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单分子生物检测方法及应用研究进展

周文超 李政昊 武杰

周文超, 李政昊, 武杰. 单分子生物检测方法及应用研究进展[J]. 中国光学(中英文), 2022, 15(5): 878-894. doi: 10.37188/CO.2022-0129
引用本文: 周文超, 李政昊, 武杰. 单分子生物检测方法及应用研究进展[J]. 中国光学(中英文), 2022, 15(5): 878-894. doi: 10.37188/CO.2022-0129
ZHOU Wen-chao, LI Zheng-hao, WU Jie. Research progress of single molecule biological detection methods and applications[J]. Chinese Optics, 2022, 15(5): 878-894. doi: 10.37188/CO.2022-0129
Citation: ZHOU Wen-chao, LI Zheng-hao, WU Jie. Research progress of single molecule biological detection methods and applications[J]. Chinese Optics, 2022, 15(5): 878-894. doi: 10.37188/CO.2022-0129

单分子生物检测方法及应用研究进展

doi: 10.37188/CO.2022-0129
基金项目: 中科院青促会会员项目(No. 2020223);国家自然科学基金面上项目(No. 61974143)
详细信息
    作者简介:

    周文超(1987—),男,山东德州人,博士,博士生导师,2014年于中国科学院长春光学精密机械与物理研究所获得博士学位,主要研究方向为高灵敏度光学生物检测。E-mail:zhouvc@ciomp.ac.cn

    李政昊(1995—),男,吉林榆树人,硕士研究生,2019年于延边大学获得学士学位,主要研究方向为光学生物检测为基础的微流控芯片设计及工艺研究。E-mail:zhenghlee@163.com

  • 中图分类号: Q632;O439

Research progress of single molecule biological detection methods and applications

Funds: Supported by Youth Innovation Promotion Association of the Chinese Academy of Sciences (No. 2020223); National Natural Science Foundation of China (No. 61974143)
  • 摘要:

    单分子生物检测技术是通过了解单分子层面上各生物分子间的动态特性,以发掘生物分子的结构与功能的高效技术。该技术的优势在于能够在单个分子上探测自由能的异质性,这是传统方法无法实现的。利用这一性能,研究人员可以解决复杂生物系统、多相催化、生物分子相互作用、酶系统和构象变化等长期存在的问题。在医疗检测方面,检测单个分子的具体信息或它们与生物因子的相互作用,不仅对癌症等各种疾病的早期诊断和治疗至关重要,而且在实时检测和精准医疗方面具有巨大的潜力。利用单分子生物检测高特异性和高精度的优势,实现对分子群中单个生物分子的实时检测,且可与阵列高通量分析相结合对临床样本进行精确诊断。本文简要介绍了单分子检测原理及其在生物传感方面的应用,在此基础上,重点概述了检测方法及相关应用,最后探讨了该研究方向的前景与发展方向。

     

  • 图 1  (a)阴离子Au25(SG)18团簇增强纳米孔检测示意图及(b)相应的电流强度[18]

    Figure 1.  (a) Schematic diagram of the anionic Au25(SG)18 cluster-enhanced nanopore detection and (b) it′s corresponding current traces[18]

    图 2  不同材料架构的零模波导(ZMW)等离子体纳米孔[20]。(a)深紫外等离子体增强Al ZMW单蛋白自身荧光。(b)用于增强单分子探测的Au-Si零模混合波导。(c)等离子体纳米孔器件结构增强单分子荧光检测,该结构由金膜制备的纳米孔和独立式氮化硅膜制备的纳米孔组成。

    Figure 2.  Various material framework of zero-mode waveguide (ZMW) plasma nanopore[20]. (a)The autofluorescence of Al ZMW monoprotein was enhanced by deep ultraviolet plasma. (b) Au-Si zero-mode hybrid waveguides for enhanced single-molecule detection. (c)The plasma nanopore device structure enhances single-molecule fluorescence detection, which consists of nanopore prepared by gold film and nanopore prepared by independent silicon nitride film.

