WO2018082405A1 - 一种多靶分子浓度检测方法 - Google Patents

一种多靶分子浓度检测方法 Download PDF

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WO2018082405A1
WO2018082405A1 PCT/CN2017/102220 CN2017102220W WO2018082405A1 WO 2018082405 A1 WO2018082405 A1 WO 2018082405A1 CN 2017102220 W CN2017102220 W CN 2017102220W WO 2018082405 A1 WO2018082405 A1 WO 2018082405A1
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probe
target
labeled
molecules
magnetic bead
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PCT/CN2017/102220
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English (en)
French (fr)
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孙树清
吴振杰
李国花
何永红
马辉
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清华大学深圳研究生院
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/137—Metal/ion, e.g. metal label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/143—Magnetism, e.g. magnetic label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/149—Particles, e.g. beads

Definitions

  • the present invention relates to a biological detection method, and more particularly to a method for simultaneously detecting the concentration of a plurality of target molecules.
  • the high-sensitivity characteristic means that the target molecule is detected at a very low concentration
  • the high-throughput characteristic means that a plurality of lower-concentration target molecules can be simultaneously detected at the same time, which shortens the detection. Time, which reduces the cost of testing, both of which are indispensable features of an excellent bioassay.
  • many detection methods are more demanding for high sensitivity and cannot meet the requirements of high throughput at the same time.
  • the technical problem to be solved by the present invention is to make up for the deficiencies of the above prior art, and to propose a multi-target molecular concentration detecting method, which can realize high-throughput detection of concentration of a plurality of target molecules, and has high sensitivity.
  • a multi-target molecular concentration detection method includes the following steps:
  • step S3 adding the plurality of labeled probes to the reaction vessel after step S2, the plurality of target molecules are combined with the corresponding second probe molecules to form a magnetic bead probe-target molecule-labeled probe complex ;
  • the magnetic bead probe is added to the reaction vessel after the step S2', and the plurality of target molecules are respectively combined with the corresponding first probe molecules to form a magnetic bead probe-target molecule-labeled probe complex.
  • step S5 adding an eluent to the reaction vessel after the step S4, causing the magnetic bead probe-target molecule-labeled probe complex to undergo structural dissociation and dissociation into the magnetic bead probe and the target a molecule and the plurality of labeled probes;
  • the surface of the slide is surface-modified by a silane solution containing one or more of an amino group, a carboxyl group, a thiol group, and a hydroxyl group to form a self-assembled film on the surface of the slide;
  • step S7 taking the solution after step S5 to form a sample on the glass slide treated in step S6, counting the plurality of labeled probes under a microscope, and calculating the number of various labeled probes in the solution, The concentration of the corresponding target molecule is calculated by the number of each labeled probe.
  • the multi-target molecular concentration detecting method of the present invention when detecting the concentration of a plurality of target molecules, different labeled particles are combined with the corresponding target molecules in a one-to-one correspondence, and subsequently dissociated.
  • the slide is modified with a silane solution containing a specific group to form a self-assembled film on the surface of the slide, and the surface of the slide exposes the above specific chemical group, so that the slide is displayed in the aqueous solution.
  • the labeled particles corresponding to each target molecule are respectively fixed, and the influence of each other is small, which ensures the specific detection of the target molecule, thereby ensuring high-throughput detection while ensuring ultrasensitive detection of each single target molecule.
  • a plurality of labeled particles can be used for high-throughput and ultra-sensitive biological detection.
  • the lower limit of the detection concentration can be as low as 3 ⁇ 10 -18 mol/L.
  • FIG. 1 is a flow chart showing the principle of detection of a detection method according to an embodiment of the present invention
  • 2a is a transmission electron micrograph of a gold nanosphere in a detection method according to an embodiment of the present invention
  • 2b is a transmission electron micrograph of gold/silver composite nanoparticles in a detection method according to an embodiment of the present invention
  • 2c is a transmission electron micrograph of a gold nanorod in a detection method according to an embodiment of the present invention.
  • 3 is an SEM image of a composite of a magnetic bead probe-target molecule-labeled probe obtained in the detection method of the embodiment of the present invention
  • 4a is an image of a gold nanorod in a dark field microscope in accordance with an embodiment of the present invention.
  • 4b is an image of a gold nanosphere in a dark field microscope in accordance with an embodiment of the present invention.
  • 4c is an image of a gold/silver composite nanoparticle in a dark field microscope in accordance with an embodiment of the present invention
  • 4d is an image of a gold nanorod, a gold nanosphere, and a gold/silver composite nanoparticle in a dark field microscope in a specific embodiment of the present invention
  • Figure 5a is a graph showing the relationship between the concentration of HIV and the actual number of detections in a specific embodiment of the present invention.
  • Figure 5b is a graph showing the relationship between the concentration of HBV and the actual number of detections in an embodiment of the present invention.
  • Figure 5c is a graph showing the relationship between the concentration of HPV and the actual number of detections in a specific embodiment of the present invention.
  • Figure 6 is a graph showing the results of detection of seven samples containing different target molecules by a detection method according to a specific embodiment of the present invention.
  • the specific embodiment provides a method for detecting the concentration of a plurality of target molecules, and the three target molecules are detected as an example, and the following steps are included:
  • the second probe molecule of the first target molecule is coupled to the surface of the first labeled particle to form a first labeled probe
  • the second probe molecule of the second target molecule is coupled to the surface of the second labeled particle to form a first
  • the second labeled probe couples the second probe molecule of the third target molecule to the surface of the third labeled particle to form a third labeled probe.
  • the first probe molecules of the three target molecules are respectively connected to three different magnetic beads, and each group of magnetic beads corresponds to a first probe of a target molecule to form a magnetic bead probe, which is to be explored.
  • Needle I was coupled to magnetic beads A
  • probe II was coupled to magnetic beads B
  • probe III was coupled to magnetic beads C.
  • coupling a second probe molecule of the second target molecule to a second labeled particle The sub-surface forms a second labeled probe
  • the second probe molecule of the third target molecule is coupled to the surface of the third labeled particle to form a third labeled probe.
