Real-time detection of 20 amino acids and discrimination of pathologically relevant peptides with functionalized nanopore.

Precise identification and quantification of amino acids is crucial for many biological applications. Here we report a copper(II)-functionalized Mycobacterium smegmatis porin A (MspA) nanopore with the N91H substitution, which enables direct identification of all 20 proteinogenic amino acids when combined with a machine-learning algorithm. The validation accuracy reaches 99.1%, with 30.9% signal recovery. The feasibility of ultrasensitive quantification of amino acids was also demonstrated at the nanomolar range. Furthermore, the capability of this system for real-time analyses of two representative post-translational modifications (PTMs), one unnatural amino acid and ten synthetic peptides using exopeptidases, including clinically relevant peptides associated with Alzheimer's disease and cancer neoantigens, was demonstrated. Notably, our strategy successfully distinguishes peptides with only one amino acid difference from the hydrolysate and provides the possibility to infer the peptide sequence.

An autocatalytic CRISPR-Cas amplification effect propelled by the LNA-modified split activators for DNA sensing.

CRISPR-Cas systems with dual functions offer precise sequence-based recognition and efficient catalytic cleavage of nucleic acids, making them highly promising in biosensing and diagnostic technologies. However, current methods encounter challenges of complexity, low turnover efficiency, and the necessity for sophisticated probe design. To better integrate the dual functions of Cas proteins, we proposed a novel approach called CRISPR-Cas Autocatalysis Amplification driven by LNA-modified Split Activators (CALSA) for the highly efficient detection of single-stranded DNA (ssDNA) and genomic DNA. By introducing split ssDNA activators and the site-directed trans-cleavage mediated by LNA modifications, an autocatalysis-driven positive feedback loop of nucleic acids based on the LbCas12a system was constructed. Consequently, CALSA enabled one-pot and real-time detection of genomic DNA and cell-free DNA (cfDNA) from different tumor cell lines. Notably, CALSA achieved high sensitivity, single-base specificity, and remarkably short reaction times. Due to the high programmability of nucleic acid circuits, these results highlighted the immense potential of CALSA as a powerful tool for cascade signal amplification. Moreover, the sensitivity and specificity further emphasized the value of CALSA in biosensing and diagnostics, opening avenues for future clinical applications.

E. coli outer membrane protein T (OmpT) nanopore for peptide sensing.

Peptide detection methods with facility and high sensitivity are essential for diagnosing disease associated with peptide biomarkers. Nanopore sensing technology had emerged as a low cost, high-throughput, and scalable tool for peptide detection. The omptins family proteins which can form ß-barrel pores have great potentials to be developed as nanopore biosensor. However, there are no study about the channel properties of E. coli OmpT and the development of OmpT as a nanopore biosensor. In this study, the OmpT biological nanopore channel was constructed with a conductance of 1.49 nS in 500 mM NaCl buffer and a three-step gating phenomenon under negative voltage higher than 100 mV and then was developed as a peptide biosensor which can detect peptide without the interfere of ssDNA and dNTPs. The OmpT constructed in this study has potential application in peptide detection, and also provides a new idea for the detection of peptides using the specific binding ability of protease.

Development of a fluorescent DNA nanomachine for ultrasensitive detection of Salmonella enteritidis without labeling and enzymes.

A capture probe complex containing a specific Salmonella enteritidis (S. enteritidis) aptamer and partly hybridized signal trigger sequence was designed with the ability to directly detect viable S. enteritidis. In the presence of the target S. enteritidis, single-stranded trigger sequences were liberated and in turn reacted with hairpins I, II, and III to initiate the triple strand migration reaction; this in turn produced numerous hairpin I·II·III complexes with scaffolds of copper nanoparticles (CuNPs) and replaced the trigger sequence which initiated the next cycle of triple migration reaction. Cyclically, the reuse of the trigger sequences and the successive, cascading production of scaffolds of CuNPs achieved the synthesis of highly fluorescent CuNPs, thus providing significantly enhanced fluorescent signals to achieve ultrasensitive detection of live S. enteritidis as low as 25 CFU/mL with a linear range of detection from 50 to 104 CFU/mL with an emission wavelength at 590 nm. By integrating the triple cascade strand migration amplification with recyclable trigger sequences, aptamer-based target recognition, and self-protection mediated by CuNPs hairpin scaffolds, this is the first report on a non-labeled, non-enzymatic, modification-free, and DNA extraction-free ultrasensitive fluorescent biosensor for the direct detection of live Salmonella, which is distinguished from dead Salmonella. It also provides a new strategy to detect viable bacteria by applying the CuNPs, thus extending the application of metal nanoparticles. Graphical abstract.

Synergistic regulation of fusion pore opening and dilation by SNARE and synaptotagmin-1.

