2008 NSTI Nanotechnology Conference and Trade Show - Nanotech 2008 - 11th Annual

Partnering Events:

TechConnect Summit
Clean Technology 2008

Use of Nanoparticles for Signal Amplification

A. Virel, L.S. Peña, V. Pavlov
CIC Biomagune, ES

Keywords:
nanoparticles, signal amplification, peroxidase, acetylcholine esterase

Abstract:
Here, we report two new concepts based on metal nanoparticles for signal amplification in biosensing. We synthesized hemin di(N,N´-acetylhydrazide) derivatives and immobilized some of them on gold nanoparticles. We demonstrated that the reconstitution of this modified hemins with apo horseradish peroxidase can be performed only in the presence of hydrogen peroxide. Further, we show how this oxidative reconstitution can be applied for the ultra-sensitive colorimetric detection of hydrogen peroxide and glucose oxidase. Our experimental data prove that hemin di(N,N´-acetylhydrazide) can not be oxidized by hydrogen peroxide in the absence of apoenzyme. All three components i.e. of apo horseradish peroxidase, hemin di(N,N´-acetylhydrazide) and H2O2 are essential for generation of peroxidase activity in the system. Thus, we discovered the first example of oxidative reconstitution in vitro. We speculate that in vivo similar redox reconstitutions also can play a role during self-assembling of enzymes and they constitute an important part of enzyme activation mechanism triggered by an enzymatic substrate. We found that the reported system can be applied for colorimetric detection of H2O2 using TMB as a substrate with detection limit 50 nM under physiological conditions. The sensitivity to hydrogen peroxide of the present colorimetric system is comparable with this of the commercially available fluorimetric system based on the oxidation of the fluorogenic substrate 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). Oxidative reconstitution approach can be easily applied for detection of any anzyme generating hydrogen peroxide, for example, glucose oxidase. The detection limit was 40 micro U/ml of glucose oxidase. The introduction of this new signal amplification cascade will allow use existing equipment and well established peroxidase detection techniques for more sensitive assays. We also demonstrated that the hydrolysis of acethylthiocholine mediated by the acetylcholine esterase, AChE, yields thiol bearing compound thiocholine. The latter at trace concentration inhibits growth of silver nanoparticles on gold seeding nanoparticles by the reduction of AgNO3 with ascorbic acid. Very low concentrations of AChE inhibit formation of silver nanoparticles and catalytic growth of silver nanoparticles is promoted by inhibition of the enzyme by 1,5–bis(4-allyldimethylammoniumphenyl)-pentane-3-one dibromide or by diethyl p-nitrophenyl phosphate (paraoxon). Simple and sensitive colorimetric homogeneous and heterogeneous colorimetric assays for AChE inhibitors were developed


Nanotech 2008 Conference Program Abstract