ABSTRACT
The goal of this work was to introduce a modified electrochemical sandwich model for target protein detection, exploiting antibody as the capturing probe, aptamer as the detection probe and methylene blue as the electrochemical active marker intercalating in the probing aptamer without previous labeling. With appropriate design of the sequence of the aptamer, the aptamer was successfully utilized instead of antibody for obtaining the electrochemical detection. A special immobilization interface consisting of nanogold-chitosan composite film was used to improve the conductivity and performance characteristics of the electrode. The capturing antibody was linked to the glassy carbon electrodes modified with composite film via a linker of glutaraldehyde. Differential pulse voltammetry was performed to produce the response signal. Thrombin was taken as the model target analyte to demonstrate the feasibility of proposed methodology. The sensor shows the linear response for thrombin in the range 1-60 nM with a detection limit of 0.5 nM. The proposed approach provides an alternative approach for sandwich protein assay using aptamers.
Subject(s)
Antibodies/immunology , Aptamers, Nucleotide/chemistry , Thrombin/analysis , Thrombin/chemistry , Aptamers, Nucleotide/genetics , Carbon/chemistry , Chitosan/chemistry , Electrochemistry , Electrodes , Glass , Gold/chemistry , Humans , Microscopy, Electron, Scanning , Nanostructures/chemistry , Osmolar Concentration , Thrombin/genetics , Thrombin/immunologyABSTRACT
In the present study, we report a novel sensitive method for the detection of adenosine using surface-enhanced Raman scattering (SERS) sensing platform based on a structure-switching aptamer. First, Ag-clad Au colloids film on a polished gold disc is prepared as enhanced substrate and modified with thiolated capture DNA. The formation of an aptamer/DNA duplex of expanded anti-adenosine aptamer and tetramethylrhodamine-labeled DNA (denoted TMR-DNA) is then developed, in which TMR-DNA could also hybridize completely with capture DNA. The introduction of adenosine thus triggers structure switching of the aptamer from aptamer/DNA duplex to aptamer/target complex. As a result, the released TMR-DNA is captured onto the SERS substrate, resulting in an increase of SERS signal. Under optimized assay conditions, a wide linear dynamic range (2.0 x 10(-8)M to 2 x 10(-6)M) was reached with low detection limit (1.0 x 10(-8)M). Moreover, high selectivity, stability and facile regeneration are achieved. The successful test demonstrates the feasibility of the strategy for adenosine assay.
Subject(s)
Adenosine/analysis , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Spectrum Analysis, Raman/methods , Gold/chemistry , Metal Nanoparticles/chemistry , Sensitivity and SpecificityABSTRACT
CeO(2)/Chitosan (CHIT) composite matrix was firstly developed for the single-stranded DNA (ssDNA) probe immobilization and the fabrication of DNA biosensor related to the colorectal cancer gene. Such matrix combined the advantages of CeO(2) and chitosan, with good biocompatibility, nontoxicity and excellent electronic conductivity, showing the enhanced loading of ssDNA probe on the surface of electrode. The preparation method is quite simple and inexpensive. The hybridization detection was accomplished by using methylene blue (MB), an electroactive lable, as the indicator. The differential pulse voltammetry (DPV) was employed to record the signal response of MB and determine the amount of colorectal cancer target DNA sequence. The experimental conditions were optimized. The established biosensor has high detection sensitivity, a relatively wide linear range from 1.59x10(-11) to 1.16x10(-7)molL(-1) and the ability to discriminate completely complementary target sequence and four-base-mismatched sequence.
