Nanopore sensing specific enhancement technique for single molecule detection

Pan Qin-Jie; Zhao Can-Dong; Chen Qi; He Yu-Hui; Miao Xiang-Shui

doi:10.7498/aps.73.20240159

Abstract

Nanopore sensors have become important tools for analyzing biomarkers, including but not limited to nucleic acids, proteins, and other biomolecules that play important roles in life. Though the nanopores themselves have no selectivity towards target molecules, higher sensitivity of nanopore sensing to the target biomarkers could be achieved with the help of the specificity enhancement technology. In this work, the basic principles of nanopore sensing are first introduced, then methods of modifying nanopore surface as well as the development and application of those selectivity enhancement technologies of nanopore sensing in recent years are reviewed. These enhancement technologies primarily fall into two categories: surface functionalization and molecular probes. Surface functionalization is further categorized based on the types of functional molecules used, while molecular probes are classified according to carrier forms. Finally, in this paper several challenges that nanopore sensing continues to encounter are discussed and some suggestions are made for its future development.

Keywords:

Authors and contacts

1.
Institute of Information Storage Materials and Devices, School of Integrated Circuits, Huazhong University of Science and Technology, Wuhan 430074, China
2.
Hubei Yangtze Memory Laboratories, Wuhan 430205, China

Corresponding author: Chen Qi, chenqi_whu@hust.edu.cn

Funds: Project supported by the National Natural Science Foundation of China (Grant No. 62374063).

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Cited By

图 1 (a)未被修饰的纳米孔和用APTMS修饰的纳米孔示意图, 插图是透射电子显微镜(TEM)拍摄的修饰前5.2 nm纳米孔图像 ; (b) 1000碱基长度的 DNA 在 pH 6.0, 7.0和8.0条件下通过被修饰的 5.2 nm 纳米孔电流迹线图, i_o为开孔电流, i_b为阻塞电流, t_D为DNA过孔时间^[26]

Figure 1. (a) Schematic of an uncoated and an APTMS-coated nanopore, inset is a TEM image of a 5.2 nm nanopore before coating; (b) 1 kbp DNA translocating through a coated 5.2 nm pore at pH 6.0, 7.0, and 8.0, where i_o is the open-pore current, i_b is the blocked-level current, and t_D is the translocation time^[26].

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图 2 (a)未被修饰的纳米孔和用SDS修饰的纳米孔示意图; (b)未修饰(蓝线)和SDS修饰纳米孔(红线)测得的代表性DNA过孔事件^[27]

Figure 2. (a) Schematic diagram of the uncoated and SDS coated pore; (b) typical representative translocation event observed with uncoated (blue) and SDS coated nanopore (red)^[27].

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图 3 (a)淀粉样蛋白β通过流体脂质双分子层修饰的纳米孔示意图; (b) 不同聚类待测分子过孔引起的阻塞电流、过孔时间(∆I, t_d)数据产生的散点图, 黄色星号表示每种聚类产生的阻塞电流平均值 (i) 球形低聚物, (ii) 短原纤维, (iii) 长原纤维, (iv) 纤维^[30]

Figure 3. (a) Schematic representation of amyloid-β translocation through a nanopore modified by a fluid lipid bilayer; (b) scattered plot of blocking currents generated by different clusters, with yellow asterisks indicating the average blocking currents generated by each cluster, (i) spherical oligomer, (ii) short protofibril, (iii) long protofibril, (iv) fiber^[30].

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图 4 (a)葡萄糖氧化酶通过未修饰的纳米孔示意图(左), EDC/NHS交联反应后葡萄糖氧化酶通过半胱氨酸修饰的纳米孔示意图(右); (b) 两种过孔事件的阻塞电流、过孔时间散点图以及直方图^[31]

Figure 4. (a) Glucose oxidase through the unmodified nanopore (left), and glucose oxidase through the cysteine-modified nanopore after EDC/NHS cross-linking reaction (right); (b) blocked current scatter plots and histograms of the two translocation events^[31].

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图 5 (a) 两种凝血酶通过官能化纳米孔进行识别示意图; (b) 待测分子引起的相对电流阻塞、孔内停留时间散点图; (c) 采用机器学习方法处理数据后获得的识别率混淆矩阵^[39]

Figure 5. (a) Schematic illustration of two thrombin species identified by functionalized nanopores; (b) scatter diagram of relative current obstruction and in-hole residence time caused by molecules to be measured; (c) the recognition rate confusion matrix obtained after processing data using machine learning methods^[39].

