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Background: Effective therapeutics and vaccines for coronavirus disease 2019 (COVID-19) are currently lacking because of the mutation and immune escape of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on the propagation characteristics of SARS-CoV-2, rapid and accurate detection of complete virions from clinical samples and the environment is critical for assessing infection risk and containing further COVID-19 outbreaks. However, currently applicable methods cannot achieve large-scale clinical application due to factors such as the high viral load, cumbersome virus isolation steps, demanding environmental conditions, and long experimental periods. In this study, we developed an immuno molecular detection method combining capture of the viral spike glycoprotein with monoclonal antibodies and nucleic acid amplification via quantitative reverse transcription PCR to rapidly and accurately detect complete virions., Results: After constructing a novel pseudovirus, screening for specific antibodies, and optimizing the detection parameters, the assay achieved a limit of detection of 9 × 10 2 transduction units/mL of viral titer with high confidence (~ 95%) and excellent stability against human serum and common virus/pseudovirus. The coefficients of variation were 1.0 ~ 2.0% for intra-assay and inter-assay analyses, respectively. Compared with reverse transcription-PCR, the immunomolecular method more accurately quantified complete virions. SARS-CoV-2/pseudovirus was more stable on plastic and paper compared with aluminum and copper in the detection of SARS-CoV-2 pseudovirus under different conditions. Complete virions were detected up to 96 h after they were applied to these surfaces (except for copper), although the titer of the virions was greatly reduced., Conclusion: Convenient, inexpensive, and accurate complete virus detection can be applied to many fields, including monitoring the infectivity of convalescent and post-discharge patients and assessing high-risk environments (isolation rooms, operating rooms, patient living environments, and cold chain logistics). This method can also be used to detect intact virions, including Hepatitis B and C viruses, human immunodeficiency virus, influenza, and the partial pulmonary virus, which may further improve the accuracy of diagnoses and facilitate individualized and precise treatments., (© 2022. The Author(s).)

Although nanopore as a single-molecule sensing platform has proven its potential in various applications, data analysis of nanopores remains challenging. Herein, we introduce a method with increased accuracy in nanopore analysis based on the central limit theorem (CLT). An optimal voltage used in detection is determined from the standard deviations of blockage currents and time constants at various voltage biases. Compared with the conventional data analysis method, blockage signals processed with the CLT result in more concentrated distributions of blockage currents and durations. It allows fitting a Gaussian to the duration histogram and avoids the influence of bin sizes on time constants in duration analysis. The proposed method is further validated by applying it to detect isolated microRNAs with solid-state nanopores. Under the optimal voltage, different nucleic acids present in the isolation process are translocated through the nanopore. By processing the event signals with the CLT, all the nucleic acids including the microRNA are well differentiated. The method proposed here should also be applicable for sensing other biomolecules with the solid-state nanopores.

Despite numerous efforts, the accurate determination of trace biomolecules with zeptomolar sensitivity remains elusive. Here, a 3D carbon nanomaze (CAM) electrode for the ultrasensitive detection of trace biomolecules such as nucleic acids, proteins, and extracellular vesicles is reported. The CAM electrode consists of an interlaced carbon fiber array on which intercrossed graphene sheets are vertically tethered in situ, permitting local confinement of trace molecules to increase molecular hybridization efficiency. Furthermore, a self‐assembled DNA tetrahedron array adopts a rigid spatial conformation to guarantee the controllable arrangement of immobilized biological probes, facilitating analytical sensitivity and reproducibility. In a proof‐of‐concept experiment on detecting microRNA‐155, a linearity of 0.1 aM to 100 nM and a sensitivity of 0.023 aM (23 zM) are achieved. With the optimal parameters, the proposed nanoelectrode demonstrates encouraging consistency with quantitative real‐time polymerase chain reaction during clinical sample detection. Through simple functionalization by appending various biomolecular probes of interest, the developed CAM platform with ultrahigh sensitivity can be exploited as a versatile tool in environmental, chemistry, biology, and healthcare fields. [ABSTRACT FROM AUTHOR]

To overcome the effect of other components of complicated biological samples on nanopore stochastic sensing, displacement chemical reaction was utilized to selectively extract the target nucleic acid from whole blood. Given its simplicity and high sensitivity for detecting nucleic acids, our developed displacement chemistry-based nanopore sensing strategy offers the potential for fieldable/point-of-care diagnostic applications. [ABSTRACT FROM AUTHOR]

