antibody NbA was the antibody numbered 1A9 in patent CN113912729A.
  • the soluble ST2 protein detection antibody is named detection antibody Cy5-NbB, and the detection antibody Cy5-NbB is the antibody numbered 2B10 in the patent CN113912729A with a Cy5 group connected.
  • the invention also provides a method for detecting the protein to be tested.
  • the sample to be tested is dropped on the biochip and fluorescence detection is performed. If the detection result shows a fluorescent color, it indicates that the protein to be tested is contained. If the detection result does not show a fluorescent color, it indicates that it does not contain the protein to be tested. Protein to be tested.
  • the protein to be tested may be a soluble ST2 protein, in which case the corresponding capture antibody is a soluble ST2 protein capture antibody; and the detection antibody is a soluble ST2 protein detection antibody.
  • the soluble ST2 protein capture antibody is named capture antibody NbA, and the capture antibody NbA can be a single domain antibody 1A9 with the amino acid sequence shown in Sequence 1.
  • the soluble ST2 protein detection antibody is named detection antibody Cy5-NbB.
  • the detection antibody Cy5-NbB can be a single domain antibody 2B10 connected with a Cy5 group.
  • the amino acid sequence of the single domain antibody 2B10 is shown in Sequence 2. .
  • the invention also provides a method for preparing a photonic crystal biochip, which includes the following steps:
  • step 2 Print a photocrystal array on the substrate in step 1 using a liquid containing photocrystal beads as ink to obtain a substrate with a photocrystal array;
  • the capture antibody is an antibody that can capture the protein to be tested
  • the detection antibody is an antibody that can bind to the protein to be tested and carries a fluorescent signal.
  • the fluorescent signal can be a Cy5 group.
  • the liquid containing photocrystal beads is prepared from 919 parts by volume of poly(methyl methacrylate-acrylic acid-styrene) latex balls, 80 parts by volume of ethylene glycol and 1 part by volume of surfactant;
  • the particle size of polymethylmethacrylate-acrylic-styrene latex balls is 300 nm; the heating and curing conditions are 110°C, 10 minutes.
  • the capture antibody is an ST2 protein capture antibody
  • the detection antibody is an ST2 protein detection antibody
  • the soluble ST2 protein capture antibody is named capture antibody NbA, and the capture antibody NbA can be a single domain antibody 1A9 with the amino acid sequence shown in Sequence 1.
  • the soluble ST2 protein detection antibody is named detection antibody Cy5-NbB.
  • the detection antibody Cy5-NbB can be a single domain antibody 2B10 connected with a Cy5 group.
  • the amino acid sequence of the single domain antibody 2B10 is shown in Sequence 2. .
  • the photonic crystal biochip of the present invention can realize fast, simple operation and trace detection of protein detection biochip by introducing photonic crystal.
  • the present invention can quickly, easily operate and detect soluble ST2 protein in trace amounts. .
  • Figure 1 shows the basic process of establishing a detection method for soluble ST2 protein on a photonic crystal biochip based on green printing.
  • Figure 2 is a microscope and scanning electron microscope picture of several photonic crystal chips prepared in Example 1, where a is a picture of the fluorescence enhancement effect of photonic crystal beads of different particle sizes, and b is a scanning electron microscope of photonic crystal beads of different particle sizes. picture.
  • Figure 4 shows the detection of 15 clinical samples using the photonic crystal detection chip of the present invention, and the overall linear trend is compared with the ELISA detection.
  • Figure 5 is a comparison chart of the fluorescence intensity of Cy5-NbB on photonic crystal arrays of different particle sizes in Example 5.
  • Figure 6 is a performance comparison chart of chips prepared using IgG antibodies, Nanobodies of the present invention and PC300 photonic crystal arrays in Example 6 respectively.
  • the green printing method in the following examples refers to the method of using inkjet printing technology to prepare responsive and patterned colloidal photonic crystal films in patent CN102161284A.
  • the capture antibody NbA in the following examples is the single domain antibody 1A9 shown in Sequence 1
  • the detection antibody Cy5-NbB is the single domain antibody 2B10 connected with the Cy5 group.
  • the amino acid sequence of the single domain antibody 2B10 is as shown in Sequence 2.
  • the public can prepare according to the method in patent CN113912729A, or entrust Nanjing Rongjiekang Biotechnology Co., Ltd. to prepare and obtain it.
  • the 1A9 sequence is:
  • the 2B10 sequence is:
  • the PBS in the following examples is Zhongshan Jinqiao ZLI-9061
  • the FBS in the following examples is gibco0025
  • the healthy human EDTA anticoagulant serum in the following examples is human serum from the Health Examination Center of Beijing Anzhen Hospital
  • the recombinant protein sST2 in the following examples is Biopsis' il-H5229.
  • the polymethylmethacrylate-acrylic-styrene latex balls in the following examples are prepared by using the method in patent 200510011219.2 at a temperature of 50°C.
