Navneet Chandra Verma

Recent advances in single-molecule technologies are transforming the field of protein analysis. Solid-state nanopores provide an effective method to linearize and thread full-length proteins in a single file. However, slowing their rapid translocation remains a challenge for accurate, time-resolved ion-current-based fingerprinting. In this work, we present a click-chemistry-based strategy for covalently attaching short oligonucleotides to cysteine residues on denatured proteins across a broad range of molecular weights. The negatively charged oligonucleotides increase the capture rate by a factor of ten compared with native proteins and induce a distinct ‘stick–slip’ motion that slows protein passage through the nanopore by more than 20-fold. These oligonucleotide tags also produce characteristic, time-resolved ion current pulses that serve as unique protein-specific signatures. To uncover the physical mechanism responsible for the protein translocation dynamics, we model our system using all-atom molecular dynamics and finite element simulations. By leveraging a supervised machine learning classifier, we demonstrate that a small number of translocation events is sufficient to identify individual proteins, achieving near-perfect classification accuracy. To demonstrate the robustness of the method, we successfully distinguish between VEGF-A isoforms (VEGF-165 and VEGF-121), which are relevant to cancer diagnostics, within a mixed protein sample. Our nanopore-based fingerprinting technique eliminates the need for affinity reagents, such as protein-specific antibodies, or motor proteins, offering a rapid, direct and cost-effective approach for single-molecule protein identification and classification.

Driven by recent advancements in nanofabrication techniques, single-molecule sensing and manipulations in nanofluidic devices are rapidly evolving. These sophisticated biosensors have already had significant impacts on basic research as well as on applications in molecular diagnostics. The nanoscale dimensions of these devices introduce new physical phenomena by confining the biomolecules in at least one dimension, creating effects such as biopolymer linearization, stretching, and separation by mass that are utilized to enhance the biomolecule sensing resolutions. At the same time, the suppressed diffusional motion allows for better single-molecule SNR (signal-to-noise ratio) sensing over time. In particular, nanofluidic devices based on nanochannels have been established as promising technologies for the linearization of ultralong genomic DNA molecules and for optical genome mapping, opening a window to directly observe and infer genome organization. More recently, nanochannels have shown promising capabilities for single-molecule protein sizing, separation, and identification. Consequently, this technology is attracting remarkable interest for applications in single-molecule proteomics. In this review, we discuss the recent advancements of nanochannel-based technologies, focusing on their applications for single-molecule sensing and the characterization of a wide range of biomolecules.

Emerging single‐molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra‐fast full‐length protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms, and low throughputs. Here, a single‐molecule method for parallel protein separation and tracking is introduced, yielding multi‐dimensional molecular properties used for their identification. Proteins are tagged by chemo‐selective dual amino‐acid specific labels and are electrophoretically separated by their mass/charge in custom‐designed thin silicon channel with subwavelength height. This approach allows analysis of thousands of individual proteins within a few minutes by tracking their motion during the migration. The power of the method is demonstrated by quantifying a cytokine panel for host‐response discrimination between viral and bacterial infections. Moreover, it is shown that two clinically‐relevant splice isoforms of Vascular endothelial growth factor (VEGF) can be accurately quantified from human serum samples. Being non‐destructive and compatible with full‐length intact proteins, this method opens up ways for antibody‐free single‐protein molecule quantification.

Gold nanocluster (GNC), because of its interesting photoluminescence properties and the easy renal clearance from the body, has got tremendous biomedical applications. Unfortunately, it has never been explored for super resolution microscopy (SRM). Here, we present a protein conjugated red emissive GNC, for the super resolution radial fluctuation (SRRF) of lysosome in HeLa cells. The diameter of the lysosome obtained in SRRF is ~59 nm, which is very close to the original diameter of the smallest lysosome in HeLa cell. Protein conjugation to GNC helped for the specific labelling to lysosome. We hope that GNC will not only replace some of the common dyes used in SRM but due to their electron beam contrast could also be used as a multimodal probe for several other correlative bioimaging techniques.

Insufficient purification and incomplete characterization pose a serious problem for attributing photoluminescence properties to carbogenic nanodots, especially those synthesized by bottom-up approaches. Here, we provide a roadmap for the successful future of these nanodots.

We introduce an orange emissive fluorescent nanodot for successful single molecule stochastic optical reconstruction microscopy (STORM), super resolution radial fluctuation (SRRF) microscopy and transmission electron microscopy (TEM).

Carbogenic fluorescent nanodots (FND) have recently drawn increasing attention in bioimaging. This is mainly due to their unique attractive photoluminescence properties. Although a vast number of reports are available, amphiphilic FND (aFND) are scarcely reported. In addition, the synthetic methods for amphiphilic FND are either tedious multistep processes or in most cases require additional surface passivation. Here, for the first time, using Maillard reaction protocol, we report an easy one pot synthesis aFND using sucrose and diaminooctane as the main precursors. The amphiphilic nature could be regulated easily upon changing the diaminooctane concentration. We demonstrate that the greater the hydrophobic nature in aFND, the more cell‐penetrating capability is observed. Flow cytometry measurements quantitatively show the fast live cell internalisation of the aFND inside HeLa cells. All the above features mark the excellent bio‐compatibility of aFND and give insight into its use as an excellent bioimaging tool.