We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.

Biosensor Technologies AIT Austrian Institute of Technology GmbH Konrad Lorenz Straße 24 3430 Tulln an der Donau Austria

CEST Competence Center for Electrochemical Surface Technologies 3430 Tulln an der Donau Austria

Department of Chemistry Ugo Schiff University of Florence via della Lastruccia 3 13 Sesto Fiorentino 50019 Firenze Italy

Department of Nanobiotechnology University of Natural Resources and Life Sciences Vienna 1190 Vienna Austria

FZU Institute of Physics Czech Academy of Sciences Na Slovance 2 182 21 Prague Czech Republic

Molecular Diagnostics Health and Environment AIT Austrian Institute of Technology GmbH 1210 Vienna Austria

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Barišić I.; Schoenthaler S.; Ke R.; Nilsson M.; Noehammer C.; Wiesinger-Mayr H. Multiplex Detection of Antibiotic Resistance Genes Using Padlock Probes. Diagn. Microbiol. Infect. Dis. 2013, 77, 118–125. 10.1016/j.diagmicrobio.2013.06.013. PubMed DOI

Wolff N.; Hendling M.; Schönthaler S.; Geiss A. F.; Barišić I. Low-Cost Microarray Platform to Detect Antibiotic Resistance Genes. Sens. Bio-Sens. Res. 2019, 23, 100266.10.1016/j.sbsr.2019.100266. DOI

Simion M.; Kleps I.; Ignat T.; Condac E.; Craciunoiu F.; Angelescu A.; Miu M.; Bragaru A.; Costache M.; Savu L.. Bioanalytical Silicon Micro-Devices for DNA. In CAS 2005 Proceedings. 2005 International Semiconductor Conference; IEEE, 2005; pp 235–238.

Rothemund P. W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297–302. 10.1038/nature04586. PubMed DOI

Zhao W.; Ali M. M.; Brook M. A.; Li Y. Rolling Circle Amplification: Applications in Nanotechnology and Biodetection with Functional Nucleic Acids. Angew. Chem., Int. Ed. 2008, 47, 6330–6337. 10.1002/anie.200705982. PubMed DOI

Houdebine L.-M.Transgenic Animal Models in Biomedical Research. In Target Discovery and Validation Reviews and Protocols; Humana Press: New Jersey, 2007.

Mullis K.; Faloona F.; Scharf S.; Saiki R.; Horn G.; Erlich H. Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction. Cold Spring Harbor Symp. Quant. Biol. 1986, 51, 263–273. 10.1101/SQB.1986.051.01.032. PubMed DOI

Ali M. M.; Li F.; Zhang Z.; Zhang K.; Kang D.-K.; Ankrum J. A.; Le X. C.; Zhao W. Rolling Circle Amplification: A Versatile Tool for Chemical Biology, Materials Science and Medicine. Chem. Soc. Rev. 2014, 43, 3324–3341. 10.1039/c3cs60439j. PubMed DOI

Poon L. L.; Wong B. W.; Ma E. H.; Chan K. H.; Chow L. M.; Abeyewickreme W.; Tangpukdee N.; Yuen K. Y.; Guan Y.; Looareesuwan S.; Peiris J. M. Sensitive and Inexpensive Molecular Test for Falciparum Malaria: Detecting Plasmodium Falciparum DNA Directly from Heat-Treated Blood by Loop-Mediated Isothermal Amplification. Clin. Chem. 2006, 52, 303–306. 10.1373/clinchem.2005.057901. PubMed DOI

Pulido M. R.; Garcia-Quintanilla M.; Martin-Pena R.; Cisneros J. M.; McConnell M. J. Progress on the Development of Rapid Methods for Antimicrobial Susceptibility Testing. J. Antimicrob. Chemother. 2013, 68, 2710–2717. 10.1093/jac/dkt253. PubMed DOI

Dufva M.; Christensen C. B. v.. Optimization of Oligonucleotide DNA Microarrays. In Microarrays; Humana Press: Totowa, NJ, 2007. PubMed

Munir A.; Waseem H.; Williams M.; Stedtfeld R.; Gulari E.; Tiedje J.; Hashsham S. Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB. Microarrays 2017, 6, 9.10.3390/microarrays6020009. PubMed DOI PMC

Edwards M. C.; Gibbs R. A. Multiplex PCR: Advantages, Development, and Applications. PCR Methods Appl. 1994, 3, S65–S75. 10.1101/gr.3.4.S65. PubMed DOI

Khodakov D.; Li J.; Zhang J. X.; Zhang D. Y. Highly Multiplexed Rapid DNA Detection with Single-Nucleotide Specificity via Convective PCR in a Portable Device. Nat. Biomed. Eng. 2021, 5, 702–712. 10.1038/s41551-021-00755-4. PubMed DOI

Deng R.; Zhang K.; Wang L.; Ren X.; Sun Y.; Li J. DNA-Sequence-Encoded Rolling Circle Amplicon for Single-Cell RNA Imaging. Chem 2018, 4, 1373–1386. 10.1016/j.chempr.2018.03.003. DOI

Ng P. C.; Kirkness E. F.. Whole Genome Sequencing. In Genetic Variation; Humana Press, 2010.

