Wang, Kai-YiKai-YiWangChuang, Szu-AnSzu-AnChuangLin, Po-ChiaoPo-ChiaoLinHuang, Li-ShingLi-ShingHuangChen, Shu-HuaShu-HuaChenOuarda, SaibSaibOuardaPan, Wen-HarnWen-HarnPanLee, Ping-YingPing-YingLeeLin, Chun-ChengChun-ChengLinChen, Yu-JuYu-JuChen2009-10-282018-07-102009-10-282018-07-102008http://ntur.lib.ntu.edu.tw//handle/246246/172145Taking 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. ? 2008 American Chemical Society.application/pdf1645324 bytesapplication/pdfen-US[SDGs]SDG3Body fluids; Glycoproteins; Immunology; Multiplexing; Nanoparticles; Nanostructured materials; Nanostructures; Analytes; C-reactive protein; Co crystallizations; Functionalized; Human serum; Magnetic nano-particles; MALDI-TOF-MS; Seed layers; Serum amyloid A; Serum amyloid P; Signal response; Simultaneous enrichment; Antigens; antigen; biological marker; C reactive protein; nanoparticle; serum amyloid A; serum amyloid P; accuracy; article; crystallization; enzyme linked immunosorbent assay; heart catheterization; human; immunoassay; magnetic nanoprobe; matrix assisted laser desorption ionization time of flight mass spectrometry; nanoprobe; protein analysis; protein expression; protein profiling; protein quantitation; screening test; sensitivity and specificity; stomach cancer; Biological Markers; C-Reactive Protein; Cardiovascular Diseases; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Heart Catheterization; Humans; Immunoassay; Magnetics; Nanoparticles; Prognosis; Proteomics; Serum; Serum Amyloid A Protein; Serum Amyloid P-Component; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stomach Neoplasmsjournal article10.1021/ac800354uhttp://ntur.lib.ntu.edu.tw/bitstream/246246/172145/1/13.pdf