    图 3  通过野生型气溶胶膜通道运输C-A3和mC-A3[33]。(a)气溶胶纳米孔模型。气溶素(灰色)嵌入脂质双层膜(深蓝色),核苷酸(红色)放置在孔的入口;(b)含有甲基胞嘧啶和胞嘧啶的甲基化和非甲基化寡核苷酸结构。红色部分为添加的甲基部分

    Figure 3.  Transporting C-A3 and mC-A3 through a wild-type aerolysin membrane channel[33]. (a) All-atom model of the full-length aerolysin nanopore system. Aerolysin (gray) was inserted into a lipid bilayer membrane (dark blue), while nucleotides (red) were placed at the entrance of the pore. (b) Structure of methylated and unmethylated oligonucleotides containing methylcytosine and cytosine, respectively. The only difference between them was the addition of a methyl group which is marked in red

    图 4  微磁珠加载示意图[35]。(a)使用磁力和亲-疏水微孔阵列高效加载微磁珠。(b)直径、间距和深度不同的微孔阵列在多次循环下的加载率。(c、d)40倍显微镜下加载前后的亮场显微图像。(e)100倍显微镜下加载后的亮场显微图像

    Figure 4.  Magnetic bead seeding[35]. (a) Schematic of the magnetic bead seeding on HIH microwell arrays. (b) The bead distribution in arrays with varying well diameters, array pitches and well depths, for multiple-seeding cycles. (c,d) ×40 magnification bright field microscopy image of a microwell array before and after magnetic bead seeding, respectively. (e) ×100 magnification bright field microscopy image of a microwell array after seeding

    图 5  数字ELISA示意图[37]。(a)抗体结合微珠捕获单个目标生物分子,然后由另一个与标记抗体结合的抗体检测。将微珠装入飞升微孔阵列中用于分离和检测单个分子。(b)产生单分子信号的飞升微孔阵列的一部分荧光图像。大多数飞升体积微孔含有一颗微珠,但这些珠中只有一小部分具有催化酶活性,表明是一种单一的结合蛋白。(c)蛋白质在本体溶液中的浓度与结合蛋白质分子的微珠的百分比关系

    Figure 5.  Schematic representation of digital ELISA[37]. (a) Antibody-coated beads captures the single target biomolecules, which are then detected by another antibody conjugated with a labeled antibody. Loading of beads into femtoliter well arrays for isolation and detection of single molecules. (b) Fluorescence image of a small section of the femtoliter well array after signals from single molecules are generated. While the majority of femtoliter chambers contain a bead from the assay, only a fraction of those beads possesses catalytic enzyme activity, indicative of a single, bound protein. (c) The concentration of protein in the bulk solution is correlated to the percentage of beads that have bound a protein molecule

    图 6  数字式单分子阵列检测技术进行MicroRNA检测[43]。(a)单个miRNA分子通过与生物素化的探测器探针杂交到探针化的顺磁珠中被捕获。随后加入链霉亲和素结合酶来标记捕获的miRNA复合物,以便在与荧光酶底物孵育时产生荧光信号。(b)单个微珠与荧光衬底一起装入飞升微孔阵列,随后用油密封。然后将孔上荧光的数量作为目标miRNA浓度的数字读数

    Figure 6.  MicroRNA detection with digital single molecule detection technology[43]. (a) Individual miRNA molecules are captured by hybridization to probecoated paramagnetic beads, along with biotinylated detector probe. The streptavidin-conjugated enzyme is subsequently added to label the captured miRNA complex, to allow generation of a fluorescent signal upon incubation with a fluorogenic enzyme substrate. (b) Individual beads are loaded along with fluorogenic substrate into a femtoliter microwell array, which is subsequently sealed with oil. The number of fluorescent “on” wells is then counted as a digital readout of the target miRNA concentration

    图 7  由谐振腔表面的全内反射激发的WGM[51]

    Figure 7.  Illustration of a WGM cavity excited by frustrated total internal reflection at a prism surface[51]

    图 8  核酸适体的病毒颗粒在固体基质上的检测[79]

    Figure 8.  Detection of virus particles of aptamer on solid substrates[79]

    图 9  两种检测病毒RNA的CRISPR方法[89]。(a)SHERLOCK 方法;(b)DETECTR方法

    Figure 9.  Two CRISPR methods for detecting viral RNA[89]. (a) SHERLOCK assay; (b) DETECTR assay

    表  1  文中不同单分子生物检测方法的优缺点对比

    Table  1.   Advantages and disadvantages of various methods of single molecule biological detection

    Single molecule methodsMain detection moleculesMain advantagesMain disadvantages
    NanoporeLong and short chains of DNA and RNA, proteins, and polypeptideRequired low sample volume; compact and simple;
    allows label free detection
    Prone to error; high cost
    SERSVirus, protein, biomarkersHigh sensitivity; high selectivityNonspecific binding with interfering molecule can give false signals
    Digital single moleculeNucleic acid, protein, small moleculeHigh specificity; quantitative; wide dynamic rangeLimited multiplexing capabilities; high cost
    WGMVirus, Protein conformation etc.High sensitivity; easy to manufacture; low costLow specificity, limited to frequency
    and wavelength
    CRISPRNucleic acidLow cost; high efficiencyNot suitable for multiple analyte detection, required sample pretreatment
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  • 收稿日期:  2022-06-14
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