  • the above target molecule may be a DNA molecule or a protein molecule.
  • the labeled particles are particles that can be counted individually under a microscope.
  • a variety of different labeled particles are: a combination of noble metal nanoparticles, fluorescent microspheres, quantum dots, and different species in upconverting nanocrystals.
  • the plurality of different labeled particles are various noble metal nanoparticles (such as gold nanospheres, gold nanorods, silver nanospheres, gold/silver composite nanoparticles, etc.), and localized surface plasmas of various noble metal nanoparticles
  • the body resonance peaks differ by 50 nm or more.
  • the absorption cross section and scattering cross section of noble metal nanoparticles are 3-5 orders of magnitude higher than ordinary organic fluorescent molecules, and have a very high signal-to-noise ratio.
  • the peak positions are 50 nm apart from each other, which can better distinguish different markers.
  • the needle in turn, distinguishes different target molecules to be detected.
  • a target molecule-magnetic bead probe complex is formed on the surface of the magnetic bead probe.
  • the step is set in an environment of rapid shaking or gentle ultrasound, and the shaking rate is 30 r/min-60r. /min, the ultrasonic frequency is 10KHz-30KHz. This not only increases the chance of the target molecule colliding with the corresponding magnetic bead probe, but also reduces the mismatch probability of only individual base pairing.
  • this step is also performed in an environment of rapid shaking or gentle ultrasound, shaking rate
  • the frequency is from 30 rpm to 60 rpm
  • the ultrasonic frequency is from 10 kHz to 30 kHz. This not only increases the chance of the target molecule colliding with the corresponding magnetic bead probe, but also reduces the mismatch probability of only individual base pairing.
  • this step is also carried out in the same rapid shaking or gentle ultrasound environment.
  • this step is also carried out in the same rapid shaking or gentle ultrasound environment.
  • the number of the magnetic bead probes in the above step (2) or the step (3') is 10 5 - 10 7 if the step (1) is passed, as in the step (1'). Then the number of magnetic beads per group is 10 5 - 10 7 .
  • the number of the three labeled probes added in the step (3) or the step (2') is 10 10 -10 12 , respectively.
  • step (3) when proceeding in the order of step (2) and step (3), after the end of the reaction of the step (2), the following steps are further included, and then the step (3) is further performed: (a) Introducing a magnet to the outside of the reaction vessel to cause the target molecule-magnetic bead probe complex to aggregate on the side of the magnet, and sucking the liquid in the reaction vessel without taking away the target molecule-magnetic bead probe composite Things. After the step (a), the total volume of the reaction system can be reduced, and then the step (3) can be further carried out, thereby further improving the efficiency of the subsequent reaction.
  • the step may specifically be: adding a PBS buffer to the reaction vessel after the step (3) or (3') reaction, and then introducing a magnet outside the reaction vessel to make the magnetic bead probe-target molecule
  • the labeled probe complex is collected on one side of the magnet, and the liquid in the reaction vessel is aspirated to remove the unreacted labeled probe, and the operation is repeated until the unreacted labeled probe is completely removed.
  • the eluent is added in an amount of 10 to 100 ⁇ L.
  • a magnet is introduced outside the reaction vessel after the step (5), and the magnetic bead probe is collected on the side of the magnet.
  • the amount of the magnetic bead probe in the solution is reduced, and then the solution is aspirated, so that only the target molecule and the labeled probe are included in the subsequently aspirated solution, and the magnetic bead probe is not introduced.
  • Counting under the microscope makes it easier to count the labeled probes, which improves work efficiency.
  • the surface of the slide is surface-modified by a silane solution containing one or more of an amino group, a carboxyl group, a thiol group and a hydroxyl group to form a self-assembled film on the surface of the slide, and the surface of the slide is exposed.
  • a silane solution containing one or more of an amino group, a carboxyl group, a thiol group and a hydroxyl group to form a self-assembled film on the surface of the slide, and the surface of the slide is exposed.
  • the specific chemical group described above allows the surface of the slide to exhibit a certain electrical property in an aqueous solution or can be combined with a labeled probe.
  • the slide is surface-modified such that one or more of an amino group, a carboxyl group, a thiol group, and a hydroxyl group are exposed on the surface of the slide.
  • an amino group a silane solution containing an amino group and an alcohol may be mixed in a ratio of 3:7 to 1:9 by volume to prepare a mixed solution, and the slide glass is immersed in the mixed solution to make the carrier Glass The surface of the sheet forms a layer of amino self-assembled film.
  • the silane solution and the alcohol are mixed in a ratio of 3:7 to 1:9 by volume to prepare a mixed solution, and the slide is immersed in the mixed solution to make the solution The surface of the slide is formed into a self-assembled film of the corresponding group.
  • the probe molecule contained in the labeled probe is a DNA sequence
  • it is negatively charged in an aqueous solution.
  • the surface of the slide is positively charged in the aqueous solution by the above-described self-assembled film forming an amino group.
  • the labeled probe and the surface of the slide are attracted by physical charges such that the labeled particles on the labeled probe are immobilized on the slide.
  • the surface of the slide is negatively charged in the aqueous solution by modifying the group forming the carboxyl group on the surface of the slide, thereby passing the physical charge.
  • the immobilized labeled particles are adsorbed on the slide.
  • the above-mentioned group (amino group, carboxyl group, sulfhydryl group, hydroxyl group) may be modified on the slide to have the above-mentioned group on the surface.
  • the group (amino group, carboxyl group, sulfhydryl group, hydroxyl group, etc.) can further react with a group in the labeled probe (a group of its own or a group whose subsequent modification is increased), and the binding of the labeled particles is ensured by chemical reaction bonding.
  • the probe molecule contained in the labeled probe is an antibody which is not charged.
  • an aminosilane-modified glass slide is used to expose the surface of the amino group
  • the labeled probe is used to expose the surface of the carboxyl group
  • the amino group in the aqueous solution is chemically bonded to the carboxyl group, thereby realizing the labeling particle fixation.