  • the particle sizes of the prepared polymethyl methacrylate-acrylic acid-styrene latex spheres are 280, 300, 310, 320, 360, and 370 nm respectively.
  • the photocrystal array is prepared as shown in steps a-c in Figure 1, specifically as follows:
  • a layer of adhesive can be polyvinyl alcohol water-based adhesive
  • a layer of adhesive can be polyvinyl alcohol water-based adhesive
  • the liquid containing photocrystal beads used for printing is polymethylmethacrylate-acrylic-styrene latex beads, ethylene glycol, and surfactant (the surfactant is silicone surfactant with German BYK product number BYK-3455). agent), the volume ratio of the mixed solution is 919:80:1. Adjust the drying speed of the photocrystal ink after printing to ensure that the photocrystal beads are assembled neatly.
  • the preparation method of the polymethylmethacrylate-acrylic-styrene latex balls is as follows: at a temperature of 50°C, the polymethyl methacrylate-acrylic acid-styrene latex balls containing particles with particle sizes of 300, 310, 320, Emulsion of monodisperse poly(styrene-methyl methacrylate-acrylic acid) triblock copolymer latex particles at 360 and 370 nm.
  • poly(methyl methacrylate-acrylic acid-styrene) latex balls with particle sizes (diameter) of 300, 310, 320, 360, and 370nm were used to prepare photocrystal array-300 and photocrystal array -310, optical crystal array-320, optical crystal array-360 and optical crystal array-370 respectively correspond to PC 300 , PC 310 , PC 320 , PC 360 and PC 370 in Figure 2.
  • a Nordson EFD dispensing machine is used to prepare a photocrystal chip that can capture soluble ST2 protein using a green printing method.
  • a 5*5 lattice with a diameter of about 240 ⁇ m and a spacing of 0.5mm is printed.
  • the spacing of each 5*5 lattice is 7mm, the specific method is shown in Figure 1:
  • the fluorescently labeled detection antibody Cy5-NbB is non-covalently fixed on the chip surface through inkjet printing and arranged on the periphery of the photocrystal array.
  • the dried (room temperature conditions, 25°C) antibody array is stored in a dry condition. Still remain active. Obtain photonic crystal detection chip.
  • PBS, FBS and EDTA anticoagulant serum of healthy people were used as solvents to prepare gradient concentration sST2 solutions as the samples to be detected.
  • the gradient concentrations of the sST2 solutions as the samples to be detected were 0.01, 0.1, 1, 10, 100 and 1000ng/mL respectively.
  • the performance of the above samples was tested using the fluorescence-coupled immunosorbent assay (referred to as PC-FLISA) incorporating photocrystals in Example 2 and the traditional enzyme-linked immunosorbent assay FLISA, respectively.
  • the results are shown in a in Figure 3 .
  • the traditional enzyme-linked immunosorbent assay FLISA can be performed by referring to the steps described in the literature Performance of coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay (FLISA) to detect malaria antigen. It can be seen from a in Figure 3 that the fluorescence intensity detected using the PC-FLISA method of the present invention is significantly higher than the fluorescence intensity detected using the traditional FLISA at the same concentration.
  • the detection results were photographed with laser confocal, and statistical mapping of fluorescence intensity was performed to determine the linear range and detection limit.
  • the results are shown in b, c, and d in Figure 3. According to the test results, the corresponding detectable antigen concentration reaches 0.01ng/ml, and the enhancement effect is stable.
  • the blood of 15 clinical patients with cardiovascular-related diseases was used as a sample and PBS was diluted ten times to obtain a sample solution to be tested.
  • the sample solution to be tested was tested using the photonic crystal detection chip of the present invention and the traditional ELISA for additional testing.
  • the results are shown in Figure 4 shown.
  • traditional ELISA detection methods please refer to the literature Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Lequin RM. Clin Chem.2005 Dec; 51(12):2415-8.doi:10.1373/clinchem.2005.051532.Epub Proceed as described in 2005 Sep 22.
  • poly(methyl methacrylate-acrylic acid-styrene) latex balls with particle sizes (diameter) of 280, 300, 320, and 360 nm were respectively used to prepare photocrystal array-280 (PC 280 ), Photocrystal Array-300 (PC 300 ), Photocrystal Array-320 (PC 320 ) and Photocrystal Array-360 (PC 360 ). Comparing the fluorescence intensity of Cy5-NbB (0.5 ⁇ L, 450ng/mL) on the above four photocrystal microdot arrays, the results are shown in Figure 5.
  • PC300 The highest emission enhancement (60.3 times) was obtained for PC300 because the blue band edge matches the emission wavelength, which can significantly enhance the spontaneous emission of the dye located in the voids of the PC matrix.
  • the blue band edge of PC280 matches the excitation wavelength, which can improve the excitation efficiency, but its forbidden band overlaps with Cy5-NbB emission, thus suppressing the emission of the dye located within the PC matrix. So PC280 only achieves 39.8 times enhancement.