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图 6 (a)使用官能化纳米孔选择性检测 miRNA-21 的方法　(i)纳米孔内壁采用金膜改性; (ii)巯基修饰的DNA1与纳米孔壁结合; (iii) MCH阻断活性位点; (iv) DNA1与纳米孔内壁结合, 特异性识别miRNA-21; (v) 引入发夹 H1 和 H2 以引发杂化链式反应. (b)官能化纳米孔在不同浓度miRNA-21 (0.1 pmol/L—0.5 nmol/L)下的I-E曲线(10 mmol/L KCl)^[42]

Figure 6. (a) Strategies for selective detection of miRNA-21 using functionalized nanopore: (i) the inner wall of the bare nanopore was modified with gold film; (ii) the sulfhydryl-modified DNA1 binds to the wall of the nanopore; (iii) the excess active site was blocked with MCH; (iv) DNA1 bound to the inner wall of the nanopore specifically recognized miRNA-21; (v) introducing hairpins H1 and H2 to initiate the hybrid chain reaction. (b) I-E curves (10 mmol/L KCl) of functionalized nanopore at different concentrations of miRNA-21 (0.1 pmol/L–0.5 nmol/L)^[42].

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图 7 (a)纳米孔修饰以及适配体与HAdV样品相互作用示意图; (b)归一化的电流整流比^[43]

Figure 7. (a) Scheme depicting the modification of the nanopore and the interaction of the aptamer with infectious HAdV samples; (b) normalized current rectification ratio^[43].

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图 8 (a)纳米移液器共价修饰流程示意图; (b)检测不同浓度N蛋白的纳米移液器的I-V曲线; (c)不同蛋白质引起的归一化离子电流变化^[47]

Figure 8. (a) Schematic description of the covalent modification procedures of nanopipettes; (b) I-V curves of the nanopipettes for detecting different concentrations of the N protein; (c) normalized ionic current changes caused by different proteins^[47].

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图 9 (a)未与配体结合(开放)和与配体结合(闭合)状态下的GBP示意图; (b)不同半乳糖浓度下的电流迹线(左), 闭合 (L1) 和开放 (L2) 构象在 30 s内的分布图(右)^[49]

Figure 9. (a) Schematic diagram of GBP in the ligand-free (open) and ligand-bound (closed) states; (b) current trace at different galactose concentrations (left), histogram of the closed (L1) and open (L2) conformations in 30 s (right)^[49].

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图 10 (a)通过DNA链杂交将Ty1固定在ClyA纳米孔上的方法示意图; (b)添加不同浓度的刺突蛋白前后 ClyA-f-Ty1 的代表性电流迹线^[50]

Figure 10. (a) Schematic diagram of the method of fixing Ty1 on ClyA nanopore by DNA strand hybridization; (b) the representative current trace of ClyA-f-Ty1 before and after the addition of different concentrations of spike protein^[50].

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图 11 (a) t-FhuA纳米孔检测Bs的示意图; (b)不同Bs浓度下的纳米孔电流迹线^[51]

Figure 11. (a) Schematic diagram of t-FhuA nanopore detection of Bs; (b) nanopore current traces at different Bs concentrations^[51].

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图 12 (a)锥形纳米孔中生物素和链霉亲和素之间的特异性结合示意图; (b) BSA, Lys以及不同浓度的链霉亲和素引起的电流下降比例^[52]

Figure 12. (a) Schematic diagram of specific binding between biotin and streptavidin in conical nanopore; (b) the proportion of current drop caused by BSA, Lys, and streptavidin at different concentrations^[52].

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图 13 (a) β-CD官能化锥形纳米孔的流程及其对映异构体的手性识别示意图; (b)加入等浓度L-His和D-His时纳米孔的电流变化比^[58]

Figure 13. (a) Process of β-CD functionalized conical nanopore and the chiral recognition diagram of its enantiomers; (b) current change ratio upon addition of L-His and D-His in equal concentration^[58].

