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Single-molecule fluorescence methods for protein biomarker analysis

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Abstract

Proteins have been considered key building blocks of life. In particular, the protein content of an organism and a cell offers significant information for the in-depth understanding of the disease and biological processes. Single-molecule protein detection/sequencing tools will revolutionize clinical (proteomics) research, offering ultrasensitivity for low-abundance biomarker (protein) detection, which is important for the realization of early-stage disease diagnosis and single-cell proteomics. This improved detection/measurement capability delivers new sets of techniques to explore new frontiers and address important challenges in various interdisciplinary areas including nanostructured materials, molecular medicine, molecular biology, and chemistry. Importantly, fluorescence-based methods have emerged as indispensable tools for single protein detection/sequencing studies, providing a higher signal-to-noise ratio (SNR). Improvements in fluorescent dyes/probes and detector capabilities coupled with advanced (image) analysis strategies have fueled current developments for single protein biomarker detections. For example, in comparison to conventional ELISA (i.e., based on ensembled measurements), single-molecule fluorescence detection is more sensitive, faster, and more accurate with reduced background, high-throughput, and so on. In comparison to MS sequencing, fluorescence-based single-molecule protein sequencing can achieve the sequencing of peptides themselves with higher sensitivity. This review summarizes various typical single-molecule detection technologies including their methodology (modes of operation), detection limits, advantages and drawbacks, and current challenges with recent examples. We describe the fluorescence-based single-molecule protein sequencing/detection based on five kinds of technologies such as fluorosequencing, N-terminal amino acid binder, nanopore light sensing, and DNA nanotechnology. Finally, we present our perspective for developing high-performance fluorescence-based sequencing/detection techniques.

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References

  1. Wu C, Garden PM, Walt DR. Ultrasensitive Detection of Attomolar Protein Concentrations by Dropcast Single Molecule Assays. J Am Chem Soc. 2020;142(28):12314–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Zhou F, Wang M, Yuan L, Cheng Z, Wu Z, Chen H. Sensitive sandwich ELISA based on a gold nanoparticle layer for cancer detection. Analyst. 2012;137(8):1779–84.

    CAS  PubMed  Google Scholar 

  3. Chatterjee T, Knappik A, Sandford E, Tewari M, Choi SW, Strong WB, et al. Direct kinetic fingerprinting and digital counting of single protein molecules. Proc Natl Acad Sci. 2020;117(37):22815–22.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Mao C-P, Wang S-C, Su Y-P, Tseng S-H, He L, Wu AA, et al. Protein detection in blood with single-molecule imaging. Science Advances. 2021;7(33):eabg6522.

  5. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7–30.

    PubMed  Google Scholar 

  6. Thaxton CS, Elghanian R, Thomas AD, Stoeva SI, Lee JS, Smith ND, et al. Nanoparticle-based bio-barcode assay redefines “undetectable” PSA and biochemical recurrence after radical prostatectomy. Proc Natl Acad Sci U S A. 2009;106(44):18437–42.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Blennow K, de Leon MJ, Zetterberg H. Alzheimer’s disease. The Lancet. 2006;368(9533):387–403.

    CAS  Google Scholar 

  8. Zecca C, Tortelli R, Panza F, Arcuti S, Piccininni M, Capozzo R, et al. Plasma β-amyloid1–42 reference values in cognitively normal subjects. J Neurol Sci. 2018;391:120–6.

    CAS  PubMed  Google Scholar 

  9. Tatebe H, Kasai T, Ohmichi T, Kishi Y, Kakeya T, Waragai M, et al. Quantification of plasma phosphorylated tau to use as a biomarker for brain Alzheimer pathology: pilot case-control studies including patients with Alzheimer’s disease and down syndrome. Mol Neurodegener. 2017;12(1):63.

    PubMed  PubMed Central  Google Scholar 

  10. Zetterberg H, Wilson D, Andreasson U, Minthon L, Blennow K, Randall J, et al. Plasma tau levels in Alzheimer’s disease. Alzheimer’s Research & Therapy. 2013;5(2):9.

    CAS  Google Scholar 

  11. Akama K, Shirai K, Suzuki S. Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System. Anal Chem. 2016;88(14):7123–9.

    CAS  PubMed  Google Scholar 

  12. Zhang L, Fan W, Jia D, Feng Q, Ren W, Liu C. Microchamber-Free Digital Flow Cytometric Analysis of T4 Polynucleotide Kinase Phosphatase Based on Single-Enzyme-to-Single-Bead Space-Confined Reaction. Anal Chem. 2021;93(44):14828–36.

