Nanopores have become one of the most important tools for single-molecule sensing, but the challenge for selective detection of specific biomolecules still exists. In this contribution, we develop a new technique for sensing carcinoembryonic antigen (CEA), one of the important cancer biomarkers, using solid-state nanopores as a tool. The method is based on the specific affinity between aptamer (Apt) modified magnetic Fe3O4–Au nanoparticles (MNPs) and CEA, and the formed CEA–Apt–MNPs and remaining Apt–MNPs can transport the nanopores by applying a positive potential after magnetic separation. Due to the obvious particle size difference between CEA–Apt–MNPs and Apt-MPs, their corresponding blockage signals could be distinguished completely by the degree of the current decline. Moreover, the frequency of the blockage signals for CEA–Apt–MNPs is proportional to the concentration of CEA within certain limits, indicating that our designed nanopore sensing strategy can quantitatively detect CEA in complex samples. This work demonstrates that our designed nanopore-based strategy can be used for CEA sensing with good selectivity and sensitivity and also can be used to analyze other protein biomarkers for early diagnosis and monitoring of cancer, though the detection limit (0.6 ng/mL) is not relatively low. In future works, we plan to improve our detection limit by the improvement of the nanopipette preparation technology and detection method.

Solid-state nanopores have been employed as useful tools for single molecule analysis due to their advantages of easy fabrication and controllable diameter, but selectivity is always a big concern for complicated samples. In this work, functionalized magnetic core−shell Fe 3 O 4 −Au nanoparticles, which acted as a molecular carrier, were introduced into nanopore electrochemical system for microRNA sensing in complicated samples with high sensitivity, selectivity and signal-to-noise ratio (SNR). This strategy is based on the specific affinity between neutral peptide nucleic acids (PNA)-modified Fe 3 O 4 −Au nanoparticles and negative miRNA, and the formation of negative Fe 3 O 4 −Au−PNA−miRNA complex, which can pass through the nanopore by application of a positive potential and eliminate neutral Fe 3 O 4 −Au−PNA complex. To detect miRNA in complicated samples, a magnet has been used to separate Fe 3 O 4 −Au−PNA−miRNA complex with good selectivity. We think this is a facile and effective method for the detection of different targets at single molecular level, including nucleic acids, proteins, and other small molecules, which will open up a new approach in the nanopore sensing field.

Stochastic collision electrochemistry is a hot topic in single molecule/particle research, which provides an opportunity to investigate the details of the single molecule/particle reaction mechanism that is always masked in ensemble-averaged measurements. In this work, we develop an electrochemical amplification strategy to monitor the electrocatalytic behavior of single G-quadruplex/hemin (GQH) for the reaction between hydrogen peroxide and hydroquinone (HQ) through the collision upon a gold nanoelectrode. The intrinsic peroxidase activities of single GQH were investigated by stochastic collision electrochemical measurements, giving further insights into understanding biocatalytic processes. Based on the unique catalytic activity of GQH, we have also designed a hybridization chain reaction strategy to detect miRNA-15 with good selectivity and sensitivity. This work provided a meaningful strategy to investigate the electrochemical amplification and the broad application for nucleic acid sensing at the single molecule/particle level.

A simple strategy was developed for nanopore modification, and was used for miRNA sensing with good sensitivity and selectivity.

Nanopipettes provide a promising confined space that enables advances in single-molecule analysis, and their unique conical tubular structure is also suitable for single-cell analysis. In this work, functionalized-nanopore-based single-entity electrochemistry (SEE) analysis tools were developed for the label-free monitoring of single-molecule glycoprotein−boronate affinity interaction for the first time, and immunoglobulin G (IgG, one of the important biomarkers for many diseases such as COVID-19 and cancers) was employed as the model glycoprotein. The principle of this method is based on a single glycoprotein molecule passing through 4-mercaptophenylboronic acid (4-MPBA)-modified nanopipettes under a bias voltage and in the meantime interacting with the boronate group from modified 4-MPBA. This translocation and affinity interaction process can generate distinguishable current blockade signals. Based on the statistical analysis of these signals, the equilibrium association constant (κ a ) of single-molecule glycoprotein−boronate affinity interaction was obtained. The results show that the κ a of IgG in the confined nanopore at the single-molecule level is much larger than that measured in the open system at the ensemble level, which is possibly due to the enhanced multivalent synergistic binding in the restricted space. Moreover, the functionalized-nanopore-based SEE analysis tools were further applied for the label-free detection of IgG, and the results indicate that our method has potential application value for the detection of glycoproteins in real samples, which also paves way for the single-cell analysis of glycoproteins.