Studies On The Strategy Of Signal Amplification For Electrochemical Detection Of Proteins And Nucleic Acids

The highly sensitive detection of nucleic acids and proteins has attracted considerable interest in a wide range of areas including bioengineering, clinical medicine, environmental monitoring, food and agricultural field. Among various analytical techniques, electrochemical methods is one of promising methods due to its superiorities of simpler and cheaper instruments that are easy to miniaturize. Because the amount of some proteins and nucleic acids is usually low in the body fluid or tissues, the development of ultrasensitive protocols for these DNA and protein is surely of desirable. The major research works focuses on signal amplification based on nanotechnology, analytical chemistry, biotechnology, and material science, nanomaterials as follows:1. Signal amplification for DNA detection based on the HRP-functionalized Fe3O4 nanoparticlesAn electrochemical approach for the sensitive detection of sequence-specific DNA has been developed. Horseradish peroxidase (HRP) assembled on the Fe3O4 nanoparticles (NPs) was utilized as signal amplification sources. High-content HRP was adsorbed on the Fe3O4 NPs via layer-by-layer (LbL) assemble technique to prepare HRP-functionalized Fe3O4 NPs. Signal probe and diluting probe were then immobilized on the HRP-functionalized Fe3O4 NPs through the bridge of Au NPs. Thereafter, the resulting DNA-Au-HRP-Fe3O4 (DAHF) bioconjugates were successfully anchored to the gold nanofilm (GNF) modified electrode surface for the construction of sandwich-type electrochemical DNA biosensor. The electrochemical behaviors of the prepared biosensor had been investigated by the cyclic voltammetry (CV), chronoamperometry (i-t), and electrochemical impedance spectroscopy (EIS). Under optimal conditions, the proposed strategy could detect the target DNA down to the level of 7 pM with a linear calibration range from 50 pM—500 nM, it exhibites excellent discrimination to two-base mismatched DNA and non-complementary DNA sequences and opens new opportunities for ultrasensitive detection of other biorecognition event.2. Magnetic particles and cadmium sulfide nanoparticles tagging for signal-amplifying detection of nucleic acidsA versatile DNA recognition system using cadmium sulfide nanoparticles (CdS NPs) as labels was proposed for ultrasensitive detection of specific sequence DNA based on target recycling. This strategy utilized the magnetic particles (MNPs) for the immobilization of linker DNA and CdS NPs and the subsequent target DNA hybridization. Using the unique characteristic of nicking endonuclease for cutting one specific strand of double strand DNA (ds DNA), the linker DNA could be transected and the target DNA be liberated for re-hybridization and hence enhanced the amount of released CdS NPs. Due to the advantage of the MNPs and signal amplification from the target recycling, the analyte DNA could be detected by the square-wave stripping voltammetry (SWV) in a wide linear range from 0.4 fM to 100 fM with the detection concentration of the target DNA down to 0.08 fM. The proposed DNA detection strategy possesses high sensitivity, satisfactory reproducibility and excellent stability, which might have potential in other DNA biological assays.3. Electrochemical DNA bioanalysis based on the stable Y junction probe A simple and facile single-step DNA sensing platform with improved selectivity and lower detection limit based on the Y junction structure was successfully developed. Firstly the capture probe DNA was efficiently immobilized onto gold nanofilm (GNF) electrodes via Au—S bonding, for the subsequent forming of Y junction structure with target DNA and reporter probe DNA labeled with methylene blue (MB). The contact of MB with the GNF electrode surface would enhance the interfacial electron communication, based on which a typical "nascent-signaling" electrochemical signal could be observed by differential pulse voltammetry (DPV). Owing to the eliminating of the background signal compared with traditional signal-on electrochemical sensors by the introduction of the Y junction structure, the present platform could significantly low the detection limits and enhance the selectivity. The results indicated that, in pH 7.4 Tris-HCl buffer solution, the peak current was linear with the concentration of target DNA in the range of 1.0pM-1.0 nM with a detection limit down to 0.24 pM. In addition, this novel DNA sensor exhibites fairly good reproducibility, stability, reusability and excellent selectivity against even a single base mismatch and opens new opportunities for ultrasensitive detection of specific sequence DNA.4. CdS nanoparticles functionalized colloidal carbon particles:preparation, characterization and application for electrochemical detection of thrombinA novel and simple method for preparing cadmium sulfide nanoparticles (CdS NPs) functionalized colloidal carbon particles (CPs) has been successfully developed by in situ growing abundant CdS NPs on the surfaces of monodisperse carbon particles (CdS/CPs). The obtained CdS/CPs conjugates as signal amplification labels were further used for the ultrasensitive determination of thrombin. The CdS/CPs conjugates were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and UV-visible absorption spectrum (UV). The protein electrical detection involves a dual binding event, based on thrombin linked to the CdS/CPs tags and glass surface by the specific aptamer-protein affinity interactions and a succedent electrochemical stripping transduction. Owing to the high-content CdS NPs on carbon particles, this assay allowed a desirable detection limit down to 60 aM, which was 1000 times lower than that of only using CdS NPs as labels in the control experiments. This protocol exhibited excellent selectivity against these common proteins such as bovine plasma albumin, lysozyme and hemoglobin. The signal amplification approach proposed here provides a facile, cost-effective method for the ultrasensitive determination of thrombin in the practical samples, which provides a potential alternative tool for the detection of protein in clinical laboratory.