Ren Ren

Single‐molecule protein profiling offers unparalleled sensitivity for detecting rare biomarkers, with significant potential for early disease diagnosis and monitoring. Nanopore sensors enable the detection of proteins in solution and are well‐suited for clinical applications due to their high sensitivity and throughput. Here, we introduce a nanopore‐based detection strategy utilising an aptamer‐protein‐aptamer sandwich structure to enhance protein detection further. The aptamer probe is extended with a double‐stranded DNA carrier to enhance protein transport and detection efficiency. Upon protein binding, dimerization of the DNA carriers occurs, allowing charge‐based classification of bound versus unbound states. Additionally, the sandwich complex produces a characteristic subpeak within individual nanopore events, with subpeak height correlating to protein size. This approach enables selective detection of dimeric proteins, including vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF), with sub‐picomolar detection limits. In principle, it can be adapted for any protein with two distinct aptamer‐binding sites, as demonstrated with thrombin. This method also facilitates real‐time monitoring of ligand‐induced receptor dimerization in complex biological fluids, offering a powerful tool for potential single‐molecule‐based diagnostics.

Introducing exogenous biomolecules into individual cells with precise control over space, time, and dosage is crucial for both fundamental and applied biological research. Glass nanopipettes have long been employed to deliver biomolecules into individual cells; yet, their reliance on the electrical charge of the target molecule and the need for penetrating the cellular membrane pose significant limitations. We demonstrate that voltage pulses applied through a glass nanopipette in proximity to the cell membrane induce localized electroporation and generate directional flow, enabling controlled delivery of both charged and neutral biomolecules into subcellular compartments, e.g., the nucleus, without the need for penetrating the cellular membrane. This approach minimizes cell damage and preserves cell viability, even after multiple rounds of injection. Our findings will serve as a reference for the design of novel nanopipette methods, contributing to the newly established field of spatiotemporal analysis of live cells.

Single‐molecule protein profiling offers unparalleled sensitivity for detecting rare biomarkers, with significant potential for early disease diagnosis and monitoring. Nanopore sensors enable the detection of proteins in solution and are well‐suited for clinical applications due to their high sensitivity and throughput. Here, we introduce a nanopore‐based detection strategy utilising an aptamer‐protein‐aptamer sandwich structure to enhance protein detection further. The aptamer probe is extended with a double‐stranded DNA carrier to enhance protein transport and detection efficiency. Upon protein binding, dimerization of the DNA carriers occurs, allowing charge‐based classification of bound versus unbound states. Additionally, the sandwich complex produces a characteristic subpeak within individual nanopore events, with subpeak height correlating to protein size. This approach enables selective detection of dimeric proteins, including vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF), with sub‐picomolar detection limits. In principle, it can be adapted for any protein with two distinct aptamer‐binding sites, as demonstrated with thrombin. This method also facilitates real‐time monitoring of ligand‐induced receptor dimerization in complex biological fluids, offering a powerful tool for potential single‐molecule‐based diagnostics.

Quantum mechanical tunnelling sensors (QMTs) have emerged as a promising technology for next‐generation single‐molecule detection. Furthermore, QMT sensors can be combined with redox species resulting in repeated oxidation and reduction (redox cycling).. We developed robust QMT probes with electrode gap distances below 2 nm. Using the generator‐collector (GC) mode, we verified that redox cycling of the ferrocyanide/ferricyanide (Fe(CN)₆^3−/4−) couple occurs both in the tunnelling regime and on the electrode surface. Our findings indicated that the current enhancement is affected by both the gap distance and surface modifications of the probes. These QMT probes exhibited remarkable sensitivity, capable of detecting Fe(CN)₆^3−/4− concentrations down to sub‐picomolar levels. Utilising this ability to modulate redox reactions, we adapted the QMT probes to serve as electrochemical sensors for detecting viral proteins. By modifying the electrode surfaces, our functionalised QMT probes achieved sub‐pM detection limits with high selectivity in biofluids such as nasopharyngeal secretions. These findings highlight the potential of QMT probes to develop into a new class of electrochemical tunnelling sensors, offering significant advancements in biomedical diagnostics.

Quantum mechanical tunnelling sensors (QMTs) have emerged as a promising technology for next‐generation single‐molecule detection. Furthermore, QMT sensors can be combined with redox species resulting in repeated oxidation and reduction (redox cycling).. We developed robust QMT probes with electrode gap distances below 2 nm. Using the generator‐collector (GC) mode, we verified that redox cycling of the ferrocyanide/ferricyanide (Fe(CN)₆^3−/4−) couple occurs both in the tunnelling regime and on the electrode surface. Our findings indicated that the current enhancement is affected by both the gap distance and surface modifications of the probes. These QMT probes exhibited remarkable sensitivity, capable of detecting Fe(CN)₆^3−/4− concentrations down to sub‐picomolar levels. Utilising this ability to modulate redox reactions, we adapted the QMT probes to serve as electrochemical sensors for detecting viral proteins. By modifying the electrode surfaces, our functionalised QMT probes achieved sub‐pM detection limits with high selectivity in biofluids such as nasopharyngeal secretions. These findings highlight the potential of QMT probes to develop into a new class of electrochemical tunnelling sensors, offering significant advancements in biomedical diagnostics.

