Research progress of single molecule biological detection methods and applications
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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。
Abstract:Single molecule biological detection technology is an efficient technology to understand the dynamic characteristics of various biomolecules at the single molecule level and explore their structure and function. The advantage of this technology is that it can detect the heterogeneity of free energy on a single molecule, which is beyond the traditional methods. Therefore, researchers use it to solve long-standing problems in complex biological systems, heterogeneous catalysis, biomolecular interactions, enzyme systems and conformational changes. In terms of medical detection, detecting specific information about single molecules or their interactions with biological factors is not only crucial for the early diagnosis and treatment of various diseases such as cancer, but also has great potential for real-time detection and precision medicine. The advantages of high specificity and high precision of single-molecule bioassays are used to real-time detection of single biomolecules in molecular populations, and can be combined with multiple high-throughput analysis for the precise diagnosis of clinical samples. In this paper, the principle of single molecule detection and the application of biosensing are introduced, and the detection methods and related applications are summarized. Finally, the prospect and development direction of this research direction are discussed.
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Key words:
- single molecule biological detection /
- nucleic acid /
- nanopore /
- protein /
- heterogeneity
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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
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 methods Main detection molecules Main advantages Main disadvantages Nanopore Long and short chains of DNA and RNA, proteins, and polypeptide Required low sample volume; compact and simple;
allows label free detectionProne to error; high cost SERS Virus, protein, biomarkers High sensitivity; high selectivity Nonspecific binding with interfering molecule can give false signals Digital single molecule Nucleic acid, protein, small molecule High specificity; quantitative; wide dynamic range Limited multiplexing capabilities; high cost WGM Virus, Protein conformation etc. High sensitivity; easy to manufacture; low cost Low specificity, limited to frequency
and wavelengthCRISPR Nucleic acid Low cost; high efficiency Not suitable for multiple analyte detection, required sample pretreatment 
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