  • the forbidden bands of PC320 and PC360 are far away from the excitation and emission wavelengths of Cy5-NbB, and they enhance the emission by about 10.8 and 1.5 times due to their large surface area and scattering effect. Based on the above results, PC300 can be used to enhance the fluorescence signal of Cy5-NbB.
  • Cy5 antibodies (IgG) and Cy5 nanobodies were used respectively according to the method in Example 2 using PC 300 or a blank substrate as the substrate to prepare a photocrystal chip and perform performance testing. The results are shown in Figure 6.
  • a is the optical bright field image and fluorescence image of Cy5 antibody (IgG) (0.5 ⁇ L, 10 ⁇ g/mL) and Cy5 nanobody (0.5 ⁇ L, 10 ⁇ g/mL) on the photocrystal array PC300 and the blank substrate respectively, where: Ab represents The photocrystal chip prepared by Cy5 antibody (IgG) and blank substrate, Nb represents the photocrystal chip prepared by Cy5 nanobody and blank substrate, Ab+PC 300 indicates the photocrystal chip prepared by Cy5 antibody (IgG) and PC 300 substrate, Nb+ PC 300 represents the photocrystal chip prepared by Cy5 nanobody and PC 300 substrate;
  • Cy5 antibody (IgG) and Cy5 Nanobody on PC 300 array and blank substrate are the corresponding intensity analysis using Cy5 antibody (IgG) and Cy5 Nanobody on PC 300 array and blank substrate. It can be seen that the fluorescence intensity of Cy5 Nanobody is much enhanced when using PC 300 array than when using Cy5 antibody.
  • (IgG) fluorescence enhancement where Ab indicates that the antibody used is Cy5 antibody (IgG), Nb indicates that the antibody used is Cy5 nanobody, Without PC 300 indicates that the substrate used is a blank substrate, and With PC 300 indicates that the substrate used is PC 300 base plate;
  • i is the colloidal sphere assembled in PC 300 dots
  • ii is the IgG antibody (Ab) immobilized on the PC 300 surface
  • iii is the scanning electron microscope image of the nanobody (Nb) immobilized on the PC 300 surface. From Figure It can be observed that the IgG antibody affects the regular arrangement and assembly of PC300 photocrystal beads, making the surface uneven.
  • PC 300 represents the PC 300 photocrystal array
  • Ab+PC 300 represents the photocrystal prepared by Cy5 antibody (IgG) and PC 300 substrate.
  • Chip, Nb+PC 300 represents the photocrystal chip prepared by Cy5 nanobody and PC 300 substrate;
  • PC 300 represents PC 300 photocrystal array
  • Ab+PC 300 represents the photocrystal chip prepared by Cy5 antibody (IgG) and PC 300 substrate
  • Nb+PC 300 represents the photocrystal chip prepared by Cy5 nanobody and PC 300 substrate;
  • e is the electric field distribution of simulated nanobodies and IgG antibodies on the photocrystal array, where Ab represents the binding of Cy5 antibodies (IgG) to the PC 300 substrate, and Nb represents the binding of Cy5 nanobodies to the PC 300 substrate;
  • f is the power outflow density distribution of IgG antibodies and Ab in the simulated photocrystal array, where Ab represents the binding of Cy5 antibody (IgG) to the PC 300 substrate, and Nb represents the binding of Cy5 nanobodies to the PC 300 substrate.
  • the photonic crystal biochip of the present invention can realize fast, simple operation and trace detection of protein detection biochip by introducing photonic crystal.
  • the present invention can quickly, easily operate and detect soluble ST2 protein in trace amounts. .
  • the photocrystal array uses poly(methyl methacrylate-acrylic acid-styrene) latex balls with a particle size of 300 nm and Cy5 groups as fluorescent signals, so that the photocrystal array and the fluorescent signal have the best match.
  • the effect can expand the collected signal 100 times, which can greatly improve the accuracy and sensitivity of detection.
  • antibodies such as IgM or IgG have large structures, and photocrystal arrays are generally nanoscale arrays, when larger antibodies are connected to nanoscale photocrystal arrays, on the one hand, the operation is more complicated, and on the other hand, the operation is more complex.
  • Antibodies with large particles also have a greater impact on the smoothness of the surface of the photocrystal array, weakening the amplification effect of the original photocrystal array on the fluorescence signal.
  • the present invention uses single domain antibodies with smaller structures (generally nano antibodies), After being combined with the photocrystal array, it has less impact on the smoothness of the surface of the photocrystal array, which is beneficial to amplifying the fluorescence signal, thereby improving the sensitivity and accuracy of detection. At the same time, by using smaller nanoscale single-domain antibodies, the number of antibodies per unit area will be greatly increased, thereby improving detection efficiency.

