WO2017016281A1 - 一种靶分子浓度的检测方法 - Google Patents

一种靶分子浓度的检测方法 Download PDF

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WO2017016281A1
WO2017016281A1 PCT/CN2016/082073 CN2016082073W WO2017016281A1 WO 2017016281 A1 WO2017016281 A1 WO 2017016281A1 CN 2016082073 W CN2016082073 W CN 2016082073W WO 2017016281 A1 WO2017016281 A1 WO 2017016281A1
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probe
target molecule
magnetic bead
labeled
target
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French (fr)
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孙树清
李国花
朱亮
何永红
马辉
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清华大学深圳研究生院
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    • 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
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • 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
    • C12Q1/6823—Release of bound markers
    • 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
    • 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

Definitions

  • the invention relates to a biological detection method, in particular to a method for detecting the concentration of a target molecule.
  • the detection method based on fluorescent label is the main means to achieve higher sensitivity detection.
  • target molecules are first immobilized on the surface of the matrix on which the probe molecules are functionalized. Thereafter, a fluorescent molecule or an enzyme having a strong optical signal marks the immobilized target molecule, and the measured optical signal intensity has a positive correlation with the concentration of the target molecule in the sample, thereby realizing the concentration measurement.
  • the fluorescent index used therein is generally low in lightness, and the poor light stability makes it extremely easy to cause photobleaching and quenching, and the detection limit is generally not lower than 10 -13 mol/L.
  • these labeling methods have certain value for many disease detection and diagnosis, they are far from sufficient to meet the early detection needs of some high-mortality diseases.
  • high-sensitivity detection technology based on single-molecule detection has received great attention, and it is expected to greatly exceed the traditional detection mode based on fluorescence intensity signal in detection sensitivity.
  • the present invention proposes a method for detecting the concentration of a target molecule.
  • a method for detecting a concentration of a target molecule includes the following steps:
  • the number of the magnetic bead probes in the step (2) or the step (3') is 10 5 - 10 7 .
  • the number of the labeled probes in the step (3) or the step (2') is 10 10 -10 12 .
  • the eluent is added in an amount of 10 to 100 ⁇ L.
  • the step (3) when the detecting method is performed in the order of the step (2) and the step (3), after the end of the reaction of the step (2), the following steps are further included, and then the step is performed ( 3):
  • the total volume of the reaction system can be reduced, and then the step (3) can be further carried out, which can further improve the efficiency of the subsequent reaction.
  • the step (4) is specifically: 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 beads
  • the probe-target molecule-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 Remove.
  • the method further comprises the steps of: introducing a magnet outside the reaction vessel after the step (5), and collecting the magnetic bead probe on one side of the magnet.
  • the amount of the magnetic bead probe in the solution is reduced, and then the solution is taken up, and then counting under the microscope, the labeled probe is more easily counted, and the working efficiency can be improved.
  • the marking particles are noble metal nanoparticles, fluorescent microspheres or upconverting nanocrystals.
  • Precious metal nanoparticles (such as gold nanospheres, gold nanorods, gold/silver composite nanoparticles, etc.) have an absorption cross section and a scattering cross section that are 3-5 orders of magnitude higher than ordinary organic fluorescent molecules, and have a very high signal-to-noise ratio. It can be used for highly sensitive detection of many markers.
  • precious metal nanoparticles can easily identify individual particles under ordinary dark field microscopy; similar countable single-particle markers and fluorescent micro-particles Balls (which are formed by coating quantum dots, fluorescent dyes into matrix microspheres) and upconverting nanocrystals, which can also easily achieve single identification under a fluorescence microscope, such single-counting particles due to extremely high letters
  • the noise ratio is much lower than that of a conventional single-molecule detector.
  • the target molecule is a DNA molecule or a protein.
  • the noble metal nanoparticles are gold nanospheres, gold nanorods or gold/silver composite nanoparticles.