    CAS  PubMed  Google Scholar 

  13. Shashkova S, Leake MC. Single-molecule fluorescence microscopy review: shedding new light on old problems. Biosci Rep. 2017;37(4):BSR20170031.

  14. Pecker LH, Lanzkron S. In the Clinic Sickle Cell Disease. Ann Int Med. 2021;174(1):ITC1–16.

    PubMed  Google Scholar 

  15. Timp W, Timp G. Beyond mass spectrometry, the next step in proteomics. Sci Adv. 2020;6(2):eaax8978.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Angel TE, Aryal UK, Hengel SM, Baker ES, Kelly RT, Robinson EW, et al. Mass spectrometry-based proteomics: existing capabilities and future directions. Chem Soc Rev. 2012;41(10):3912–28.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Swaminathan J, Boulgakov AA, Hernandez ET, Bardo AM, Bachman JL, Marotta J, et al. Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures. Nat Biotechnol. 2018;36(11):1076–82.

    CAS  Google Scholar 

  18. Chait BT. Mass spectrometry: bottom-up or top-down? Science. 2006;314(5796):65–6.

    CAS  PubMed  Google Scholar 

  19. Rissin DM, Kan CW, Campbell TG, Howes SC, Fournier DR, Song L, et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol. 2010;28(6):595–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Moerner WE, Kador L. Optical detection and spectroscopy of single molecules in a solid. Phys Rev Lett. 1989;62(21):2535–8.

    CAS  PubMed  Google Scholar 

  21. Wilson DH, Rissin DM, Kan CW, Fournier DR, Piech T, Campbell TG, et al. The Simoa HD-1 Analyzer: A Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and Multiplexing. SLAS Technology. 2016;21(4):533–47.

    CAS  Google Scholar 

  22. Chen Y, Shimoni O, Huang G, Wen S, Liao J, Duong HTT, et al. Upconversion nanoparticle-assisted single-molecule assay for detecting circulating antigens of aggressive prostate cancer. Cytometry A. 2022;101(5):400–10.

    CAS  PubMed  Google Scholar 

  23. Farka Z, Mickert MJ, Hlavacek A, Skladal P, Gorris HH. Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers. Anal Chem. 2017;89(21):11825–30.

    CAS  PubMed  Google Scholar 

  24. Cohen L, Cui N, Cai Y, Garden PM, Li X, Weitz DA, et al. Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay. ACS Nano. 2020;14(8):9491–501.

    CAS  PubMed  Google Scholar 

  25. Wu C, Dougan TJ, Walt DR. High-Throughput, High-Multiplex Digital Protein Detection with Attomolar Sensitivity. ACS Nano. 2022;16(1):1025–35.

  26. Hao N, Zhang JXJ. Microfluidic Screening of Circulating Tumor Biomarkers toward Liquid Biopsy. Sep Purif Rev. 2018;47(1):19–48.

    CAS  Google Scholar 

  27. Shang L, Cheng Y, Zhao Y. Emerging Droplet Microfluidics. Chem Rev. 2017;117(12):7964–8040.

    CAS  PubMed  Google Scholar 

  28. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods. 2013;10(10):1003–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, et al. High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number. Anal Chem. 2011;83(22):8604–10.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Mazutis L, Gilbert J, Ung WL, Weitz DA, Griffiths AD, Heyman JA. Single-cell analysis and sorting using droplet-based microfluidics. Nat Protoc. 2013;8(5):870–91.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Prakadan SM, Shalek AK, Weitz DA. Scaling by shrinking: empowering single-cell “omics” with microfluidic devices. Nat Rev Genet. 2017;18(6):345–61.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Shahi P, Kim SC, Haliburton JR, Gartner ZJ, Abate AR. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding. Sci Rep. 2017;7(1):44447.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Cui N, Zhang H, Schneider N, Tao Y, Asahara H, Sun Z, et al. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders. Sci Rep. 2016;6(1):22575.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Abbaspourrad A, Zhang H, Tao Y, Cui N, Asahara H, Zhou Y, et al. Label-free single-cell protein quantification using a drop-based mix-and-read system. Sci Rep. 2015;5(1):12756.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Huang Q, Li N, Zhang H, Che C, Sun F, Xiong Y, et al. Critical Review: digital resolution biomolecular sensing for diagnostics and life science research. Lab Chip. 2020;20(16):2816–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Fan Y, Hao R, Han C, Zhang B. Counting Single Redox Molecules in a Nanoscale Electrochemical Cell. Anal Chem. 2018;90(23):13837–41.

    CAS  PubMed  Google Scholar 

  37. Barulin A, Claude J-B, Patra S, Bonod N, Wenger J. Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides. Nano Lett. 2019;19(10):7434–42.

