WO2018068448A1 - 一种利用微阵列微流控芯片筛选适配体的方法 - Google Patents

一种利用微阵列微流控芯片筛选适配体的方法 Download PDF

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WO2018068448A1
WO2018068448A1 PCT/CN2017/073024 CN2017073024W WO2018068448A1 WO 2018068448 A1 WO2018068448 A1 WO 2018068448A1 CN 2017073024 W CN2017073024 W CN 2017073024W WO 2018068448 A1 WO2018068448 A1 WO 2018068448A1
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screening
aptamer
microarray
chip
solution
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许丹科
李慧
刘晓辉
陈竹
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Nanjing University
Nanjing Tech University
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Nanjing Tech University
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays
    • 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
    • G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins

Definitions

  • the invention belongs to the technical field of biological detection, and particularly relates to a method for screening an aptamer by using a microarray microfluidic chip.
  • the aptamer has similar properties to the antibody, but in many respects it also exhibits superior characteristics to the antibody: (1) more specific, higher affinity for the target molecule than the antibody; (2) suitable Ligands are easier to obtain than antibodies (synthetically, independent of animals and cells), and can be synthesized in large quantities and quickly in vitro, making preparation methods simpler and faster; (3) screening for different types of target molecules Including biotoxic molecules and molecules with only haptenity, broadening its application range; (4) non-immunogenicity, can be used repeatedly in vivo; (5) stability is better than antibodies, which is conducive to storage. With the advancement of aptamer technology, aptamers will likely be a useful complement to antibody technology in many fields. It is based on various advantages of aptamers that aptamers have shown broad application prospects in analysis, clinical, environmental, molecular recognition and drug screening.
  • nanomaterial-based aptamer screening technology can eliminate the immobilization of proteins on the surface of solid phase substrates, which can effectively reduce the number of screening rounds and speed up the screening process.
  • the method based on microfluidic technology to improve the screening effect has been extensively studied. These methods include: microfluidic aptamer screening for capillary electrophoresis, sol-gel microfluidic aptamer screening, surface plasmon resonance (SPR) microfluidic aptamer screening, microfluidic screening based on magnetic bead particles, And microfluidic screening techniques based on microspheres of various materials.
  • SPR surface plasmon resonance
  • agarose droplet PCR amplification technology and polyemulsion PCR amplification (ePCR) technology, which can directly select positive clones for sequencing, avoiding traditional aptamers.
  • ePCR polyemulsion PCR amplification
  • the cloning process of the screening process is cumbersome.
  • the above methods are intended to make the screening of aptamers more time-saving, automated, efficient and reduce the cost of the entire screening.
  • Our job is to develop a new aptamer screening technique that is more stable, the screening process is repeatable, and the screening steps are quantifiable.
  • the method optimizes the process of aptamer screening to make the aptamer screening process more simple and standardized.
  • the technical problem to be solved by the present invention is to provide a novel microarray microfluidic chip for applying to an aptamer screening method.
  • a method for screening an aptamer using a microarray microfluidic chip wherein the microarray microfluidic chip is prepared as follows:
  • the PDMS prepolymer and the curing agent are mixed at a mass ratio of 5:1 to 100:1, preferably 10:1, and after being uniformly mixed, they are placed in a vacuum desiccator, and connected to a circulating water vacuum pump for vacuuming for 30 minutes to remove bubbles. , and then poured onto the microfluidic channel template, after curing, PDMS microfluidic channel is obtained, and lactoferrin and negative protein microarray with concentration of 0.1-10 mg/mL are prepared on the glass substrate, and the PDMS microfluidic channel is obtained. Simultaneously treating the glass substrate with the spotted glass substrate, the two kinds of substrates are closely adhered to obtain a microarray microfluidic chip.
  • PCR amplification process the solution obtained in the step (2) is divided into 3 to 10 parts of the same solution having a volume of 23 ⁇ L, and 25 ⁇ L of 2 ⁇ Taq polymerase, 1 ⁇ L, and 20 ⁇ M TAMRA-labeled forward primer are sequentially added to each portion. And 1 ⁇ L, 20 ⁇ M biotinylated backward primer, mixed uniformly and then subjected to PCR amplification;
  • PCR product After product of this step was diluted 5 to 40 times as a template again amplification: PCR product after taking 5 ⁇ L diluted with 1 ⁇ L 2 ⁇ SYBR Premix Ex Taq TM enzyme before 1 ⁇ L 20 ⁇ M TAMRA-labeled forward primer, 1 ⁇ L 20 ⁇ M biotin The backward primers and 18 ⁇ L of ultrapure water were uniformly mixed. 10 ⁇ L of the sample was taken into the microplate every one round of PCR amplification, and the fluorescence signal was detected by BioTek. The highest fluorescence signal was used as the optimal number of rounds, and the remaining products were expanded. Increase the number of rounds;
  • the PCR product obtained in the step (3) is thoroughly mixed with 800 ⁇ L to 5 mL of Promega magnetic beads, and the supernatant is removed after rapid shaking for 0.5 to 2 hours; and 25 ⁇ L of the 0.01-0.1 M NaOH solution is further vortexed. 3 to 15 min, dissociate the double strands attached to the magnetic beads into the solution; then take the supernatant and add 12.5 ⁇ L, 0.1 M HCl 25 in sequence.
  • ⁇ L, DPEC water and 62.5 ⁇ L 2 ⁇ PBSM buffer solution the obtained neutral solution is used as a secondary library for the next round of screening, and its content is 40 pmol to 100 pmol;
  • the second round of screening using a syringe pump at a flow rate of 1 ⁇ 5 ⁇ L / min, the library was sequentially transferred to the negative screen at a temperature of 25 ⁇ 40 ° C at 1 ⁇ 5 ⁇ L / min, the positive sieve channel; then 5 ⁇ 30 ⁇ L / Min flow rate 150 ⁇ L, 1 ⁇ PBS buffer solution to remove unreacted ssDNA sequence in the positive sieve channel; gently tear off the PDMS layer, scan the chip with Luxscan ⁇ 10K / A microarray scanner; finally use DPEC water at 95 ° C The lactoferrin-bound ssDNA sequence on the chip was eluted by heating for 3 to 10 minutes, and the resulting solution was dried at 60 ° C to a volume of 25 to 250 ⁇ L, which was left for PCR amplification;
  • the library has 40 random bases in the middle, the nucleotide sequence at the 5' end of the random base is as shown in SEQ ID NO. 1, and the nucleotide sequence at the 3' end of the random base is SEQ ID. As shown in NO.2,
  • the forward primer has a nucleotide sequence as shown in SEQ ID NO. 3; and the backward primer has a nucleotide sequence as shown in SEQ ID NO.
  • An aptamer for detecting lactoferrin content the nucleotide sequence of which is shown in SEQ ID NO. 5 to 65.
  • the aptamer is produced by a systemic evolution of ligands by exponential enrichment (SELEX), which can be used to screen specificity from a random single-stranded nucleic acid sequence library.
  • SELEX systemic evolution of ligands by exponential enrichment
  • the nucleic acid ligand (aptamer) which is highly affinity with the target substance, the invention is based on the large-scale, high-throughput integration of the microarray technology (Microarray), the small volume and the like, and the microfluidic technology (Microfluidic) automatic fluid transport, the molecule Advantages such as enhanced reaction efficiency and closed reaction chambers that avoid external pollution will be suitable
  • the negative screening process and the positive screening process of ligand screening are combined on a microfluidic chip to quickly and efficiently perform aptamer screening.
  • the method of screening the aptamers by this microarray microfluidic chip was designed for the first time.
  • Protein microarray production methods, screening libraries and protein array process monitoring are common; in addition, PCR process, PCR product purification, secondary library regeneration process can be used. Therefore, the method of screening aptamers using microarray microfluidic chips has a good general purpose and important research significance.
  • the invention mainly discloses a method for screening an aptamer by a microarray microfluidic chip, which combines a positive and negative screening process on a microfluidic chip, combines the advantages of microfluidic and microarray technology, and has a simple screening process.
  • a high affinity aptamer is obtained conveniently and after 7 rounds of screening.
  • the present invention also discloses specific steps for screening of lactoferrin aptamers, including detailed processes such as chip preparation, positive and negative screening processes, and PCR amplification.
  • the aptamer screened by the method has good specificity to the target protein and high affinity; and the aptamer is more easily obtained than the antibody, and can be synthesized in a large amount and quickly in vitro, and the preparation method is simpler and faster, so the method Aptamers are expected to be a useful complement to antibody technology in many fields.
  • the chip mode of the positive and negative screen collection is convenient, fast and efficient, and can be used for screening other proteins and nucleic acid aptamers, and provides a good idea design and reference for other aptamer screening workers.
  • FIG. 1 Microarray microfluidics is applicable to the aptamer screening flow chart.
  • Figure 2 is a graph of the results of the first screening, (a) the amount of each round of library, (b) the fluorescence signal intensity of the aptamer on the chip, (c) the imaging image of the aptamer on the chip, (d) ) Image after chip cleaning.
  • Figure 3 shows the results of the second screening, (a) the amount of each round of library, (b) the fluorescence signal intensity of the aptamer on the chip, (c) the imaging image of the aptamer on the chip, (d) ) Image image after chip cleaning.
  • Figure 4 shows the results of the third screening, (a) the amount of each round of library, (b) the fluorescence signal intensity of the aptamer on the chip, (c) the imaging image of the aptamer on the chip, (d) ) Image image after chip cleaning.
  • Figure 5 is a linear plot of other aptamers for lactoferrin fluorescence polarization detection.
