Single-Molecule Investigation of the Protein–Aptamer Interactions and Sensing Application Inside the Single Glass Nanopore

Abstract

Solid-state nanopores offer a nanoconfined space for a single-molecule sensing strategy. Evaluating the behavior of proteins and protein-related interactions at the single-molecule level is becoming more and more important for a better understanding of biological processes and diseases. In this work, the aptamer-functionalized nanopore was prepared as the sensing platform for kinetic analysis of the carcinoembryonic antigen (CEA) with its aptamers, which is an important cancer biomarker. CEA molecules were captured by the aptamers immobilized on the inner surface of the nanopore, and there was a complicated interaction between the CEA molecules and the aptamer, which is the process of association and dissociation. This could be used to measure the dynamics of aptamer–protein interactions without labeling. The kinetic analysis could be evaluated at the single-molecule level to interpret the dissociation constants of the binding and dissociation processes. Results showed that the translocation of CEA molecules in a functionalized nanopore had a deep blockades degree and long duration compared with nanopore modified with bare gold, which could be used for CEA sensing. This protein and protein-related interaction we designed provides new insights for evaluating the binding affinity, which will be beneficial for protein sensing and immunoassays.

Iontronic Sensing Based on Confined Ion Transport

“…The kinetic analysis was conducted at the singlemolecule level to interpret the dissociation constants of the binding and dissociation processes. 215 Single-Cell Studies. Single-cell analysis is of utmost importance, as it enables analysis of cellular heterogeneity, observing dynamic processes, studying cellular development, and unraveling disease complexities.…”

“…Iontronic sensing on the basis of confined ion transport has emerged as an effective platform for analyzing single molecules using the principle of RPS. The RPS mechanism allows the detection of single molecules, such as ions/molecules, , and macromolecules like DNA/RNA/proteins, ,− with extraordinary sensitivity, enabling the extraction of their structural information. As single-molecule detection methods gain increasing importance in diagnostic applications, iontronics play a significant role in advancing the capabilities of single-molecule sensing.…”

“…The dynamic interactions between CEA molecules and the aptamer, involving association and dissociation, facilitated the measurement of aptamer–protein dynamics without the need for labeling. The kinetic analysis was conducted at the single-molecule level to interpret the dissociation constants of the binding and dissociation processes …”

Recent Developments in Single-Entity Electrochemistry

“…Moreover, the nanopipette-based nanoelectrode configuration offers a small tip opening size combined with a large inner electrochemically active area, which aids in minimizing damage to the cell while retaining a high detection sensitivity. Recent progress includes functionalization of the nanoelectrode surface with aptamers, , ligands, and tannic acid to analyze a range of biomolecules from living cells. Another approach that facilitates intracellular delivery and detection is the use of a double barrel pipet where one barrel is filled with carbon for electrochemical detection and the other barrel is filled with biomolecules for delivery .…”

Nanopore sensing specific enhancement technique for single molecule detection

“…在此基础上, Rotem等 [38] 将寡核苷酸作为适配器连接到α寡核苷纳米孔上, 通过适配器与各种核酸适配体结合, 允许相同的纳 米孔结构对多种分析物进行检测, 并用该纳米孔对 凝血酶浓度进行了定量分析. 当前, 使用机器学习 辅助纳米孔识别是一个可行的方案, 如图5所示, Reynaud等 [39] 使用核酸适配体官能化的纳米孔在 机器学习的辅助下成功区分了α凝血酶和 g 血凝 血酶, 最高准确率达到了98.8%. 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] Fig.…”