  • the beneficial effects of the invention are as follows: the labeled particles in the invention have a one-to-one correspondence with the target molecules, and the labeled particles can be counted individually under the microscope, and the concentration of the target molecules can be determined by counting the labeled particles, and the single target molecule It also produces an identifiable signal that has a significant sensitivity advantage over the need for multiple markers (corresponding to the target molecule) to produce an identifiable signal in an intensity-based detection method. Furthermore, the liquid phase reaction environment of the present invention The capture kinetics of the target molecule is greatly optimized, so that more target molecules can be captured to the magnetic bead probe, which is beneficial to further improvement of sensitivity.
  • the invention is an ultra-sensitive biological detection method. In one embodiment of the present invention, a genetic sequence of HIV is detected by using a gold nanorod as a labeled probe, and the detection limit can be as low as 1.3 ⁇ 10 ⁇ 18 mol/L.
  • Fig. 1 is a flow chart showing the principle of reaction of a preferred embodiment of the present invention.
  • FIG 3 is an SEM image of a magnetic bead probe-target molecule-labeled probe complex when the target molecule concentration is 100 nM in a preferred embodiment of the invention.
  • FIG. 4 is a partial enlarged view of an image of a gold nanorod in a dark field in a preferred embodiment of the present invention.
  • Figure 5 is a calibration plot of the relationship between target molecule concentration and the average number of gold nanorods in a preferred embodiment of the invention.
  • the invention provides a method for detecting a concentration of a target molecule, comprising the following steps:
  • the labeled particles refer to particles that can be counted individually under a microscope.
  • the flow chart of the detection principle of the present invention is shown in Fig. 1, wherein: 1 indicates a magnetic bead, 2 indicates a first probe molecule, 3 indicates a target molecule, 4 indicates a labeled particle, and 5 indicates a second probe molecule.
  • a known HIV-associated gene (5'-AGAAGATATTTGGAATAACATGACCTGGATGCA-3', a target molecule) is exemplified.
  • the present invention will be described in detail using gold nanorods as labeling particles.
  • the method for detecting the concentration of a target molecule includes the following steps:
  • the first probe molecule is modified with an amino group at the 3' end, and the specific sequence is 5'-TTATTCCAAATATCTTCT-NH2-3. ', coupled to a magnetic bead to form a magnetic bead probe; the second probe molecule is modified with a thiol group at the 5' end, the specific sequence is 5'-HS-TGCATCCAGGTCATG-3', which is coupled to the gold nanorod A labeled probe is formed thereon, and an electron micrograph of the gold nanorod used therein is shown in FIG.
  • step (3) 10 12 labeled probes were added to the test tube of step (2), and reacted for 2 hours, and the target molecule was combined with the second probe molecule to form a magnetic bead probe-target molecule-labeled probe complex.
  • the gold nanorods are coated on the surface, which proves that the strong interaction between the double-stranded DNA can indeed form a stable structure between the magnetic beads and the gold nanorods, indicating that the label is reliable, which is the gold nanorod probe available.
  • the random selection of 30 images can be used to determine the concentration of target molecules.
  • the lower limit of detection of the method in DNA detection is about 1.3X10 -18 mol/L, which is 4-5 orders of magnitude higher than the detection technique based on fluorescence intensity. High application value.
  • the same standard DNA sample of 8 ⁇ 10 -17 mol/L was detected and compared with the above-mentioned calibration curve, as shown in Fig. 5, the concentration was 6.7 ⁇ 2.1. ⁇ 10 -17 mol/L, the error is about 16%, indicating that the detection method of the present invention is highly reliable.
  • the magnetic beads having the superparamagnetic characteristics and coupled with the first probe molecules are used as the capture matrix, and the target molecules 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 the 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, which utilizes the superparamagnetism of the magnetic beads.
  • the excess particles participating in the label separate the reaction system, and by elution, the particles participating in the label are released from the surface of the magnetic beads into the solution, and the labeled particles in the solution are counted to determine the concentration of the target molecule.
  • the target molecule may be first 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 be first 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 protein, and accordingly, the probe molecule is an antibody and the eluent is a urea solution.

