Multiplexed immunoassay: quantitation and profiling of serum biomarkers using magnetic nanoprobes and MALDI-TOF MS.
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.
Wang
Kai-Yi
KY
Institute of Chemistry, Academia Sinica, Taipei, Taiwan, ROC.
Chuang
Szu-An
SA
Lin
Po-Chiao
PC
Huang
Li-Shing
LS
Chen
Shu-Hua
SH
Ouarda
Saib
S
Pan
Wen-Harn
WH
Lee
Ping-Ying
PY
Lin
Chun-Cheng
CC
Chen
Yu-Ju
YJ
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
D015415:Biomarkers; D000685:Serum Amyloid A Protein; D000683:Serum Amyloid P-Component; D002097:C-Reactive Protein