Mahmoudian L.; Kaji N.; Tokeshi M.; Nilsson M.; Baba Y. Rolling Circle Amplification and Circle-to-Circle Amplification of a Specific Gene Integrated with Electrophoretic Analysis on a Single Chip. Anal. Chem. 2008, 80, 2483–2490. 10.1021/ac702289j. PubMed DOI

Dahl F.; Banér J.; Gullberg M.; Mendel-Hartvig M.; Landegren U.; Nilsson M. Circle-to-Circle Amplification for Precise and Sensitive DNA Analysis. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4548–4553. 10.1073/pnas.0400834101. PubMed DOI PMC

Li X.-H.; Zhang X.-L.; Wu J.; Lin N.; Sun W.-M.; Chen M.; Ou Q.-S.; Lin Z.-Y. Hyperbranched Rolling Circle Amplification (HRCA)-Based Fluorescence Biosensor for Ultrasensitive and Specific Detection of Single-Nucleotide Polymorphism Genotyping Associated with the Therapy of Chronic Hepatitis B Virus Infection. Talanta 2019, 191, 277–282. 10.1016/j.talanta.2018.08.064. PubMed DOI

Xu X.; Wang L.; Li X.; Cui W.; Jiang W. Multiple Sealed Primers-Mediated Rolling Circle Amplification Strategy for Sensitive and Specific Detection of DNA Methyltransferase Activity. Talanta 2019, 194, 282–288. 10.1016/j.talanta.2018.09.113. PubMed DOI

Yan J.; Hu C.; Wang P.; Liu R.; Zuo X.; Liu X.; Song S.; Fan C.; He D.; Sun G. Novel Rolling Circle Amplification and DNA Origami-Based DNA Belt-Involved Signal Amplification Assay for Highly Sensitive Detection of Prostate-Specific Antigen (PSA). ACS Appl. Mater. Interfaces 2014, 6, 20372–20377. 10.1021/am505913d. PubMed DOI

Ouyang X.; Li J.; Liu H.; Zhao B.; Yan J.; Ma Y.; Xiao S.; Song S.; Huang Q.; Chao J.; Fan C. Rolling Circle Amplification-Based DNA Origami Nanostructrures for Intracellular Delivery of Immunostimulatory Drugs. Small 2013, 9, 3082–3087. 10.1002/smll.201300458. PubMed DOI

Ke R.; Mignardi M.; Pacureanu A.; Svedlund J.; Botling J.; Wählby C.; Nilsson M. In Situ Sequencing for RNA Analysis in Preserved Tissue and Cells. Nat. Methods 2013, 10, 857–860. 10.1038/nmeth.2563. PubMed DOI

Cheglakov Z.; Weizmann Y.; Braunschweig A. B.; Wilner O. I.; Willner I. Increasing the Complexity of Periodic Protein Nanostructures by the Rolling-Circle-Amplified Synthesis of Aptamers. Angew. Chem., Int. Ed. 2008, 47, 126–130. 10.1002/anie.200703688. PubMed DOI

Juette M. F.; Terry D. S.; Wasserman M. R.; Zhou Z.; Altman R. B.; Zheng Q.; Blanchard S. C. The Bright Future of Single-Molecule Fluorescence Imaging. Curr. Opin. Chem. Biol. 2014, 20, 103–111. 10.1016/j.cbpa.2014.05.010. PubMed DOI PMC

Holzmeister P.; Acuna G. P.; Grohmann D.; Tinnefeld P. Breaking the Concentration Limit of Optical Single-Molecule Detection. Chem. Soc. Rev. 2014, 43, 1014–1028. 10.1039/C3CS60207A. PubMed DOI

Kühnemund M.; Hernández-Neuta I.; Sharif M. I.; Cornaglia M.; Gijs M. A. M.; Nilsson M. Sensitive and Inexpensive Digital DNA Analysis by Microfluidic Enrichment of Rolling Circle Amplified Single-Molecules. Nucleic Acids Res. 2017, 45, e5910.1093/nar/gkw1324. PubMed DOI PMC