    CAS  PubMed  Google Scholar 

  38. Fan S, Webb JEA, Yang Y, Nieves DJ, Goncales VR, Tran J, et al. Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy. Angew Chem Int Ed Engl. 2019;58(41):14495–8.

    CAS  PubMed  Google Scholar 

  39. Vukojevic V, Heidkamp M, Ming Y, Johansson B, Terenius L, Rigler R. Quantitative single-molecule imaging by confocal laser scanning microscopy. Proc Natl Acad Sci U S A. 2008;105(47):18176–81.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Macdonald PJ, Ruan Q, Tetin SY. Direct single-molecule counting for immunoassay applications. Anal Biochem. 2019;566:139–45.

    CAS  PubMed  Google Scholar 

  41. Stroock AD, Dertinger SK, Ajdari A, Mezic I, Stone HA, Whitesides GM. Chaotic mixer for microchannels. Science. 2002;295(5555):647–51.

    CAS  PubMed  Google Scholar 

  42. Axelrod D, T P Burghardt a, Thompson NL. Total Internal Reflection Fluorescence. Ann Rev Biophys Bioeng. 1984;13(1):247–68.

    CAS  Google Scholar 

  43. Khorasaninejad M, Chen WT, Devlin RC, Oh J, Zhu AY, Capasso F. Metalenses at visible wavelengths: Diffraction-limited focusing and subwavelength resolution imaging. Science. 2016;352(6290):1190–4.

    CAS  PubMed  Google Scholar 

  44. Zhang C-Y, Yeh H-C, Kuroki MT, Wang T-H. Single-quantum-dot-based DNA nanosensor. Nat Mater. 2005;4(11):826–31.

    CAS  PubMed  Google Scholar 

  45. Mickert MJ, Farka Z, Kostiv U, Hlaváček A, Horák D, Skládal P, et al. Measurement of Sub-femtomolar Concentrations of Prostate-Specific Antigen through Single-Molecule Counting with an Upconversion-Linked Immunosorbent Assay. Anal Chem. 2019;91(15):9435–41.

    CAS  PubMed  Google Scholar 

  46. Aebersold R, Mann M. Mass-spectrometric exploration of proteome structure and function. Nature. 2016;537(7620):347–55.

    CAS  PubMed  Google Scholar 

  47. Shendure J, Balasubramanian S, Church GM, Gilbert W, Rogers J, Schloss JA, et al. DNA sequencing at 40: past, present and future. Nature. 2017;550(7676):345–53.

    CAS  PubMed  Google Scholar 

  48. Abascal F, Juan D, Jungreis I, Kellis M, Martinez L, Rigau M, et al. Loose ends: almost one in five human genes still have unresolved coding status. Nucleic Acids Res. 2018;46(14):7070–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Hu ZL, Huo MZ, Ying YL, Long YT. Biological Nanopore Approach for Single-Molecule Protein Sequencing. Angewandte Chemie-International Edition. 2021;60(27):14738–49.

    CAS  PubMed  Google Scholar 

  50. Reed BD, Meyer MJ, Abramzon V, Ad O, Ad O, Adcock P, et al. Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device. Science. 2022;378(6616):186–92.

    CAS  PubMed  Google Scholar 

  51. Wang R, Gilboa T, Song J, Huttner D, Grinstaff MW, Meller A. Single-Molecule Discrimination of Labeled DNAs and Polypeptides Using Photoluminescent-Free TiO2 Nanopores. ACS Nano. 2018;12(11):11648–56.

    CAS  PubMed  Google Scholar 

  52. Dai M, Yin P. Methods and compositions relating to super-resolution imaging and modification. US patent 10006917. 2018.

  53. De Lannoy CV, Filius M, van Wee R, Joo C, de Ridder D. Evaluation of FRET X for single-molecule protein fingerprinting. iSci. 2021;24(11):103239.

    Google Scholar 

  54. Swaminathan J, Boulgakov AA, Marcotte EM. A theoretical justification for single molecule peptide sequencing. PLoS Comput Biol. 2015;11(2): e1004080.

    PubMed  PubMed Central  Google Scholar 

  55. Gavrilyuk J, Ban H, Nagano M, Hakamata W, Barbas CF 3rd. Formylbenzene Diazonium Hexafluorophosphate Reagent for Tyrosine-Selective Modification of Proteins and the Introduction of a Bioorthogonal Aldehyde. Bioconjug Chem. 2012;23(12):2321–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Ban H, Gavrilyuk J, Barbas CF III. Tyrosine bioconjugation through aqueous ene-type reactions: a click-like reaction for tyrosine. J Am Chem Soc. 2010;132(5):1523–5.