  • the microfluidic channel template was fabricated by the combination of lithography mask and chemical etching, and was reserved for the subsequent PDMS channel preparation; the PDMS precursor with mass ratio of 10:1 was weighed and fully mixed with the curing agent. After vacuuming, it was poured onto a microfluidic channel template to obtain a PDMS microfluidic channel; a lactoferrin and a negative protein microarray with a concentration of 5 mg/mL were prepared on a glass substrate using a spotting instrument, and PDMS and dots were applied. The sample glass substrate is simultaneously plasma treated, and then closely adhered together as a screening chip for each subsequent screening;
  • the solution eluted on the chip was divided into 6 parts of the same solution in a volume of 23 ⁇ L, and each was added 25 ⁇ L, 2 ⁇ Taq polymerase, 1 ⁇ L, 20 ⁇ M TAMRA-labeled forward primer and 1 ⁇ L, 20 ⁇ M biotinylated.
  • the primers were mixed and homogenized and then subjected to PCR amplification; the thermal cycle of PCR was as follows: 94 ° C for 5 min; cycle 94 ° C for 30 s, 60.5 ° C for 30 s, 72 ° C for 30 s for 10 rounds, 72 ° C for 5 min to terminate the reaction; once again amplified as a template 10 times: after taking 5 ⁇ L dilution, 2 ⁇ SYBR Premix Ex Taq TM enzyme, 1 ⁇ L, PCR products 25 ⁇ L front 20 ⁇ M TAMRA-labeled forward primer, 1 ⁇ L, 20 ⁇ M biotinylated forward primer and 18 ⁇ L of ultrapure water was well mixed, and 10 ⁇ L of the sample was taken into the microplate for each round of PCR amplification.
  • the fluorescence signal was detected by BioTek. The highest fluorescence signal was used as the optimal number of rounds, and the remaining products were amplified by the number of rounds.
  • the specific steps of screening the lactoferrin aptamer include detailed processes such as chip preparation, positive and negative screening process, and PCR amplification, as described below, wherein:
  • TARMA modified forward primer 5'-TARMA-GACAGGCAGGACACCGTAAC-3'
  • Biotinylated backward primer 5'Biotin-GAAGAGGAGGGAGGTAGCAG-3'
  • Chip preparation Firstly, a microfluidic channel template was fabricated by using a combination of photolithography mask and chemical etching as a PDMS channel to make an abrasive tool; a PDMS precursor and a mass ratio of 10:1 were weighed and cured. The agent is thoroughly mixed, vacuumed and poured onto a microfluidic channel template to obtain a PDMS microfluidic channel; a lactoferrin and a negative protein microarray having a concentration of 5 mg/mL are prepared on a glass substrate using a spotting apparatus. And for PDMS and spotted glass base The sheets are simultaneously plasma treated and then closely attached together as a screening chip for each subsequent screening;
  • PCR amplification process The solution obtained in the step (2) is divided into 3 parts of the same solution having a volume of 23 ⁇ L, and 25 ⁇ L, 2 ⁇ Taq polymerase, 1 ⁇ L, 20 ⁇ M TAMRA-labeled forward primer and 1 ⁇ L are sequentially added in each portion. 20 ⁇ M biotinylated backward primers were mixed and homogenized for PCR amplification; the thermal cycling process of PCR was as follows: 94 ° C for 5 min; cycle 94 ° C for 30 s, 60.5 ° C for 30 s, 72 ° C for 30 s for 10 rounds, 72 ° C for 5 min to terminate the reaction.
  • product of this step obtained was diluted 10-fold as template again amplified: after taking 5 ⁇ L diluted PCR product with 25 ⁇ L, 2 ⁇ SYBR Premix Ex Taq TM enzyme, 1 ⁇ L, before 20 ⁇ M TAMRA-labeled forward primer, 1 ⁇ L, 20 ⁇ M biological The normalized backward primer and 18 ⁇ L DPEC water were well mixed. PCR amplification was performed for 10 ⁇ L of the sample to the microplate. The fluorescence signal was detected by BioTek. The highest fluorescence signal was used as the optimal number of rounds. Amplify the number of rounds;
  • PCR product obtained in the step (4) was thoroughly mixed with 600 ⁇ L of Promega magnetic beads, and the supernatant was removed by shaking for 1 hour on a shaker; then 25 ⁇ L, 50 mM NaOH was vortexed for 5 min to connect the magnetic beads. Double-strand dissociation; aspirate the supernatant and sequentially add 12.5 ⁇ L of 100 mM HCl, 25 ⁇ L of H 2 O, 62.5 ⁇ L of 2 ⁇ PBSM, and the resulting solution is used as a secondary library for the next round of screening, and its content is about 40 pmol;
  • the second round of screening the library was pumped into the negative sieve channel at a flow rate of 2.5 ⁇ L/min, and reacted at room temperature for 50 min; then the pump was used to input the library into the positive sieve channel at a flow rate of 2.5 ⁇ L/min, at room temperature.
  • the reaction was carried out for 50 min; then 150 ⁇ L of 1 ⁇ PBS buffer solution was passed at a flow rate of 15 ⁇ L/min to remove unreacted chains in the positive sieve channel; the PDMS layer was gently peeled off, and the chip was scanned using a Luxscan ⁇ 10K/A microarray scanner; The protein-bound chain was eluted with non-nuclear water at 95 ° C for 5 min, and the resulting solution was dried at 60 ° C to a volume of 92 ⁇ L, which was left for PCR amplification;
  • the specific steps of screening the lactoferrin aptamer include detailed processes such as chip preparation, positive and negative screening process, and micro PCR amplification, as described below, wherein:
  • TARMA modified forward primer 5'-TARMA-GACAGGCAGGACACCGTAAC-3'
  • Biotinylated backward primer 5'-Biotin-GAAGAGGAGGGAGGTAGCAG-3'
  • Chip preparation Firstly, a microfluidic channel template was fabricated by using a combination of photolithography mask and chemical etching as a PDMS channel to make an abrasive tool; a PDMS precursor and a mass ratio of 10:1 were weighed and cured. The agent was thoroughly mixed, vacuumed and poured onto a microfluidic channel template to obtain a PDMS microfluidic channel; a lactoferrin and a negative protein microarray with a concentration of 2.5 mg/mL were prepared on a glass substrate using a spotting apparatus. And the PDMS and the spotted glass substrate are simultaneously plasma-treated, and then closely adhered together as a screening chip for the next round of screening;
  • PCR amplification process The solution obtained in the step (3) is divided into 4 parts of the same solution having a volume of 23 ⁇ L, and 25 ⁇ L, 2 ⁇ Taq polymerase, 1 ⁇ L, 20 ⁇ M TAMRA-labeled forward primer and 1 ⁇ L are sequentially added in each portion. 20 ⁇ M biotinylated backward primers were mixed and homogenized for PCR amplification; the thermal cycling process of PCR was as follows: 94 ° C for 5 min; cycle 94 ° C for 30 s, 60.5 ° C for 30 s, 72 ° C for 30 s for 10 rounds, 72 ° C for 5 min to terminate the reaction.
  • product of this step obtained was diluted 10-fold as template again amplified: after taking 5 ⁇ L diluted PCR product with 25 ⁇ L, 2 ⁇ SYBR Premix Ex Taq TM enzyme, 1 ⁇ L, before 20 ⁇ M TAMRA-labeled forward primer, 1 ⁇ L, 20 ⁇ M biological The primed primers and 18 ⁇ L of ultrapure water were well mixed. PCR amplification was performed for 10 ⁇ L of each sample to the microplate. The fluorescence signal was detected by BioTek. The highest fluorescence signal was used as the optimal number of rounds. Amplify the number of rounds;
  • the PCR product obtained in the step (4) was thoroughly mixed with 800 ⁇ L of Promega magnetic beads, and the supernatant was removed after shaking for 1 hour on a shaker; and 25 ⁇ L, 1 M NaOH was vortexed for 5 min to connect the magnetic beads. Double-strand dissociation; aspirate the supernatant and sequentially add 12.5 ⁇ L of 2M HCl, 25 ⁇ L of H 2 O, 62.5 ⁇ L of 2 ⁇ PBSM, and the resulting solution was used as a second round of screening secondary library at a concentration of 90 pmol;
  • the second round of screening the library was pumped into the negative sieve channel at a flow rate of 2.5 ⁇ L/min, and reacted at room temperature for 50 min; then the pump was used to input the library into the positive sieve channel at a flow rate of 2.5 ⁇ L/min, at room temperature.
  • the reaction was carried out for 50 min; then 150 ⁇ L of 1 ⁇ PBS buffer solution was passed at a flow rate of 15 ⁇ L/min to remove unreacted chains in the positive sieve channel; the PDMS layer was gently peeled off, and the chip was scanned using a Luxscan ⁇ 10K/A microarray scanner; The protein-bound chain was eluted with non-nuclear water at 95 ° C for 5 min, and the resulting solution was dried at 60 ° C to a volume of 92 ⁇ L, which was left for PCR amplification;
  • the specific steps of screening the lactoferrin aptamer include detailed processes such as chip preparation, positive and negative screening process, and micro PCR amplification, as described below, wherein:
  • TARMA modified forward primer 5'-TARMA-GACAGGCAGGACACCGTAAC-3'
  • Biotinylated backward primer 5'-Biotin-GAAGAGGAGGGAGGTAGCAG-3'
  • Chip preparation Firstly, a microfluidic channel template was fabricated by using a combination of photolithography mask and chemical etching as a PDMS channel to make an abrasive tool; a PDMS precursor and a mass ratio of 10:1 were weighed and cured. The agent is thoroughly mixed, vacuumed and poured onto a microfluidic channel template to obtain a PDMS microfluidic channel; a lactoferrin and a negative protein microarray having a concentration of 5 mg/mL are prepared on a glass substrate using a spotting apparatus. The PDMS and the spotted glass substrate are simultaneously plasma-treated, and then closely adhered together as a screening chip for each subsequent screening;
  • Screening preparation the screening chip obtained in the step (1) was placed in a constant temperature water bath to incubate the protein at 37 ° C for 2 h; then 20 mg/ml BSA and 0.01 mM random sequence short-chain ssDNA (20 nt) were introduced at 37 ° C conditions. Blocked for 1 h; then rinse the channel with 150 ⁇ L, 1 ⁇ PBST solution;
  • PCR amplification process The solution obtained in the step (3) is divided into 4 parts of the same solution having a volume of 23 ⁇ L, and 25 ⁇ L, 2 ⁇ Taq polymerase, 1 ⁇ L, 20 ⁇ M TAMRA-labeled forward primer and 1 ⁇ L are sequentially added in each portion. 20 ⁇ M biotinylated backward primers were mixed and homogenized for PCR amplification; the thermal cycling process of PCR was as follows: 94 ° C for 5 min; cycle 94 ° C for 30 s, 60.5 ° C for 30 s, 72 ° C for 30 s for 10 rounds, 72 ° C for 5 min to terminate the reaction.
