Single-particle (digital) immunoassays offer significantly lower limits of detection (LODs) than traditional immunoassays, making them suitable for the detection of low-abundance biomarkers. The most common approach for digital detection is based on counting individual labels. Here, we introduce a novel dot-blot particle-linked immunosorbent assay (PLISA) with digital readout utilizing laser ablation (LA) of photon upconversion nanoparticle (UCNP) labels from the nitrocellulose substrate. Compared to conventional LA, our approach allows desorption of intact nanoparticles and their precise counting by single-particle inductively coupled plasma mass spectrometry (SP ICP MS), thus counting individual UCNP-labeled immunocomplexes. Digital signal processing filters instrument noise and nanoparticle aggregates, minimizing potential errors. The immunoassay and LA SP ICP MS readout were optimized using human serum albumin, a kidney damage biomarker, as a model analyte, obtaining LODs of 0.18 and 0.12 ng/mL for the reference upconversion luminescence (UCL) and LA SP ICP MS readout, respectively. Building upon these optimized conditions, we developed PLISA for prostate-specific antigen, the key prostate cancer biomarker, with LODs of 2.4, 1.4, and 0.3 pg/mL for the UCL, analog, and digital LA SP ICP MS readout, respectively. The LOD in the sub-pg/mL range highlighted the advantage of particle counting and its ability to detect low-abundance biomarkers, as superior performance was achieved compared to the UCL and analog LA ICP MS readout. Finally, clinical serum samples of patients tested for prostate cancer were analyzed, and a strong correlation with the reference electrochemiluminescence method confirmed the potential of LA SP ICP MS for clinical diagnostics.

Department of Biochemistry Faculty of Science Masaryk University Kamenice 5 Brno 625 00 Czech Republic

Department of Chemistry Faculty of Science Masaryk University Kamenice 5 Brno 625 00 Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 5 Brno 625 00 Czech Republic

Institute of Analytical Chemistry of the Czech Academy of Sciences Veveří 97 Brno 602 00 Czech Republic

Urology Clinic University Hospital Brno Jihlavská 20 Brno 625 00 Czech Republic

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Tian W., Wang L., Lei H., Sun Y., Xiao Z.. Antibody Production and Application for Immunoassay Development of Environmental Hormones: A Review. Chem. Biol. Technol. Agric. 2018;5(1):5. doi: 10.1186/s40538-018-0117-0. DOI

Kreisig T., Hoffmann R., Zuchner T.. Homogeneous Fluorescence-Based Immunoassay Detects Antigens Within 90 Seconds. Anal. Chem. 2011;83(11):4281–4287. doi: 10.1021/ac200777h. PubMed DOI

Rupprecht K. R., Nair R. K., Harwick L. C., Grote J., Beligere G. S., Rege S. D., Chen Y.-Y., Lin Z., Fishpaugh J. R.. Development of a Dot-Blot Assay for Screening Monoclonal Antibodies to Low-Molecular-Mass Drugs. Anal. Biochem. 2010;407(2):160–164. doi: 10.1016/j.ab.2010.08.003. PubMed DOI

Máčala J., Makhneva E., Hlaváček A., Kopecký M., Gorris H. H., Skládal P., Farka Z.. Upconversion Nanoparticle-Based Dot-Blot Immunoassay for Quantitative Biomarker Detection. Anal. Chem. 2024;96(25):10237–10245. doi: 10.1021/acs.analchem.4c00837. PubMed DOI PMC

Sil B. K., Jamiruddin M. R., Haq M. A., Khondoker M. U., Jahan N., Khandker S. S., Ali T., Oishee M. J., Kaitsuka T., Mie M., Tomizawa K., Kobatake E., Haque M., Adnan N.. AuNP Coupled Rapid Flow-Through Dot-Blot Immuno-Assay for Enhanced Detection of SARS-CoV-2 Specific Nucleocapsid and Receptor Binding Domain IgG. Int. J. Nanomedicine. 2021;16:4739–4753. doi: 10.2147/IJN.S313140. PubMed DOI PMC