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图 14 (a) PNRSS结构示意图, 孔隙限制处的反应部分(棕色)被苯硼酸(PBA)修饰. PBA 作为固定反应物, 结合目标分析物(如儿茶酚胺)并报告传感事件, 蓝色、绿色和黄色区域分别代表 PNRSS 链的延伸部分、牵引部分和系绳部位; (b)振幅 SD 与阻塞百分比I_b的散点图, 蓝色 (L-N)、橙色 (D-N)、黄色 (L-E) 和紫色 (D-E)^[61]

Figure 14. (a) PNRSS structure diagram, the reactive portion (brown) at the pore limit is modified by phenylboric acid (PBA). PBA acts as a fixed reactant, binds to target analytes (such as catecholamine) and reports sensing events, the blue, green, and yellow areas represent the PNRSS chain extension, traction, and tether, respectively; (b) scatter plot of amplitude SD versus percentage blockade I_b, blue (L-N), orange (D-N), yellow (L-E), and purple (D-E)^[61].

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图 15 (a)与MS结合的短双链DNA分子通过纳米孔示意图; (b)与MS结合的ssBio34 (红色)和dsBio34 (蓝色)事件频率与施加电压的关系^[68]

Figure 15. (a) Schematic diagram of translocation of short double-stranded DNA molecules bound to MS through nanopores; (b) event rate vs. applied voltage for ssBio34 (red) and dsBio34 (blue) with MS bound^[68].

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图 16 (a)固态纳米孔用于CRP检测示意图, 右上图为15 nm孔径的纳米孔TEM图像; (b) CRP、适配体和 CRP-适配体复合物通过纳米孔的阻塞电流比以及过孔时间的散点图与直方图^[72]

Figure 16. (a) Schematic diagram of solid-state nanopores used for CRP detection, the up right is a TEM image of an aperture of 15 nm in diameter; (b) scatter plots and histograms of blocking current ratios and times of translocation of CRP, aptamer, and CRP-aptamer complexes through nanopore^[72].

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图 17 利用气溶素(Aerolysin)纳米孔进行多重生物标志物鉴别以及不同生物标志物引起的相应阻塞电流事件示意图^[73]

Figure 17. Schematic illustration of multiplexed biomarker discrimination with the aerolysin nanopore and the corresponding blocking current events induced by different biomarkers^[73].

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图 18 (a)石英纳米移液管选择性检测CEA分子示意图, 左边的分子代表Apt-MNPs, 右边的分子代表CEA-Apt-MNP复合物; (b)在+400 mV下1 mol/L KCl的CEA和Apt-MNP混合溶液的CEA-Apt-MNPs(左)和Apt-MNP(右)相应阻塞信号的电流迹线; (c)使用适配体官能化MNPs分离CEA的策略^[84]

Figure 18. (a) Schematic illustration of selective detection of CEA molecules with a quartz nanopipette, the left molecule represents the Apt-MNPs, and the right one refers to CEA-Apt-MNP complexes; (b) current-time trace for the presence of a mixed solution of CEA and Apt–MNPs with 1 mol/L KCl at +400 mV, the arrow points to the current traces of the corresponding blockage signals of CEA-Apt-MNPs (left) and Apt-MNPs (right); (c) the strategy for separation of CEA using aptamer functionalized MNPs^[84].

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图 19 基于夹心法分析, 点击化学以及纳米孔传感来检测生物标志物的方法示意图^[85]

Figure 19. Schematic diagram of a method for detecting biomarkers based on sandwich assay, click chemistry, and nanopore sensing^[85].

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图 20 (a)载体制备示意图; (b) α-Syn低聚物与DNA载体结合示意图. (c) DNA载体与不同α-Syn低聚物结合的统计结果 (i) 每个样品的典型电流迹线, 峰值电流随着α-Syn低聚物聚合时间的延长而逐渐增大; (ii) 每个样品的3个代表性信号^[93]

Figure 20. (a) Schematic diagram of carrier preparation; (b) schematic diagram of α-Syn oligomer binding to DNA carrier; (c) statistical results of binding of DNA vectors to different α-Syn oligomers: (i) typical current trace for each sample; the peak current increases gradually with the extension of polymerization time of α-Syn oligomer; (ii) three representative signals for each sample^[93].