  • product of this step obtained was diluted 10-fold as template again amplified: after taking 5 ⁇ L diluted PCR product with 25 ⁇ L, 2 ⁇ SYBR Premix Ex Taq TM enzyme, 1 ⁇ L, before 20 ⁇ M TAMRA-labeled forward primer, 1 ⁇ L, 20 ⁇ M biological The normalized backward primer and 18 ⁇ L DPEC water were well mixed. PCR amplification was performed for 10 ⁇ L of the sample to the microplate. The fluorescence signal was detected by BioTek. The highest fluorescence signal was used as the optimal number of rounds. Amplify the number of rounds;
  • the PCR product obtained in the step (4) was thoroughly mixed with 800 ⁇ L of Promega magnetic beads, and the supernatant was removed after shaking for 1 hour on a shaker; and 25 ⁇ L, 50 mM NaOH was vortexed for 5 min to connect the magnetic beads. Double-strand dissociation; the supernatant was aspirated and sequentially added with 12.5 ⁇ L of 100 mM HCl, 25 ⁇ L of H 2 O, 62.5 ⁇ L of 2 ⁇ PBSM, and the resulting solution was used as a secondary round of screening secondary library with a content of 60 pmol;
  • the second round of screening the library was pumped into the negative sieve channel at a flow rate of 5 ⁇ L/min, and reacted at room temperature for 50 min; then the library was fed into the positive sieve channel at a flow rate of 5 ⁇ L/min, under normal temperature conditions.
  • the reaction was carried out for 50 min; then 150 ⁇ L of 1 ⁇ PBS buffer solution was passed at a flow rate of 30 ⁇ L/min to remove unreacted chains in the positive sieve channel; the PDMS layer was gently torn off, and the chip was scanned using a Luxscan ⁇ 10K/A microarray scanner; The nuclear water was eluted by heating at 95 ° C for 5 min, and the resulting solution was dried at 60 ° C to a volume of 92 ⁇ L, which was left for PCR amplification;
  • TARMA modified former primer 5'-TARMA ⁇ GACAGGCAGGACACCGTAAC-3'
  • Biotinylated post-primer 5'-Biotin-GAAGAGGAGGGAGGTAGCAG-3'
  • Chip preparation Firstly, a microfluidic channel template was fabricated by using a combination of photolithography mask and chemical etching as a PDMS channel to make an abrasive tool; a PDMS precursor and a mass ratio of 10:1 were weighed and cured. The agent is thoroughly mixed, vacuumed and poured onto a microfluidic channel template to obtain a PDMS microfluidic channel; a lactoferrin and a negative protein microarray having a concentration of 5 mg/mL are prepared on a glass substrate using a spotting apparatus. The PDMS and the spotted glass substrate are simultaneously plasma-treated, and then closely adhered together as a screening chip for each subsequent screening;
  • PCR amplification process The solution obtained in the step (3) is divided into 4 parts of the same solution having a volume of 23 ⁇ L, and 25 ⁇ L, 2 ⁇ Taq polymerase, 1 ⁇ L, 20 ⁇ M TAMRA-labeled forward primer and 1 ⁇ L are sequentially added in each portion. 20 ⁇ M biotinylated backward primers were mixed and homogenized for PCR amplification; the thermal cycling process of PCR was as follows: 94 ° C for 5 min; cycle 94 ° C for 30 s, 60.5 ° C for 30 s, 72 ° C for 30 s for 10 rounds, 72 ° C for 5 min to terminate the reaction.
  • the PCR product obtained in the step (4) is thoroughly mixed with 800 ⁇ L of Promega magnetic beads, and the supernatant is removed after shaking for 1 hour on a shaker; and 25 ⁇ L, 50 mM NaOH is vortexed for 5 minutes to connect the magnetic beads.
  • the double-stranded dissociation; the supernatant was aspirated and sequentially added with 12.5 ⁇ L of 100 mM HCl, 25 ⁇ L of DPEC water, and 62.5 ⁇ L of 2 ⁇ PBSM, and the resulting solution was used as a secondary library for the second round of screening, and its content was about 60 pmol;
  • the second round of screening the library was pumped into the negative sieve channel at a flow rate of 5 ⁇ L/min, and reacted at room temperature for 50 min; then the library was fed into the positive sieve channel at a flow rate of 5 ⁇ L/min, under normal temperature conditions.
  • the reaction was carried out for 50 min; then 150 ⁇ L of 1 ⁇ PBS buffer solution was passed at a flow rate of 30 ⁇ L/min to remove unreacted chains in the positive sieve channel; the PDMS layer was gently torn off, and the chip was scanned using a Luxscan ⁇ 10K/A microarray scanner; The nuclear water was eluted by heating at 95 ° C for 5 min, and the resulting solution was dried at 60 ° C to a volume of 92 ⁇ L, which was left for PCR amplification;
  • the sequence of the resulting test aptamer is shown in SEQ ID NO. 5 to SEQ ID NO.
  • FITC fluorescein isothiocyanate
  • base sequence AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT
  • step (1) The mixture of the step (1) was placed in a 96-well plate at 37 ° C for 15 min, and directly scanned with a BioTeK microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 528 nm.
  • Example 8 Method for detecting lactoferrin content
  • FITC fluorescein isothiocyanate
  • step (1) The mixture of the step (1) was placed in a 96-well plate at 37 ° C for 15 min, and directly scanned with a BioTeK microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 528 nm.
  • Example 9 Method for detecting lactoferrin content
  • FITC fluorescein isothiocyanate
  • base sequence AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT
  • step (1) The mixture of the step (1) was placed in a 96-well plate at 37 ° C for 15 min, and directly scanned with a BioTeK microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 528 nm.
  • Example 10 Method for detecting lactoferrin content
  • FITC fluorescein isothiocyanate
  • step (1) The mixture of the step (1) was placed in a 96-well plate at 37 ° C for 15 min, and directly scanned with a BioTeK microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 528 nm.
  • Example 11 Method for detecting lactoferrin content
  • FITC fluorescein isothiocyanate
  • step (1) The mixture of the step (1) was placed in a 96-well plate at 37 ° C for 15 min, and directly scanned with a BioTeK microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 528 nm.
  • Example 12 Method for detecting lactoferrin content
  • FITC fluorescein isothiocyanate
  • step (1) The mixture of the step (1) was placed in a 96-well plate at 37 ° C for 15 min, and directly scanned with a BioTeK microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 528 nm.
  • Example 13 Screening of ⁇ -lactalbumin aptamers
  • ⁇ -lactalbumin aptamer screening includes detailed processes such as chip preparation, positive and negative screening process, and PCR amplification, as described below, wherein:
  • TARMA modified forward primer 5'-TARMA-GACAGGCAGGACACCGTAAC-3'
  • Biotinylated backward primer 5'Biotin-GAAGAGGAGGGAGGTAGCAG-3'
  • Chip preparation Firstly, a microfluidic channel template was fabricated by using a combination of photolithography mask and chemical etching as a PDMS channel to make an abrasive tool; a PDMS precursor and a mass ratio of 10:1 were weighed and cured. The agent is thoroughly mixed, vacuumed and poured onto a microfluidic channel template to obtain a PDMS microfluidic channel; a lactoferrin and a negative protein microarray having a concentration of 5 mg/mL are prepared on a glass substrate using a spotting apparatus. The PDMS and the spotted glass substrate are simultaneously plasma-treated, and then closely adhered together as a screening chip for each subsequent screening;
  • ⁇ -lactalbumin-bound ssDNA sequence was eluted by heating with DPEC water at 95 ° C for 5 min, and the resulting solution was dried to a volume of 92 ⁇ L under high purity nitrogen at 50 ° C;
  • PCR amplification process The solution obtained in the step (2) is divided into 4 parts of the same solution each having a volume of 23 ⁇ L, and 25 ⁇ L of each, 2 ⁇ Taq polymerase, 1 ⁇ L, 20 ⁇ M TAMRA-labeled forward primer and 1 ⁇ L are sequentially added. 20 ⁇ M biotinylated backward primers were mixed and homogenized for PCR amplification; the thermal cycling process of PCR was as follows: 94 ° C for 5 min; cycle 94 ° C for 30 s, 60.5 ° C for 30 s, 72 ° C for 30 s for 10 rounds, 72 ° C for 5 min to terminate the reaction.
