In-situ fabrication of self-supported cobalt molybdenum sulphide on carbon paper for bifunctional water electrocatalysis.

The fabrication of highly efficient yet stable noble-metal-free bifunctional electrocatalysts that can simultaneously catalyse both hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) remains challenging. Herein, we employ the heterostructure coupling strategy, showcasing an aerosol-assisted chemical vapour deposition (AACVD) aided synthetic approach for the in-situ growth of cobalt molybdenum sulphide nanocomposites on carbon paper (CoMoS@CP) as a bifunctional electrocatalyst. The AACVD allows the rational incorporation of Co in the Mo-S binary structure, which modulates the morphology of CoMoS@CP, resulting in enhanced HER activity (ŋ10 = 171 mV in acidic and ŋ10 = 177 mV in alkaline conditions). Furthermore, the CoS2 species in the CoMoS@CP ternary structure extends the OER capability, yielding an ŋ100 of 455 mV in 1 M KOH. Lastly, we found that the synergistic effect of the Co-Mo-S interface elevates the bifunctional performance beyond binary counterparts, achieving a low cell voltage (1.70 V at 10 mA cm-2) in overall water splitting test and outstanding catalytic stability (∼90 % performance retention after 50-/30-h continuous operation at 10 and 100 mA cm-2, respectively). This work has opened up a new methodology for the controllable synthesis of self-supported transition metal-based electrocatalysts for applications in overall water splitting.

Selective Single-Molecule Nanopore Detection of mpox A29 Protein Directly in Biofluids.

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.

Multiplexed detection of viral antigen and RNA using nanopore sensing and encoded molecular probes.

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.

Nanopore Detection Using Supercharged Polypeptide Molecular Carriers.

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.

Single-Molecule Binding Assay Using Nanopores and Dimeric NP Conjugates.

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.

Single-molecule amplification-free multiplexed detection of circulating microRNA cancer biomarkers from serum.

MicroRNAs (miRNAs) play essential roles in post-transcriptional gene expression and are also found freely circulating in bodily fluids such as blood. Dysregulated miRNA signatures have been associated with many diseases including cancer, and miRNA profiling from liquid biopsies offers a promising strategy for cancer diagnosis, prognosis and monitoring. Here, we develop size-encoded molecular probes that can be used for simultaneous electro-optical nanopore sensing of miRNAs, allowing for ultrasensitive, sequence-specific and multiplexed detection directly in unprocessed human serum, in sample volumes as small as 0.1 µl. We show that this approach allows for femtomolar sensitivity and single-base mismatch selectivity. We demonstrate the ability to simultaneously monitor miRNAs (miR-141-3p and miR-375-3p) from prostate cancer patients with active disease and in remission. This technology can pave the way for next generation of minimally invasive diagnostic and companion diagnostic tests for cancer.

Reversing current rectification to improve DNA-sensing sensitivity in conical nanopores.

Herein, we report the ultrasensitive DNA detection through designing an elegant nanopore biosensor as the first case to realize the reversal of current rectification direction for sensing. Attributed to the unique asymmetric structure, the glass conical nanopore exhibits the sensitive response to the surface charge, which can be facilely monitored by ion current rectification curves. In our design, an enzymatic cleavage reaction was employed to alter the surface charge of the nanopore for DNA sensing. The measured ion current rectification was strongly responsive to DNA concentrations, even reaching to the reversed status from the negative ratio (-6.5) to the positive ratio (+16.1). The detectable concentration for DNA was as low as 0.1 fM. This is an ultrasensitive and label-free DNA sensing approach, based on the rectification direction-reversed amplification in a single glass conical nanopore.

Small molecule electro-optical binding assay using nanopores.

The identification of short nucleic acids and proteins at the single molecule level is a major driving force for the development of novel detection strategies. Nanopore sensing has been gaining in prominence due to its label-free operation and single molecule sensitivity. However, it remains challenging to detect small molecules selectively. Here we propose to combine the electrical sensing modality of a nanopore with fluorescence-based detection. Selectivity is achieved by grafting either molecular beacons, complementary DNA, or proteins to a DNA molecular carrier. We show that the fraction of synchronised events between the electrical and optical channels, can be used to perform single molecule binding assays without the need to directly label the analyte. Such a strategy can be used to detect targets in complex biological fluids such as human serum and urine. Future optimisation of this technology may enable novel assays for quantitative protein detection as well as gene mutation analysis with applications in next-generation clinical sample analysis.

A temperature, pH and sugar triple-stimuli-responsive nanofluidic diode.

In this article, we have demonstrated for the first time a triple stimuli-responsive nanofluidic diode that can rectify ionic current under multiple external stimuli including temperature, pH, and sugar. This diode was fabricated by immobilizing poly[2-(dimethylamino)ethyl methacrylate]-co-[4-vinyl phenylboronic acid] (P(DMAEMA-co-VPBA)) onto the wall of a single glass conical nanopore channel via surface-initiator atom transfer radical polymerization (SI-ATRP). The copolymer brushes contain functional groups sensitive to pH, temperature and sugar that can induce charge and configuration change to affect the status of the pore wall. The experimental results confirmed that the P(DMAEMA-co-VPBA) brush modified nanochannel regulated the ionic current rectification successfully under three different external stimuli. This biomimetically inspired research simulates the complex biological multi-functions of ion channels and promotes the development of "smart" biomimetic nanochannel systems for actuating and sensing applications.

A conformation and charge co-modulated ultrasensitive biomimetic ion channel.

For the first time, a biomimetic ion channel co-modulated simultaneously by conformation and charge using a single stimulus has been demonstrated, and, based on the synergetic effect of this channel, an ultrasensitive nanopore sensor for ATP with a limit of detection down to sub-pM was developed.

Surface charge modulated aptasensor in a single glass conical nanopore.

In this work, we have proposed a label-free nanopore-based biosensing strategy for protein detection by performing the DNA-protein interaction inside a single glass conical nanopore. A lysozyme binding aptamer (LBA) was used to functionalize the walls of glass nanopore via siloxane chemistry and negatively charged recognition sites were thus generated. The covalent modification procedures and their recognition towards lysozyme of the single conical nanopore were characterized via ionic current passing through the nanopore membrane, which was measured by recording the current-voltage (I-V) curves in 1mM KCl electrolyte at pH=7.4. With the occurring of recognition event, the negatively charged wall was partially neutralized by the positively charged lysozyme molecules, leading to a sensitive change of the surface charge-dependent current-voltage (I-V) characteristics. Our results not only demonstrate excellent selectivity and sensitivity towards the target protein, but also suggest a route to extend this nanopore-based sensing strategy to the biosensing platform designs of a wide range of proteins based on a charge modulation.