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图 21 (a) DNA载体的2D示意图, 具有凝血酶和AChE的特异性探针; (b)在–200 mV下记录的电流时间迹线, 并在5 kHz下滤波, 清楚显示3个电平, 第1个与DNA载体相关, 第2个与凝血酶相关, 第3个与AChE相关^[65]

Figure 21. (a) 2D schematic of the λ-DNA carrier with two independent aptamer probes specific to thrombin and AChE; (b) typical current-time trace recorded at –200 mV and re-filtered at 5 kHz clearly showing three levels, the 1st associated with the DNA carrier, 2nd with thrombin and 3rd from AChE^[65].

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图 22 (a)带有分子信标的DNA载体制备及其与相应miRNA结合示意图; (b)存在miRNA (let-7a, miR-375-3p和miR-141-3p)的情况下, DNA载体过孔的代表性光子和电流时间迹线, 代表 5.6, 10 和 38.5 kbp DNA 片段长度的过孔事件的3个电信号用实心圆、方形和星号标记, 光信号中相应同步事用圆圈标记^[95]

Figure 22. (a) Schematic representation of the preparation of size-coded DNA probes and their binding to respective miRNA targets; (b) representative photon and current time traces of DNA carrier translocations in the presence of miRNAs (let-7a, miR-375-3p, and miR-141-3p), three electrical signals representing translocation events of 5.6, 10, and 38.5 kbp DNA fragment lengths are marked with a filled circle, square, and asterisk, the corresponding synchronization in the optical signal is marked with a circle^[95].

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图 23 目标DNA存在的情况下, DNA哑铃纳米开关的拓扑变化从“开放”状态转变到“封闭”状态以及代表性电流迹线示意图^[98]

Figure 23. Schematic diagram of the topological change of the DNA dumbbell nanoswitch from the “open” state to the “closed” state in the presence of the target nucleic acid and representative current traces^[98].

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图 24 (a) opTET2 和 opTET2/SA 的原理图, 红色T代表碱基的生物素修饰; (b) opTET结构的原子力显微镜(AFM) 图像; (c) DNA 四面体opTET2 和 opTET2/SA 通过 30 nm 纳米孔过孔事件的散点图与直方图^[102]

Figure 24. (a) Schematic diagram of opTET2 and opTET2/SA, and the red T representing biotin modification of the base; (b) AFM images of opTET structures; (c) scatter plots and histograms of DNA tetrahedron opTET2 and opTET2/SA translocation events through 30 nm nanopore^[102].

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表 1 目标生物分子对应的特异性增强技术以及检测极限

Table 1. Specific enhancement techniques and detection limits corresponding to target biomolecules.

特异性增强技术	目标生物分子	纳米孔类型	检测极限
表面官能化	α-thrombin γ-thrombin	SiN_x	N/A^[39]
	miRNA-21	Quartz	0.1 pmol/L^[42]
	HAdV SARS-CoV-2	PET	6 pfu/mL^[43] 10⁴ copies/mL^[43]
	N protein	Nanopipette	73.204 pg/mL^[47]
	Neuraminidase	ClyA	38 nmol/L^[49]
	SARS-CoV-2 Spike Protein	ClyA	2.3 nmol/L^[50]
	Barstar	t-FhuA	12.6 nmol/L^[51]
	Streptavidin	PET	N/A^[52]
	L-His, D-His	PET	N/A^[58]
	L-Trp	PI	N/A^[59]
	norepinephrine epinephrine	MspA	1 μmol/L^[61]
	ribonucleotide	MspA	N/A^[62]
分子探针	miR155	SiN_x	10 nmol/L^[68]
	C-reactive protein	SiN_x	0.3 ng/μL^[72]
	alpha-1 antitrypsin Tau 381 BACE1	Aerolysin	77.9 fmol/L^[73] 6.79 fmol/L^[73] 86.4 fmol/L^[73]
	miRNA-21	Glass	5 nmol/L^[83]
	Carcinoembryonic-antigen	Quartz	0.01 nmol/L^[84]
	alpha fetoprotein	α-HL	1 fmol/L^[85]
	ESAT-6/CFP-10	α-HL	10 amol/L^[86]
	α-Syn	Quartz	2.2 pmol/L^[93]
	Thrombin AChE	Quartz	N/A^[65]
	miR-375-3p miR-141-3p	SiN_x	8 fmol/L^[95] 5 fmol/L^[95]
	E. coli DH5α 16 SrRNA Salmonella 16 SrRNA A. Baumanii 16 SrRNA	Glass	N/A^[98]
	Streptavidin	SiN_x	N/A^[102]

DownLoad: CSV

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