  • product of this step obtained was diluted 10-fold as template again amplified: after taking 5 ⁇ L diluted PCR product with 25 ⁇ L, 2 ⁇ SYBR Premix Ex Taq TM enzyme, 1 ⁇ L, before 20 ⁇ M TAMRA-labeled forward primer, 1 ⁇ L, 20 ⁇ M biological The normalized backward primer and 18 ⁇ L DPEC water were well mixed. PCR amplification was performed for 10 ⁇ L of the sample to the microplate. The fluorescence signal was detected by BioTek. The highest fluorescence signal was used as the optimal number of rounds. Amplify the number of rounds;
  • the PCR product obtained in the step (4) was thoroughly mixed with 800 ⁇ L of Promega magnetic beads, and the supernatant was removed after shaking for 1 hour on a shaker; and 25 ⁇ L, 50 mM NaOH was vortexed for 5 min to connect the magnetic beads. Double-strand dissociation; aspirate the supernatant and sequentially add 12.5 ⁇ L of 100 mM HCl, 25 ⁇ L of H 2 O, 62.5 ⁇ L of 2 ⁇ PBSM, and the resulting solution is used as a secondary library for the next round of screening, and its content is about 40 pmol;
  • the second round of screening the library was pumped into the negative sieve channel at a flow rate of 2.5 ⁇ L/min, and reacted at room temperature for 50 min; then the pump was used to input the library into the positive sieve channel at a flow rate of 2.5 ⁇ L/min, at room temperature.
  • the reaction was carried out for 50 min; then 150 ⁇ L of 1 ⁇ PBS buffer solution was passed at a flow rate of 15 ⁇ L/min to remove unreacted chains in the positive sieve channel; the PDMS layer was gently peeled off, and the chip was scanned using a Luxscan ⁇ 10K/A microarray scanner; The protein-bound chain was eluted with non-nuclear water at 95 ° C for 5 min, and the resulting solution was dried at 60 ° C to a volume of 92 ⁇ L, which was left for PCR amplification;
  • ⁇ -lactoglobulin aptamer screening includes detailed processes such as chip preparation, positive and negative screening process, and PCR amplification, as described below, wherein:
  • TARMA modified forward primer 5'-TARMA-GACAGGCAGGACACCGTAAC-3'
  • Biotinylated backward primer 5'Biotin-GAAGAGGAGGGAGGTAGCAG-3'
  • Chip preparation Firstly, a microfluidic channel template was fabricated by using a combination of photolithography mask and chemical etching as a PDMS channel to make an abrasive tool; a PDMS precursor and a mass ratio of 10:1 were weighed and cured. The agent is thoroughly mixed, vacuumed and poured onto a microfluidic channel template to obtain a PDMS microfluidic channel; a sampler is used to prepare a ⁇ -lactoglobulin and a negative protein microparticle at a concentration of 5 mg/mL on a glass substrate. The array, and the PDMS and the spotted glass substrate are simultaneously plasma treated, and then closely adhered together as a screening chip for each subsequent screening;
  • ⁇ -lactoglobulin-conjugated ssDNA sequence was eluted by heating with DPEC water at 95 ° C for 5 min, and the resulting solution was dried to a volume of 92 ⁇ L under high purity nitrogen at 50 ° C;
  • PCR amplification process The solution obtained in the step (2) is divided into 4 parts of the same solution each having a volume of 23 ⁇ L, and 25 ⁇ L of each, 2 ⁇ Taq polymerase, 1 ⁇ L, 20 ⁇ M TAMRA-labeled forward primer and 1 ⁇ L are sequentially added. 20 ⁇ M biotinylated backward primers were mixed and homogenized for PCR amplification; the thermal cycling process of PCR was as follows: 94 ° C for 5 min; cycle 94 ° C for 30 s, 60.5 ° C for 30 s, 72 ° C for 30 s for 10 rounds, 72 ° C for 5 min to terminate the reaction.
  • product of this step obtained was diluted 10-fold as template again amplified: after taking 5 ⁇ L diluted PCR product with 25 ⁇ L, 2 ⁇ SYBR Premix Ex Taq TM enzyme, 1 ⁇ L, before 20 ⁇ M TAMRA-labeled forward primer, 1 ⁇ L, 20 ⁇ M biological The normalized backward primer and 18 ⁇ L DPEC water were well mixed. PCR amplification was performed for 10 ⁇ L of the sample to the microplate. The fluorescence signal was detected by BioTek. The highest fluorescence signal was used as the optimal number of rounds. Amplify the number of rounds;
  • the PCR product obtained in the step (4) was thoroughly mixed with 800 ⁇ L of Promega magnetic beads, and the supernatant was removed after shaking for 1 hour on a shaker; and 25 ⁇ L, 50 mM NaOH was vortexed for 5 min to connect the magnetic beads. Double-strand dissociation; aspirate the supernatant and sequentially add 12.5 ⁇ L of 100 mM HCl, 25 ⁇ L of H 2 O, 62.5 ⁇ L of 2 ⁇ PBSM, and the resulting solution is used as a secondary library for the next round of screening, and its content is about 40 pmol;