  • the physical charge adsorption or chemical reaction combination ensures that the subsequent labeled particles are fixed on the carrier sheet, so that the corresponding labeled particles of each target molecule are respectively fixed, preventing different labeled particles from aggregating each other, and affecting the accuracy of detection.
  • FIG. 1 represents a magnetic bead
  • 2 represents a first probe molecule of a first target molecule
  • 3 represents a first probe molecule of a second target molecule
  • 4 represents a first probe molecule of a third target molecule
  • 5 Represents the first target molecule
  • 6 represents the second target molecule
  • 7 represents the third target molecule
  • 8 represents the first marker particle
  • 9 represents the second marker particle
  • 10 represents the third marker particle
  • 11 denotes a second probe molecule of the first target molecule
  • 12 denotes a second probe molecule of the second target molecule
  • 13 denotes a second probe molecule of the third target molecule.
  • the three related genes are known as examples, respectively:
  • HPV Human papillomavirus
  • HBV hepatitis B virus
  • Gold nanorods, gold nanospheres, and gold/silver composite nanoparticles are respectively labeled particles.
  • the first probe molecule is modified with an amino group at the 3' end, the specific sequence is 5'-TTATTCCAAATATCTTCT-NH2-3', and the second probe molecule is modified with a thiol group at the 5' end, and the specific sequence is 5'-HS -TGCATCCAGGTCATG-3'.
  • the first probe molecule is modified with an amino group at the 3' end, the specific sequence is 5'-GTGTGGATAATAGAGAATGTATATCTATGGAAAAAAAAAA-NH2-3', and the second probe molecule is modified with a thiol group at the 5' end, and the specific sequence is 5'-HS -AAAAAAAAAAACAGAAAATGCTAGTGCTTATGCAGCAAAT-3'.
  • the first probe molecule is modified with an amino group at the 3' end, the specific sequence is 5'-ATAACTFAAAGCCAAAAAAAAAA-NH2-3', and the second probe molecule is modified with a thiol group at the 5' end, and the specific sequence is 5'-HS -AAAAAAAAAATACCACATCATCCAT-3'.
  • the first probe molecules of the three target molecules are respectively coupled to the magnetic beads to form a magnetic bead probe
  • the second probe molecules of the HIV, HPV and HBV target molecules are respectively coupled to the gold nanorods
  • the gold nanospheres and A labeled probe was formed on the gold/silver composite nanoparticles
  • the transmission electron microscope images of the gold nanospheres, gold/silver composite nanoparticles, and gold nanorods used are shown in Figs. 2a, 2b, and 2c, respectively.
  • the three target molecules can be paired with the labeled particles.
  • HIV, HPV, and HBV target molecules can correspond to gold nanospheres, gold/silver composite nanoparticles, gold nanorods, or gold nanospheres and gold nanorods.
  • the gold/silver composite nanoparticles can also correspond to gold nanorods, gold nanospheres, and gold/silver composite nanoparticles, and various combinations can be used as long as the three labeled particles are different from each other.
  • step (3) Add 10 12 labeled probes to the test tube of step (2), place the test tube on the shaker, and set the shaker speed to 50 r/min (also in ultrasonic 20 KHz ultrasonic environment) After reacting for 2 hours, the three target molecules bind to the corresponding second probe molecules to form a magnetic bead probe-target molecule-labeled probe complex.
  • the magnetic bead probe-target molecule-labeled probe complex obtained in this step was made into an SEM sample (as shown in Fig. 3) and observed.
  • a shows the magnetic sphere in the complex of the HIV target molecule
  • b shows the magnetic sphere in the complex of the HPV target molecule
  • c shows the magnetic sphere in the complex of the HBV target molecule
  • df is A magnified picture of a portion of the magnetic sphere shown by ac.
  • a large number of (10 3 ) gold nanorods, gold nanospheres and gold/silver composite nanoparticles are coated on the surface of each magnetic bead to verify the strongness between the double-stranded DNA. The interaction does create a stable structure between the magnetic beads and the labeled particles, indicating that the marking is reliable.
  • This is the experimental basis for gold nanorods, gold nanospheres and gold/silver composite nanoparticles as probes for label detection.
  • a magnet is introduced outside the test tube wall to cause the magnetic bead probe to gather on the side of the test tube having the magnet.
  • Figure 4a is an image of a gold nanorod in a dark field microscope showing red.
  • Figure 4b is an image of a gold nanosphere in a dark field microscope showing green.
  • Figure 4c shows the gold/silver composite The image of the rice particles in the dark field microscope shows blue.
  • Figure 4d is an image of a dark field microscope in the presence of gold nanorods, gold nanospheres, and gold/silver composite nanoparticles, including red, green, and blue.
  • Fig. 5a is a graph showing the relationship between the concentration of HIV and the actual number of detections
  • Fig. 5b is a graph showing the relationship between the concentration of HBV and the actual number of detections
  • Fig. 5c is a graph showing the relationship between the concentration of HPV and the actual number of detections. It can be seen from the figure that the actual detection data has a good correspondence with the concentration, and the linearity of the detection is good.
  • the concentration calibration curve can be determined, and the definition of the specific embodiment can be further calculated according to the standard deviation of the detection limit of 3 times.
  • the detection limit of the DNA detection of the three target molecules is about 3 ⁇ 10 -18.
  • Mol/L is 4-5 orders of magnitude higher than the fluorescence intensity-based detection technology, and the detection error of the three target molecules is within 15%, which is relatively reliable, which shows that the invention has extremely high application value.
  • Samples 1-3 are samples of target molecule sequences containing only HIV, HBV and HPV, respectively.
  • Samples 4-6 contained two components: Sample 4 contained 10 fM of HIV and HBV, Sample 5 contained 10 fM of HIV and HPV, and Sample 6 contained 10 fM of HBV and HPV. All three components in sample 7 contained 10 fM.
  • the method of the present embodiment has a better specificity when detecting the simultaneous addition of three kinds of labeled particles. The method of the present embodiment can ensure specificity while achieving high throughput detection.
  • the magnetic beads having the superparamagnetic property and coupled with the first probe molecules are used as the capture matrix, and the target molecules to be detected in the target molecule solution are captured on the surface of the magnetic beads, which is easy to count single particles.