Lee H., Ko M., Lee J., Gupta A., Yoon H. R., Ryu S.-H., Kim S. K., Rha E., Kwon K. K., Lee D.-H., Kim H., Jung H.-C., Lee S.-G.. Development of a Sensitive Immunoassay for Detecting Persistent Contaminant Dichlorodiphenyltrichloroethane (DDT) in Food Samples Using a Monoclonal Antibody. Food Control. 2024;160:110320. doi: 10.1016/j.foodcont.2024.110320. DOI

Costa J. G., Vilariño M. J.. Semiquantitative Dot Blot with the GRA8 Antigen to Differentiate the Stages of Toxoplasmosis Infection. J. Microbiol. Methods. 2018;149:9–13. doi: 10.1016/j.mimet.2018.04.015. PubMed DOI

Tsuji M., Yu H.-S., Ishihara Y., Isse T., Ikeda-Ishihara N., Tuchiya T., Kawamoto T.. A Simple Method for Detection of Multiple Chemical-Specific IgGs in Serum Based on Dot Blotting. Health. N. Hav. 2016;08(15):1645–1653. doi: 10.4236/health.2016.815160. DOI

Kimura M., Aviv A.. Measurement of Telomere DNA Content by Dot Blot Analysis. Nucleic Acids Res. 2011;39(12):e84–e84. doi: 10.1093/nar/gkr235. PubMed DOI PMC

Farka Z., Brandmeier J. C., Mickert M. J., Pastucha M., Lacina K., Skládal P., Soukka T., Gorris H. H.. Nanoparticle-Based Bioaffinity Assays: From the Research Laboratory to the Market. Adv. Mater. 2024;36(3):2307653. doi: 10.1002/adma.202307653. PubMed DOI

Rahi S., Lanjekar V., Ghormade V.. Development of a Rapid Dot-Blot Assay for Ochratoxin A (OTA) Detection Using Peptide Conjugated Gold Nanoparticles for Bio-Recognition and Detection. Food Control. 2022;136:108842. doi: 10.1016/j.foodcont.2022.108842. DOI

Zhang P., Lu H., Chen J., Han H., Ma W.. Simple and Sensitive Detection of HBsAg by Using a Quantum Dots Nanobeads Based Dot-Blot Immunoassay. Theranostics. 2014;4(3):307–315. doi: 10.7150/thno.8007. PubMed DOI PMC

Resch-Genger U., Gorris H. H.. Perspectives and Challenges of Photon-Upconversion Nanoparticles - Part I: Routes to Brighter Particles and Quantitative Spectroscopic Studies. Anal. Bioanal. Chem. 2017;409(25):5855–5874. doi: 10.1007/s00216-017-0499-z. PubMed DOI

Hlaváček A., Farka Z., Mickert M. J., Kostiv U., Brandmeier J. C., Horák D., Skládal P., Foret F., Gorris H. H.. Bioconjugates of Photon-Upconversion Nanoparticles for Cancer Biomarker Detection and Imaging. Nat. Protoc. 2022;17(4):1028–1072. doi: 10.1038/s41596-021-00670-7. PubMed DOI

Mickert M. J., Farka Z., Kostiv U., Hlaváček A., Horák D., Skládal P., Gorris H. H.. Measurement of Sub-Femtomolar Concentrations of Prostate-Specific Antigen through Single-Molecule Counting with an Upconversion-Linked Immunosorbent Assay. Anal. Chem. 2019;91(15):9435–9441. doi: 10.1021/acs.analchem.9b02872. PubMed DOI

Farka Z., Juřík T., Kovář D., Trnková L., Skládal P.. Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges. Chem. Rev. 2017;117(15):9973–10042. doi: 10.1021/acs.chemrev.7b00037. PubMed DOI

Esteban-Fernández D., Bierkandt F. S., Linscheid M. W.. MeCAT Labeling for Absolute Quantification of Intact Proteins Using Label-Specific Isotope Dilution ICP-MS. J. Anal. At. Spectrom. 2012;27(10):1701. doi: 10.1039/c2ja30137g. DOI

Tanner S. D., Bandura D. R., Ornatsky O., Baranov V. I., Nitz M., Winnik M. A.. Flow Cytometer with Mass Spectrometer Detection for Massively Multiplexed Single-Cell Biomarker Assay. Pure Appl. Chem. 2008;80(12):2627–2641. doi: 10.1351/pac200880122627. DOI