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Abstract

一种光子晶体生物芯片、使用光子晶体生物芯片检测待测蛋白的方法及制备光子晶体生物芯片的方法，其中，光子晶体生物芯片包括反应基底层，反应基底层上设有至少一个稳定捕获区和至少一个不稳定检测区；稳定捕获区包含光子晶体阵列及至少一种捕获抗体；不稳定检测区包含光子晶体阵列及至少一种检测抗体。

Description

一种光子晶体生物芯片及其蛋白检测方法 技术领域
本发明属于借助于测试材料的化学或物理性质来测试或分析材料领域，具体涉及一种光子晶体生物芯片及其蛋白检测方法。
背景技术
ST2即人生长刺激表达基因2，是IL-1受体家族一员，主要有跨模型ST2L(membrane-bound receptor form,ST2 receptor)和可溶型sST2(soluble ST2)两种亚型，ST2L是IL-33的功能性受体，IL-33/ST2L信号通路已被证明具有心脏保护作用，而sST2与IL-33结合将导致IL-33/ST2L信号通路被下调。sST2主要由各级血管内皮细胞在应激或损伤下产生，其浓度已被证实在急性心肌梗塞和急性心力衰竭患者中升高，在主动脉夹层患者中明显升高且具有较高特异性与敏感性。人体循环sST2的定量测定，可用于心血管疾病患者辅助鉴别诊断及预后评估。现阶段临床仍未广泛开展sST2相关的检测项目，作为新兴生物标志物目前临床仍缺乏普遍适用的sST2检测方法。
在涉及信号转换的检测方法中，通过将可检测到的信号进行扩大增强，可以有效提升检测方法的灵敏度。在荧光传感器检测领域中,光子晶体有着广泛的应用。光子晶体荧光传感器基于光子晶体荧光增强效应，在传统的荧光检测体系中引入光子晶体结构，增强荧光信号强度，提升检测灵敏度，降低检测限度。光子晶体具有周期性结构，荧光分子会进入光子局域位置时，提高电磁场激发光与荧光分子之间的相互作用，增强自发辐射的荧光信号，易于检测。荧光标记法是生物分子检测的重要检测方法，但仍然存在检测分子浓度低，背景噪声高，样品微量等问题，严重影响到检测结果的可靠性和准确性。检测器中光子晶体的加入，荧光增强效应可增强检测信号，提高检测器件的灵敏度，实现对DNA、蛋白质等的高灵敏度检测与分析，对医疗疾病诊断有着重要作用。专利CN102161284A中公开了的利用喷墨打印技术制备具有响应性及图案化胶体光子晶体膜的方法。专利200510011219.2中公开了聚甲基丙烯酸甲酯-丙烯酸-苯乙烯乳胶球制备方法。专利CN113912729A中公开了纳米抗体的制备方法。
发明公开
本发明所要解决的技术问题是如何得到一种能够快速和/或简便和/或灵敏地进行蛋白检测的光子晶体生物芯片。
本发明提供一种光子晶体生物芯片，包括反应基底层，所述反应基底层上设有至少一个稳定捕获区和至少一个不稳定检测区；所述稳定捕获区包含光子晶体阵列及至少一种捕获抗体；所述不稳定检测区包含光子晶体阵列及至少一种检测抗体，所述检测抗体被荧光基团标记。
所述荧光基团选自FAM、VIC、HEX、TRT、CY3、CY5、ROX、JOE、FITC、TET、NED、TAMRA、LC RED460、LC RED705、Quasar705或Texas Red中的至少一种。
所述捕获抗体为能够捕获待测蛋白的抗体，所述检测抗体为能够与待测蛋白结合并标记有荧光基团的抗体，所述荧光信号可以为Cy5基团。
所述反应基底层为高分子片或高分子膜。
所述反应基底层由聚酯、聚苯乙烯、聚甲基丙烯酸、聚丙烯或聚氯乙烯制成。
所述光子晶体为聚甲基丙烯酸甲酯-丙烯酸-苯乙烯乳胶球。
所述聚甲基丙烯酸甲酯-丙烯酸-苯乙烯乳胶球的粒径(直径)为300nm。
所述芯片为用于检测可溶性ST2蛋白的光子晶体生物芯片，所述捕获抗体为可溶性ST2蛋白捕获抗体；所述检测抗体为可溶性ST2蛋白检测抗体。
将所述可溶性ST2蛋白捕获抗体命名为捕获抗体NbA，所述捕获抗体NbA为专利CN113912729A中的编号为1A9的抗体。
将所述可溶性ST2蛋白检测抗体命名为检测抗体Cy5-NbB，所述检测抗体Cy5-NbB为连接有Cy5基团的专利CN113912729A中的编号为2B10的抗体。
本发明还提供一种检测待测蛋白的方法，将待测样本滴在所述的生物芯片上，进行荧光检测，若检测结果显示荧光色表明含有待测蛋白，若不显示荧光色表明不含有待测蛋白。