  • the labeled particles are coupled to a second probe molecule, and the target molecules captured on the surface of the magnetic beads are labeled to form a magnetic bead probe-target molecule-labeled probe complex. Excessive particles not involved in the labeling are separated from the reaction system by the superparamagnetism of the magnetic beads. By elution, the particles participating in the label are released from the surface of the magnetic beads into the solution.
  • the slide is modified by a silane solution containing a specific group to form a self-assembled film on the surface of the slide, and the surface of the slide exposes the above specific chemical group, so that the slide is displayed in the aqueous solution. It has a certain electrical property or can react with the labeled probe through a group.
  • the probe in the labeled probe is fixed on the slide by electrical attraction or group binding reaction, so that the plurality of labeled particles can be counted separately. Achieve concentration determination of a variety of target molecules.
  • the target molecule may also be captured on the labeled particle coupled to the second probe molecule, and then reacted with the magnetic bead coupled to the first probe molecule to form a magnetic bead probe.
  • - Target molecule - labeled probe complex may also be captured on the labeled particle coupled to the second probe molecule, and then reacted with the magnetic bead coupled to the first probe molecule to form a magnetic bead probe.
  • the target molecule can also be a plurality of proteins, and accordingly, the probe molecule is an antibody and the eluent is a urea solution.

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Abstract

一种多靶分子浓度检测方法，包括以下步骤：S1，将多种靶分子（5，6，7）各自对应的第一探针分子（2，3，4）偶联到磁珠（1）上，形成磁珠探针；将多种靶分子（5，6，7）各自对应的第二探针分子（11，12，13）分别偶联到多种不同的标记粒子（8，9，10）表面，形成多种标记探针；S2，形成靶分子-磁珠探针复合物；S3，形成磁珠探针-靶分子-标记探针复合物；S4，将未参与反应的标记探针除去；S5，在经过步骤S4后的反应容器中加入洗脱液，使得磁珠探针-靶分子-标记探针复合物发生结构解离；S6，通过包含氨基、羧基、巯基、羟基中的一种或者多种的硅烷溶液对载玻片进行表面修饰；S7，计算靶分子（5，6，7）的浓度。该多靶分子浓度检测方法，可实现高通量检测多种靶分子（5，6，7）的浓度，且灵敏度也较高。

Description

一种多靶分子浓度检测方法 【技术领域】
本发明涉及生物检测方法，特别是涉及一种同时检测多种靶分子浓度的方法。
【背景技术】
高灵敏、高通量的生物检测在疾病的诊断和治疗(尤其是疾病发展的早期)、细菌病毒检测以及临床基础研究中具有重要意义。高灵敏度的特性是指在靶分子浓度非常低的情况下，比较可靠地将其检测到；而高通量的特性是指可一次同时检测多种浓度较低的靶分子，既缩短了检测的时间，又降低了检测的成本，以上两者均是优秀的生物检测方法中不可或缺的特性。目前，很多检测方法更多的追求高灵敏度的特点而无法同时满足高通量的要求。如近年来引起人们极大关注的基于单分子探测的高灵敏探测技术，该技术有望在检测灵敏度上，大大超越传统的基于荧光强度信号的检测模式。然而，普通的单分子探测技术很难同时测定多种靶分子，无法满足高通量的要求，而且该技术往往需要昂贵的仪器支持，如配置有单光子探测能力的共聚焦荧光显微镜、全内反射荧光显微镜等，在成本上往往过于昂贵，很难应用于实际的高灵敏检测中。专利申请CN105132533A中提出的靶分子浓度检测方法，针对单分子检测时具有较高的灵敏度，但同时检测多种靶分子时，无法很好地实现各种靶分子的特异性检测。