Abstract

一种靶分子浓度的检测方法，包括如下步骤：(1)将第一探针分子偶联到磁珠表面形成磁珠探针，将第二探针分子偶联到标记粒子表面形成标记探针；(2)将磁珠探针和靶分子溶液混合，靶分子被捕获至所述磁珠探针的表面；(3)加入标记探针，靶分子与第二探针分子结合，形成磁珠探针-靶分子-标记探针复合物；(4)将未参与反应的标记探针除去；(5)加入洗脱液，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)吸取经过步骤(5)后的溶液在载玻片上制成样本，在显微镜下对标记探针进行计数并计算溶液中总的标记探针的数量从而计算靶分子的浓度。所述检测方法可靠性和灵敏度高。

Description

一种靶分子浓度的检测方法 技术领域
本发明涉及生物检测方法，特别是涉及一种靶分子浓度的检测方法。
背景技术
高灵敏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。目前，基于荧光标记的探测方法是实现较高灵敏检测的主要手段。在典型的商业化的标记探测技术如生物芯片(chip array)、液相生物芯片(suspension array)、酶联免疫放大(ELISA)中，靶分子首先被固定在探针分子功能化的基质表面，其后，具有强光学信号的荧光类分子或酶等对固定上的靶分子进行标记，测得的光学信号强度与样品中靶分子的浓度具有正相关性，从而实现其浓度测定。在这种标记检测方法中，其中所用的荧光类标志物光学亮度普遍偏低，光稳定性差使其极易产生光致漂白和淬灭，其探测极限一般都不低于10-13mol/L，虽然这些标记检测方法对于许多疾病检测诊断等具有一定的价值，但还远远不足以满足一些高死亡率疾病的早期检测需求。近年来，基于单分子探测的高灵敏探测技术得到了人们极大的关注，并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。普通的单分子探测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。
发明内容
为了弥补上述现有技术的不足，本发明提出一种靶分子浓度的检测方法。
本发明的技术问题通过以下的技术方案予以解决：
一种靶分子浓度的检测方法，包括如下步骤：
(1)将第一探针分子偶联到磁珠表面形成磁珠探针，将第二探针分子偶联到标记粒子表面形成标记探针；
(2)在反应容器中，将所述磁珠探针和靶分子的溶液混合，所述靶分子与所述第一探针分子反应，所述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；
(3)将所述标记探针加入所述反应容器，所述靶分子与所述第二探针分子 结合，形成磁珠探针-靶分子-标记探针复合物；
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，将所述标记探针和靶分子的溶液混合，所述靶分子与所述第二探针分子反应，所述靶分子被捕获至所述标记探针的表面；
(3’)将所述磁珠探针加入所述反应容器，所述靶分子与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；
(4)将未参与反应的所述标记探针除去；
(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、所述靶分子和所述标记探针；
(6)吸取经过步骤(5)后的溶液在载玻片上制成样本，在显微镜下对所述标记探针进行计数并计算所述溶液中总的标记探针的数量，通过所述总的标记探针的数量计算所述靶分子的浓度。
优选地，所述步骤(2)或者所述步骤(3’)中的所述磁珠探针的加入个数为105-107个。
优选地，所述步骤(3)或者所述步骤(2’)中的所述标记探针的加入个数为1010-1012个。
优选地，所述步骤(5)中，所述洗脱液的加入量为10-100μL。
优选地，当所述检测方法按所述步骤(2)和步骤与(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。
优选地，所述步骤(4)具体为：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。
优选地，在所述步骤(6)的吸取之前，还包括如下步骤：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。
经过以上技术方案，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，再进行显微镜下计数，会更容易对标记探针进行计数，可以提高工作效率。
优选地，所述标记粒子为贵金属纳米粒子、荧光微球或者上转换纳米晶。
