In this report, water-soluble carbon dots (CDs) with stable fluorescence (FL) were designed and synthesized by using environment-friendly reduced L-glutathione (GSH) as the precursor and ethylenediamine (EDA) as the passivating agent through a one-step hydro-thermal method. The CDs, with bright blue FL, had the quantum yield (QY) of 40% and showed excellent monodispersity and solubility in water. The FL intensity was remarkably enhanced when a racemic modification of L-/D-arginine (Arg) was added into the CDs solution. Therefore, the CDs could be considered as a sensitive probe for the detection of Arg. Under optimized conditions, the CDs exhibited a linear fluorescence response in the range of 3–124 μM (R² = 0.9987) Arg with the detection limit of 2.85 × 10⁻⁸ M. In addition, no evident difference was observed when comparing the FL results of L-Arg and D-Arg. Therefore, this method could not be used in the chiral recognition of two enantiomers. Under optimum conditions, the mechanism and the reasons for the enhancement of FL were investigated. The developed method exhibited great stability, selectivity and accuracy in determining the Arg content in human urine sample and the recovery range was from 97.0% to 102.2%. Thus, our proposed method has potential application in biological molecular recognition.

Protein is an important component of all the cells and tissues of the human body and is the material basis of life. Its content, sequence, and spatial structure have a great impact on proteomics and human biology. It can reflect the important information of normal or pathophysiological processes and promote the development of new diagnoses and treatment methods. However, the current techniques of proteomics for protein analysis are limited by chemical modifications, large sample sizes, or cumbersome operations. Solving this problem requires overcoming huge challenges. Nanopore single molecule detection technology overcomes this shortcoming. As a new sensing technology, it has the advantages of no labeling, high sensitivity, fast detection speed, real-time monitoring, and simple operation. It is widely used in gene sequencing, detection of peptides and proteins, markers and microorganisms, and other biomolecules and metal ions. Therefore, based on the advantages of novel nanopore single-molecule detection technology, its application to protein sequence detection and structure recognition has also been proposed and developed. In this paper, the application of nanopore single-molecule detection technology in protein detection in recent years is reviewed, and its development prospect is investigated.

Nanopore detection technology has now developed into an indispensable tool for single molecule experiments, in which useful information on molecular properties can be obtained from the ion current flow induced by individual molecules, but the relatively high background noise affects the data analysis. Therefore, a nanopore signal noise reduction method based on wavelet transform and Kalman filter is proposed, which can achieve multi-scale decomposition and optimal estimation of the nanopore signal. The real measurement shows that the Kalman filter based on the wavelet mode maxima method reduces the root mean square (RMS) of the background noise by 17.8%, and the noise reduction effect is better than the traditional Kalman filter method.

D-Penicillamine (D-PA), as the dominant enantiomer, is usually used in many clinical drugs, but the other enantiomer, L-penicillamine (L-PA) is a toxic enantiomer. Therefore, it is of great significance to analyze and detect L-PA in biomedical or environmental samples. CdTe quantum dots (CdTe QDs) were modified by N-acetyl-L-cysteine (NALC) as novel fluorescent probes and were successfully utilized for the determination of L-penicillamine. The fluorescence of the CdTe QDs decreased significantly in the presence of cupric ions (Cu²⁺) due to the binding of Cu²⁺ to NALC on the surface of the CdTe QDs, but the addition of L-PA immediately resulted in efficient fluorescence recovery of the modulated CdTe QDs. The experimental results showed that the pH of the buffer solution and the concentration of Cu²⁺ affected the fluorescence intensity upon adding L-PA. Under the optimum conditions, the recovered fluorescence intensity was linearly proportional to the increasing L-PA concentration in the range of 2 × 10⁻⁷ to 4 × 10⁻⁶ mol L⁻¹, with correlation coefficient R² = 0.9980. So, a new, simple and rapid method for the determination of L-PA has been developed and its potential has been demonstrated in human serum samples and environmental samples with satisfactory results.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has exposed the urgency of research on rapid and efficient virus detection and strategies to inhibit its replication. Previous studies have mostly focused on traditional immunoassay or optical methods, but they have limitations in terms of sensitivity, timeliness, and in-depth analysis of molecular interaction mechanisms. Solid-state nanopore single-molecule detection methods, which can monitor molecular conditions in real time at the single-molecule level, bring new opportunities to solve this problem. The nucleocapsid protein (N protein) of SARS-CoV-2 was systematically investigated under different conditions, such as external drive voltage, pH, nanopore size, and N protein concentration. The translocation of the N protein through the nanopore was then analyzed. Subsequently, we analyzed the translocation characteristics of the N protein, RNA, and N protein–RNA complexes. With the aid of EMSA experiments, we conclusively confirmed that RNA binds to the N protein. Building on this finding, we further explored small molecules that could affect the nanopore translocation of N protein–RNA complexes, such as gallocatechin gallate (GCG), epigallocatechin gallate (EGCG), and the influenza A viral inhibitor Nucleozin. The results show that GCG can disrupt the liquid-phase condensation of the N protein–RNA complex and inhibit the replication of the N protein. Meanwhile, the structural isomer EGCG of GCG and the small molecule Nucleozin can also block RNA-triggered N protein liquid–liquid phase separation (LLPS). Our results confirmed that GCG, EGCG, and Nucleozin exhibit antagonistic effects on the N protein, with differences in their effective concentrations and the potency of their antagonism. Herein, using solid-state nanopore single-molecule detection technology, we developed an experimental method that can effectively detect RNA-induced changes in N protein properties and the regulatory effects of small molecules on the LLPS of N protein–RNA complexes. These findings not only provide highly valuable insights for in-depth research on the molecular interactions involved in viral replication, but also open up promising new avenues for future responses to similar viral outbreaks, the development of a rapid and effective detection method based on solid-state nanopores and single-molecule detection, and antiviral therapies targeting N protein–RNA interactions.