【发明内容】
本发明所要解决的技术问题是：弥补上述现有技术的不足，提出一种多靶分子浓度检测方法，可实现高通量检测多种靶分子的浓度，且灵敏度也较高。
本发明的技术问题通过以下的技术方案予以解决：
一种多靶分子浓度检测方法，包括以下步骤：
S1，将多种靶分子各自对应的第一探针分子偶联到磁珠上，形成磁珠探针；将多种靶分子各自对应的第二探针分子分别偶联到多种不同的标记粒子表面，形成多种标记探针；所述多种不同的标记粒子被观测时能彼此区分开；
S2，在反应容器中，将所述磁珠探针和多种靶分子的溶液混合，所述多种靶分子与相应的第一探针分子反应，所述多种靶分子被捕获至所述磁珠探针的表面形成靶分子-磁珠探针复合物；
S3，将所述多种标记探针加入经过步骤S2后的反应容器中，所述多种靶分子与相应的第二探针分子结合，形成磁珠探针-靶分子-标记探针复合物；
或者分别将步骤S2，S3替换为如下步骤S2’，S3’，
S2’，在反应容器中，将多种标记探针的混合液和多种靶分子的溶液混合，多种靶分子与相应的第二探针分子反应，多种靶分子被分别捕获至多种标记探针的表面；
S3’，将所述磁珠探针加入经过步骤S2’后的反应容器中，多种靶分子分别与相应的第一探针分子结合，形成磁珠探针-靶分子-标记探针复合物；
S4，将未参与反应的所述标记探针除去；
S5，在经过步骤S4后的反应容器中加入洗脱液，使得所述磁珠探针-靶分子-标记探针复合物发生结构解离，解离成所述磁珠探针、所述靶分子和所述多种标记探针；
S6，通过包含氨基、羧基、巯基、羟基中的一种或者多种的硅烷溶液对载玻片进行表面修饰，使所述载玻片的表面形成一层自组装膜；
S7，吸取经过步骤S5后的溶液在经过步骤S6处理过的载玻片上制成样本，在显微镜下对所述多种标记探针进行计数并计算所述溶液中各种标记探针的数量，通过各标记探针的数量计算相应的靶分子的浓度。
本发明与现有技术对比的有益效果是：
本发明的多靶分子浓度检测方法，在对多种靶分子的浓度进行检测时，将不同的标记粒子与相应的靶分子一一对应地结合，后续解离。配合包含特定基团的硅烷溶液对载玻片进行修饰，在载玻片的表面形成一层自组装膜，玻片的表面裸露出上述特定的化学基团，使得载玻片在水溶液中显出一定的电性或者可以与标记探针通过基团结合反应，这样，当包含标记探针的溶液在载玻片上制成样本时，标记探针中探针通过电性吸引或者基团结合反应而固定在载玻片上。这样，各靶分子对应的标记粒子分别固定，彼此的影响较小，很好地保证了靶分子的特异性检测，从而在实现高通量检测的同时确保各单一靶分子的超灵敏检测。本发明中使用多种标记粒子可以高通量超灵敏的生物检测，对多种靶分子检测时，检测浓度下限均可以低至3×10-18mol/L。
【附图说明】
图1是本发明具体实施方式的检测方法的检测原理流程图；
图2a是本发明具体实施方式的检测方法中金纳米球的透射电子显微镜图；
图2b是本发明具体实施方式的检测方法中金/银复合纳米粒子的透射电子显微镜图；
图2c是本发明具体实施方式的检测方法中金纳米棒的透射电子显微镜图；
图3是本发明具体实施方式的检测方法中得到的磁珠探针-靶分子-标记探针的复合物的SEM图；
图4a是本发明具体实施方式中金纳米棒在暗场显微镜中的图像；
图4b是本发明具体实施方式中金纳米球在暗场显微镜中的图像；
图4c是本发明具体实施方式中金/银复合纳米粒子在暗场显微镜中的图像；
图4d是本发明具体实施方式中金纳米棒、金纳米球及金/银复合纳米粒子同时存在时在暗场显微镜中的图像；
图5a是本发明具体实施方式中HIV的浓度与实际探测数目的关系图；
图5b是本发明具体实施方式中HBV的浓度与实际探测数目的关系图；
图5c是本发明具体实施方式中HPV的浓度与实际探测数目的关系图；
图6是本发明具体实施方式的检测方法对包含不同靶分子的7个样品进行检测的结果示意图。
【具体实施方式】
下面结合具体实施方式并对照附图对本发明做进一步详细说明。
本具体实施方式提供一种检测多种靶分子浓度的方法，以检测三种靶分子为例进行说明，包括如下步骤：
(1)将三种靶分子的第一探针分子同时偶联到同一种磁珠表面形成磁珠探针(即将探针Ⅰ、Ⅱ、Ⅲ同时偶联到同一种磁珠A上)，将第一种靶分子的第二探针分子偶联到第一种标记粒子表面形成第一标记探针，将第二种靶分子的第二探针分子偶联到第二种标记粒子表面形成第二标记探针，将第三种靶分子的第二探针分子偶联到第三种标记粒子表面形成第三标记探针。
或者将所述步骤(1)替换为如下步骤(1’)，
(1’)将三种靶分子的第一探针分子分别接到三组不同的磁珠上，每组磁珠上对应一种靶分子的第一探针，形成磁珠探针，即将探针Ⅰ偶联到磁珠A上，探针Ⅱ偶联到磁珠B上、探针Ⅲ偶联到磁珠C上。将第一种靶分子的第二探针分子偶联到第一种标记粒子表面形成第一标记探针，将第二种靶分子的第二探针分子偶联到第二种标记粒 子表面形成第二标记探针，将第三种靶分子的第二探针分子偶联到第三种标记粒子表面形成第三标记探针。
上述靶分子可为DNA分子或者蛋白质分子。标记粒子为在显微镜下可以单个计数的粒子。多种不同的标记粒子为：贵金属纳米粒子、荧光微球、量子点、上转换纳米晶中的不同种的组合。优选地，多种不同的标记粒子均为多种贵金属纳米粒子(如金纳米球、金纳米棒、银纳米球，金/银复合纳米粒子等)，且多种贵金属纳米粒子的局域表面等离子体共振峰位相差50nm以上。贵金属纳米粒子的吸收截面和散射截面比普通的有机荧光分子高出3-5个量级，具有极高的信噪比，彼此之间峰位相差50nm，这样可更好地区分不同的标记探针，进而区分不同的待检测靶分子。