所述待测蛋白可以为可溶性ST2蛋白，此时对应的所述捕获抗体为可溶性ST2蛋白捕获抗体；所述检测抗体为可溶性ST2蛋白检测抗体。
将所述可溶性ST2蛋白捕获抗体命名为捕获抗体NbA，所述捕获抗体NbA可以为氨基酸序列为序列1所示的单域抗体1A9。
将所述可溶性ST2蛋白检测抗体命名为检测抗体Cy5-NbB，所述检测抗体Cy5-NbB可以为连接有Cy5基团的单域抗体2B10，所述单域抗体2B10的氨基酸序列如序列2所示。
所述的光子晶体生物芯片在检测或者辅助检测可溶性ST2蛋白中的应用也应在本发明的保护范围之内。
本发明还提供一种光子晶体生物芯片的制备方法，包括如下步骤：
1)在反应基底层的表面事先均匀涂覆一层胶黏剂，得到基材；
2)在步骤1的基材上，以含有光晶小球的液体为墨水打印光晶阵列，得到带有光晶阵列的基材；
3)将步骤2中带有光晶阵列的基材干燥后，加热固化；
4)固化完成后，活化光晶阵列，加入捕获抗体使其与光晶阵列结合，形成稳定捕获区；
5)使用BSA进行封闭；
6)在光晶阵列的外围固定检测抗体，形成不稳定检测区。
所述捕获抗体为能够捕获待测蛋白的抗体，所述检测抗体为能够与待测蛋白结合并带有荧光信号的抗体，所述荧光信号可以为Cy5基团。
所述含有光晶小球的液体由919体积份的聚(甲基丙烯酸甲酯-丙烯酸-苯乙烯)乳胶球、80体积份的乙二醇和1体积份的表面活性剂配制而成；所述聚甲基丙烯酸甲酯-丙烯酸-苯乙烯乳胶球的粒径为300nm；所述加热固化条件为110℃，10min。
所述捕获抗体为ST2蛋白捕获抗体；所述检测抗体为ST2蛋白检测抗体。
将所述可溶性ST2蛋白捕获抗体命名为捕获抗体NbA，所述捕获抗体NbA可以为氨基酸序列为序列1所示的单域抗体1A9。
将所述可溶性ST2蛋白检测抗体命名为检测抗体Cy5-NbB，所述检测抗体Cy5-NbB可以为连接有Cy5基团的单域抗体2B10，所述单域抗体2B10的氨基酸序列如序列2所示。
本发明的光子晶体生物芯片，能够通过引入光子晶体，实现快速、操作简单、微量检测的蛋白检测生物芯片，在用于可溶性ST2蛋白检测时，本发明能够快速、操作简单、微量检测可溶性ST2蛋白。
附图说明
图1为基于绿色打印建立光子晶体生物芯片可溶性ST2蛋白的检测方法的基本流程。
图2为实施例1制备的几种光子晶体芯片的显微镜和扫描电镜图片，其中， a为不同粒径光晶小球对荧光的增强效果图片，b为不同粒径的光晶小球扫描电镜图片。
图3为实施例3中的不同梯度浓度的可溶性ST2蛋白采用的本发明的方法和传统酶联免疫吸附测定法ELISA的检测的结果对比，其中，a为带有光子晶体的检测芯片标准曲线各浓度的荧光图；b为不带有光子晶体的检测芯片标准曲线中各浓度的荧光统计，R 2＝0.96；c为带有光子晶体的检测芯片标准曲线的荧光统计,R 2＝0.975；d为带有光子晶体的检测芯片随时间延长荧光强度的变化。
图4为利用本发明的光子晶体检测芯片对15例临床样本检测，整体线性趋势与ELISA检测对比。
图5为实施例5中Cy5-NbB在不同粒径的光子晶体阵列上的荧光强度比对图。
图6为实施例6中分别使用IgG抗体与本发明的纳米抗体与PC300光子晶体阵列制备的芯片的性能比对图。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到，下述实施例中所述的室温均是指25℃。
实验样本：下述实施例中以15名均于2021年10月到2021年11月期间就诊于北京安贞医院的患者，均为自愿参与。
下述实施例中的绿色打印法是指专利CN102161284A中的利用喷墨打印技术制备具有响应性及图案化胶体光子晶体膜的方法。下述实施例中的捕获抗体NbA为序列1所示的单域抗体1A9，检测抗体Cy5-NbB为连接有Cy5基团的单域抗体2B10，所述单域抗体2B10的氨基酸序列如序列2所示，公众可以按照专利CN113912729A中的方法制备，或者委托南京融捷康生物科技有限公司制备获取。
其中，1A9序列为：
Figure PCTCN2022142780-appb-000001
2B10序列为：
Figure PCTCN2022142780-appb-000002
下述实施例中的PBS为中杉金桥ZLI-9061，下述实施例中的FBS为gibco0025，下述实施例中的健康人EDTA抗凝血清为来源于北京安贞医院健康体检中心的人血清，下述实施例中的重组蛋白sST2为百普赛斯的il-H5229。下述实施例中的聚甲基丙烯酸甲酯-丙烯酸-苯乙烯乳胶球为在温度为50℃下，将采用专利200510011219.2中的方法制备得到的含有单分散聚(苯乙烯-甲基丙烯酸甲酯-丙烯酸)三嵌段共聚物乳胶粒的乳液；制备的聚甲基丙烯酸甲酯-丙烯酸-苯乙烯乳胶球的粒径分别为280、300、310、320、360、370nm。