Cruz-Alonso M., Fernandez B., Álvarez L., González-Iglesias H., Traub H., Jakubowski N., Pereiro R.. Bioimaging of Metallothioneins in Ocular Tissue Sections by Laser Ablation-ICP-MS Using Bioconjugated Gold Nanoclusters as Specific Tags. Microchim. Acta. 2018;185(1):64. doi: 10.1007/s00604-017-2597-1. PubMed DOI

Laborda F., Bolea E., Jiménez-Lamana J.. Single Particle Inductively Coupled Plasma Mass Spectrometry: A Powerful Tool for Nanoanalysis. Anal. Chem. 2014;86(5):2270–2278. doi: 10.1021/ac402980q. PubMed DOI

Degueldre C., Favarger P.-Y.. Colloid Analysis by Single Particle Inductively Coupled Plasma-Mass Spectroscopy: A Feasibility Study. Colloids Surf. A Physicochem. Eng. Asp. 2003;217(1–3):137–142. doi: 10.1016/S0927-7757(02)00568-X. DOI

Bolea E., Jimenez M. S., Perez-Arantegui J., Vidal J. C., Bakir M., Ben-Jeddou K., Gimenez-Ingalaturre A. C., Ojeda D., Trujillo C., Laborda F.. Analytical Applications of Single Particle Inductively Coupled Plasma Mass Spectrometry: A Comprehensive and Critical Review. Anal. Methods. 2021;13(25):2742–2795. doi: 10.1039/D1AY00761K. PubMed DOI

Laborda F., Bolea E., Jiménez-Lamana J.. Single Particle Inductively Coupled Plasma Mass Spectrometry: A Powerful Tool for Nanoanalysis. Anal. Chem. 2014;86(5):2270–2278. doi: 10.1021/ac402980q. PubMed DOI

Benešová I., Dlabková K., Zelenák F., Vaculovič T., Kanický V., Preisler J.. Direct Analysis of Gold Nanoparticles from Dried Droplets Using Substrate-Assisted Laser Desorption Single Particle-ICPMS. Anal. Chem. 2016;88(5):2576–2582. doi: 10.1021/acs.analchem.5b02421. PubMed DOI

Stiborek M., Jindřichová L., Meliorisová S., Bednařík A., Prysiazhnyi V., Kroupa J., Houška P., Adamová B., Navrátilová J., Kanický V., Preisler J.. Infrared Laser Desorption of Intact Nanoparticles for Digital Tissue Imaging. Anal. Chem. 2022;94(51):18114–18120. doi: 10.1021/acs.analchem.2c05216. PubMed DOI

Cao Y., Mo G., Feng J., He X., Tang L., Yu C., Deng B.. Based on ZnSe Quantum Dots Labeling and Single Particle Mode ICP-MS Coupled with Sandwich Magnetic Immunoassay for the Detection of Carcinoembryonic Antigen in Human Serum. Anal. Chim. Acta. 2018;1028:22–31. doi: 10.1016/j.aca.2018.04.039. PubMed DOI

Liu R., Xing Z., Lv Y., Zhang S., Zhang X.. Sensitive Sandwich Immunoassay Based on Single Particle Mode Inductively Coupled Plasma Mass Spectrometry Detection. Talanta. 2010;83(1):48–54. doi: 10.1016/j.talanta.2010.08.037. PubMed DOI

Prysiazhnyi V., Bednařík A., Žalud M., Hegrová V., Neuman J., Preisler J.. Fate of Gold Nanoparticles in Laser Desorption/Ionization Mass Spectrometry: Toward the Imaging of Individual Nanoparticles. J. Am. Soc. Mass Spectrom. 2023;34(4):570–578. doi: 10.1021/jasms.2c00300. PubMed DOI PMC

Žalud M., Prysiazhnyi V., Bednařík A., Preisler J.. Detection of Single Ag Nanoparticles Using Laser Desorption/Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2023;34(7):1459–1466. doi: 10.1021/jasms.3c00137. PubMed DOI PMC

