Research Progress in Virus Detection Based on Nanopore Sensors
Wu Lingzhi1*, Qi Ke1, Zhang Xuanhao1, Tang Lihua2
1(College of Science, Nanjing University of Posts and Telecommunications, Nanjing 210023, China) 2(School of Computer Science, Nanjing University of Posts and Telecommunications, Nanjing 210023, China)
Abstract：In recent years, infectious diseases caused by various pathogenic microorganisms have frequently emerged, posing a serious threat to human health and medical safety. There has been a growing demand for large-scale clinical diagnosis of viruses. With its unique single-molecule detection capability, simple operation process, and potential portability, nanopore technology is emerging as an important tool for virus detection. This article reviewed advances of nanopores on viral detection from several aspects, including key technologies and diagnostic applications. The typical cases of the nanopore platforms, such as nanopore sequencing, single particle counting, and single molecular identification, were further summarized on different scenarios of the pathogen surveillance, along with a brief overview of the nanopores integrated with other technologies, like machine learning and microfluidics, further enhancing detection capabilities and expanding their application. These technologies will be help for developing new approaches for precise epidemic prevention and control.
[1] Eisfeld AJ, Biswas A, Guan L, et al. Pathogenicity and transmissibility of bovine H5N1 influenza virus [J]. Nature, 2024, 633(8029): 426-432. [2] Chow IC, Wong SS. Immunological imprinting and risks of influenza B virus infection [J]. Nat Immunol, 2024, 25(8): 1319-1321. [3] Moutinho S. Little-known virus is on the rise in South America [J]. Science, 2024, 384(6700): 1052-1053. [4] Bennett AJ, Paskey AC, Ebinger A, et al. Relatives of rubella virus in diverse mammals [J]. Nature, 2020, 586(7829): 424-428. [5] Rheinberger HJ. From infectious filtrate [J]. Science, 2000, 289(5480): 731-731. [6] Olitsky PK, Boëz L. Studies on the physical and chemical properties of the virus of foot-and-mouth disease: II. cataphoresis and filtration [J]. J Exp Med, 1927, 45(4): 685-699. [7] Qiu Y, Li Q, Lee BE, et al. UV inactivation of human infectious viruses at two full-scale wastewater treatment plants in Canada [J]. Water Res, 2018, 147: 73-81. [8] Stanley W. Crystalline tobacco-mosaic virus protein [J]. Am J Bot, 1937, 24(2): 59-68. [9] Servellita V, Sotomayor Gonzalez A, Lamson DM, et al. Adeno-associated virus type 2 in US children with acute severe hepatitis [J]. Nature, 2023, 617(7961): 574-580. [10] Kim H, Kang N, An K, et al. MRPrimerV: a database of PCR primers for RNA virus detection [J]. Nucleic Acids Res, 2017, 45(D1): D475-D481. [11] Ratinho L, Meyer N, Greive S, et al. Nanopore sensing of protein and peptide conformation for point-of-care applications [J]. Nature Communications, 2025, 16(1): 3211. [12] Yang SL, DeFalco L, Anderson DE, et al. Comprehensive mapping of SARS-CoV-2 interactions in vivo reveals functional virus-host interactions [J]. Nature Communications, 2021, 12(1): 5113. [13] Bull RA, Adikari TN, Ferguson JM, et al. Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis [J]. Nature Communications, 2020, 11(1): 6272. [14] Quick J, Loman NJ, Duraffour S, et al. Real-time, portable genome sequencing for Ebola surveillance [J]. Nature, 2016, 530(7589): 228-232. [15] Dong M, Kshirsagar A, Politza AJ, et al. High fidelity machine-learning-assisted false positive discrimination in loop-mediated isothermal amplification using nanopore-based sizing and counting [J]. ACS Nano, 2024, 18(9): 7170-7179. [16] Tang Z, Nouri R, Dong M, et al. Rapid detection of novel coronavirus SARS-CoV-2 by RT-LAMP coupled solid-state nanopores [J]. Biosens Bioelectron, 2022, 197: 113759. [17] 武灵芝, 罗星月, 邓浩丰, 等. 纳米孔在蛋白质单分子水平研究中的应用 [J]. 