(2)在反应容器中，将所述磁珠探针和三种靶分子的溶液混合，所述三种靶分子与相应的所述第一探针分子反应，所述三种靶分子被捕获至所述磁珠探针的表面形成靶分子-磁珠探针复合物。
优选地，为减轻其中两种靶分子对另一种靶分子与其相应磁珠探针的结合效率的影响，该步骤设置在快速摇晃或者轻柔超声的环境下进行，摇晃速率为30r/min-60r/min，超声频率为10KHz-30KHz。这样既增加了靶分子与相应磁珠探针相遇碰撞的机会，也可以降低只有个别碱基配对的错配概率。
(3)将所述第一、第二及第三标记探针加入所述反应容器，所述三种靶分子与相应的所述第二探针分子结合，形成磁珠探针-靶分子-标记探针复合物。
同样地，为减轻其中两种标记探针对另一种标记探针与其相应靶分子-磁珠探针的结合效率的影响，该步骤也设置在快速摇晃或者轻柔超声的环境下进行，摇晃速率为30r/min-60r/min，超声频率为10KHz-30KHz。这样既增加了靶分子与相应磁珠探针相遇碰撞的机会，也可以降低只有个别碱基配对的错配概率。
或者分别将所述步骤(2)和(3)替换为如下步骤(2’)和(3’)，
(2’)在反应容器中，将所述第一、第二及第三标记探针的混合液和三种靶分子的溶液混合，所述三种靶分子与相应的所述第二探针分子反应，所述三种靶分子分别被捕获至所述第一、第二及第三标记探针的表面。
同样地，为减轻相互之间造成结合效率下降的影响，该步骤也在同样的快速摇晃或者轻柔超声的环境下进行。
(3’)将所述磁珠探针加入所述反应容器，所述三种靶分子分别与相应的所述第一 探针分子结合，形成磁珠探针-靶分子-标记探针复合物。
同样地，为减轻相互之间造成结合效率下降的影响，该步骤也在同样的快速摇晃或者轻柔超声的环境下进行。
上述步骤(2)或者步骤(3’)中的所述磁珠探针的加入个数，如为经过步骤(1)，则为105-107个，如为经过步骤(1’)，则每组磁珠的数目为105-107个。
步骤(3)或者步骤(2’)中的所述三种标记探针的加入个数分别为1010-1012个。
优选地，当按照步骤(2)和步骤(3)的顺序进行时，在所述步骤(2)的反应结束后，还包括如下步骤，然后再进行所述步骤(3)：(a)在所述反应容器外侧引入磁铁，使所述靶分子-磁珠探针复合物在所述磁铁一侧聚集，吸取所述反应容器中的液体而不带走所述靶分子-磁珠探针复合物。经过步骤(a)之后可以减小反应体系的总体积，然后再进行步骤(3)，能进一步提升后续反应的效率。
(4)将未参与反应的所述标记探针除去。
该步骤，具体可为：在经过步骤(3)或者(3’)反应后的反应容器中加入PBS缓冲液，然后，在所述反应容器外引入磁铁，使所述磁珠探针-靶分子-标记探针复合物在所述磁铁一侧聚集，吸取所述反应容器中的液体将所述未反应的标记探针除去，重复操作直到所述未反应的标记探针完全除去。
(5)在经过步骤(4)后的反应容器中加入洗脱液，使得所述磁珠探针-靶分子-标记探针复合物发生结构解离，解离成所述磁珠探针、所述靶分子和所述第一、第二、第三标记探针。该步骤中，洗脱液的加入量为10-100μL。
(6)在经过步骤(5)后的反应容器外引入磁铁，将所述磁珠探针在所述磁铁一侧聚集。这样，先将磁珠探针聚集后，溶液中磁珠探针的量减少后，再吸取溶液，使得后续吸取的溶液中仅包括靶分子和标记探针，而不引入磁珠探针，后续进行显微镜下计数，会更容易对标记探针进行计数，可以提高工作效率。
(7)通过包含氨基、羧基、巯基、羟基中的一种或者多种的硅烷溶液对载玻片进行表面修饰，使载玻片的表面形成一层自组装膜，载玻片的表面裸露出上述特定的化学基团，使载玻片表面在水溶液中可以显出一定的电性或者可以与标记探针结合反应。
该步骤中，对玻片进行表面修饰处理，使得载玻片表面裸露出氨基、羧基、巯基、羟基中的一种或者多种。例如，以氨基为例，可将包含氨基的硅烷溶液与酒精按体积比3:7～1:9的比例混合配制成混合溶液，将载玻片浸泡于所述混合溶液中，使所述载玻 片的表面形成一层氨基自组装膜。同样地，如为包含其它基团的硅烷溶液，将硅烷溶液与酒精按体积比3:7～1:9的比例混合配制成混合溶液，将载玻片浸泡于所述混合溶液中，使所述载玻片的表面形成一层相应基团的自组装膜。
标记探针中所含的探针分子为DNA序列时，其在水溶液中显负电。通过上述形成氨基基团的自组装膜，使得载玻片表面在水溶液中带上正电。这样，当标记探针置于载玻片上时，标记探针与载玻片表面通过物理电荷相吸，使得标记探针上的标记粒子固定在玻片上。此外，对于标记探针中所含的探针分子在水溶液中显正电的情形，则通过修饰载玻片表面形成羧基的基团，使载玻片表面在水溶液中带负电，从而通过物理电荷吸附固定标记粒子在玻片上。而对于标记探针中所含的探针分子在水溶液中不带电荷的情形，则对玻片进行上述基团(氨基、羧基、巯基、羟基)的修饰后，可使其表面带有上述基团(氨基、羧、巯基、羟基等)，进而可以与标记探针中的基团(自身的基团或者后续修饰增加的基团)反应，通过化学反应结合保证标记粒子的固定。例如，靶分子为蛋白质时，标记探针中所含的探针分子为抗体，其不带电荷。此时使用氨基硅烷修饰玻片使表面裸露氨基，通过修饰标记探针使其表面裸露羧基，水溶液中氨基与羧基化学结合，从而实现标记粒子固定。
通过上述修饰处理，通过物理电荷吸附或者化学反应结合保证后续标记粒子在载波片上固定住，从而各靶分子对应的标记粒子分别固定，防止不同标记粒子相互聚集，影响探测的准确度。
(8)吸取经过步骤(6)后的溶液在经过步骤(7)后的载玻片上制成样本，在显微镜下对所述三种标记探针进行计数并计算所述溶液中各标记探针的总数量，通过标记探针的数量计算相应的靶分子的浓度。
上述的检测原理流程图如图1所示。其中，1表示磁珠，2表示第一种靶分子的第一探针分子，3表示第二种靶分子的第一探针分子，4表示第三种靶分子的第一探针分子，5表示第一种靶分子，6表示第二种靶分子，7表示第三种靶分子，8表示第一种标记物粒子，9表示第二种标记物粒子，10表示第三种标记物粒子，11表示第一种靶分子的第二探针分子，12表示第二种靶分子的第二探针分子，13表示第三种靶分子的第二探针分子。