Fusion pore opening is a transient intermediate state of synaptic vesicle exocytosis, which is highly dynamic and precisely regulated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and synaptotagmin-1 (Syt1). Yet, the regulatory mechanism is not fully understood. In this work, using single-channel membrane fusion electrophysiology, we determined that SNAREpins are important for driving fusion pore opening and dilation but incapable of regulating the dynamics. When Syt1 was added, the closing frequency of fusion pores significantly increased, while the radius of fusion pores mildly decreased. In response to Ca2+, SNARE/Syt1 greatly increased the radius of fusion pores and reduced their closing frequency. Moreover, the residue F349 in the C2B domain of Syt1, which mediates Syt1 oligomerization, was required for clamping fusion pore opening in the absence of Ca2+, probably by extending the distance between the two membranes. Finally, in Ca2+-triggered fusion, the primary interface between SNARE and Syt1 plays a critical role in stabilizing and dilating the fusion pore, while the polybasic region of Syt1 C2B domain has a mild effect on increasing the radius of the fusion pore. In summary, our results suggest that Syt1, SNARE, and the anionic membrane synergically orchestrate the dynamics of fusion pore opening in synaptic vesicle exocytosis.

Direct single-molecule detection of CoA-SH and ATP by the membrane proteins TMEM120A and TMEM120B.

Membrane proteins are vital resources for developing biosensors. TMEM120A is a membrane protein associated with human pain transmission and lipid metabolism, and recent studies have demonstrated its ability to transport ions and bind to coenzyme A (COA-SH), indicating its potential to develop into a single-molecule sensor based on electrical methods. In this study, we investigated the ion transport properties of TMEM120A and its homolog TMEM120B on an artificial lipid bilayer using single-channel recording. The results demonstrate that both proteins can fuse into the lipid bilayer and generate stable ion currents under a bias voltage. Based on the stable ion transport capabilities of TMEM120A and TMEM120B, as well as the feature of TMEM120A binding with COA-SH, we developed these two proteins into a single-molecule sensor for detecting COA-SH and structurally similar molecules. We found that both COA-SH and ATP can reversibly bind to single TMEM120A and TMEM120B proteins embedded in the lipid bilayer and temporarily block ion currents during the binding process. By analyzing the current blocking signal, COA-SH and ATP can be identified at the single-molecule level. In conclusion, our work has provided two single-molecule biosensors for detecting COA-SH and ATP, offering insights for exploring and developing bio-inspired small molecule sensors.

Direct and Continuous Monitoring of Multicomponent Antibiotic Gentamicin in Blood at Single-Molecule Resolution.

Point-of-care monitoring of small molecules in biofluids is crucial for clinical diagnosis and treatment. However, the inherent low degree of recognition of small molecules and the complex composition of biofluids present significant obstacles for current detection technologies. Although nanopore sensing excels in the analysis of small molecules, the direct detection of small molecules in complex biofluids remains a challenge. In this study, we present a method for sensing the small molecule drug gentamicin in whole blood based on the mechanosensitive channel of small conductance in Pseudomonas aeruginosa (PaMscS) nanopore. PaMscS can directly detect gentamicin and distinguish its main components with only a monomethyl difference. The 'molecular sieve' structure of PaMscS enables the direct measurement of gentamicin in human whole blood within 10 min. Furthermore, a continuous monitoring device constructed based on PaMscS achieved continuous monitoring of gentamicin in live rats for approximately 2.5 h without blood consumption, while the drug components can be analyzed in situ. This approach enables rapid and convenient drug monitoring with single-molecule level resolution, which can significantly lower the threshold for drug concentration monitoring and promote more efficient drug use. Moreover, this work also lays the foundation for the future development of continuous monitoring technology with single-molecule level resolution in the living body.

High-fidelity biosensing of dNTPs and nucleic acids by controllable subnanometer channel PaMscS.

Current tools for dNTP analysis mainly rely on expensive fluorescent labeling, mass spectrometry or electrochemistry. Single-molecule assay by protein nanopores with an internal diameter of ca. 1-3.6 nm provides a useful tool for dNTP sensing. However, the most commonly used protein nanopores require additional modifications to enable dNTP detection. In this study, the PaMscS channel (mechanosensitive channel of small conductance from Pseudomonas aeruginosa) embedded in the bilayer lipid membrane (BLM) of E. coli polar lipid extract was applied as a nanopore for single molecular sensing. Two mutants of PaMscS nanopores on the side portal region (PaMscS W130A and PaMscS K180R) were selected for direct dNTP or pyrophosphoric acid (PPi) detection without aptamer or protein modification. Notably, the PaMscS mutant pore can be adjusted by regulation of osmolarity differences, which is crucial for the optimal detection of specific molecules. In addition, we established a PaMscS-based diagnosis method for the rapid sensing of disease-associated nucleic acids by monitoring the consumption of dNTPs, with 86% specificity and 100% sensitivity among 22 clinical samples. This protein nanopore, without aptamer or modification, paves a new way for dNTPs, PPi direct sensing and nucleic acid detection with low cost but high versatility.

