title: Multiplexed immunoassay: quantitation and profiling of serum biomarkers using magnetic nanoprobes and MALDI-TOF MS.
abstract: Taking advantage of efficient affinity extraction by surface-functionalized magnetic nanoparticles (MNPs) and accurate MALDI-TOF MS readout, we present a multiplexed immunoassay for simultaneous enrichment and quantitation of multiple disease-associated antigens, serum amyloid A (SAA), C-reactive protein (CRP), and serum amyloid P (SAP) from human serum. To obtain reproducible MALDI signal response with direct on-MNP detection, the seed-layer method improved homogeneity of the cocrystallization of MNPs and captured antigens. Our methodology demonstrated good quantitation linearity of targeted analytes (R(2) approximately 0.97) with reduced signal variation (RSD < 10%). The lower limit of quantitation is in the nanogram level with overall assay precision (intraday, 7.0%; interday, 11.3%) and accuracy (intraday, 6.3%; interday, 17.5%) including steps of nanoprobe extraction and MALDI-TOF MS analysis. This triplexed immunoassay showed overexpression of SAA and CRP in patients with cardiac catheterization or gastric cancer (P < 0.05), consistent with single-analyte ELISA and previous studies. Compared to the determination of disease onset by single protein quantitation, our multiplexed immunoassay revealed a distinct triplexed pattern in the control group, patients with gastric cancer, and cardiac catheterization. On the basis of the advantages of flexibility in nanoprobe preparation, high specificity and sensitivity, and rapid screening by MALDI-TOF MS, this platform may provide a new methodology for disease diagnosis.
authors.affiliation: Institute of Chemistry, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Kai-Yi
authors.initials: KY
authors.lastname: Wang
authors.forename: Szu-An
authors.initials: SA
authors.lastname: Chuang
authors.forename: Po-Chiao
authors.initials: PC
authors.lastname: Lin
authors.forename: Li-Shing
authors.initials: LS
authors.lastname: Huang
authors.forename: Shu-Hua
authors.initials: SH
authors.lastname: Chen
authors.forename: Saib
authors.initials: S
authors.lastname: Ouarda
authors.forename: Wen-Harn
authors.initials: WH
authors.lastname: Pan
authors.forename: Ping-Ying
authors.initials: PY
authors.lastname: Lee
authors.forename: Chun-Cheng
authors.initials: CC
authors.lastname: Lin
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Chen
mesh_terms: D015415:Biomarkers / Q000097:blood*; D002097:C-Reactive Protein / Q000032:analysis*; D006328:Cardiac Catheterization; D002318:Cardiovascular Diseases / Q000097:blood; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007118:Immunoassay; D008280:Magnetics; D053758:Nanoparticles; D011379:Prognosis; D040901:Proteomics; D044967:Serum / Q000737:chemistry*; D000685:Serum Amyloid A Protein / Q000032:analysis*; D000683:Serum Amyloid P-Component / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013274:Stomach Neoplasms / Q000097:blood
chemical_list: D015415:Biomarkers; D000685:Serum Amyloid A Protein; D000683:Serum Amyloid P-Component; D002097:C-Reactive Protein