    CAS  PubMed  Google Scholar 

  57. Bach K, Beerkens BLH, Zanon PRA, Hacker SM. Light-Activatable, 2,5-Disubstituted Tetrazoles for the Proteome-wide Profiling of Aspartates and Glutamates in Living Bacteria. ACS Cent Sci. 2020;6(4):546–54.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Hernandez ET, Swaminathan J, Marcotte EM, Anslyn EV. Solution-phase and solid-phase sequential, selective modification of side chains in KDYWEC and KDYWE as models for usage in single-molecule protein sequencing. New J Chem. 2017;41(2):462–9.

    CAS  PubMed  Google Scholar 

  59. Taylor MT, Nelson JE, Suero MG, Gaunt MJ. A protein functionalization platform based on selective reactions at methionine residues. Nature. 2018;562(7728):563–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Lin Shixian, Yang Xiaoyu, Jia Shang, Weeks Amy M, MH, Peter S. Lee, Rita V. Nichiporuk ATI, James A. Wells, F. Dean Toste CJC. Redox-based reagents for chemoselective methionine bioconjugation. Sci. 2017;355(6325):597–602.

    CAS  Google Scholar 

  61. Christian AH, Jia S, Cao W, Zhang P, Meza AT, Sigman MS, et al. A Physical Organic Approach to Tuning Reagents for Selective and Stable Methionine Bioconjugation. J Am Chem Soc. 2019;141(32):12657–62.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Jia S, He D, Chang CJ. Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation. J Am Chem Soc. 2019;141(18):7294–301.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Howard CJ, Floyd BM, Bardo AM, Swaminathan J, Marcotte EM, Anslyn EV. Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics. ACS Chem Biol. 2020;15(6):1401–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Perona JJ, Hadd A. Structural diversity and protein engineering of the aminoacyl-tRNA synthetases. Biochemistry. 2012;51(44):8705–29.

    CAS  PubMed  Google Scholar 

  65. Dougan DA, Reid BG, Horwich AL, Bukau B. ClpS, a Substrate Modulator of the ClpAP Machine. Mol Cell. 2002;9(3):673–83.

    CAS  PubMed  Google Scholar 

  66. Erbse A, Schmidt R, Bornemann T, Schneider-Mergener J, Mogk A, Zahn R, et al. ClpS is an essential component of the N-end rule pathway in Escherichia coli. Nature. 2006;439(7077):753–6.

    CAS  PubMed  Google Scholar 

  67. Roman-Hernandez G, Hou JY, Grant RA, Sauer RT, Baker TA. The ClpS adaptor mediates staged delivery of N-end rule substrates to the AAA+ ClpAP protease. Mol Cell. 2011;43(2):217–28.

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Nishimura K, van Wijk KJ. Organization, function and substrates of the essential Clp protease system in plastids. Biochim Biophys Acta. 2015;1847(9):915–30.

    CAS  PubMed  Google Scholar 

  69. Tullman J, Callahan N, Ellington B, Kelman Z, Marino JP. Engineering ClpS for selective and enhanced N-terminal amino acid binding. Appl Microbiol Biotechnol. 2019;103(6):2621–33.

    CAS  PubMed  Google Scholar 

  70. Rodriques SG, Marblestone AH, Boyden ES. A theoretical analysis of single molecule protein sequencing via weak binding spectra. PLoS ONE. 2019;14(3): e0212868.

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Alfaro JA, Bohlander P, Dai MJ, Filius M, Howard CJ, van Kooten XF, et al. The emerging landscape of single-molecule protein sequencing technologies. Nat Methods. 2021;18(6):604–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Tullman J, Marino JP, Kelman Z. Leveraging nature’s biomolecular designs in next-generation protein sequencing reagent development. Appl Microbiol Biotechnol. 2020;104(17):7261–71.

    CAS  PubMed  Google Scholar 

  73. Ferro ES, Rioli V, Castro LM, Fricker LD. Intracellular peptides: From discovery to function. EuPA Open Proteom. 2014;3:143–51.

    CAS  Google Scholar 

  74. Gonzales T, Robert-Baudouy J. Bacterial aminopeptidases: properties and functions. FEMS Microbiol Rev. 1996;18(4):319–44.

    CAS  PubMed  Google Scholar 

  75. Sanderink G-J, Artur Y, Siest G. Human Aminopeptidases: A Review of the Literature. 1988;26(12):795–808.

  76. Brinkerhoff H, Kang AS, Liu J, Aksimentiev A, Dekker C. Multiple rereads of single proteins at single–amino acid resolution using nanopores. Science. 2021;374(6574):1509–13.