贵金属纳米粒子(如金纳米球、金纳米棒、金/银复合纳米粒子等)，其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，使其可用于诸多标记相关的高灵敏检测，尤为重要的是，贵金属纳米粒子甚至可以在普通的暗场显微镜下轻易实现单个颗粒的辨识；类似的可计数的单粒子标志物还有荧光微球(其是将量子点、荧光染料包覆进入基质微球形成的)和上转换纳米晶，其也能轻易的在荧光显微镜下实现单个识别，这类可单个计数的粒子由于极高的信噪比，其探测成本相对于普通的单分子探测器件要低得多。
优选地，所述靶分子为DNA分子或者蛋白质。
优选地，所述贵金属纳米粒子为金纳米球、金纳米棒或金/银复合纳米粒子。
本发明的有益效果还有：本发明中的标记粒子与靶分子具有一一对应关系，且标记粒子可以在显微镜下单个计数，对标记粒子的计数即可实现靶分子的浓度测定，单个靶分子也能产生可以识别的信号，相比于基于强度的探测方法中需要多个标志物(对应于靶分子)才能产生可识别的信号具有明显的灵敏度优势，此外，本发明的液相的反应环境大大优化了靶分子的捕获动力学过程，从而能使得更多的靶分子被捕获到磁珠探针，有利于灵敏度的进一步提升。本发明是一种超灵敏的生物检测方法，在本发明的一个实施例中，以金纳米棒为标记探针对艾滋病毒的某种基因序列进行检测，其检测浓度下限可以低至1.3X10-18mol/L。
附图说明
图1是本发明的优选实施例的反应原理流程示意图。
图2是本发明的优选实施例中所用的金纳米棒的SEM图。
图3是本发明的优选实施例中当靶分子浓度为100nM时的磁珠探针-靶分子-标记探针复合物的SEM图。
图4是本发明的优选实施例中的金纳米棒在暗场中的图像的局部放大图。
图5是本发明的优选实施例中的靶分子浓度与金纳米棒平均数目关系的定标曲线图。
具体实施方式
下面对照附图并结合优选的实施方式对本发明作进一步说明。
本发明提供一种靶分子浓度的检测方法，包括如下步骤：
(1)将第一探针分子偶联到磁珠表面形成磁珠探针，将第二探针分子偶联到标记粒子表面形成标记探针；
(2)在反应容器中，将所述磁珠探针和靶分子的溶液混合，所述靶分子与所述第一探针分子反应，所述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；
(3)将所述标记探针加入所述反应容器，所述靶分子与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；
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，将所述标记探针和靶分子的溶液混合，所述靶分子与所述第二探针分子反应，所述靶分子被捕获至所述标记探针的表面；
(3’)将所述磁珠探针加入所述反应容器，所述靶分子与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；
(4)将未参与反应的所述标记探针除去；
(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、所述靶分子和所述标记探针；
(6)吸取经过步骤(5)后的溶液在载玻片上制成样本，在显微镜下对所述标记探针进行计数并计算所述溶液中总的标记探针的数量，通过所述总的标记探针的数量计算所述靶分子的浓度。
其中，标记粒子是指在显微镜下可以单个计数的粒子。本发明的检测原理流程图如图1所示，其中：1表示磁珠，2表示第一探针分子，3表示靶分子，4表示标记粒子，5表示第二探针分子。
在一个优选的实施例中，以已知的一种艾滋病毒的相关基因(5’-AGAAGATATTTGGAATAACATGACCTGGATGCA-3’，即靶分子)为例， 以金纳米棒为标记粒子，对本发明进行详细说明。
靶分子浓度的检测方法，包括如下步骤：
(1)根据以上艾滋病毒的相关基因，按常规的方法设计两个探针分子(DNA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’，将其偶联在磁珠上形成磁珠探针；第二探针分子在5’端以巯基修饰，具体序列为5’-HS-TGCATCCAGGTCATG-3’，将其偶联在金纳米棒上形成标记探针，其中所用的金纳米棒的电镜图如图2所示。
(2)将106个磁珠探针加入到含有60微升的靶分子溶液(靶分子浓度为100nM)的试管中反应1个小时，靶分子与第一探针分子反应，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)将1012个标记探针加入步骤(2)的试管中，反应2个小时，靶分子与第二探针分子结合，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，可看到每个磁珠上都有数目庞大(～7000)的金纳米棒包覆在其表面上，验证了双链DNA之间的强相互作用确实能使磁珠和金纳米棒之间形成稳定结构，说明标记很可靠，这一点是金纳米棒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(本例中为二次去粒子水)并在70℃的水浴锅中加热5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，用移液枪小心移取4微升的上清液滴落在洁净的载玻片上，用盖玻片压盖在其上形成显微镜样本，利用暗场显微镜对样本中的红色亮点进行计数，暗场显微图如图4所示。