Castro R. B. de, de Souza A. C. A., Pavione N. da R. T., Badaró de Moraes J. V., Ribeiro I. C., Agripino J. de M., Bressan G. C., Vasconcellos R. de S., Silva-Júnior A., Fietto J. L. R.. Urea, Salts, and Tween 20 Influence on Adsorption of IgG and Leishmania RNTPDase2 to Nitrocellulose. Anal. Biochem. 2022;646:114648. doi: 10.1016/j.ab.2022.114648. PubMed DOI

Makhneva E., Sklenárová D., Brandmeier J. C., Hlaváček A., Gorris H. H., Skládal P., Farka Z.. Influence of Label and Solid Support on the Performance of Heterogeneous Immunoassays. Anal. Chem. 2022;94(47):16376–16383. doi: 10.1021/acs.analchem.2c03543. PubMed DOI

Wild, D. The Immunoassay Handbook - Theory and Applications of Ligand Binding, ELISA and Related Techniques, 4th ed.. Elsevier: Oxford, 2013.

Guo L., Zhao Y., Huang Q., Huang J., Tao Y., Chen J., Li H.-Y., Liu H.. Electrochemical Protein Biosensors for Disease Marker Detection: Progress and Opportunities. Microsyst. Nanoeng. 2024;10(1):65. doi: 10.1038/s41378-024-00700-w. PubMed DOI PMC

Rissin D. M., Kan C. W., Campbell T. G., Howes S. C., Fournier D. R., Song L., Piech T., Patel P. P., Chang L., Rivnak A. J., Ferrell E. P., Randall J. D., Provuncher G. K., Walt D. R., Duffy D. C.. Single-Molecule Enzyme-Linked Immunosorbent Assay Detects Serum Proteins at Subfemtomolar Concentrations. Nat. Biotechnol. 2010;28(6):595–599. doi: 10.1038/nbt.1641. PubMed DOI PMC

Ploussard G., Fossati N., Wiegel T., D’Amico A., Hofman M. S., Gillessen S., Mottet N., Joniau S., Spratt D. E.. Management of Persistently Elevated Prostate-Specific Antigen After Radical Prostatectomy: A Systematic Review of the Literature. Eur. Urol. Oncol. 2021;4(2):150–169. doi: 10.1016/j.euo.2021.01.001. PubMed DOI

Eshetu B., Worede A., Fentie A., Chane E., Fetene G., Wondifraw H., Shimelis M., Girma M., Hadgu R., Demeke K., Fasil A.. Assessment of Electrolyte Imbalance and Associated Factors Among Adult Diabetic Patients Attending the University of Gondar Comprehensive Specialized Hospital, Ethiopia: A Comparative Cross-Sectional Study. Diabetes Metab. Syndr. Obes. 2023;16:1207–1220. doi: 10.2147/DMSO.S404788. PubMed DOI PMC

Dodd A., Ducroq D., Neale S., Wise M., Mitchem K., Armston A., Barth J., El-Farhan N., Rees D., Evans C.. The Effect of Serum Matrix and Gender on Cortisol Measurement by Commonly Used Immunoassays. Ann. Clin. Biochem. 2014;51(3):379–385. doi: 10.1177/0004563213514567. PubMed DOI

Su Y., Zhou L.. Review of Single-Molecule Immunoassays: Non-Chip and on-Chip Assays. Anal. Chim. Acta. 2024;1322:342885. doi: 10.1016/j.aca.2024.342885. PubMed DOI

Duffy D. C.. Digital Detection of Proteins. Lab Chip. 2023;23(5):818–847. doi: 10.1039/D2LC00783E. PubMed DOI

Wu Y., Fu Y., Guo J., Guo J.. Single-Molecule Immunoassay Technology: Recent Advances. Talanta. 2023;265:124903. doi: 10.1016/j.talanta.2023.124903. PubMed DOI

Farka Z., Mickert M. J., Pastucha M., Mikušová Z., Skládal P., Gorris H. H.. Advances in Optical Single-Molecule Detection: En Route to Supersensitive Bioaffinity Assays. Angew. Chem., Int. Ed. 2020;59(27):10746–10773. doi: 10.1002/anie.201913924. PubMed DOI PMC

Huang Z., Wang C., Liu R., Su Y., Lv Y.. Self-Validated Homogeneous Immunoassay by Single Nanoparticle in-Depth Scrutinization. Anal. Chem. 2020;92(3):2876–2881. doi: 10.1021/acs.analchem.9b05596. PubMed DOI

