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Surface plasmon resonance aided electrochemical immunosensor for CK-MB determination in undiluted serum samples.

ABSTRACT: This article presents a simple chronoamperometric immunosensor for the quantitative assessment of creatine kinase MB (CK-MB) in 50 microL undiluted serum samples. The immunosensor consists of gold working and counter electrodes patterned onto a glass chip by thin-film photolithography and an external Ag|AgCl reference electrode. The detection limit (DL) of the chronoamperometric method is 13 ng mL(-1) (DL = 2xRMSD/S, where RMSD is the residual mean standard deviation of the measured points around a calibration curve with a slope of S). In spiked serum samples, the response was linear up to 300 ng mL(-1) of CK-MB. A surface plasmon resonance (SPR) system with simultaneous electrochemical detection (EC-SPR) aided the development of the sandwich immunoassay. Real-time monitoring of the SPR signal was used to optimize the capture antibody immobilization, CK-MB and detection antibody binding, as well as to minimize the nonspecific adsorption of serum proteins to the sensor surface. The detection antibody has been labeled with alkaline phosphatase (ALP) enzyme for sensitive electrochemical detection. ALP catalyzes the hydrolysis of ascorbic acid phosphate and generates ascorbic acid, which is measured chronoamperometrically. The electrochemical immunoassay for CK-MB was less sensitive to nonspecific adsorption related interferences, had a better detection limit, and required a lower volume of sample than the SPR method.

SUBMITTER: Garay F

PROVIDER: S-EPMC2930610 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Surface plasmon resonance aided electrochemical immunosensor for CK-MB determination in undiluted serum samples.

Garay Fernando F Kisiel Greggory G Fang Aiping A Lindner Erno E

Analytical and bioanalytical chemistry 20100508 5

This article presents a simple chronoamperometric immunosensor for the quantitative assessment of creatine kinase MB (CK-MB) in 50 microL undiluted serum samples. The immunosensor consists of gold working and counter electrodes patterned onto a glass chip by thin-film photolithography and an external Ag|AgCl reference electrode. The detection limit (DL) of the chronoamperometric method is 13 ng mL(-1) (DL = 2xRMSD/S, where RMSD is the residual mean standard deviation of the measured points aroun ...[more]

PMID: 20449577

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Project description:Palm kernel cake (PKC) is the solid residue following oil extraction of palm kernels and useful to fatten animals either as a single feed with only minerals and vitamins supplementation, or mixed with other feedstuffs such as corn kernels or soy beans. The occurrence of mycotoxins (aflatoxins, ochratoxins, zearalenone, and fumonisins) in feed samples affects the animal's health and also serves as a secondary contamination to humans via consumption of eggs, milk and meats. Of these, aflatoxin B? (AFB?) is the most toxically potent and a confirmed carcinogen to both humans and animals. Methods such as High Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) are common in the determination of mycotoxins. However, these methods usually require sample pre-treatment, extensive cleanup and skilled operator. Therefore, in the present work, a rapid method of electrochemical immunosensor for the detection of AFB? was developed based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). Multi-walled carbon nanotubes (MWCNT) and chitosan (CS) were used as the electrode modifier for signal enhancement. N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) activated the carboxyl groups at the surface of nanocomposite for the attachment of AFB?-BSA antigen by covalent bonding. An indirect competitive reaction occurred between AFB?-BSA and free AFB? for the binding site of a fixed amount of anti-AFB? antibody. A catalytic signal based on horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H?O?) and 3,3',5,5'-tetramethylbenzidine (TMB) mediator was observed as a result of attachment of the secondary antibody to the immunoassay system. As a result, the reduction peak of TMB(Ox) was measured by using differential pulse voltammetry (DPV) analysis. Based on the results, the electrochemical surface area was increased from 0.396 cm² to 1.298 cm² due to the electrode modification with MWCNT/CS. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.0001 to 10 ng/mL with limit of detection of 0.1 pg/mL. Good recoveries were obtained for the detection of spiked feed samples (PKC, corn kernels, soy beans). The developed method could be used for the screening of AFB? in real samples.

Dataset Information

Surface plasmon resonance aided electrochemical immunosensor for CK-MB determination in undiluted serum samples.

Publications

Surface plasmon resonance aided electrochemical immunosensor for CK-MB determination in undiluted serum samples.

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