如下优选的实例中，以已知的三种相关基因(即靶分子)为例，分别为：
艾滋病毒(HIV)的相关基因序列：
5’-AGAAGATATTTGGAATAACATGACCTGGATGCA-3’，
人类乳头瘤病毒(HPV)的相关基因序列：
5’-CCATAGATATACATTCTCTATTATCCACCTGCATTTGCTGCATAAGCACTAGCATTTT-3’，
乙型肝炎病毒(HBV)的相关序列：
5’-TTGGCGGGCAGTTATATGGATGATGTGGTA-3’。
以金纳米棒、金纳米球、金/银复合纳米粒子分别为标记粒子。
检测时，包括如下步骤：
(1)根据以上靶分子的序列，按常规的方法针对每种靶分子分别设计两个探针分子(DNA序列)。
对于HIV序列：第一探针分子在3’端以氨基修饰，具体序列为5’-TTATTCCAAATATCTTCT-NH2-3’，第二探针分子在5’端以巯基修饰，具体序列为5’-HS-TGCATCCAGGTCATG-3’。
对于HPV序列：第一探针分子在3’端以氨基修饰，具体序列为5’-GTGTGGATAATAGAGAATGTATATCTATGGAAAAAAAAAA-NH2-3’，第二探针分子在5’端以巯基修饰，具体序列为5’-HS-AAAAAAAAAAACAGAAAATGCTAGTGCTTATGCAGCAAAT-3’。
对于HBV序列：第一探针分子在3’端以氨基修饰，具体序列为5’-ATAACTFAAAGCCAAAAAAAAAAAA-NH2-3’，第二探针分子在5’端以巯基修饰，具体序列为5’-HS-AAAAAAAAAATACCACATCATCCAT-3’。
分别将三种靶分子的第一探针分子偶联在磁珠上形成磁珠探针，分别将HIV、HPV、HBV靶分子的第二探针分子偶联在金纳米棒、金纳米球及金/银复合纳米粒子上形成标记探针，其中所用的金纳米球、金/银复合纳米粒子、金纳米棒的透射电子显微镜的图片分别如图2a、2b、2c所示。三种靶分子可与标记粒子自由配对，例如HIV、HPV、HBV靶分子可一一对应金纳米球、金/银复合纳米粒子、金纳米棒，也可一一对应金纳米球、金纳米棒、金/银复合纳米粒子，还可一一对应金纳米棒、金纳米球、金/银复合纳米粒子，各种组合均可行，只要确保三种标记粒子彼此不同即可。
(2)将106个磁珠探针加入到含有三种靶分子的溶液60微升(靶分子浓度分别为100nM)的试管中，将试管置于摇床上，摇床转速设为50r/min(也可在超声20KHZ的超 声环境下进行)，反应1个小时，三种靶分子与相应的第一探针分子反应，被捕获至磁珠探针的表面，待反应结束后，在试管壁外引入一块磁铁，小心吸取试管中的液体而不致带走靶分子-磁珠探针复合物。
(3)分别将1012个的三种标记探针加入步骤(2)的试管中，将试管置于摇床上，摇床转速设为50r/min(也可在超声20KHz的超声环境下进行)，反应2个小时，三种靶分子与对应的第二探针分子结合，形成磁珠探针-靶分子-标记探针复合物。
(4)在试管中加入100μL的磷酸盐(PBS)缓冲液，轻轻摇晃数下，然后在试管壁外引入磁铁，使磁珠探针-靶分子-标记探针复合物在磁铁一侧聚集，吸取试管中的液体将未反应的标记探针除去，重复这个步骤六次以完全除去未完全反应的标记探针。
将该步骤得到的磁珠探针-靶分子-标记探针的复合物制成SEM样本(如图3所示)予以观测。图3中a示意了检测HIV靶分子的复合物中的磁球，b示意了检测HPV靶分子的复合物中的磁球，c示意了检测HBV靶分子的复合物中的磁球，d-f是对a-c所示磁球的局部进行放大的图片。从图中可得到，每个磁珠上都有数目庞大(103)的金纳米棒、金纳米球及金/银复合纳米粒子包覆在其表面上，验证了双链DNA之间的强相互作用确实能使磁珠和标记粒子之间形成稳定结构，说明标记很可靠。这一点是金纳米棒、金纳米球及金/银复合纳米粒子作为探针可用于标记检测的实验基础。
(5)在试管中加入20微升的洗脱液(本例中为超纯水)并在70℃的水浴锅中加热10分钟，使磁珠探针-靶分子-标记探针复合物发生结构解离，解离成磁珠探针、靶分子和标记探针。
(6)在试管壁外引入磁铁，使磁珠探针在试管有磁铁的一侧聚集。
(7)将3-氨基丙基三乙氧基硅烷与酒精按1:9的比例混合配置成玻片修饰液，将载玻片浸泡于混合溶液中6h以上，使玻片的表面形成一层氨基自组装膜，在水溶液中玻片的表面带有正电。之后将载玻片进行超声清洗，并在真空干燥箱中干燥。
(8)用移液枪小心移取3微升的上清液滴落在经硅烷修饰且洁净的载玻片上，用盖玻片压盖在其上形成显微镜样本，由于DNA分子在水溶液中显负电，而玻片的表面修饰后在水溶液中显正电，两者互相吸引，保证贵金属颗纳米粒子固定在玻片上，利用暗场显微镜分别对样本中的红色(金纳米棒)、绿色(金纳米球)及蓝色(金/银复合纳米粒子)亮点进行计数，暗场显微图如图4所示。图4a为金纳米棒在暗场显微镜中的图像，显示红色。图4b为金纳米球在暗场显微镜中的图像，显示绿色。图4c为金/银复合纳 米粒子在暗场显微镜中的图像，显示蓝色。图4d为金纳米棒、金纳米球及金/银复合纳米粒子同时存在时在暗场显微镜中的图像，包括红色、绿色和蓝色。
对随机选取的50幅图片进行统计，即可用于对靶分子的浓度测定。在实验中，对一系列浓度的靶分子DNA进行检测，检测结果如图5a～5c所示。图5a为HIV的浓度与实际探测数目的关系图，图5b为HBV的浓度与实际探测数目的关系图，图5c为HPV的浓度与实际探测数目的关系图。从图中可知，实际探测数据与浓度有较好地对应关系，检测的线性度较好。
根据浓度检测结果可制定出浓度定标曲线，依据检测极限的3倍标准差定义，可进一步计算得到本具体实施方式的方法在三种靶分子DNA探测中的检测下限大约为3×10-18mol/L，比一般基于荧光强度的探测技术高出4-5个量级，并且三种靶分子的检测误差均在15％以内，比较可靠，由此可见该发明具有极高的应用价值。
