The properties of glucose biosensors based on dendrimer layers on a gold support, which depend on the method of immobilization of glucose oxidase (GOX), were studied by amperometry. The kinetic parameters of enzymatic reactions, response time, sensitivity, detection limit, linear range, and enzyme turnover were determined. We showed that a more stable and sensitive sensor was obtained when GOX was immobilized on the dendrimer by crosslinking with glutaraldehyde in vacuum. This biosensor was stable for at least eight weeks. The response time was approximately 1.3 min, the detection limit of glucose was 25 micro M, and the apparent Michaelis-Menten constant was relative low ( K(m)=1.1+/-0.1 mM) in comparison with that for GOX in solution. The reason for these differences is discussed. The example of the application of the developed biosensors for the detection of mercury is also presented. The inhibitory effect of mercury on GOX activity was observed at mercury concentration of 100 nM.
We applied the acoustic transverse shear mode (TSM) method for study of the surface properties of a DNA aptasensor that specifically binds human immunoglobulin E (IgE). The biotinylated 45-mer DNA aptamers were immobilized on the surface of a self-assembled layer composed of a mixture of polyamidoamine dendrimers of the fourth generation with 1-hexadecanetiol covered by neutravidin. Using the TSM method, we studied the kinetics of changes of the series resonant frequency, f(s), and the motional resistance, R(m), of a quartz crystal transducer, used as a support for formation of the sensing layer. We have shown that attachment of the biotinylated DNA aptamers onto the surface covered by neutravidin results in a decrease of f(s), but in an increase of R(m). Similar changes of f(s) and R(m) were observed following addition of IgE. This suggests the contribution of friction forces to the crystal oscillation, which was taken into account in the calculation of the mass changes at the sensor surface following binding processes.
We tested a new design of an enzyme biosensor based on acetylcholinesterase (AChE) and choline oxidase (ChO) immobilized on the supported monomolecular layer composed of poly(amidoamine) (PAMAM) dendrimers of the fourth generation (G4) mixed with 1-hexadecanethiol (HDT). The resulting enzymatic activity, measured amperometrically, was substantially depressed in the presence of the organophosphate pesticide dimethyl-2,2-dichlorovinylphosphate (DDVP, Dichlorvos), carbamate pesticides carbofuran and carbamate drug eserine. The detection limits (1.3 Â 10 À 3 ppb for DDVP, 0.01 ppb for carbofuran and 0.03 for eserine) were considerably lower than so far reported for AChE based amperometric and potentiometric sensors. The relative simple protocol of biosensor preparation, high sensitivity and stability is very promising for determination of environmental pollutants in field conditions.
Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, k(off), compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.