Wu H.; Wang C.; Wu S. Single-Cell Sequencing for Drug Discovery and Drug Development. Curr. Top. Med. Chem. 2017, 17, 1769–1777. 10.2174/1568026617666161116145358. PubMed DOI

Xu L.; Brito I. L.; Alm E. J.; Blainey P. C. Virtual Microfluidics for Digital Quantification and Single-Cell Sequencing. Nat. Methods 2016, 13, 759–762. 10.1038/nmeth.3955. PubMed DOI PMC

Kühnemund M.; Witters D.; Nilsson M.; Lammertyn J. Circle-to-Circle Amplification on a Digital Microfluidic Chip for Amplified Single Molecule Detection. Lab Chip 2014, 14, 2983–2992. 10.1039/C4LC00348A. PubMed DOI

Hatch A.; Sano T.; Misasi J.; Smith C. L. Rolling Circle Amplification of DNA Immobilized on Solid Surfaces and Its Application to Multiplex Mutation Detection. Genet. Anal.: Biomol. Eng. 1999, 15, 35–40. 10.1016/S1050-3862(98)00014-X. PubMed DOI

Schweitzer B.; Wiltshire S.; Lambert J.; O’Malley S.; Kukanskis K.; Zhu Z.; Kingsmore S. F.; Lizardi P. M.; Ward D. C. Immunoassays with Rolling Circle DNA Amplification: A Versatile Platform for Ultrasensitive Antigen Detection. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10113–10119. 10.1073/pnas.170237197. PubMed DOI PMC

Björkesten J.; Patil S.; Fredolini C.; Lönn P.; Landegren U. A Multiplex Platform for Digital Measurement of Circular DNA Reaction Products. Nucleic Acids Res. 2020, 48, e7310.1093/nar/gkaa419. PubMed DOI PMC

Kühnemund M.; Witters D.; Nilsson M.; Lammertyn J. Circle-to-Circle Amplification on a Digital Microfluidic Chip for Amplified Single Molecule Detection. Lab Chip 2014, 14, 2983–2992. 10.1039/C4LC00348A. PubMed DOI

Chen H.; Hou Y.; Qi F.; Zhang J.; Koh K.; Shen Z.; Li G. Detection of Vascular Endothelial Growth Factor Based on Rolling Circle Amplification as a Means of Signal Enhancement in Surface Plasmon Resonance. Biosens. Bioelectron. 2014, 61, 83–87. 10.1016/j.bios.2014.05.005. PubMed DOI

Yao C.; Xiang Y.; Deng K.; Xia H.; Fu W. Sensitive and Specific HBV Genomic DNA Detection Using RCA-Based QCM Biosensor. Sens. Actuators, B 2013, 181, 382–387. 10.1016/j.snb.2013.01.063. DOI

de la Torre T. Z. G.; Herthnek D.; Strømme M. A Magnetic Nanobead-Based Read-Out Procedure for Rapid Detection of DNA Molecules. J. Nanosci. Nanotechnol. 2017, 17, 2861–2864. 10.1166/jnn.2017.13877. PubMed DOI

Tang X.; Wang Y.; Zhou L.; Zhang W.; Yang S.; Yu L.; Zhao S.; Chang K.; Chen M. Strand Displacement-Triggered G-Quadruplex/Rolling Circle Amplification Strategy for the Ultra-Sensitive Electrochemical Sensing of Exosomal MicroRNAs. Microchim. Acta 2020, 187, 172.10.1007/s00604-020-4143-9. PubMed DOI

Xiang Y.; Zhu X.; Huang Q.; Zheng J.; Fu W. Real-Time Monitoring of Mycobacterium Genomic DNA with Target-Primed Rolling Circle Amplification by a Au Nanoparticle-Embedded SPR Biosensor. Biosens. Bioelectron. 2015, 66, 512–519. 10.1016/j.bios.2014.11.021. PubMed DOI

Huang Y.-Y.; Hsu H.-Y.; Huang C.-J. C. A Protein Detection Technique by Using Surface Plasmon Resonance (SPR) with Rolling Circle Amplification (RCA) and Nanogold-Modified Tags. Biosens. Bioelectron. 2007, 22, 980–985. 10.1016/j.bios.2006.04.017. PubMed DOI

He P.; Liu L.; Qiao W.; Zhang S. Ultrasensitive Detection of Thrombin Using Surface Plasmon Resonance and Quartz Crystal Microbalance Sensors by Aptamer-Based Rolling Circle Amplification and Nanoparticle Signal Enhancement. Chem. Commun. 2014, 50, 1481–1484. 10.1039/C3CC48223E. PubMed DOI

