{"doi":"10.1002/advs.202505207","title":"Label‐Free Single‐Molecule Immunoassay","abstract":"Single-molecule immunoassay is a reliable technique for the detection and quantification of low-abundance blood biomarkers, which are essential for early disease diagnosis and biomedical research. However, current single-molecule methods predominantly rely on endpoint detection and necessitate signal amplification via labeling, which brings a variety of unwanted effects, like matrix effect and autofluorescence interference. This study introduces a real-time mass imaging-based label-free single-molecule immunoassay (LFSMiA). Featuring plasmonic scattering microscopy-based mass imaging, a 2-step sandwich assay format enables background reduction, minimization of matrix effect by dynamic tracking of single binding events, and fully leveraging real-time data for improved measurement precision through a Bayesian Gaussian process model, the LFSMiA enables ultra-sensitive and direct protein detection at the single-molecule level in neat blood sample matrices. LFSMiA measurement is demonstrated for interleukin-6 and prostate-specific antigen in buffer, undiluted serum, and whole blood with sub-femtomolar detection limits and eight logs of dynamic ranges. Moreover, comparable performance is achieved with an inexpensive miniaturized setup. To show its translational potential to clinical settings and point-of-care diagnostics, N-terminal pro-B-type natriuretic peptide is examined in patient whole blood samples using the LFSMiA and results in a strong linear correlation (r > 0.99) with standard clinical lab results.","journal":"Advanced Science","year":2025,"id":526032,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.958,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1401109,"name":"Chao Chen","orcid":"0000-0001-8173-218X","position":1,"is_corresponding":false},{"id":1401110,"name":"Shuang Zhou","orcid":"0000-0003-4157-0774","position":2,"is_corresponding":false},{"id":680355,"name":"Guangzhong Ma","orcid":"0000-0002-1197-659X","position":3,"is_corresponding":false},{"id":1178731,"name":"Mohammad Javad Haji Najafi Chemerkouh","orcid":"0000-0001-6994-9394","position":4,"is_corresponding":false},{"id":589378,"name":"Christine L.H. Snozek","orcid":"0000-0003-0769-7165","position":5,"is_corresponding":false},{"id":232182,"name":"Eric H. Yang","orcid":"0000-0003-4889-7454","position":6,"is_corresponding":false},{"id":343219,"name":"Jiapei Jiang","orcid":null,"position":7,"is_corresponding":false},{"id":1179151,"name":"Brandyn Braswell","orcid":null,"position":8,"is_corresponding":false},{"id":680356,"name":"Zijian Wan","orcid":"0000-0001-7923-6451","position":9,"is_corresponding":false},{"id":858091,"name":"Xinyu Zhou","orcid":"0000-0003-1399-828X","position":10,"is_corresponding":false},{"id":341790,"name":"Shaopeng Wang","orcid":"0000-0002-2680-0503","position":11,"is_corresponding":false},{"id":1401108,"name":"Xiaoyan Zhou","orcid":"0000-0002-8289-1139","position":0,"is_corresponding":true}],"reference_count":41,"raw_metadata":null,"created_at":"2026-07-19T02:50:25.860105Z","pmid":"40538199","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}