对随机选取的30幅图片进行统计，即可用于对靶分子的浓度测定，在实验中，我们对一系列浓度的靶分子DNA进行检测，制定出其浓度定标曲线，如图 5所示，依据检测极限的3倍标准差定义，可算得该方法在DNA探测中的检测下限大约为1.3X10-18mol/L，比一般基于荧光强度的探测技术高出4-5个量级，有极高的应用价值。
依据上述的检测方法，对8×10-17摩尔/升的同一种标准DNA样品进行检测，并与上述测得的定标曲线进行比对，如图5所示，得出浓度为6.7±2.1×10-17摩尔/升，误差在16％左右，说明本发明的检测方法可靠性很高。
在以上实施例中，是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，对溶液中的标记粒子进行计数实现靶分子的浓度测定。在其他一些实施例中，还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技术人员来说，在不脱离本发明构思的前提下，还可以做出若干等同替代或明显变型，而且性能或用途相同，都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种靶分子浓度的检测方法，其特征在于，包括如下步骤：
    (1)将第一探针分子偶联到磁珠表面形成磁珠探针，将第二探针分子偶联到标记粒子表面形成标记探针；
    (2)在反应容器中，将所述磁珠探针和靶分子溶液混合，所述靶分子与所述第一探针分子反应，所述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；
    (3)将所述标记探针加入所述反应容器，所述靶分子与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；
    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，将所述标记探针和靶分子溶液混合，所述靶分子与所述第二探针分子反应，所述靶分子被捕获至所述标记探针的表面；
    (3’)将所述磁珠探针加入所述反应容器，所述靶分子与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；
    (4)将未参与反应的所述标记探针除去；
    (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、所述靶分子和所述标记探针；
    (6)吸取经过步骤(5)后的溶液在载玻片上制成样本，在显微镜下对所述标记探针进行计数并计算所述溶液中总的标记探针的数量，通过所述总的标记探针的数量计算所述靶分子的浓度。
  2. 如权利要求1所述的靶分子浓度的检测方法，其特征在于：所述步骤(2)或者所述步骤(3’)中的所述磁珠探针的加入个数为105-107个。
  3. 如权利要求1所述的靶分子浓度的检测方法，其特征在于：所述步骤(3)或者所述步骤(2’)中的所述标记探针的加入个数为1010-1012个。
  4. 如权利要求1所述的靶分子浓度的检测方法，其特征在于：所述步骤(5)中，所述洗脱液的加入量为10-100μL。
  5. 如权利要求1所述的靶分子浓度的检测方法，其特征在于：当所述检测方法按所述步骤(2)和步骤与(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。
  6. 如权利要求1所述的靶分子浓度的检测方法，其特征在于：所述步骤(4)具体为：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。
  7. 如权利要求1所述的靶分子浓度的检测方法，其特征在于：在所述步骤(6)的吸取之前，还包括如下步骤：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。
  8. 如权利要求1-7任意一项所述的靶分子浓度的检测方法，其特征在于：所述标记粒子为贵金属纳米粒子、荧光微球或者上转换纳米晶。
  9. 如权利要求1-7任意一项所述的靶分子浓度的检测方法，其特征在于：所述靶分子为DNA分子或者蛋白质。
  10. 如权利要求8所述的靶分子浓度的检测方法，其特征在于：所述贵金属纳米粒子为金纳米球、金纳米棒或金/银复合纳米粒子。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021087006A1 (en) * 2019-10-28 2021-05-06 Genotix Biotechnologies Inc. Analyte detection and quantification by discrete enumeration of particle complexes