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Assad ON, Di Fiori N, Squires AH, Meller A. Two color DNA barcode detection in photoluminescence suppressed silicon nitride nanopores. Nano Lett. 2015;15(1):745–52.

    CAS  PubMed  Google Scholar 

  78. Sawafta F, Clancy B, Carlsen AT, Huber M, Hall AR. Solid-state nanopores and nanopore arrays optimized for optical detection. Nanoscale. 2014;6(12):6991–6.

    CAS  PubMed  Google Scholar 

  79. Dette C, Perez-Osorio MA, Kley CS, Punke P, Patrick CE, Jacobson P, et al. TiO2 anatase with a bandgap in the visible region. Nano Lett. 2014;14(11):6533–8.

    CAS  PubMed  Google Scholar 

  80. Schneider J, Matsuoka M, Takeuchi M, Zhang J, Horiuchi Y, Anpo M, et al. Understanding TiO2 photocatalysis: mechanisms and materials. Chem Rev. 2014;114(19):9919–86.

    CAS  PubMed  Google Scholar 

  81. Tang H, Prasad K, Sanjinès R, Schmid PE, Lévy F. Electrical and optical properties of TiO2anatase thin films. J Appl Phys. 1994;75(4):2042–7.

    CAS  Google Scholar 

  82. Chandrasekaran AR, Punnoose JA, Zhou L, Dey P, Dey BK, Halvorsen K. DNA nanotechnology approaches for microRNA detection and diagnosis. Nucleic Acids Res. 2019;47(20):10489–505.

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Chen T, Ren L, Liu X, Zhou M, Li L, Xu J, et al. DNA Nanotechnology for Cancer Diagnosis and Therapy. International Journal of Molecular Sciences. 2018;19(6):1671.

  84. Chi Q, Yang Z, Xu K, Wang C, Liang H. DNA Nanostructure as an Efficient Drug Delivery Platform for Immunotherapy. Front Pharmacol. 2019;10:1585.

    CAS  PubMed  Google Scholar 

  85. Hu Q, Li H, Wang L, Gu H, Fan C. DNA Nanotechnology-Enabled Drug Delivery Systems. Chem Rev. 2019;119(10):6459–506.

    CAS  PubMed  Google Scholar 

  86. Schnitzbauer J, Strauss MT, Schlichthaerle T, Schueder F, Jungmann R. Super-resolution microscopy with DNA-PAINT. Nat Protoc. 2017;12(6):1198–228.

    CAS  PubMed  Google Scholar 

  87. Floyd BM, Marcotte EM. Protein Sequencing, One Molecule at a Time. Annu Rev Biophys. 2022;51:181–200.

    PubMed  PubMed Central  Google Scholar 

  88. Dai M, Jungmann R, Yin P. Optical imaging of individual biomolecules in densely packed clusters. Nat Nanotechnol. 2016;11(9):798–807.

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Bustamante C, Alexander L, Maciuba K, Kaiser CM. Single-Molecule Studies of Protein Folding with Optical Tweezers. Annu Rev Biochem. 2020;89:443–70.

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Filius M, Kim SH, Severins I, Joo C. High-Resolution Single-Molecule FRET via DNA eXchange (FRET X). Nano Lett. 2021;21(7):3295–301.

    CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by the National Natural Science Foundation of China (22150410330, 22074059).

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  1. Haihan He, Chuhong Wu, and Muhammad Saqib contributed equally to this work.

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  1. Department of Chemistry, Southern University of Science and Technology, Shenzhen, 518055, China

    Haihan He, Chuhong Wu, Muhammad Saqib & Rui Hao

  2. Research Center for Chemical Biology and Omics Analysis, School of Science, Southern University of Science and Technology, Shenzhen, 518055, China

    Haihan He, Chuhong Wu, Muhammad Saqib & Rui Hao

  3. Institute of Chemistry, Khwaja Fareed University of Engineering & Information Technology, Rahim Yar Khan 64200, Pakistan

    Muhammad Saqib

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Haihan He, Chuhong Wu, and Muhammad Saqib contributed equally. The manuscript was written and proofread by all authors.

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Published in the topical collection Young Investigators in (Bio-)Analytical Chemistry 2023 with guest editors Zhi-Yuan Gu, Beatriz Jurado-Sánchez, Thomas H. Linz, Leandro Wang Hantao, Nongnoot Wongkaew, and Peng Wu.

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He, H., Wu, C., Saqib, M. et al. Single-molecule fluorescence methods for protein biomarker analysis. Anal Bioanal Chem 415, 3655–3669 (2023). https://doi.org/10.1007/s00216-022-04502-9

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