生物工程学报, 2025, 41(1): 92-116. [18] Ren M, Liu D, Qin F, et al. Single-molecule resolution of macromolecules with nanopore devices [J]. Adv Colloid Interface Sci, 2025: 103417. [19] Ritmejeris J, Chen X, Dekker C. Single-molecule protein sequencing with nanopores [J]. Nat Rev Bioeng, 2025, 3(4): 303-316. [20] He S, Li Y, Fang S, et al. Solid-state nanopore single-molecule capture: advances and challenges [J]. Small, 2025, 21(44): e07736. [21] Arjmandi N, Van Roy W, Lagae L, et al. Measuring the electric charge and zeta potential of nanometer-sized objects using pyramidal-shaped nanopores [J]. Anal Chem, 2012, 84(20): 8490-8496. [22] Arjmandi N, Van Roy W, Lagae L. Measuring mass of nanoparticles and viruses in liquids with nanometer-scale pores [J]. Anal Chem, 2014, 86(10): 4637-4641. [23] Tourancheau A, Mead EA, Zhang XS, et al. Discovering multiple types of DNA methylation from bacteria and microbiome using nanopore sequencing [J]. Nature Methods, 2021, 18(5): 491-498. [24] Ma H, Wang Y, Li YX, et al. Label-free mapping of multivalent binding pathways with ligand-receptor-anchored nanopores [J]. J Am Chem Soc, 2024, 146(41): 28014-28022. [25] Confederat S, Lee S, Vang D, et al. Next-generation nanopore sensors based on conductive pulse sensing for enhanced detection of nanoparticles [J]. Small, 2024, 20(4): 2305186. [26] Kafetzopoulou L, Pullan S, Lemey P, et al. Metagenomic sequencing at the epicenter of the Nigeria 2018 Lassa fever outbreak [J]. Science, 2019, 363(6422): 74-77. [27] 张得芳, 马秋月, 尹佟明, 等. 第三代测序技术及其应用 [J]. 中国生物工程杂志, 2013, 33(5): 125-131. [28] 曹必溥, 缪丽华, 郭宝颜, 等. 纳米孔测序技术应用于食品微生物检测的可行性分析 [J]. 中国生物工程杂志, 2018, 38(12): 91-98. [29] Van Puyvelde S, Argimon S. Sequencing in the time of Ebola [J]. Nat Rev Microbiol, 2019, 17(1): 5. [30] Kakuk B, Dörmő Á, Csabai Z, et al. In-depth temporal transcriptome profiling of monkeypox and host cells using nanopore sequencing [J]. Sci Data, 2023, 10(1): 262. [31] Arima A, Tsutsui M, Yoshida T, et al. Digital pathology platform for respiratory tract infection diagnosis via multiplex single-particle detections [J]. ACS Sens, 2020, 5(11): 3398-3403. [32] Zhou K, Li L, Tan Z, et al. Characterization of hepatitis B virus capsids by resistive-pulse sensing [J]. J Am Chem Soc, 2011, 133(6): 1618-1621. [33] Arima A, Tsutsui M, Harlisa IH, et al. Selective detections of single-viruses using solid-state nanopores [J]. Sci Rep, 2018, 8(1): 16305. [34] Taniguchi M, Minami S, Ono C, et al. Combining machine learning and nanopore construction creates an artificial intelligence nanopore for coronavirus detection [J]. Nature Communications, 2021, 12(1): 3726. [35] Arima A, Harlisa IH, Yoshida T, et al. Identifying single viruses using biorecognition solid-state nanopores [J]. J Am Chem Soc, 2018, 140(48): 16834-16841. [36] Cai S, Ren R, He J, et al. Selective single-moleculenanopore detection of mpox A29 protein directly in biofluids [J]. Nano Lett, 2023, 23(24): 11438-11446. [37] Liu S, Zhao Y, Parks JW, et al. Correlated electrical and optical analysis of single nanoparticles and biomolecules on a nanopore-gated optofluidic chip [J]. Nano Lett, 2014, 14(8): 4816-4820. [38] Quick J, Grubaugh ND, Pullan ST, et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples [J]. Nat Protoc, 2017, 12(6): 1261-1276. [39] Deng X, Achari A, Federman S, et al. Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance [J]. Nat Microbiol, 2020, 5(3): 443-454. [40] Di Paola N, Sanchez-Lockhart M, Zeng X, et al. Viral genomics in Ebola virus research [J]. Nat Rev Microbiol, 2020, 18(7): 365-378. [41] Lu H, Giordano F, Ning Z. Oxford Nanopore MinION sequencing and genome assembly [J]. Genomics, Proteomics Bioinf, 2016, 14(5): 265-279. [42] Kilianski A, Roth PA, Liem AT, et al. Use of unamplified RNA/cDNA-hybrid nanopore sequencing for rapid detection and characterization of RNA viruses [J]. Emerging Infect Dis, 2016, 22(8): 1448. [43] Meredith LW, Hamilton WL, Warne B, et al. Rapid implementation of SARS-CoV-2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study [J]. Lancet Infect Dis, 2020, 20(11): 1263-1271. [44] Kim D, Lee JY, Yang JS, et al. The architecture of SARS-CoV-2 transcriptome [J]. Cell, 2020, 181(4): 914-921. [45] Dekker C. Solid-state nanopores [J]. Nat