Shi D.; Huang J.; Chuai Z.; Chen D.; Zhu X.; Wang H.; Peng J.; Wu H.; Huang Q.; Fu W. Isothermal and Rapid Detection of Pathogenic Microorganisms Using a Nano-Rolling Circle Amplification-Surface Plasmon Resonance Biosensor. Biosens. Bioelectron. 2014, 62, 280–287. 10.1016/j.bios.2014.06.066. PubMed DOI

Liebermann T.; Knoll W. Surface-Plasmon Field-Enhanced Fluorescence Spectroscopy. Colloids Surf., A 2000, 171, 115–130. 10.1016/S0927-7757(99)00550-6. DOI

Arima Y.; Teramura Y.; Takiguchi H.; Kawano K.; Kotera H.; Iwata H.. Surface Plasmon Resonance and Surface Plasmon Field-Enhanced Fluorescence Spectroscopy for Sensitive Detection of Tumor Markers. In Biosensors and Biodetection; Humana Press, 2009; pp 3–20. PubMed

Lechner B.; Hageneder S.; Schmidt K.; Kreuzer M. P.; Conzemius R.; Reimhult E.; Barišić I.; Dostalek J. In Situ Monitoring of Rolling Circle Amplification on a Solid Support by Surface Plasmon Resonance and Optical Waveguide Spectroscopy. ACS Appl. Mater. Interfaces 2021, 13, 32352–32362. 10.1021/acsami.1c03715. PubMed DOI

Vasilev K.; Knoll W.; Kreiter M. Fluorescence Intensities of Chromophores in Front of a Thin Metal Film. J. Chem. Phys. 2004, 120, 3439–3445. 10.1063/1.1640341. PubMed DOI

Ekgasit S.; Yu F.; Knoll W. Fluorescence Intensity in Surface-Plasmon Field-Enhanced Fluorescence Spectroscopy. Sens. Actuators, B 2005, 104, 294–301. 10.1016/j.snb.2004.05.021. DOI

Zhang Y.; Noji H. Digital Bioassays: Theory, Applications, and Perspectives. Anal. Chem. 2017, 89, 92–101. 10.1021/acs.analchem.6b04290. PubMed DOI

Huang Y.-Y.; Hsu H.-Y.; Huang C.-J. C. A Protein Detection Technique by Using Surface Plasmon Resonance (SPR) with Rolling Circle Amplification (RCA) and Nanogold-Modified Tags. Biosens. Bioelectron. 2007, 22, 980–985. 10.1016/j.bios.2006.04.017. PubMed DOI

Forinová M.; Pilipenco A.; Víšová I.; Lynn N. S.; Dostálek J.; Mašková H.; Hönig V.; Palus M.; Selinger M.; Kočová P.; Dyčka F.; Štěrba J.; Houska M.; Vrabcová M.; Horák P.; Anthi J.; Tung C.-P.; Yu C.-M.; Chen C.-Y.; Huang Y.-C.; Tsai P.-H.; Lin S.-Y.; Hsu H.-J.; Yang A.-S.; Dejneka A.; Vaisocherová-Lísalová H. Functionalized Terpolymer-Brush-Based Biointerface with Improved Antifouling Properties for Ultra-Sensitive Direct Detection of Virus in Crude Clinical Samples. ACS Appl. Mater. Interfaces 2021, 13, 60612–60624. 10.1021/acsami.1c16930. PubMed DOI

Hucknall A.; Rangarajan S.; Chilkoti A. In Pursuit of Zero: Polymer Brushes That Resist the Adsorption of Proteins. Adv. Mater. 2009, 21, 2441–2446. 10.1002/adma.200900383. DOI

Rodriguez-Emmenegger C.; Brynda E.; Riedel T.; Houska M.; Šubr V.; Alles A. B.; Hasan E.; Gautrot J. E.; Huck W. T. S. Polymer Brushes Showing Non-Fouling in Blood Plasma Challenge the Currently Accepted Design of Protein Resistant Surfaces. Macromol. Rapid Commun. 2011, 32, 952–957. 10.1002/marc.201100189. PubMed DOI

Vietz C.; Schütte M. L.; Wei Q.; Richter L.; Lalkens B.; Ozcan A.; Tinnefeld P.; Acuna G. P. Benchmarking Smartphone Fluorescence-Based Microscopy with DNA Origami Nanobeads: Reducing the Gap toward Single-Molecule Sensitivity. ACS Omega 2019, 4, 637–642. 10.1021/acsomega.8b03136. PubMed DOI PMC

Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design