实施例1 光晶小球粒径的选择
如图1中的步骤a-c所示的方法制备光晶阵列，具体如下：
a)在PET基材表面事先均匀涂覆一层胶黏剂(可以为聚乙烯醇水性胶黏剂)，用以固着后续打印上来的光晶小球。
b)以含有光晶小球的液体为光晶墨水，用SONOguide仪器在步骤的基材上进行喷墨打印，单个5×5矩阵阵列边长仅不超过1mm。打印所用的含有光晶小球的液体为聚甲基丙烯酸甲酯-丙烯酸-苯乙烯乳胶球与乙二醇、表面活性剂(表面活性剂为德国毕克货号为BYK-3455的有机硅表面活性剂)的混合溶液，混合溶液的体积比例是919:80:1。以调节光晶墨水打印后的干燥速度，保证光晶小球的组装齐整。所述聚甲基丙烯酸甲酯-丙烯酸-苯乙烯乳胶球的制备方法为：在温度为50℃下，将采用专利号为ZL200510011219.2的方法制备得到的含有粒径为300、310、320、360、370nm的单分散聚(苯乙烯-甲基丙烯酸甲酯-丙烯酸)三嵌段共聚物乳胶粒的乳液。
c)干燥后将芯片至于烘箱中110℃高温处理10min，实现固化。
按照上述制备方法，分别采用粒径(直径)为300、310、320、360、370nm 的聚(甲基丙烯酸甲酯-丙烯酸-苯乙烯)乳胶球，制备得到光晶阵列-300、光晶阵列-310、光晶阵列-320、光晶阵列-360和光晶阵列-370，分别对应图2中的PC 300，PC 310，PC 320，PC 360和PC 370。
采用普通光学显微镜检查打印的光晶点阵，结果如图2中的a的所示，能够看到角度依赖的红色光晶色。采用扫描电镜展示点阵表面形貌，结果如图2中的b的所示，小球组装排列整齐，紧密程度合适。向光晶芯片表面滴加Cy5纯染料，干燥后通过共聚焦显微镜拍摄并直接定量荧光强度对比增强效果，并检测反射光谱，确认禁带与Cy5发射最匹配的光晶小球的粒径为300nm。
实施例2 可捕获可溶性ST2蛋白的光晶芯片的制备：
利用Nordson EFD点胶机采用绿色打印法制备可捕获可溶性ST2蛋白的光晶芯片，上述打印步骤中的打印直径约240μm，间距0.5mm的5*5点阵，每个5*5点阵间距为7mm，具体方法如图1所示：
1)按照实施例1中的方法制备光晶阵列-300；
2)将制备的光晶阵列-300以Sulfo-NHS(SIGMA24510)/EDC(SIGMA22981)酯活化光晶小球表面的羧基，加入纳米抗体NbA，使其与纳米抗体提供的氨基脱水缩合，将作为捕获抗体的NbA与光晶阵列共价结合，固定在光晶阵列表面；
3)使用5％BSA室温下进行封闭，用以降低检测识别过程中的非特异性结合。
4)将带有荧光标记的检测抗体Cy5-NbB，通过喷墨打印非共价固定在芯片表面，排列在光晶阵列外围，干燥(室温条件，25℃)后的抗体点阵在干燥储存下仍能保持活性。得到光子晶体检测芯片。
使用光子晶体检测芯片进行检测时，向芯片滴加20μl的待检测溶液，将外圈的荧光检测抗体NbB阵列溶解，待检测物质被NbB识别并结合，溶液中水分不断蒸发体系的体积减小，由于光晶阵列相对PET基材表面更加亲水，液滴向中心回缩，待检测物与NbB的复合物被中圈光晶阵列上的NbA捕获并固定，经过光子晶体的角度依赖型荧光增强，荧光信号被聚集放大。将完成检测所需的全部试剂打印在毫米见方的芯片表面，最大化减少了完成检测所需的操作步骤。
实施例3
分别以PBS，FBS和健康人EDTA抗凝血清为溶剂配制梯度浓度sST2溶液作为待检测样本，sST2溶液作为待检测样本的梯度浓度分别为0.01、0.1、1、10、 100和1000ng/mL。
分别将上述样品用实施例2中的加入光晶的荧光偶联免疫吸附测定法(简称PC-FLISA)和传统酶联免疫吸附测定法FLISA进行性能检测，结果如图3中的a所示。其中，传统酶联免疫吸附测定法FLISA可参照文献Performance of coumarin-derived dendrimer-based fluorescence-linked immunosorbent assay(FLISA)to detect malaria antigen中记载的步骤进行。从图3中的a可以看出同一浓度使用本发明的PC-FLISA方法检测的荧光强度明显高于传统的FLISA检测的荧光强度。