Nanotechnol, 2007, 2(4): 209-215. [46] Harms ZD, Haywood DG, Kneller AR, et al. Single-particle electrophoresis in nanochannels [J]. Anal Chem, 2015, 87(1): 699-705. [47] Zhou J, Zlotnick A, Jacobson SC. Disassembly of single virus capsids monitored in real time with multicycle resistive-pulse sensing [J]. Anal Chem, 2021, 94(2): 985-992. [48] Harms ZD, Mogensen KB, Nunes PS, et al. Nanofluidic devices with two pores in series for resistive-pulse sensing of single virus capsids [J]. Anal Chem, 2011, 83(24): 9573-9578. [49] Kondylis P, Zhou J, Harms ZD, et al. Nanofluidic devices with 8 pores in series for real-time, resistive-pulse analysis of hepatitis B virus capsid assembly [J]. Anal Chem, 2017, 89(9): 4855-4862. [50] Nouri R, Jiang Y, Lian XL, et al. Sequence-specific recognition of HIV-1 DNA with solid-state CRISPR-Cas12a-assisted nanopores (SCAN) [J]. ACS Sens, 2020, 5(5): 1273-1280. [51] Karawdeniya BI, Bandara YND, Khan AI, et al. Adeno-associated virus characterization for cargo discrimination through nanopore responsiveness [J]. Nanoscale, 2020, 12(46): 23721-23731. [52] Tsutsui M, Wada M, Arima A, et al. Identifying viral vector characteristics by nanopore sensing [J]. ACS Nano, 2024, 18(24): 15695-15704. [53] Tsutsui M, Tsunekawa Y, Wada M, et al. Enhanced discriminability of viral vectors in viscous nanopores [J]. Small Methods, 2025: 2401321. [54] Andreano E, Paciello I, Piccini G, et al. Hybrid immunity improves B cells and antibodies against SARS-CoV-2 variants [J]. Nature, 2021, 600(7889): 530-535. [55] Murakami K, Kubota SI, Tanaka K, et al. High-precision rapid testing of omicron SARS-CoV-2 variants in clinical samples using AI-nanopore [J]. Lab Chip, 2023, 23(22): 4909-4918. [56] Sampad MJN, Zhang H, Yuzvinsky TD, et al. Optical trapping assisted label-free and amplification-free detection of SARS-CoV-2 RNAs with an optofluidic nanopore sensor [J]. Biosens Bioelectron, 2021, 194: 113588. [57] Sampad MJN, Saiduzzaman S, Walker ZJ, et al. Label-free and amplification-free viral RNA quantification from primate biofluids using a trapping-assisted optofluidic nanopore platform [J]. Proc Natl Acad Sci, 2024, 121(16) : e2400203121. [58] Zhang Y, Chen X, Wang C, et al. Nanoparticle-assisted detection of nucleic acids in a polymeric nanopore with a large pore size [J]. Biosens Bioelectron, 2022, 196: 113697. [59] Oh S, Lee MK, Chi SW. Single-molecule-based detection of conserved Influenza A virus RNA promoter using a protein nanopore [J]. ACS Sens, 2019, 4(11): 2849-2853. [60] Niedzwiecki DJ, Iyer R, Borer PN, et al. Sampling a biomarker of the human immunodeficiency virus across a synthetic nanopore [J]. ACS Nano, 2013, 7(4): 3341-3350. [61] Ren R, Cai S, Fang X, et al. Multiplexed detection of viral antigen and RNA using nanopore sensing and encoded molecular probes [J]. Nature Communications, 2023, 14(1): 7362. [62] Bošković F, Zhu J, Tivony R, et al. Simultaneous identification of viruses and viral variants with programmable DNA nanobait [J]. Nat Nanotechnol, 2023, 18(3): 290-298. [63] Peinetti AS, Lake RJ, Cong W, et al. Direct detection of human adenovirus or SARS-CoV-2 with ability to inform infectivity using DNA aptamer-nanopore sensors [J]. Sci Adv, 2021, 7(39). eabh2848. [64] Sethi K, Dailey GP, Zahid OK, et al. Direct detection of conserved viral sequences and other nucleic acid motifs with solid-state nanopores [J]. ACS Nano, 2021, 15(5): 8474-8483. [65] Bock C, Datlinger P, Chardon F, et al. High-content CRISPR screening [J]. Nat Rev Methods Primers, 2022, 2(1): 1-23. [66] Ahamed MA, Khalid MAU, Dong M, et al. Sensitive and specific CRISPR-Cas12a assisted nanopore with RPA for Monkeypox detection [J]. Biosens Bioelectron, 2024, 246: 115866. [67] Liu L, Xu Z, Awayda K, et al. Gold nanoparticle-labeled CRISPR-Cas13a assay for the sensitive solid-state nanopore molecular counting [J]. Adv Mater Technol, 2022, 7(3): 2101550. [68] Nouri R, Jiang Y, Tang Z, et al. Detection of SARS-CoV-2 with solid-state CRISPR-Cas12a-assisted nanopores [J]. Nano Lett, 2021, 21(19): 8393-8400.