  • the second round of screening the library was pumped into the negative sieve channel at a flow rate of 2.5 ⁇ L/min, and reacted at room temperature for 50 min; then the pump was used to input the library into the positive sieve channel at a flow rate of 2.5 ⁇ L/min, at room temperature.
  • the reaction was carried out for 50 min; then 150 ⁇ L of 1 ⁇ PBS buffer solution was passed at a flow rate of 15 ⁇ L/min to remove unreacted chains in the positive sieve channel; the PDMS layer was gently peeled off, and the chip was scanned using a Luxscan ⁇ 10K/A microarray scanner; The protein-bound chain was eluted with non-nuclear water at 95 ° C for 5 min, and the resulting solution was dried at 60 ° C to a volume of 92 ⁇ L, which was left for PCR amplification;

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Abstract

提供一种利用微阵列微流控芯片筛选乳铁蛋白适配体的方法，该方法将正负筛过程集合在一块微流控芯片上，筛选7轮后即获得高亲和力的适配体。还提供一种用于检测乳铁蛋白含量的适配体及其在检测乳铁蛋白中的应用。

Description

一种利用微阵列微流控芯片筛选适配体的方法 技术领域
本发明属于生物检测技术领域，具体涉及一种利用微阵列微流控芯片筛选适配体的方法。
背景技术
适配体具有与抗体相类似的性质，但是在许多方面还体现出了优于抗体的特点：(1)特异性更强，对目标靶分子的亲和性比抗体更高；(2)适配体较抗体更容易获得(可人工合成，不依赖于动物和细胞)，并且可以大量、快速的在体外合成，制备方法更为简单快捷；(3)可以针对不同种类的目标靶分子进行筛选，包括生物毒性分子以及只具有半抗原性的分子，拓宽了其应用范围；(4)无免疫原性，可在体内反复使用；(5)稳定性优于抗体，利于储存。随着适配体技术的不断进步，适配体在许多领域将有可能成为抗体技术有益的补充。正是基于适配体的各种优点，适配体在分析、临床、环境、分子识别及药物筛选等方面都显示出了广阔的应用前景。
目前，基于纳米材料的适配体筛选技术可免除蛋白质在固相基片表面的固定，可有效减少筛选轮数，加速筛选流程。同时，基于微流控技术来提高筛选效果的方法获得了广泛的研究。这些方法包括：毛细管电泳的微流控适配体筛选，溶胶凝胶微流控适配体筛选，表面等离子共振(SPR)微流控适配体筛选，基于磁珠粒子的微流控筛选，以及基于各种材料微球的微流控筛选技术。科学工作者为了提高PCR扩增效率，采用琼脂糖液滴PCR扩增技术，聚乳液PCR扩增(ePCR)技术，此技术可以将阳性克隆子可以直接挑选出来进行测序，避免了传统适配体筛选过程克隆测序过程的繁琐。上述这些方法目的在于为了使适配体的筛选更加省时，自动化，高效并且可以降低整个筛选的成本。我们的工作在于发展一种更加稳定、筛选过程可重复、筛选步骤可量化的适配体筛选新技术。本方法优化了适配体筛选的过程使得适配体筛选过程更加简单化、标准化。
发明内容
本发明所要解决的技术问题是提供一种新型微阵列微流控芯片应用于适配体筛选的方法。
为解决上述技术问题，本发明采用的技术方案如下：
一种利用微阵列微流控芯片筛选适配体的方法，所述的微阵列微流控芯片按照如下方法制备：
将PDMS前聚体与固化剂按照质量比为5∶1～100∶1的比例混合，优选10∶1，混合均匀后将其放入真空干燥器中，连接循环水真空泵进行抽真空30min除气泡，再浇到微流控通道模板上，经固化后得到PDMS微流控通道，在玻璃基片上点制浓度为0.1～10mg/mL的乳铁蛋白和阴性蛋白微阵列，对PDMS微流控通道和点样后的玻璃基片同时等离子处理，将上述两种基片紧密贴合，得到微阵列微流控芯片。
上述利用微阵列微流控芯片筛选适配体的方法，具体操作步骤如下：
(1)筛选准备：将微阵列微流控芯片放入恒温水浴锅中，25～40℃下孵育蛋白1～12h；再通入5～20mg/ml BSA和0.01～0.5mM随机序列短链ssDNA(20nt)在25～40℃条件下封闭1～3h，之后用150μL 1×PBST溶液清洗正负筛通道；
(2)第一轮筛选：将0.1～10nmol，125μL原始文库在95℃加热3～15min，并立即在冰上冷冻10s～5min；用注射泵以1～5μL/min流速将文库通入正筛通道，在25～40℃条件下反应30～120min；之后以5～30μL/min流速通入150μL 1×PBS缓冲溶液除去正筛通道中未结合的链；撕掉PDMS层，使用微阵列扫描仪扫描芯片；最后用DPEC水在95℃下加热3～10min洗脱芯片上乳铁蛋白结合的ssDNA，所得溶液在40～60℃条件下用高纯氮吹干至体积为25～250μL；
(3)PCR扩增过程：将步骤(2)所得溶液分为体积均为23μL的3～10份相同溶液，每份依次加入25μL、2×Taq聚合酶，1μL、20μM TAMRA标记的前向引物和1μL、20μM生物素化的后向引物，混合均匀后进行PCR扩增；
该步骤获得的产物作为模板稀释5～40倍后进行再一次扩增：取5μL稀释后的PCR产物与1μL 2×SYBR Premix Ex TaqTM酶、1μL 20μM TAMRA标记的前向引物、1μL 20μM生物素化的后向引物以及18μL超纯水混合均匀，PCR扩增每进行一轮便取10μL样品至微孔板中用BioTek检测其荧光信号，荧光信号最高者作为最佳轮数，剩余产物均扩增该轮数；
(4)分离纯化：将步骤(3)得到的PCR产物与800μL～5mL Promega磁珠充分混匀，在快速摇动0.5～2h后除去上清液；再加入25μL、0.01～0.1M NaOH溶液涡旋3～15min，使磁珠上连接的双链解离到溶液中；之后取上层清液并依次加入12.5μL、0.1M HCl 25 μL，DPEC水和62.5μL 2×PBSM缓冲溶液，所得的中性溶液作为下一轮筛选的次级文库，其含量为40pmol～100pmol；
(5)第二轮筛选：用注射泵以1～5μL/min流速将文库在25～40℃条件下以1～5μL/min依次输通入负筛，正筛通道；接着以5～30μL/min流速通入150μL，1×PBS缓冲溶液除去正筛通道中未反应的ssDNA序列；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用DPEC水在95℃下加热3～10min洗脱芯片上乳铁蛋白结合的ssDNA序列，所得溶液在60℃吹干至体积为25～250μL，留作PCR扩增；
(7)重复步骤(4)、(5)、(6)至第八轮筛选；
(8)将筛选的第五、六、七轮PCR扩增产物测序，重复上述的筛选过程，随机选取样品测序，分析测序结果，得到重复序列大于2的所有序列，再用IDT软件对得到的重复序列进行二级结构分析，IDT软件根据序列自动生成可能的二级结构和序列的ΔG，将对序列末端序列进行二次裁剪以获得更为有效的序列和更稳定的二级结构(ΔG值最小)，获得最佳的适配体。
步骤(2)中，所述文库的中间为40个随机碱基，随机碱基5’端的核苷酸序列如SEQ ID NO.1所示，随机碱基3’端的核苷酸序列如SEQ ID NO.2所示，
步骤(3)中，所述的前向引物，其核苷酸序列如SEQ ID NO.3所示；所述的后向引物，其核苷酸序列如SEQ ID NO.4所示。
一种用于检测乳铁蛋白含量的适配体，所述适配体的核苷酸序列如SEQ ID NO.5～65所示。
上述用于检测乳铁蛋白含量的适配体在检测乳铁蛋白中的应用在本发明的保护范围之内。
本发明原理：适配体的产生是借由一种指数富集配体的系统进化技术(systematic evolution ofligands by exponential enrichment，SELEX)，利用该技术可以从随机单链核酸序列库中筛选到特异性与靶物质高度亲和的核酸配体(aptamer)，本发明基于微阵列技术(Microarray)的大规模、高通量集成，占用体积小等特点与微流控技术(Microfluidic)自动化流体输送，分子反应效率增强和可避免外界污染的封闭式反应腔室等优势，将适 配体筛选的负筛过程和正筛过程集合在一块微流控芯片上，可快速高效完成适配体的筛选。通过筛选芯片正筛通道特异性捕获ssDNA序列后的微阵列荧光强度变化，可以直接直观的监测筛选过程中次级文库随着每一轮负筛选、正筛选的进化效果。本方法筛选7轮后即获得高亲和力的适配体。
此微阵列微流控芯片筛选适配体的方法是我们首次设计采用的。蛋白质微阵列的制作方法、筛选文库与蛋白阵列过程监控通用；另外，PCR过程，PCR产物纯化，次级文库的再生过程均可通用。所以，利用微阵列微流控芯片筛选适配体的方法具有很好的通用型和重要的研究意义。
有益效果：
本发明主要公开了一种微阵列微流控芯片筛选适配体的方法，该筛选芯片将正负筛过程集合在一块微流控芯片上，结合微流控和微阵列技术优点，筛选过程简单便捷且筛选7轮后即获得高亲和力的适配体。同时本发明还公开了乳铁蛋白适配体筛选的具体步骤，包括芯片制备、正负筛过程、PCR扩增等详细过程。该方法筛选出的适配体对靶标蛋白的特异性好，亲和性高；且该适配体较抗体更容易获得，可以大量、快速的在体外合成，制备方法更为简单快捷，因此该适配体在许多领域将有望成为抗体技术有益的补充。此外，该正负筛集合的芯片模式方便、快捷、高效，可以用于其它蛋白、核酸适配体的筛选，为其他适配体筛选工作者提供了很好的思路设计和参考。
附图说明
图1微阵列微流控适用于适配体筛选流程图。
图2第一次筛选得到的结果图，(a)每一轮文库的用量，(b)适配体在芯片上的荧光信号强度，(c)适配体在芯片上的成像图，(d)芯片清洗后的图像。
图3第二次筛选得到的结果图，(a)每一轮文库的用量，(b)适配体在芯片上的荧光信号强度，(c)适配体在芯片上的成像图，(d)芯片清洗后的图像图。
图4第三次筛选得到的结果图，(a)每一轮文库的用量，(b)适配体在芯片上的荧光信号强度，(c)适配体在芯片上的成像图，(d)芯片清洗后的图像图。
图5其他适配体对乳铁蛋白荧光偏振检测线性图。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：微阵列微流控芯片的制备
芯片制备：利光刻掩模与化学腐蚀相结合的技术制作微流控通道模板，留作后面的PDMS通道制备；称取质量比为10∶1的PDMS前聚体与固化剂充分混合，抽真空后浇到微流控通道模板上经固化后得到PDMS微流控通道；使用点样仪在玻璃基片点制浓度为5mg/mL的乳铁蛋白和阴性蛋白微阵列，并对PDMS和点样后的玻璃基片同时等离子处理，之后紧密贴合共同作为筛选芯片，用于接下来的每轮筛选；
