Self-assembled PEG monolayer based SPR immunosensor for label-free detection of insulin.
A simple and rapid continuous-flow immunosensor based on surface plasmon resonance (SPR) has been developed for detection of insulin as low as 1 ng ml-1 (ppb) with a response time of less than 5 min. At first, a heterobifunctional oligo(ethyleneglycol)-dithiocarboxylic acid derivative (OEG-DCA) containing dithiol and carboxyl end groups was used to functionalize the thin Au-film of SPR chip. Insulin was covalently bound to the Au-thiolate monolayer of OEG-DCA for activating the sensor surface to immunoaffinity interactions. An on-line competitive immunosensing principle is examined for detection of insulin, in which the direct affinity binding of anti-insulin antibody to the insulin on sensor surface is examined in the presence and absence of various concentrations of insulin. Immunoreaction of anti-insulin antibody with the sensor surface was optimized with reference to antibody concentration, sample analysis time and flow-rate to provide the desired detection limit and determination range. With the immunosensor developed, the lowest detectable concentration of insulin is 1 ng ml-1 and the determination range covers a wide concentration of 1-300 ng ml-1. The developed OEG-monolayer based sensor chip exhibited high resistance to non-specific adsorption of proteins, and an uninterrupted highly sensitive detection of insulin from insulin-impregnated serum samples has been demonstrated. After an immunoreaction cycle, active sensor surface was regenerated simply by a brief flow of an acidic buffer (glycine.HCl; pH 2.0) for less than 1 min. A same sensor chip was found reusable for more than 25 cycles without an appreciable change in the original sensor activity.
Gobi
K Vengatajalabathy
KV
Art, Science and Technology Center for Cooperative Research, Kyushu University, Kasuga, Fukuoka 816-8580, Japan. gobi@astec.kyushu-u.ac.jp
Iwasaka
Hiroyuki
H
Miura
Norio
N
D000906:Antibodies; D006801:Humans; D007328:Insulin / Q000032:analysis* / Q000097:blood; D011092:Polyethylene Glycols; D020349:Surface Plasmon Resonance / Q000295:instrumentation*
D000906:Antibodies; D007328:Insulin; D011092:Polyethylene Glycols