Quantitative detection of human tumor necrosis factor α by a resonance raman enzyme-linked immunosorbent assay.
Tumor necrosis factor α is an inflammatory cytokine which has been linked with many infectious and inflammatory diseases. Detection and quantification of this key biomarker is commonly achieved by use of an enzyme-linked immunosorbent assay (ELISA). This fundamental technique uses the spectroscopic detection of a chromogen such as 3,3',5,5'-tetramethylbenzidine (TMB). Horseradish peroxidase (HRP), bound to the detection antibody, catalyzes the oxidation of TMB by hydrogen peroxide to generate colored products which may be measured spectrophotometrically. In this study we have used a conventional ELISA kit and shown that, by replacing the traditional colorimetric detection with resonance Raman spectroscopy, we can achieve 50 times lower detection limits and the potential for multiplexed analysis is increased. In this approach, the laser wavelength was tuned to be in resonance with an electronic transition of the oxidized TMB. The relative intensity of the enhanced Raman bands is proportional to the amount of TMB, thus providing a means of improved quantification. Furthermore, TMB is one of the most widely used chromogenic substrates for HRP-based detection and commercial ELISA test kits, indicating that this detection technique is applicable to a large number of target analytes.
Centre for Molecular Nanometrology, WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, United Kingdom.
Stacey
S
Laing
Aaron
A
Hernandez-Santana
Jörg
J
Sassmannshausen
Darren L
DL
Asquith
Iain B
IB
McInnes
Karen
K
Faulds
Duncan
D
Graham
D000818:Animals; D001560:Benzidines / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D013059:Spectrum Analysis, Raman; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000097:blood
D001560:Benzidines; D014409:Tumor Necrosis Factor-alpha; C021758:3,3',5,5'-tetramethylbenzidine; D006735:Horseradish Peroxidase