This study introduces a novel, environmentally friendly albumin isolation method using graphene oxide (GO). GO selectively extracts albumin from serum samples, leveraging the unique interactions between GO’s oxygen-containing functional groups and serum proteins. This method achieves high purification efficiency without the need for hazardous chemicals. Comprehensive characterization of GO and reduced graphene oxide (rGO) through techniques such as X-ray diffraction (XRD) analysis, Raman spectroscopy, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) confirmed the structural and functional group transformations crucial for protein binding. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses demonstrated over 95% purity of isolated albumin, with minimal contamination from other serum proteins. The developed method, optimized for pH and incubation conditions, showcases a green, cost-effective, and simple alternative for albumin purification, promising broad applicability in biomedical research and clinical applications.

Single-molecule antigen detection using nanopores offers a promising alternative for accurate virus testing to contain their transmission. However, the selective and efficient identification of small viral proteins directly in human biofluids remains a challenge. Here, we report a nanopore sensing strategy based on a customized DNA molecular probe that combines an aptamer and an antibody to enhance the single-molecule detection of mpox virus (MPXV) A29 protein, a small protein with an M.W. of ca. 14 kDa. The formation of the aptamer-target-antibody sandwich structures enables efficient identification of targets when translocating through the nanopore. This technique can accurately detect A29 protein with a limit of detection of ∼11 fM and can distinguish the MPXV A29 from vaccinia virus A27 protein (a difference of only four amino acids) and Varicella Zoster Virus (VZV) protein directly in biofluids. The simplicity, high selectivity, and sensitivity of this approach have the potential to contribute to the diagnosis of viruses in point-of-care settings.

We report on single-molecule nanopore sensing combined with position-encoded DNA molecular probes, with chemistry tuned to simultaneously identify various antigen proteins and multiple RNA gene fragments of SARS-CoV-2 with high sensitivity and selectivity. We show that this sensing strategy can directly detect spike (S) and nucleocapsid (N) proteins in unprocessed human saliva. Moreover, our approach enables the identification of RNA fragments from patient samples using nasal/throat swabs, enabling the identification of critical mutations such as D614G, G446S, or Y144del among viral variants. In particular, it can detect and discriminate between SARS-CoV-2 lineages of wild-type B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.1.539 (Omicron) within a single measurement without the need for nucleic acid sequencing. The sensing strategy of the molecular probes is easily adaptable to other viral targets and diseases and can be expanded depending on the application required.

The analysis at the single-molecule level of proteins and their interactions can provide critical information for understanding biological processes and diseases, particularly for proteins present in biological samples with low copy numbers. Nanopore sensing is an analytical technique that allows label-free detection of single proteins in solution and is ideally suited to applications, such as studying protein-protein interactions, biomarker screening, drug discovery, and even protein sequencing. However, given the current spatiotemporal limitations in protein nanopore sensing, challenges remain in controlling protein translocation through a nanopore and relating protein structures and functions with nanopore readouts. Here, we demonstrate that supercharged unstructured polypeptides (SUPs) can be genetically fused with proteins of interest and used as molecular carriers to facilitate nanopore detection of proteins. We show that cationic SUPs can substantially slow down the translocation of target proteins due to their electrostatic interactions with the nanopore surface. This approach enables the differentiation of individual proteins with different sizes and shapes via characteristic subpeaks in the nanopore current, thus facilitating a viable route to use polypeptide molecular carriers to control molecular transport and as a potential system to study protein-protein interactions at the single-molecule level.

The ability to measure biomarkers, both specifically and selectively at the single‐molecule level in biological fluids, has the potential to transform the diagnosis, monitoring, and therapeutic intervention of diseases. The use of nanopores has been gaining prominence in this area, not only for sequencing but more recently in screening applications. The selectivity of nanopore sensing can be substantially improved with the use of labels, but substantial challenges remain, especially when trying to differentiate between bound from unbound targets. Here highly sensitive and selective molecular probes made from nanoparticles (NPs) that self‐assemble and dimerize upon binding to a biological target are designed. It is shown that both single and paired NPs can be successfully resolved and detected at the single‐molecule nanopore sensing and can be used for applications such as antigen/antibody detection and microRNA (miRNA) sequence analysis. It is expected that such technology will contribute significantly to developing highly sensitive and selective strategies for the diagnosis and screening of diseases without the need for sample processing or amplification while requiring minimal sample volume.