Huang Z., Li Z., Jiang M., Liu R., Lv Y.. Homogeneous Multiplex Immunoassay for One-Step Pancreatic Cancer Biomarker Evaluation. Anal. Chem. 2020;92(24):16105–16112. doi: 10.1021/acs.analchem.0c03780. PubMed DOI

Hu S., Liu R., Zhang S., Huang Z., Xing Z., Zhang X.. A New Strategy for Highly Sensitive Immunoassay Based on Single-Particle Mode Detection by Inductively Coupled Plasma Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2009;20(6):1096–1103. doi: 10.1016/j.jasms.2009.02.005. PubMed DOI

Liu R., Xing Z., Lv Y., Zhang S., Zhang X.. Sensitive Sandwich Immunoassay Based on Single Particle Mode Inductively Coupled Plasma Mass Spectrometry Detection. Talanta. 2010;83(1):48–54. doi: 10.1016/j.talanta.2010.08.037. PubMed DOI

Huang Z., Zhao X., Hu J., Zhang C., Xie X., Liu R., Lv Y.. Single-Nanoparticle Differential Immunoassay for Multiplexed Gastric Cancer Biomarker Monitoring. Anal. Chem. 2022;94(37):12899–12906. doi: 10.1021/acs.analchem.2c03013. PubMed DOI

Cao Y., Feng J., Tang L., Mo G., Mo W., Deng B.. Detection of Three Tumor Biomarkers in Human Lung Cancer Serum Using Single Particle Inductively Coupled Plasma Mass Spectrometry Combined with Magnetic Immunoassay. Spectrochim. Acta Part B At. Spectrosc. 2020;166:105797. doi: 10.1016/j.sab.2020.105797. DOI

Cao Y., Mo G., Feng J., He X., Tang L., Yu C., Deng B.. Based on ZnSe Quantum Dots Labeling and Single Particle Mode ICP-MS Coupled with Sandwich Magnetic Immunoassay for the Detection of Carcinoembryonic Antigen in Human Serum. Anal. Chim. Acta. 2018;1028:22–31. doi: 10.1016/j.aca.2018.04.039. PubMed DOI

Cao Y., Deng B.. Detection Of Myeloperoxidase And Osteopontin In Human Serum Using Single Particle-ICP-MS With MoS2 And ZnS Quantum Dots. At. Spectrosc. 2023;44(02):65–75. doi: 10.46770/AS.2023.042. DOI

Tvrdoňová M., Vlčnovská M., Vaníčkova L. P., Kanický V., Adam V., Ascher L., Jakubowski N., Vaculovičová M., Vaculovič T.. Gold Nanoparticles as Labels for Immunochemical Analysis Using Laser Ablation Inductively Coupled Plasma Mass Spectrometry. Anal. Bioanal. Chem. 2019;411(3):559–564. doi: 10.1007/s00216-018-1300-7. PubMed DOI

Vlčnovská M., Štossová A., Kuchyňka M., Dillingerová V., Polanská H., Masařík M., Hrstka R., Adam V., Kanický V., Vaculovič T., Vaculovičová M.. Comparison of Metal Nanoparticles (Au, Ag, Eu, Cd) Used for Immunoanalysis Using LA-ICP-MS Detection. Molecules. 2021;26(3):630. doi: 10.3390/molecules26030630. PubMed DOI PMC

Aydin S.. A Short History, Principles, and Types of ELISA, and Our Laboratory Experience with Peptide/Protein Analyses Using ELISA. Peptides (N.Y.) 2015;72:4–15. doi: 10.1016/j.peptides.2015.04.012. PubMed DOI

Dong J., Zhu Z., Li L., Xing P., Li S., Ouyang L., Liu X., Guo W., Zheng H., Qian R.. Evaluation of Miniaturized Ultrasonic Nebulization for High-Efficiency Sampling in Characterization of Silver Nanoparticles by Single Particle Inductively Coupled Plasma Mass Spectrometry. J. Anal. At. Spectrom. 2024;39(11):2791–2798. doi: 10.1039/D4JA00320A. DOI