实施例2：PCR扩增
将芯片上洗脱下来的溶液分为体积均为23μL的6份相同溶液，每份依次加入25μL，2×Taq聚合酶，1μL，20μM TAMRA标记的前向引物和1μL，20μM生物素化的后向引物，混合均匀后进行PCR扩增；PCR的热循环过程如下：94℃5min；循环94℃30s，60.5℃30s，72℃30s过程10轮，72℃5min终止反应；该步骤获得的产物稀释10倍后作为模板进行再一次扩增：取5μL稀释后PCR产物与25μL，2×SYBR Premix Ex TaqTM酶，1μL，20μM TAMRA标记的前向引物，1μL，20μM生物素化的后向引物以及18μL超纯水充分混合均匀，PCR扩增每进行一轮便取10μL样品至微孔板中用BioTek检测其荧光信号，荧光信号最高者作为最佳轮数，剩余产物均扩增该轮数。
实施例3：乳铁蛋白适配体筛选
乳铁蛋白适配体筛选的具体步骤，包括芯片制备、正负筛过程、PCR扩增等详细过程，具体如下所述，其中：
文库：5′-TAMRA-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3′
TARMA修饰的前向引物：5′-TARMA-GACAGGCAGGACACCGTAAC-3′
生物素化的后向引物：5′Biotin-GAAGAGGAGGGAGGTAGCAG-3′
(1)芯片制备：利首先利光刻掩模与化学腐蚀相结合的技术制作微流控通道模板作为PDMS通道制做磨具；后称取质量比为10∶1的PDMS前聚体与固化剂充分混合，抽真空后浇到微流控通道模板上经固化后得到PDMS微流控通道；使用点样仪在玻璃基片点制浓度为5mg/mL的乳铁蛋白和阴性蛋白微阵列，并对PDMS和点样后的玻璃基 片同时等离子处理，之后紧密贴合共同作为筛选芯片，用于接下来的每轮筛选；
(2)筛选准备：将步骤(1)得到的筛选芯片放入恒温水浴箱中37℃下孵育蛋白2h；再通入20mg/ml BSA和0.01mM随机序列短链ssDNA(20nt)在37℃条件下封闭1h；之后用150μL，1×PBST溶液清洗正负筛通道；
(3)第一轮筛选：将0.5nmol，125μL原始文库在95℃加热5min，并立即在冰上冷冻10s；再用注射泵以2.5μL/min流速将文库输进正筛通道，在常温条件下反应50min；之后以15μL/min流速通入150μL，1×PBS缓冲溶液以除去正筛通道中未结合的ssDNA序列；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用DPEC水在95℃下加热5min洗脱乳铁蛋白结合的ssDNA序列，所得溶液在50℃条件下高纯氮吹干至体积为69μL；
(4)PCR扩增过程：将步骤(2)所得溶液分为体积均为23μL的3份相同溶液，每份依次加入25μL，2×Taq聚合酶，1μL，20μM TAMRA标记的前向引物和1μL，20μM生物素化的后向引物，混合均匀后进行PCR扩增；PCR的热循环过程如下：94℃5min；循环94℃30s，60.5℃30s，72℃30s过程10轮，72℃5min终止反应；该步骤获得的产物稀释10倍后作为模板进行再一次扩增：取5μL稀释后PCR产物与25μL，2×SYBR Premix Ex TaqTM酶，1μL，20μM TAMRA标记的前向引物，1μL，20μM生物素化的后向引物以及18μL DPEC水充分混合均匀，PCR扩增每进行一轮便取10μL样品至微孔板中用BioTek检测其荧光信号，荧光信号最高者作为最佳轮数，剩余产物均扩增该轮数；
(5)分离纯化：将步骤(4)得到的PCR产物与600μL Promega磁珠充分混匀，在摇床上快速摇动1h后除去上清液；再加入25μL，50mM NaOH涡旋5min将磁珠上连接的双链解离；吸取上层清液并依次加入12.5μL 100mM HCl，25μL H2O，62.5μL 2×PBSM中和，所得的溶液作为下一轮筛选的次级文库，其含量约为40pmol；
(6)第二轮筛选：用泵以2.5μL/min流速将文库输进负筛通道，在常温条件下反应50min；再用泵以2.5μL/min流速将文库输进正筛通道，在常温条件下反应50min；接着以15μL/min流速通入150μL 1×PBS缓冲溶液除去正筛通道中未反应的链；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用无核水在95℃下加热5min洗脱蛋白结合的链，所得溶液在60℃吹干至体积为92μL，留作PCR扩增；
(7)重复步骤(4)、(5)、(6)至第八轮筛选；
(8)将筛选的第五、六、七轮PCR扩增产物送至上海生工测序，每轮随机选取120条测序，再用IDT软件对得到的链进行二级结构分析，最终获得最佳的适配体。
实施例4：乳铁蛋白适配体筛选
乳铁蛋白适配体筛选的具体步骤，包括芯片制备、正负筛过程、微量PCR扩增等详细过程，具体如下所述，其中：
文库：5′-TAMRA-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3′
TARMA修饰的前向引物：5′-TARMA-GACAGGCAGGACACCGTAAC-3′
生物素化的后向引物：5′-Biotin-GAAGAGGAGGGAGGTAGCAG-3′
(1)芯片制备：利首先利光刻掩模与化学腐蚀相结合的技术制作微流控通道模板作为PDMS通道制做磨具；后称取质量比为10∶1的PDMS前聚体与固化剂充分混合，抽真空后浇到微流控通道模板上经固化后得到PDMS微流控通道；使用点样仪在玻璃基片点制浓度为2.5mg/mL的乳铁蛋白和阴性蛋白微阵列，并对PDMS和点样后的玻璃基片同时等离子处理，之后紧密贴合共同作为筛选芯片，用于接下来的每轮筛选；
(2)筛选准备：将步骤(1)得到的筛选芯片放入水浴锅中37℃下孵育蛋白3h；再通入20mg/ml BSA和0.01mM随机序列短链ssDNA(20nt)在37℃条件下封闭2h；之后用150μL，1×PBST溶液清洗通道；
(3)第一轮筛选：将1nmol，125μL原始文库在95℃加热5min，并立即在冰上冷冻10s；再用泵以2.5μL/min流速将文库输进正筛通道，在常温条件下反应50min；之后以15μL/min流速通入150μL，1×PBS缓冲溶液以除去正筛通道中未结合的ssDNA序列；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用DPEC水在95℃下加热5min洗脱乳铁蛋白结合的链，所得溶液在50℃条件下高纯氮吹干至体积为92μL；
(4)PCR扩增过程：将步骤(3)所得溶液分为体积均为23μL的4份相同溶液，每份依次加入25μL，2×Taq聚合酶，1μL，20μM TAMRA标记的前向引物和1μL，20μM生物素化的后向引物，混合均匀后进行PCR扩增；PCR的热循环过程如下：94℃5min；循环94℃30s，60.5℃30s，72℃30s过程10轮，72℃5min终止反应；该步骤获得的产物稀释10倍后作为模板进行再一次扩增：取5μL稀释后PCR产物与25μL，2×SYBR Premix Ex TaqTM酶，1μL，20μM TAMRA标记的前向引物，1μL，20μM生物素化的后向引物以及18μL超纯水充分混合均匀，PCR扩增每进行一轮便取10μL 样品至微孔板中用BioTek检测其荧光信号，荧光信号最高者作为最佳轮数，剩余产物均扩增该轮数；
(5)分离纯化：将步骤(4)得到的PCR产物与800μL Promega磁珠充分混匀，在摇床上快速摇动1h后除去上清液；再加入25μL，1M NaOH涡旋5min将磁珠上连接的双链解离；吸取上层清液并依次加入12.5μL 2M HCl，25μL H2O，62.5μL 2×PBSM中和，所得的溶液作为第二轮筛选的次级文库，其浓度为90pmol；
(6)第二轮筛选：用泵以2.5μL/min流速将文库输进负筛通道，在常温条件下反应50min；再用泵以2.5μL/min流速将文库输进正筛通道，在常温条件下反应50min；接着以15μL/min流速通入150μL 1×PBS缓冲溶液除去正筛通道中未反应的链；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用无核水在95℃下加热5min洗脱蛋白结合的链，所得溶液在60℃吹干至体积为92μL，留作PCR扩增；
(7)重复步骤(4)、(5)、(6)至第八轮筛选；
(8)将筛选的第五、六、七轮PCR扩增产物送至上海生工测序，每轮随机选取120条测序，再用IDT软件对得到的链进行二级结构分析，最终获得最佳的适配体。
实施例5：乳铁蛋白适配体筛选
乳铁蛋白适配体筛选的具体步骤，包括芯片制备、正负筛过程、微量PCR扩增等详细过程，具体如下所述，其中：
文库：5′-TAMRA-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3′
TARMA修饰的前向引物：5′-TARMA-GACAGGCAGGACACCGTAAC-3′
生物素化的后向引物：5′-Biotin-GAAGAGGAGGGAGGTAGCAG-3′
(1)芯片制备：利首先利光刻掩模与化学腐蚀相结合的技术制作微流控通道模板作为PDMS通道制做磨具；后称取质量比为10∶1的PDMS前聚体与固化剂充分混合，抽真空后浇到微流控通道模板上经固化后得到PDMS微流控通道；使用点样仪在玻璃基片点制浓度为5mg/mL的乳铁蛋白和阴性蛋白微阵列，并对PDMS和点样后的玻璃基片同时等离子处理，之后紧密贴合共同作为筛选芯片，用于接下来的每轮筛选；
(2)筛选准备：将步骤(1)得到的筛选芯片放入恒温水浴箱中37℃下孵育蛋白2h；再通入20mg/ml BSA和0.01mM随机序列短链ssDNA(20nt)在37℃条件下封闭1h；之后用150μL，1×PBST溶液清洗通道；
(3)第一轮筛选：将1nmol，125μL原始文库在95℃加热5min，并立即在冰上冷 冻10s；再用泵以5μL/min流速将文库输进正筛通道，在常温条件下反应50min；之后以30μL/min流速通入150μL，1×PBS缓冲溶液以除去正筛通道中未结合的ssDNA序列；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用无核水在95℃下加热5min洗脱乳铁蛋白结合的链，所得溶液在50℃条件下高纯氮吹干至体积为92μL；
(4)PCR扩增过程：将步骤(3)所得溶液分为体积均为23μL的4份相同溶液，每份依次加入25μL，2×Taq聚合酶，1μL，20μM TAMRA标记的前向引物和1μL，20μM生物素化的后向引物，混合均匀后进行PCR扩增；PCR的热循环过程如下：94℃5min；循环94℃30s，60.5℃30s，72℃30s过程10轮，72℃5min终止反应；该步骤获得的产物稀释10倍后作为模板进行再一次扩增：取5μL稀释后PCR产物与25μL，2×SYBR Premix Ex TaqTM酶，1μL，20μM TAMRA标记的前向引物，1μL，20μM生物素化的后向引物以及18μL DPEC水充分混合均匀，PCR扩增每进行一轮便取10μL样品至微孔板中用BioTek检测其荧光信号，荧光信号最高者作为最佳轮数，剩余产物均扩增该轮数；