为验证本具体实施方式的检测方法的特异性，还对包含不同靶分子的7个样品进行检测，检测结果如图6所示。样品1-3分别为只含有HIV,HBV及HPV的靶分子序列的样品。样品4-6含两种组分：样品4含10fM的HIV和HBV，样品5含10fM的HIV和HPV，样品6含10fM的HBV和HPV。样品7中三种组分均含有10fM。从图6可知，本具体实施方式的方法对于同时加入三种标记粒子进行检测时，特异性较好。本具体实施方式的方法在实现高通量检测的同时，能够保证特异性。
在以上实施例中，以具有超顺磁特性且偶联有第一探针分子的磁珠为捕获基质，将靶分子溶液中的待测靶分子捕获到磁珠表面，将易于单粒子计数的标记粒子偶联上第二探针分子，对被捕获在磁珠表面的靶分子进行标记，形成磁珠探针-靶分子-标记探针复合物。利用磁珠的超顺磁性，将未参与标记的多余粒子分离出反应体系。而通过洗脱，参与标记的粒子则从磁珠表面上释放出来进入溶液中。同时通过包含特定基团的硅烷溶液对载玻片进行修饰，在载玻片的表面形成一层自组装膜，玻片的表面裸露出上述特定的化学基团，使得载玻片在水溶液中显出一定的电性或者可以与标记探针通过基团结合反应。这样，当包含标记探针的溶液在载玻片上制成样本时，标记探针中探针通过电性吸引或者基团结合反应而固定在载玻片上，从而可分别对多种标记粒子进行计数实现多种靶分子的浓度测定。在其他一些实施例中，也可以先将靶分子捕获在偶联有第二探针分子的标记粒子上，然后再与偶联有第一探针分子的磁珠进行反应，形成磁珠探针-靶分子-标记探针复合物。
在其他实施例中，靶分子还可以为多种蛋白质，相应地，探针分子为抗体，洗脱液为尿素溶液。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下做出若干替代或明显变型，而且性能或用途相同，都应当视为属于本发明的保护范围。
Figure PCTCN2017102220-appb-000001
Figure PCTCN2017102220-appb-000002
Figure PCTCN2017102220-appb-000003

Claims (9)

  1. 一种多靶分子浓度检测方法，其特征在于：包括以下步骤：
    S1，将多种靶分子各自对应的第一探针分子偶联到磁珠上，形成磁珠探针；将多种靶分子各自对应的第二探针分子分别偶联到多种不同的标记粒子表面，形成多种标记探针；所述多种不同的标记粒子被观测时能彼此区分开；
    S2，在反应容器中，将所述磁珠探针和多种靶分子的溶液混合，所述多种靶分子与相应的第一探针分子反应，所述多种靶分子被捕获至所述磁珠探针的表面形成靶分子-磁珠探针复合物；
    S3，将所述多种标记探针加入经过步骤S2后的反应容器中，所述多种靶分子与相应的第二探针分子结合，形成磁珠探针-靶分子-标记探针复合物；
    或者分别将步骤S2，S3替换为如下步骤S2’，S3’，
    S2’，在反应容器中，将多种标记探针的混合液和多种靶分子的溶液混合，多种靶分子与相应的第二探针分子反应，多种靶分子被分别捕获至多种标记探针的表面；
    S3’，将所述磁珠探针加入经过步骤S2’后的反应容器中，多种靶分子分别与相应的第一探针分子结合，形成磁珠探针-靶分子-标记探针复合物；
    S4，将未参与反应的所述标记探针除去；
    S5，在经过步骤S4后的反应容器中加入洗脱液，使得所述磁珠探针-靶分子-标记探针复合物发生结构解离，解离成所述磁珠探针、所述靶分子和所述多种标记探针；
    S6，通过包含氨基、羧基、巯基、羟基中的一种或者多种的硅烷溶液对载玻片进行表面修饰，使所述载玻片的表面形成一层自组装膜；
    S7，吸取经过步骤S5后的溶液在经过步骤S6处理过的载玻片上制成样本，在显微镜下对所述多种标记探针进行计数并计算所述溶液中各种标记探针的数量，通过各标记探针的数量计算相应的靶分子的浓度。
  2. 根据权利要求1所述的多靶分子浓度检测方法，其特征在于：步骤S6中，将所述硅烷溶液与酒精按体积比3:7～1:9的比例混合配制成混合溶液，将载玻片浸泡于所述混合溶液中，使所述载玻片的表面形成一层包含氨基、羧基、巯基、羟基中的一种或者多种的自组装膜。
  3. 根据权利要求1所述的多靶分子浓度检测方法，其特征在于：步骤S2或者步骤S2’或者步骤S3或者步骤S3’中，所述反应容器置于摇晃状态或者超声振动状态下，摇晃速率为30r/min-60r/min，超声频率为10KHz-30KHz。
  4. 根据权利要求1所述的多靶分子浓度检测方法，其特征在于：步骤S7中，溶液在吸取之前还包括：在经过步骤S5后的反应容器外引入磁铁，将所述磁珠探针在所述磁铁一侧聚集。
  5. 根据权利要求1所述的多靶分子浓度检测方法，其特征在于：步骤S1中，所述多种不同的标记粒子为：贵金属纳米粒子、荧光微球、量子点、上转换纳米晶中的不同种的组合。
  6. 根据权利要求1所述的多靶分子浓度检测方法，其特征在于：步骤S1中，所述多种不同的标记粒子均为多种贵金属纳米粒子，且多种贵金属纳米粒子的局域表面等离子体共振峰位相差50nm以上。
  7. 根据权利要求6所述的多靶分子浓度检测方法，其特征在于：步骤S1中，为三种靶分子，分别为HIV的基因序列、HPV的基因序列、HBV的基因序列；对应的三种标记粒子选自：金纳米球、金纳米棒、金/银复合纳米粒子。
  8. 根据权利要求1所述的多靶分子浓度检测方法，其特征在于：步骤S1中，第一探针分子偶联到磁珠上包括同时偶联到同一种磁珠或者分别偶联到不同种的磁珠上。
  9. 根据权利要求1所述的多靶分子浓度检测方法，其特征在于：步骤S1中，靶分子为DNA分子或者蛋白质分子。
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