以激光共聚焦拍摄检测结果，进行荧光强度统计作图，确定线性范围和检出限，结果如图3中的b、c和d所示。根据检测结果，相应的可检出的抗原浓度达到0.01ng/ml，增强效果稳定。
实施例4
以15名心血管相关疾病临床患者的血液为样本PBS十倍稀释得到待测样本溶液，将待测样本溶液分别采用本发明的光子晶体检测芯片和传统ELISA进行检测进行加测，结果如图4所示。传统ELISA检测法可以可参照文献Enzyme immunoassay(EIA)/enzyme-linked immunosorbent assay(ELISA).Lequin RM.Clin Chem.2005 Dec；51(12):2415-8.doi:10.1373/clinchem.2005.051532.Epub 2005 Sep 22.中记载的步骤进行。
从图4中可以看出采用本发明的检测样本整体线性趋势与金标准ELISA不相上下。多次重复检测下，相同样本之间，本发明的PC-FLISA法的检测结果稳定。
实施例5 不同粒径纳米微球的对比数据
按照实施例1方法，分别采用粒径(直径)为280、300、320、360nm的聚(甲基丙烯酸甲酯-丙烯酸-苯乙烯)乳胶球，制备得到光晶阵列-280(PC 280)、光晶阵列-300(PC 300)、光晶阵列-320(PC 320)和光晶阵列-360(PC 360)。比较Cy5-NbB(0.5μL,450ng/mL)在上述四种光晶微点阵列上的荧光强度，结果如图5所示。PC300获得了最高的发射增强(60.3倍)，因为蓝带边缘与发射波长匹配，可以显着增强位于PC基质空隙中的染料的自发发射。PC280的蓝带边缘与激发波长相匹配，可以提高激发效率，但其禁带与Cy5-NbB发射重叠，从而抑制了位于PC基质内的染料的发射。所以PC280只实现了39.8倍的增强。PC320和PC360的禁带远离Cy5-NbB的激发和发射波长，由于它们的大表面 积和散射效应，它们将发射增强了约10.8和1.5倍。基于上述结果，PC300能够更高的用于增强Cy5-NbB的荧光信号。
实施例6、IgG抗体(Ab)与纳米抗体(Nb)的对比数据
分别使用Cy5抗体(IgG)和Cy5纳米抗体按照实施例2中的方法以PC 300或者空白基板为基板，制备光晶芯片，并进行性能检测，结果如图6所示。
图6中：
a为Cy5抗体(IgG)(0.5μL，10μg/mL)、Cy5纳米抗体(0.5μL，10μg/mL)分别在光晶阵列PC300和空白基板上的光学明场图像和荧光图像，其中：Ab表示Cy5抗体(IgG)与空白基板制备的光晶芯片，Nb表示Cy5纳米抗体与空白基板制备的光晶芯片，Ab+PC 300表示Cy5抗体(IgG)与PC 300基板制备的光晶芯片，Nb+PC 300表示Cy5纳米抗体与PC 300基板制备的光晶芯片；
b为在PC 300阵列和空白基板上用Cy5抗体(IgG)和Cy5纳米抗体进行相应强度分析，可以看出在使用PC 300阵列的情况下Cy5纳米抗体的荧光强度的增强远高于使用Cy5抗体(IgG)的荧光增强程度，其中Ab表示使用的抗体为Cy5抗体(IgG)，Nb表示使用的抗体为Cy5纳米抗体，Without PC 300表示使用的基板为空白基板，With PC 300表示使用的基板为PC 300基板；
c中i为在PC 300点中组装的胶体球，ii为固定在PC 300表面上的IgG抗体(Ab)和iii为固定在PC 300表面上的纳米抗体(Nb)的扫描电镜图像，从图中可以观察到IgG抗体影响到了PC300光晶小球的规律排列组装，使得表面不平滑，PC 300表示PC 300光晶阵列，Ab+PC 300表示Cy5抗体(IgG)与PC 300基板制备的光晶芯片，Nb+PC 300表示Cy5纳米抗体与PC 300基板制备的光晶芯片；
d为分别偶联IgG抗体和纳米抗体的PC 300、以及单纯PC 300的反射光谱，偶联IgG抗体使得反射光谱显著下降，降低了荧光增强效果，PC 300表示PC 300光晶阵列，Ab+PC 300表示Cy5抗体(IgG)与PC 300基板制备的光晶芯片，Nb+PC 300表示Cy5纳米抗体与PC 300基板制备的光晶芯片；
e为在光晶阵列上模拟纳米抗体和IgG抗体的电场分布，其中，Ab表示将Cy5抗体(IgG)结合在PC 300基板上，Nb表示将Cy5纳米抗体结合在PC 300基板上；