• Electrochemical, nanopore and field-effect transistors biosensor has been developed. • Aptamer, DNAzyme, DNA origami and nanomaterials has been used for amplification. • The biosensor exhibits an excellent performance for amplification detection. • We discussed the challenges of detection strategies in clinical application. Due to the wide range of electrochemical devices available, DNA nanostructures and material-based technologies have been greatly broadened. They have been actively used to create a variety of beautiful nanostructures owing to their unmatched programmability. Currently, a variety of electrochemical devices have been used for rapid sensing of biomolecules and other diagnostic applications. Here, we provide a brief overview of recent advances in DNA-based biomolecular assays. Biosensing platform such as electrochemical biosensor, nanopore biosensor, and field-effect transistor biosensors (FET), which are equipped with aptamer, DNA walker, DNAzyme, DNA origami, and nanomaterials, has been developed for amplification detection. Under the optimal conditions, the proposed biosensor has good amplification detection performance. Further, we discussed the challenges of detection strategies in clinical applications and offered the prospect of this field. [ABSTRACT FROM AUTHOR]

[Display omitted] • Establishment of a cost-effective nanopore platform for NCL identification at low LOD with trace amount of sample. • Discrimination of single-molecule topologies of DNA/RNA G4 with distinct sequences while closed in sequence length. • Proof of the well-binding properties of NCL with G4 folded from both DNA and RNA. • Recognition of NCL and NCL/G4 complex in human serum. DNA/RNA quadruplexes (G4) are significant secondary structures that exist in human genes, playing essential part in transcription and replication. Nucleolin (NCL) is a ubiquitous protein mainly found in the human nucleus. It is frequently overexpressed in cancer cells and displays a remarkable preference for DNA/RNA G4 binding, playing a crucial role in controlling nucleic acid metabolism and chromatin structure. Additionally, NCL is involved in microRNA (miRNA) biogenesis.This work presents the discrimination of DNA/RNA G4 topologies harboring close sequence length with size-matched nanopore devices. The single-molecule identification of NCL under various parameters was implemented, and a low detection limit of 0.5 nM was achieved, NCL could specifically bind with DNA G4 AS1411 and RNA G4 (rG4) from pre-miRNA 92b and pre-miRNA 149, forming stable complexes that demonstrate distinct nanopore translocation properties. The augmentation of the molar ratio of G4 to NCL induces elevated complex formation. Recognition of both NCL and NCL-G4 complex in human serum is realized. Our work established a precise platform for the differentiation of nucleic acid secondary structure with similar conformation, we also proved the binding behaviors between NCL and DNA/RNA G4, as well as the serum interference test, which is valuable for the investigation of the interactions between proteins and nucleic acids as well as for further understanding the mechanism of the NCL-related diseases processes. [ABSTRACT FROM AUTHOR]

With the emergence of microRNAs as key biomarkers for disease diagnosis such as lung cancer, various techniques have been settled for their detection. However, these current methods require different amplification steps since numerous challenges for detecting circulating miRNAs are attributable to their intrinsic properties accounting for tiny sizes, high sequence similarity, and low abundance. Duplex specific nuclease (DSN)-based microRNA amplification has recently gained interest in biosensing applications thanks to its catalytic activity based on target recycling. In this context, we designed a highly selective, sensitive, and multiplexed fluorescence-based biosensor combining DSN enzyme and magnetic beads to detect three distinct microRNAs, including microRNA-21, microRNA-210, and microRNA-486-5p. By exploiting the above approach, we were able to detect as low as 98 aM, 120 aM, and 300 aM of mir-21, miR-210, and miR-486-5p, respectively. Furthermore, this recommended strategy displays a high selectivity toward an entirely matched target than the off-target. These results are ascribed to the potent DSN enzyme activity and to the locked nucleic acid (LNA)-modified DNA probe that boosted the hetero-duplex probe/target stability. Lastly, our proposed method was applied to detect microRNAs in the serum samples and displayed a high efficacy to discriminate between healthy controls and lung cancer patients. Furthermore, the analytical accuracy of the proposed strategy was validated with the computed tomography (CT) technique of the chest. Thus based on these findings, this strategy could open new directions for detecting microRNAs associated with several diseases. [Display omitted] • A multiplexed fluorescence-based biosensor combining DSN enzyme and LNA probe tethered MBs for microRNAs detection was settled. • The developed method exhibited a high sensitivity and selectiovity for lung cancer (LC) diagnosis. • The engendered limit of detection is equal to 98 aM, 120 aM, and 300 aM of mir-21, miR-210, and miR-486-5p, respectively. • Via using this serum 3-microRNAs signature, we were able to distinguish LC disease patients from healthy volunteers. • Our proposed method was validated by computed tomography and revealed a high correlation. [ABSTRACT FROM AUTHOR]