The structure and unzipping behavior of dumbbell and hairpin DNA revealed by real-time nanopore sensing.

Hairpin structures play an essential role in DNA replication, transcription, and recombination. Single-molecule studies enable the real-time measurement and observation of the energetics and dynamics of hairpin structures, including folding and DNA-protein interactions. Nanopore sensing is emerging as a powerful tool for DNA sensing and sequencing, and previous research into hairpins using an α-hemolysin (α-HL) nanopore suggested that hairpin DNA enters from its stem side. In this work, the translocation and interaction of hairpin and dumbbell DNA samples with varying stems, loops, and toeholds were investigated systematically using a Mycobacterium smegmatis porin A (MspA) nanopore. It was found that these DNA constructs could translocate through the pore under a bias voltage above +80 mV, and blockage events with two conductance states could be observed. The events of the lower blockage were correlated with the loop size of the hairpin or dumbbell DNA (7 nt to 25 nt), which could be attributed to non-specific collisions with the pore, whereas the dwell time of events with the higher blockage were correlated with the stem length, thus indicating effective translocation. Furthermore, dumbbell DNA with and without a stem opening generated different dwell times when driven through the MspA nanopore. Finally, a new strategy based on the dwell time difference was developed to detect single nucleotide polymorphisms (SNPs). These results demonstrated that the unzipping behaviors and DNA-protein interactions of hairpin and dumbbell DNA could be revealed using nanopore technology, and this could be further developed to create sensors for the secondary structures of nucleic acids.

Low-Cost and Scalable Platform with Multiplexed Microwell Array Biochip for Rapid Diagnosis of COVID-19.

Sensitive detection of SARS-CoV-2 is of great importance for inhibiting the current pandemic of COVID-19. Here, we report a simple yet efficient platform integrating a portable and low-cost custom-made detector and a novel microwell array biochip for rapid and accurate detection of SARS-CoV-2. The instrument exhibits expedited amplification speed that enables colorimetric read-out within 25 minutes. A polymeric chip with a laser-engraved microwell array was developed to process the reaction between the primers and the respiratory swab RNA extracts, based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP). To achieve clinically acceptable performance, we synthesized a group of six primers to identify the conserved regions of the ORF1ab gene of SARS-CoV-2. Clinical trials were conducted with 87 PCR-positive and 43 PCR-negative patient samples. The platform demonstrated both high sensitivity (95.40%) and high specificity (95.35%), showing potentials for rapid and user-friendly diagnosis of COVID-19 among many other infectious pathogens.

Genomic monitoring of SARS-CoV-2 uncovers an Nsp1 deletion variant that modulates type I interferon response.

The SARS-CoV-2 virus, the causative agent of COVID-19, is undergoing constant mutation. Here, we utilized an integrative approach combining epidemiology, virus genome sequencing, clinical phenotyping, and experimental validation to locate mutations of clinical importance. We identified 35 recurrent variants, some of which are associated with clinical phenotypes related to severity. One variant, containing a deletion in the Nsp1-coding region (Δ500-532), was found in more than 20% of our sequenced samples and associates with higher RT-PCR cycle thresholds and lower serum IFN-ß levels of infected patients. Deletion variants in this locus were found in 37 countries worldwide, and viruses isolated from clinical samples or engineered by reverse genetics with related deletions in Nsp1 also induce lower IFN-ß responses in infected Calu-3 cells. Taken together, our virologic surveillance characterizes recurrent genetic diversity and identified mutations in Nsp1 of biological and clinical importance, which collectively may aid molecular diagnostics and drug design.

Detection of Circulating Tumor Cells in Breast Cancer Patients by Nanopore Sensing with Aptamer-Mediated Amplification.

Circulating tumor cells (CTCs) have been utilized in the diagnosis and prognosis of tumor. However, the CTC concentration is extremely low to be detected in peripheral blood. Many existing methods suffer from either expensive labeling or complex operation. In this study, we constructed a label- and enzyme-free and sensitive method to detect the breast cancer CTCs. First of all, a probe containing a breast cancer cell-specific aptamer and a complementary single-stranded DNA (trigger DNA P1) were designed. When the target cells are present, the aptamer binds to the CTCs and releases P1 which triggers the strand displacement amplification. This process generates three-way junction structure DNA, the specific translocation signals of which are identified by nanopore assay. The detection limit of tumor cells is 5 in the current experimental setup and can be further reduced. Furthermore, the method is demonstrated in a clinical sample test with high recovery rate and accuracy. Our results suggest that this method could be applied to early diagnosis of metastatic recurrence and prognosis determination.