Multiplexed immunoassay: Quantitation and profiling of serum biomarkers using magnetic nanoprobes and MALDI-TOF MS

Kai Yi Wang, Szu An Chuang, Po Chiao Lin, Li-Shing Huang, Shu Hua Chen, Saib Ouarda, Wen Harn Pan, Ping Ying Lee, Chun Cheng Lin*, Yu Ju Chen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

69 Scopus citations


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 (R2 ≈ 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.

Original languageEnglish
Pages (from-to)6159-6167
Number of pages9
JournalAnalytical Chemistry
Issue number16
StatePublished - 15 Aug 2008


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