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Real-time label-free detection of complement activation products in human serum by white light reflectance spectroscopy.

ABSTRACT: We present a label-free, real-time sensor based on white light reflectance spectroscopy for quantitating the complement activation product C3b and its metabolites as a biomarker in human serum. Our novel sensor allows real-time monitoring of biomolecular reactions (in this case, antigen-antibody reactions) taking place on a reflective surface within a flow cell. Detection was based on monitoring the increase in film thickness caused by its immunoreaction with a specific antibody; this reaction was seen as a shift in the wavelength at which constructive interference was observed. Quantitation of C3b was achieved by immobilizing a specific mouse monoclonal antibody onto the refractive surface and monitoring the rate of the signal changes occurring during the first 60s of the immunoreaction between the antibody and known concentrations of purified C3b or dilutions of complement-activated human serum. The lowest detectable concentration of purified C3b was 20 ng/mL, and complement activation products in human serum samples could be detected at dilutions as high as 6000-fold. The advantages of the method include its relatively low cost, short analysis time, and high assay sensitivity and reliability. Thus, this novel assay method can be used to monitor serum C3b produced as a result of complement activation in a variety of normal and pathologic conditions.

SUBMITTER: Petrou PS

PROVIDER: S-EPMC2742705 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Real-time label-free detection of complement activation products in human serum by white light reflectance spectroscopy.

Petrou Panagiota S PS Ricklin Daniel D Zavali Maria M Raptis Ioannis I Kakabakos Sotirios E SE Misiakos Konstantinos K Lambris John D JD

Biosensors & bioelectronics 20090503 11

We present a label-free, real-time sensor based on white light reflectance spectroscopy for quantitating the complement activation product C3b and its metabolites as a biomarker in human serum. Our novel sensor allows real-time monitoring of biomolecular reactions (in this case, antigen-antibody reactions) taking place on a reflective surface within a flow cell. Detection was based on monitoring the increase in film thickness caused by its immunoreaction with a specific antibody; this reaction w ...[more]

PMID: 19481435

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Project description:In the last decade, researchers have been searching for innovative platforms, methods, and techniques able to address recurring problems with the current cancer detection methods. Early disease detection, fast results, point-of-care sensing, and cost are among the most prevalent issues that need further exploration in this field. Herein, studies are focused on overcoming these problems by developing an electrochemical device able to detect telomerase as a cancer biomarker. Electrochemical platforms and techniques are more appealing for cancer detection, offering lower costs than the established cancer detection methods, high sensitivity inherent to the technique, rapid signal processing, and their capacity of being miniaturized. Therefore, Au interdigital electrodes and electrochemical impedance spectroscopy were used to detect telomerase activity in acute T cell leukemia. Different cancer cell concentrations were evaluated, and a detection limit of 1.9 × 105 cells/mL was obtained. X-ray photoelectron spectroscopy was used to characterize the telomerase substrate (TS) DNA probe self-assembled monolayer on gold electrode surfaces. Atomic force microscopy displayed three-dimensional images of the surface to establish a height difference of 9.0 nm between the bare electrode and TS-modified Au electrodes. The TS probe is rich in guanines, thus forming secondary structures known as G-quadruplex that can be triggered with a fluorescence probe. Confocal microscopy fluorescence images showed the formation of DNA G-quadruplex because of TS elongation by telomerase on the Au electrode surface. Moreover, electrodes exposed to telomerase containing 2',3'-dideoxyguanosine-5'-triphosphate (ddGTP) did not exhibit high fluorescence, as ddGTP is a telomerase inhibitor, thus making this device suitable for telomerase inhibitors capacity studies. The electrochemical method and Au microchip device may be developed as a biosensor for a point-of-care medical device.

Dataset Information

Real-time label-free detection of complement activation products in human serum by white light reflectance spectroscopy.

Publications

Real-time label-free detection of complement activation products in human serum by white light reflectance spectroscopy.

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