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105132533B (zh) * 2015-07-24 2018-11-23 清华大学深圳研究生院 一种靶分子浓度的检测方法
CN105699262A (zh) * 2016-01-22 2016-06-22 清华大学深圳研究生院 一种带电微纳米颗粒的超低浓度探测方法
CN106568975A (zh) * 2016-11-03 2017-04-19 清华大学深圳研究生院 一种多靶分子浓度检测方法
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CN111999225A (zh) * 2019-12-19 2020-11-27 瑞芯智造(深圳)科技有限公司 一种微纳颗粒浓度的检测方法
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CN112964868B (zh) * 2021-02-05 2022-06-28 华中农业大学 一种基于磁分离同时检测多种目标物的生化分析方法
CN113252632B (zh) * 2021-06-25 2021-10-19 成都瀚辰光翼生物工程有限公司 样本浓度处理方法、装置、样本处理设备及可读存储介质

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1764729A (zh) * 2003-01-24 2006-04-26 人类遗传标记控股有限公司 使用嵌入核酸检测核酸中甲基化改变的分析
WO2007024676A2 (en) * 2005-08-19 2007-03-01 Nanosphere, Inc. Methods for preparing hybrid substrates comprising dna and antibodies and uses thereof
WO2011047087A2 (en) * 2009-10-13 2011-04-21 Nanostring Technologies, Inc. Protein detection via nanoreporters
CN102229984A (zh) * 2011-05-13 2011-11-02 青岛科技大学 一种基于熵驱动的dna杂交反应和荧光显微镜计数进行单分子检测的新方法
CN104007087A (zh) * 2014-05-13 2014-08-27 北京大学 一种透明平整片状基底表面的金纳米材料计数方法
CN104498600A (zh) * 2014-12-10 2015-04-08 东南大学 一种基于磁珠与核酸水解分离发光标记物的核酸检测方法
CN104531851A (zh) * 2014-12-10 2015-04-22 东南大学 一种基于磁珠与多糖水解分离发光标记物的核酸检测方法
CN104655615A (zh) * 2014-07-08 2015-05-27 青岛科技大学 基于CuS纳米粒子阳离子交换增强化学发光的生物传感器
CN105132533A (zh) * 2015-07-24 2015-12-09 清华大学深圳研究生院 一种靶分子浓度的检测方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103882136A (zh) * 2014-03-31 2014-06-25 苏州大学 一种简便灵敏通用型的基因检测方法

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1764729A (zh) * 2003-01-24 2006-04-26 人类遗传标记控股有限公司 使用嵌入核酸检测核酸中甲基化改变的分析
WO2007024676A2 (en) * 2005-08-19 2007-03-01 Nanosphere, Inc. Methods for preparing hybrid substrates comprising dna and antibodies and uses thereof
WO2011047087A2 (en) * 2009-10-13 2011-04-21 Nanostring Technologies, Inc. Protein detection via nanoreporters
CN102229984A (zh) * 2011-05-13 2011-11-02 青岛科技大学 一种基于熵驱动的dna杂交反应和荧光显微镜计数进行单分子检测的新方法
CN104007087A (zh) * 2014-05-13 2014-08-27 北京大学 一种透明平整片状基底表面的金纳米材料计数方法
CN104655615A (zh) * 2014-07-08 2015-05-27 青岛科技大学 基于CuS纳米粒子阳离子交换增强化学发光的生物传感器
CN104498600A (zh) * 2014-12-10 2015-04-08 东南大学 一种基于磁珠与核酸水解分离发光标记物的核酸检测方法
CN104531851A (zh) * 2014-12-10 2015-04-22 东南大学 一种基于磁珠与多糖水解分离发光标记物的核酸检测方法
CN105132533A (zh) * 2015-07-24 2015-12-09 清华大学深圳研究生院 一种靶分子浓度的检测方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021087006A1 (en) * 2019-10-28 2021-05-06 Genotix Biotechnologies Inc. Analyte detection and quantification by discrete enumeration of particle complexes

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