f为模拟光晶阵列中IgG抗体和Ab的功率流出密度分布，其中，Ab表示将Cy5抗体(IgG)结合在PC 300基板上，Nb表示将Cy5纳米抗体结合在PC 300基板上。
从上述结果可以看出，由于IgM或者IgG等抗体的结构较大，而光晶阵列一般都是纳米级的阵列，这样在纳米级的光晶阵列上连接较大的抗体时，一方 面其操作起来更加复杂，另一方面大颗粒的抗体对光晶阵列表面光滑度的影响也较大，减弱了原本的光晶阵列对荧光信号的放大效果，本发明使用的单域抗体结构较小，一般都为纳米的抗体，其与光晶阵列结合后，对光晶阵列表面的光滑度的影响更小，有利于对荧光信号的放大，从而提高检测的灵敏度和准确度。同时，采用体积较小的纳米级单域抗体，单位面积上的抗体数量也会大大的增加，从而能够提高检测的效率。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明的光子晶体生物芯片，能够通过引入光子晶体，实现快速、操作简单、微量检测的蛋白检测生物芯片，在用于可溶性ST2蛋白检测时，本发明能够快速、操作简单、微量检测可溶性ST2蛋白。
本发明中采用光晶阵列采用粒径为300nm的聚(甲基丙烯酸甲酯-丙烯酸-苯乙烯)乳胶球与Cy5基团作为荧光信号配合使用，使得光晶阵列与荧光信号有着最好的匹配效果，能够将采集的信号扩大100倍，能够大大的提高检测的精准度和灵敏度。
由于IgM或者IgG等抗体的结构较大，而光晶阵列一般都是纳米级的阵列，这样在纳米级的光晶阵列上连接较大的抗体时，一方面其操作起来更加复杂，另一方面大颗粒的抗体对光晶阵列表面光滑度的影响也较大，减弱了原本的光晶阵列对荧光信号的放大效果，本发明使用结构较小的单域抗体(一般都为纳米的抗体)，其与光晶阵列结合后，对光晶阵列表面的光滑度的影响更小，有利于对荧光信号的放大，从而提高检测的灵敏度和准确度。同时，采用体积较小的纳米级单域抗体，单位面积上的抗体数量也会大大的增加，从而能够提高检测的效率。

Claims (10)

  1. 光子晶体生物芯片，其特征在于，所述光子晶体生物芯片包括反应基底层，所述反应基底层上设有至少一个稳定捕获区和至少一个不稳定检测区；所述稳定捕获区包含光子晶体阵列及至少一种捕获抗体；所述不稳定检测区包含光子晶体阵列及至少一种检测抗体，所述检测抗体被荧光基团标记。
  2. 根据权利要求1所述的光子晶体生物芯片，其特征在于，所述反应基底层为高分子片或高分子膜。
  3. 根据权利要求2所述的光子晶体生物芯片，其特征在于，所述反应基底层由聚酯、聚苯乙烯、聚甲基丙烯酸、聚丙烯或聚氯乙烯制成。
  4. 根据权利要求1所述的光子晶体生物芯片，其特征在于，所述光子晶体为聚(甲基丙烯酸甲酯-丙烯酸-苯乙烯)乳胶球。
  5. 根据权利要求4所述的光子晶体生物芯片，其特征在于，所述聚(甲基丙烯酸甲酯-丙烯酸-苯乙烯)乳胶球的粒径为300nm。
  6. 根据权利要求5所述的光子晶体生物芯片，其特征在于，所述光子晶体生物芯片为用于检测可溶性ST2蛋白的光子晶体生物芯片，所述捕获抗体为可溶性ST2蛋白捕获抗体；所述检测抗体为可溶性ST2蛋白检测抗体。
  7. 一种检测待测蛋白的方法，其特征在于，所述方法包括将待测样本滴在权利要求1-6中任一所述的光子晶体生物芯片上，进行荧光检测，根据荧光检测结果确定所述待测样本是否含有待测蛋白。
  8. 一种制备光子晶体生物芯片的方法，其特征在于，所述方法包括如下步骤：
    1)在反应基底层的表面均匀涂覆一层胶黏剂，得到基材；
    2)在所述基材上，以含有光晶小球的液体为墨水打印光晶阵列，得到带有光晶阵列的基材；
    3)将所述带有光晶阵列的基材干燥后，加热固化；
    4)固化完成后，活化光晶阵列，加入捕获抗体使其与光晶阵列结合，形成稳定捕获区；
    5)使用BSA进行封闭；
    6)在光晶阵列的外围固定检测抗体，形成不稳定检测区。
  9. 根据权利要求8所述的方法，其特征在于，所述含有光晶小球的液体由919体积份的聚(甲基丙烯酸甲酯-丙烯酸-苯乙烯)乳胶球、80体积份的乙二醇和1体积份的表面活性剂配制而成；所述聚(甲基丙烯酸甲酯-丙烯酸-苯乙烯)乳胶球的粒径为300nm；所述加热固化条件为110℃，10min。
  10. 根据权利要求8所述的方法，其特征在于，所述捕获抗体为可溶性ST2蛋白捕获抗体；所述检测抗体为可溶性ST2蛋白检测抗体。
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