Protein Detection Using Arrayed Microsensor Chips: Tuning Sensor Footprint to Achieve Ultrasensitive Readout of CA-125 in Serum and Whole Blood

Jagotamoy Das and Shana O. Kelley
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, and Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Canada
Anal. Chem., Article ASAP
DOI: 10.1021/ac102917f
Publication Date (Web): January 18, 2011
Copyright © 2011 American Chemical Society

Abstract

Multiplexed assays that can measure protein biomarkers and internal standards are highly desirable given the potential to reduce false positives and negatives. We report here the use of a chip-based platform that achieves multiplexed immunosensing of the ovarian cancer biomarker CA-125 without the need for covalent labeling or sandwich complexes. The sensor chips allow the straightforward comparison of detectors of different sizes, and we used this feature to scan the microscale size regime for the best sensor size and optimize the limit of detection exhibited down to 0.1 U/mL. The assay has a straightforward design, with readout being performed in a single step involving the introduction of a noncovalently attached redox reporter group. The detection system reported exhibits excellent specificity, with analysis of a specific cancer biomarker, CA-125, performed in human serum and whole blood. The multiplexing of the system allows the analysis of the biomarker to be performed in parallel with an abundant serum protein for internal calibration.

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Characterization of Iron Dinitrosyl Species Formed in the Reaction of Nitric Oxide with a Biological Rieske Center

Abstract

Reactions of nitric oxide with cysteine-ligated iron−sulfur cluster proteins typically result in disassembly of the iron−sulfur core and formation of dinitrosyl iron complexes (DNICs). Here we report the first evidence that DNICs also form in the reaction of NO with Rieske-type [2Fe-2S] clusters. Upon treatment of a Rieske protein, component C of toluene/o-xylene monooxygenase from Pseudomonas sp. OX1, with an excess of NO(g) or NO-generators S-nitroso-N-acetyl-d,l-pencillamine and diethylamine NONOate, the absorbance bands of the [2Fe-2S] cluster are extinguished and replaced by a new feature that slowly grows in at 367 nm. Analysis of the reaction products by electron paramagnetic resonance, Mssbauer, and nuclear resonance vibrational spectroscopy reveals that the primary product of the reaction is a thiolate-bridged diiron tetranitrosyl species, [Fe₂(μ-SCys)₂(NO)₄], having a Roussin’s red ester (RRE) formula, and that mononuclear DNICs account for only a minor fraction of nitrosylated iron. Reduction of this RRE reaction product with sodium dithionite produces the one-electron-reduced RRE, having absorptions at 640 and 960 nm. These results demonstrate that NO reacts readily with a Rieske center in a protein and suggest that dinuclear RRE species, not mononuclear DNICs, may be the primary iron dinitrosyl species responsible for the pathological and physiological effects of nitric oxide in such systems in biology.

Source: Journal of The American Chemical Society