(5)分离纯化：将步骤(4)得到的PCR产物与800μL Promega磁珠充分混匀，在摇床上快速摇动1h后除去上清液；再加入25μL，50mM NaOH涡旋5min将磁珠上连接的双链解离；吸取上层清液并依次加入12.5μL 100mM HCl，25μL H2O，62.5μL 2×PBSM中和，所得的溶液作为第二轮筛选的次级文库其含量为60pmol；
(6)第二轮筛选：用泵以5μL/min流速将文库输进负筛通道，在常温条件下反应50min；再用泵以5μL/min流速将文库输进正筛通道，在常温条件下反应50min；接着以30μL/min流速通入150μL 1×PBS缓冲溶液除去正筛通道中未反应的链；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用无核水在95℃下加热5min洗脱蛋白结合的链，所得溶液在60℃吹干至体积为92μL，留作PCR扩增；
(7)重复步骤(4)、(5)、(6)至第八轮筛选；
(8)将筛选的第五、六、七轮PCR扩增产物送至上海生工做克隆测序，每轮随机选取120个克隆子进行测序，再用IDT软件对得到的链进行二级结构分析，最终获得最佳的适配体。
实施例6：乳铁蛋白适配体筛选
乳铁蛋白适配体筛选的具体步骤，包括芯片制备、正负筛过程、微量PCR扩增等 详细过程，具体如下所述，其中：
文库：5′-TAMRA-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3′
TARMA修饰的前项引物：5′-TARMA～GACAGGCAGGACACCGTAAC-3′
生物素化的后项引物：5′-Biotin-GAAGAGGAGGGAGGTAGCAG-3′
(1)芯片制备：利首先利光刻掩模与化学腐蚀相结合的技术制作微流控通道模板作为PDMS通道制做磨具；后称取质量比为10∶1的PDMS前聚体与固化剂充分混合，抽真空后浇到微流控通道模板上经固化后得到PDMS微流控通道；使用点样仪在玻璃基片点制浓度为5mg/mL的乳铁蛋白和阴性蛋白微阵列，并对PDMS和点样后的玻璃基片同时等离子处理，之后紧密贴合共同作为筛选芯片，用于接下来的每轮筛选；
(2)筛选准备：将步骤(1)得到的筛选芯片放入水浴锅中37℃下孵育蛋白2h；再通入20mg/ml BSA和0.01mM随机序列短链ssDNA(20nt)在37℃条件下封闭1h；之后用150μL，1×PBST溶液清洗通道；
(3)第一轮筛选：将1nmol，125μL原始文库在95℃加热5min，并立即在冰上冷冻10；再用注射泵以2.5μL/min流速将文库通入负筛、正筛通道，在常温条件下反应50min；接着以15μL/min流速通入150μL 1×PBS缓冲溶液除去正筛通道中未结合的ssDNA序列；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用无核水在95℃下加热5min洗脱蛋白结合的ssDNA，所得溶液在50℃条件下高纯氮吹干至体积为92μL；
(4)PCR扩增过程：将步骤(3)所得溶液分为体积均为23μL的4份相同溶液，每份依次加入25μL，2×Taq聚合酶，1μL，20μM TAMRA标记的前向引物和1μL，20μM生物素化的后向引物，混合均匀后进行PCR扩增；PCR的热循环过程如下：94℃5min；循环94℃30s，60.5℃30s，72℃30s过程10轮，72℃5min终止反应；该步骤获得的产物稀释10倍后作为模板进行再一次扩增：取5μL稀释后PCR产物与25μL，2×SYBRPremix Ex TaqTM酶，1μL，20μM TAMRA标记的前向引物，1μL，20μM生物素化的后向引物以及18μL超纯水充分混合均匀，PCR扩增每进行一轮便取10μL样品至微孔板中用BioTek检测其荧光信号，荧光信号最高者作为最佳轮数，剩余产物均扩增该轮数；
(5)分离纯化：将步骤(4)得到的PCR产物与800μL Promega磁珠充分混匀，在摇床上快速摇动1h后除去上清液；再加入25μL，50mM NaOH涡旋5min将磁珠上连 接的双链解离；吸取上层清液并依次加入12.5μL 100mM HCl，25μL DPEC水，62.5μL 2×PBSM中和，所得的溶液作为第二轮筛选的次级文库，其含量约为60pmol；
(6)第二轮筛选：用泵以5μL/min流速将文库输进负筛通道，在常温条件下反应50min；再用泵以5μL/min流速将文库输进正筛通道，在常温条件下反应50min；接着以30μL/min流速通入150μL 1×PBS缓冲溶液除去正筛通道中未反应的链；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用无核水在95℃下加热5min洗脱蛋白结合的链，所得溶液在60℃吹干至体积为92μL，留作PCR扩增；
(7)重复步骤(4)、(5)、(6)至第八轮筛选。
(8)将筛选的第五、六、七轮PCR扩增产物送至上海生工做克隆测序，每轮随机选取120个克隆子进行测序，再用IDT软件对得到的链进行二级结构分析，最终获得最佳的适配体。
得到的检测适配体的序列如SEQ ID NO.5～SEQ ID NO.65所示。
实施例7：荧光偏振法对乳铁蛋白标准样品进行检测：
(1)在10μL，250nM FITC(异硫氰酸荧光素)标记的适配体N2(碱基序列：AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT)溶液中加入100μL浓度为25μg/mL的乳铁蛋白标准样品，充分混匀。
(2)将步骤(1)的混合液置于96微孔板中37℃条件下反应15min，用BioTeK酶标仪直接扫描，激发波长为480nm，发射波长为528nm。
实施例8：检测乳铁蛋白含量的方法
(1)在10μL，250nM FITC(异硫氰酸荧光素)标记的适配体N2(碱基序列：AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT)溶液中加入稀释25-100倍的牛奶样品，充分混匀。
(2)将步骤(1)的混合液置于96微孔板中37℃条件下反应15min，用BioTeK酶标仪直接扫描，激发波长为480nm，发射波长为528nm。
(3)对比标准曲线得到牛奶样品中乳铁蛋白的浓度。
实施例9：检测乳铁蛋白含量的方法
(1)在10μL，250nM FITC(异硫氰酸荧光素)标记的适配体N6(碱基序列：AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT)溶液中 加入稀释25-100倍的牛奶样品，充分混匀。
(2)将步骤(1)的混合液置于96微孔板中37℃条件下反应15min，用BioTeK酶标仪直接扫描，激发波长为480nm，发射波长为528nm。
(3)对比标准曲线得到牛奶样品中乳铁蛋白的浓度，具体见图5(a)。
实施例10：检测乳铁蛋白含量的方法
(1)在10μL，250nM FITC(异硫氰酸荧光素)标记的适配体N14(碱基序列：AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT)溶液中加入稀释25-100倍的牛奶样品，充分混匀。
(2)将步骤(1)的混合液置于96微孔板中37℃条件下反应15min，用BioTeK酶标仪直接扫描，激发波长为480nm，发射波长为528nm。
(3)对比标准曲线得到牛奶样品中乳铁蛋白的浓度，具体见图5(b)。
实施例11：检测乳铁蛋白含量的方法
(1)在10μL，250nM FITC(异硫氰酸荧光素)标记的适配体N16(碱基序列：AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT)溶液中加入稀释25-100倍的牛奶样品，充分混匀。
(2)将步骤(1)的混合液置于96微孔板中37℃条件下反应15min，用BioTeK酶标仪直接扫描，激发波长为480nm，发射波长为528nm。
(3)对比标准曲线得到牛奶样品中乳铁蛋白的浓度，具体见图5(c)。
实施例12：检测乳铁蛋白含量的方法
(1)在10μL，250nM FITC(异硫氰酸荧光素)标记的适配体N31(碱基序列：AGGCAGGACACCGTAACCGGTGCATCTATGGCTACTAGCTTTTCCTGCCT)溶液中加入稀释25-100倍的牛奶样品，充分混匀。
(2)将步骤(1)的混合液置于96微孔板中37℃条件下反应15min，用BioTeK酶标仪直接扫描，激发波长为480nm，发射波长为528nm。
(3)对比标准曲线得到牛奶样品中乳铁蛋白的浓度，具体见图5(d)。
实施例13：α-乳白蛋白适配体筛选
α-乳白蛋白适配体筛选的具体步骤，包括芯片制备、正负筛过程、PCR扩增等详细过程，具体如下所述，其中：
文库：5′-TAMRA-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3′
TARMA修饰的前向引物：5′-TARMA-GACAGGCAGGACACCGTAAC-3′
生物素化的后向引物：5′Biotin-GAAGAGGAGGGAGGTAGCAG-3′
(1)芯片制备：利首先利光刻掩模与化学腐蚀相结合的技术制作微流控通道模板作为PDMS通道制做磨具；后称取质量比为10∶1的PDMS前聚体与固化剂充分混合，抽真空后浇到微流控通道模板上经固化后得到PDMS微流控通道；使用点样仪在玻璃基片点制浓度为5mg/mL的乳铁蛋白和阴性蛋白微阵列，并对PDMS和点样后的玻璃基片同时等离子处理，之后紧密贴合共同作为筛选芯片，用于接下来的每轮筛选；
(2)筛选准备：将步骤(1)得到的筛选芯片放入恒温水浴箱中37℃下孵育蛋白2h；再通入20mg/ml BSA和0.01mM随机序列短链ssDNA(20nt)在37℃条件下封闭1h；之后用150μL，1×PBST溶液清洗正负筛通道；
(3)第一轮筛选：将0.5nmol，125μL原始文库在95℃加热5min，并立即在冰上冷冻10s；再用注射泵以2.5μL/min流速将文库输进正筛通道，在常温条件下反应50min；之后以15μL/min流速通入150μL，1×PBS缓冲溶液以除去正筛通道中未结合的ssDNA序列；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用DPEC水在95℃下加热5min洗脱α-乳白蛋白结合的ssDNA序列，所得溶液在50℃条件下高纯氮吹干至体积为92μL；
