TY - JOUR
T1 - Development of colorimetric enzyme-ball for signal amplification of enzyme-linked immunosorbent assay
AU - Choi, Jin Ha
AU - Lim, Yong Taik
AU - Oh, Byung Keun
N1 - Publisher Copyright:
© 2014 by American Scientific Publishers.
PY - 2014
Y1 - 2014
N2 - Enzyme-linked immunosorbent assay (ELISA) is one of the most conventional protein assay methods and a well-established technique that offers multiple advantages such as selectivity, rapidity, and simplicity. However, colorimetric assay methods normally have lower sensitivity (i.e., picomolar concentrations) than other assay techniques. Bovine serum albumin (BSA) nanoparticles are able to encapsulate various biomaterials such as proteins and genes, as well as chemotherapeutic agents. In this study, we developed a novel colorimetric nanoprobe, enzyme-balls consisting of BSA and horseradish peroxidase (HRP), for signal amplification in ELISA. The colorimetric HRP enzyme-ball was synthesized by a desolvation technique using a mixed solution of BSA and HRP. It was then modified with streptavidin and a targeting antibody for the detection of the targeted protein. It has replaced the HRP-labeled antibody as a signaling probe for use in conventional ELISA. The localized, encapsulated HRP enzymes in the BSA nanoball were used to amplify the signal of the target probe. Prostate specific antigen (PSA), used as a model protein, was captured on the plate by the immobilized capturing antibody through the immune reaction that resulted from the immunoreaction, and the enzyme-ball was attached to the PSA-positive region. For the detection of PSA, we used the reaction of HRPencapsulated enzyme-balls with tetramethylbenzidine (TMB), a substrate of HRP. We then stopped the reaction with 2 M H2SO4. The resulting products were analyzed by ultraviolet-visible (UV-vis) spectroscopy at 450 nm. This novel nanoprobe enabled us to detect the targeted protein at femtomolar concentrations in a short period. This method, based on the use of HRP-conjugated antibodies, is very simple and highly sensitive for use in established ELISA techniques and can be used for detecting specific protein markers associated with tumors, bacterial infections, or other diseases.
AB - Enzyme-linked immunosorbent assay (ELISA) is one of the most conventional protein assay methods and a well-established technique that offers multiple advantages such as selectivity, rapidity, and simplicity. However, colorimetric assay methods normally have lower sensitivity (i.e., picomolar concentrations) than other assay techniques. Bovine serum albumin (BSA) nanoparticles are able to encapsulate various biomaterials such as proteins and genes, as well as chemotherapeutic agents. In this study, we developed a novel colorimetric nanoprobe, enzyme-balls consisting of BSA and horseradish peroxidase (HRP), for signal amplification in ELISA. The colorimetric HRP enzyme-ball was synthesized by a desolvation technique using a mixed solution of BSA and HRP. It was then modified with streptavidin and a targeting antibody for the detection of the targeted protein. It has replaced the HRP-labeled antibody as a signaling probe for use in conventional ELISA. The localized, encapsulated HRP enzymes in the BSA nanoball were used to amplify the signal of the target probe. Prostate specific antigen (PSA), used as a model protein, was captured on the plate by the immobilized capturing antibody through the immune reaction that resulted from the immunoreaction, and the enzyme-ball was attached to the PSA-positive region. For the detection of PSA, we used the reaction of HRPencapsulated enzyme-balls with tetramethylbenzidine (TMB), a substrate of HRP. We then stopped the reaction with 2 M H2SO4. The resulting products were analyzed by ultraviolet-visible (UV-vis) spectroscopy at 450 nm. This novel nanoprobe enabled us to detect the targeted protein at femtomolar concentrations in a short period. This method, based on the use of HRP-conjugated antibodies, is very simple and highly sensitive for use in established ELISA techniques and can be used for detecting specific protein markers associated with tumors, bacterial infections, or other diseases.
KW - Bovine serum albumin (BSA)
KW - ELISA
KW - Enzyme-ball
KW - Horseradish peroxidase (HRP)
UR - https://www.scopus.com/pages/publications/84920262539
U2 - 10.1166/sam.2014.2225
DO - 10.1166/sam.2014.2225
M3 - Article
AN - SCOPUS:84920262539
SN - 1947-2935
VL - 6
SP - 2572
EP - 2576
JO - Science of Advanced Materials
JF - Science of Advanced Materials
IS - 11
ER -