(4)PCR扩增过程：将步骤(2)所得溶液分为体积均为23μL的4份相同溶液，每份依次加入25μL，2×Taq聚合酶，1μL，20μM TAMRA标记的前向引物和1μL，20μM生物素化的后向引物，混合均匀后进行PCR扩增；PCR的热循环过程如下：94℃5min；循环94℃30s，60.5℃30s，72℃30s过程10轮，72℃5min终止反应；该步骤获得的产物稀释10倍后作为模板进行再一次扩增：取5μL稀释后PCR产物与25μL，2×SYBR Premix Ex TaqTM酶，1μL，20μM TAMRA标记的前向引物，1μL，20μM生物素化的后向引物以及18μL DPEC水充分混合均匀，PCR扩增每进行一轮便取10μL样品至微孔板中用BioTek检测其荧光信号，荧光信号最高者作为最佳轮数，剩余产物均扩增该轮数；
(5)分离纯化：将步骤(4)得到的PCR产物与800μL Promega磁珠充分混匀，在摇床上快速摇动1h后除去上清液；再加入25μL，50mM NaOH涡旋5min将磁珠上连接的双链解离；吸取上层清液并依次加入12.5μL 100mM HCl，25μL H2O，62.5μL 2×PBSM中和，所得的溶液作为下一轮筛选的次级文库，其含量约为40pmol；
(6)第二轮筛选：用泵以2.5μL/min流速将文库输进负筛通道，在常温条件下反应50min；再用泵以2.5μL/min流速将文库输进正筛通道，在常温条件下反应50min；接着以15μL/min流速通入150μL 1×PBS缓冲溶液除去正筛通道中未反应的链；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用无核水在95℃下加热5min洗脱蛋白结合的链，所得溶液在60℃吹干至体积为92μL，留作PCR扩增；
(7)重复步骤(4)、(5)、(6)至第八轮筛选；
(8)将筛选的第五、六、七轮PCR扩增产物送至上海生工测序，每轮随机选取120条测序，再用IDT软件对得到的链进行二级结构分析，最终获得最佳的适配体。
实施例14：β-乳球蛋白适配体筛选
β-乳球蛋白适配体筛选的具体步骤，包括芯片制备、正负筛过程、PCR扩增等详细过程，具体如下所述，其中：
文库：5′-TAMRA-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3′
TARMA修饰的前向引物：5′-TARMA-GACAGGCAGGACACCGTAAC-3′
生物素化的后向引物：5′Biotin-GAAGAGGAGGGAGGTAGCAG-3′
(1)芯片制备：利首先利光刻掩模与化学腐蚀相结合的技术制作微流控通道模板作为PDMS通道制做磨具；后称取质量比为10∶1的PDMS前聚体与固化剂充分混合，抽真空后浇到微流控通道模板上经固化后得到PDMS微流控通道；使用点样仪在玻璃基片点制浓度为5mg/mL的β-乳球蛋白和阴性蛋白微阵列，并对PDMS和点样后的玻璃基片同时等离子处理，之后紧密贴合共同作为筛选芯片，用于接下来的每轮筛选；
(2)筛选准备：将步骤(1)得到的筛选芯片放入恒温水浴箱中37℃下孵育蛋白2h；再通入20mg/ml BSA和0.01mM随机序列短链ssDNA(20nt)在37℃条件下封闭1h；之后用150μL，1×PBST溶液清洗正负筛通道；
(3)第一轮筛选：将0.5nmol，125μL原始文库在95℃加热5min，并立即在冰上冷冻10s；再用注射泵以2.5μL/min流速将文库输进正筛通道，在常温条件下反应50min；之后以15μL/min流速通入150μL，1×PBS缓冲溶液以除去正筛通道中未结合的ssDNA序列；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用DPEC水在95℃下加热5min洗脱β-乳球蛋白结合的ssDNA序列，所得溶液在50℃条件下高纯氮吹干至体积为92μL；
(4)PCR扩增过程：将步骤(2)所得溶液分为体积均为23μL的4份相同溶液，每 份依次加入25μL，2×Taq聚合酶，1μL，20μM TAMRA标记的前向引物和1μL，20μM生物素化的后向引物，混合均匀后进行PCR扩增；PCR的热循环过程如下：94℃5min；循环94℃30s，60.5℃30s，72℃30s过程10轮，72℃5min终止反应；该步骤获得的产物稀释10倍后作为模板进行再一次扩增：取5μL稀释后PCR产物与25μL，2×SYBR Premix Ex TaqTM酶，1μL，20μM TAMRA标记的前向引物，1μL，20μM生物素化的后向引物以及18μL DPEC水充分混合均匀，PCR扩增每进行一轮便取10μL样品至微孔板中用BioTek检测其荧光信号，荧光信号最高者作为最佳轮数，剩余产物均扩增该轮数；
(5)分离纯化：将步骤(4)得到的PCR产物与800μL Promega磁珠充分混匀，在摇床上快速摇动1h后除去上清液；再加入25μL，50mM NaOH涡旋5min将磁珠上连接的双链解离；吸取上层清液并依次加入12.5μL 100mM HCl，25μL H2O，62.5μL 2×PBSM中和，所得的溶液作为下一轮筛选的次级文库，其含量约为40pmol；
(6)第二轮筛选：用泵以2.5μL/min流速将文库输进负筛通道，在常温条件下反应50min；再用泵以2.5μL/min流速将文库输进正筛通道，在常温条件下反应50min；接着以15μL/min流速通入150μL 1×PBS缓冲溶液除去正筛通道中未反应的链；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用无核水在95℃下加热5min洗脱蛋白结合的链，所得溶液在60℃吹干至体积为92μL，留作PCR扩增；
(7)重复步骤(4)、(5)、(6)至第八轮筛选；
(8)将筛选的第五、六、七轮PCR扩增产物送至上海生工测序，每轮随机选取120条测序，再用IDT软件对得到的链进行二级结构分析，最终获得最佳的适配体。
Figure PCTCN2017073024-appb-000001
Figure PCTCN2017073024-appb-000002
Figure PCTCN2017073024-appb-000003
Figure PCTCN2017073024-appb-000004
Figure PCTCN2017073024-appb-000005
Figure PCTCN2017073024-appb-000006
Figure PCTCN2017073024-appb-000007
Figure PCTCN2017073024-appb-000008
Figure PCTCN2017073024-appb-000009
Figure PCTCN2017073024-appb-000010
Figure PCTCN2017073024-appb-000011
Figure PCTCN2017073024-appb-000012

Claims (6)

  1. 一种利用微阵列微流控芯片筛选适配体的方法，其特征在于，所述的微阵列微流控芯片按照如下方法制备：
    将PDMS前聚体与固化剂按照质量比为5∶1～100∶1的比例混合，混合均匀后，将其放入真空干燥器中，连接循环水真空泵进行抽真空30min除气泡，再浇到微流控通道模板上，经固化后得到PDMS微流控通道，在玻璃基片上点制浓度为0.1～10mg/mL的乳铁蛋白和阴性蛋白微阵列，对PDMS微流控通道和点样后的玻璃基片同时等离子处理，将上述两种基片紧密贴合，得到微阵列微流控芯片。
  2. 根据权利要求1所述的利用微阵列微流控芯片筛选适配体的方法，其特征在于，包括如下步骤：
    (1)筛选准备：将微阵列微流控芯片放入恒温水浴锅中，25～40℃下孵育蛋白1～12h；再通入5～20mg/ml BSA和0.01～0.5mM随机序列短链ssDNA(20nt)在25～40℃条件下封闭1～3h，之后用150μL 1×PBST溶液清洗正负筛通道；
    (2)第一轮筛选：将0.1～10nmol，125μL原始文库在95℃加热3～15min，并立即在冰上冷冻10s～5min；用注射泵以1～5μL/min流速将文库通入正筛通道，在25～40℃条件下反应30～120min；之后以5～30μL/min流速通入150μL 1×PBS缓冲溶液除去正筛通道中未结合的链；撕掉PDMS层，使用微阵列扫描仪扫描芯片；最后用DPEC水在95℃下加热3～10min洗脱芯片上乳铁蛋白结合的ssDNA，所得溶液在40～60℃条件下用高纯氮吹干至体积为25～250μL；
    (3)PCR扩增过程：将步骤(2)所得溶液分为体积均为23μL的3～10份相同溶液，每份依次加入25μL、2×Taq聚合酶，1μL、20μM TAMRA标记的前向引物和1μL、20μM生物素化的后向引物，混合均匀后进行PCR扩增；
    该步骤获得的产物作为模板稀释5～40倍后进行再一次扩增：取5μL稀释后的PCR产物与1μL2×SYBR Premix Ex TaqTM酶、1μL20μM TAMRA标记的前向引物、1μL20μM生物素化的后向引物以及18μL超纯水混合均匀，PCR扩增每进行一轮便取10μL样品至微孔板中用BioTek检测其荧光信号，荧光信号最高者作为最佳轮数，剩余产物均扩增该轮数；
    (4)分离纯化：将步骤(3)得到的PCR产物与800μL～5mL Promega磁珠充分混匀，在快速摇动0.5～2h后除去上清液；再加入25μL、0.01～0.1M NaOH溶液涡旋3～15min，使磁珠上连接的双链解离到溶液中；之后取上层清液并依次加入12.5μL、0.1M HCl25 μL，DPEC水和62.5μL2×PBSM缓冲溶液，所得的中性溶液作为下一轮筛选的次级文库，其含量为40pmol～100pmol；
    (5)第二轮筛选：用注射泵以1～5μL/min流速将文库在25～40℃条件下以1～5μL/min依次输通入负筛，正筛通道；接着以5～30μL/min流速通入150μL，1×PBS缓冲溶液除去正筛通道中未反应的ssDNA序列；轻轻撕掉PDMS层，使用Luxscan～10K/A微阵列扫描仪扫描芯片；最后用DPEC水在95℃下加热3～10min洗脱芯片上乳铁蛋白结合的ssDNA序列，所得溶液在60℃吹干至体积为25～250μL，留作PCR扩增；
    (7)重复步骤(4)、(5)、(6)至第八轮筛选；
    (8)将筛选的第五、六、七轮PCR扩增产物测序，重复上述的筛选过程，随机选取样品测序，分析测序结果，得到重复序列大于2的所有序列，再用IDT软件对得到的重复序列进行二级结构分析，获得最佳的适配体。
  3. 根据权利要求2所述的利用微阵列微流控芯片筛选适配体的方法，其中，步骤(2)中，所述文库的中间为40个随机碱基，随机碱基5’端的核苷酸序列如SEQ ID NO.1所示，随机碱基3’端的核苷酸序列如SEQ ID NO.2所示，
  4. 根据权利要求2所述的利用微阵列微流控芯片筛选适配体的方法，其中，步骤(3)中，所述的前向引物，其核苷酸序列如SEQ ID NO.3所示；
    所述的后向引物，其核苷酸序列如SEQ ID NO.4所示。
  5. 一种用于检测乳铁蛋白含量的适配体，其特征在于，所述适配体的核苷酸序列如SEQ ID NO.5～65所示。
  6. 权利要求5所述用于检测乳铁蛋白含量的适配体在检测乳铁蛋白中的应用。
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