Study Of DNA Biosensors Based On Dendrimers And Au Nanoparticles

Sequence-specific detection of DNA targets associated with genetic or pathogenic diseases and forensic applications has become increasingly important in molecular diagnostics. Various methods have been used to detect sequence-selective DNA hybridization, including optical, electrochemical, and piezoelectric transduction techniques. The electrochemical method has attracted particular attention because of its high sensitivity, low cost, and compatibility with microfabrication technology of all these technique. The recent developments of electrochemical DNA biosensor have been reviewed in several reports.In recent years, the dendritic polymers (dendrimers) belong to a new class of synthetic, macromolecule possessing a regularly branched treelike structure. The dendrimers can be used to modify electrode surface due to their good biocompatibility and adequate functional groups for chemical fixation. The fourth-generation polyamidoamine (G4 PAMAM) polymers, is of particular interest because of its nanoscopic spherical structure and good biocompatibility. G4 PAMAM dendrimer possesses 64 primary amine groups on the surface and has a globular shape with a diameter about 4.5 nm. Using G4 PAMAM to immbilize DNA showed high sensitivity and selectivity for DNA hybridization assay.In this reasearch, we investigate the suitability of the fourth-PAMAM dendrimers and Au nanoparticles for the development of the DNA biosensor using self-assembly and covalent immobilization technique. It provided a basic theoretical reseach for clinic diagnoses of many diseases at early stage. Three types of DNA biosensors were prepared which could be summarized as follows:1. A novel DNA assay based on gold nanoparticles (Au NPs) and polyamindoamine (PAMAM) modified Au electrode was established. A gold electrode was first modified with 3-mercaptopropionic acid, and then reacted with an amino-terminated polyamidoamine to obtain a PAMAM thin film modified Au electrode. Then the PAMAM thin film modified Au electrode was further modified with Au NPs. And then thiolated capure DNA probe was immobilized onto the propered electrode via sulfur-gold affinity. The characteristics of the immobilization and hybridization of DNA were studied by cyclic voltammetry (CV), difference pulse voltammetry (DPV). The hybridization events were monitored by differential pulse voltammetry (DPV) using methtlene blue (MB) as an indicator. The dynamic detection range of the sequence-specific DNA was from 2.7×10-11 to 2.7×10-14 mo1·L-1 . With the enhancement effect of AuNPs, a detection limit as low as 1.4×10-14 mol L-1 was achieved. This DNA biosensor exhibited excellent selectivity against two-base mismatched DNA.2. A nocel and sensitive electrochemical approach for sequence-specific DNA detection based on polyamindoamine (PAMAM) modified Au electrode and signal amplidication with Au nanoparticles (Au NPs) is reported. A gold electrode was first modified with 3-mercaptopropionic acid, and then reacted with an amino-terminated polyamidoamine to obtain a thin film. Single-stranded 3'-biotin end-labeled oligonucleotide was immobilized onto the film to obtain a stable recognition layer through biotin-avidin combination to detect complementary target using signal amplification with Au NPs and [Ru(NH3)6]3+ as redox electroactive indicators. The properties of the avidin/PAMAM/3-mercapropionic acid (RSH)/Au, the characteristics of the immobilization and hybridization of DNA were studied by cyclic voltammetry (CV), difference pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The dynamic detection range of the sequence-specific DNA was from 2.7×10-11 to 2.7×10-14 mo1·L-1 and the detection limit was 1.4×10-14 mo1·L-1. The DNA sensor not only showed low detection limit, but also exhibited excellent selectivity against two-base mismatched DNA.3. An electrochemical sending steategy for highly sensitive detection of sequence-specific DNA was developed based on signal amplification with gold nanoparticles. A gold electrodfe was first modified with gold nanoparticles (Au NPs), and then modified with 4-ATP to obtain a thin film. Single-stranded 3'-biotin end–labeled oligonucleotide was immobilized onto the film to obtain a stable recognition layer through biotin-avidin combination to detect complementary target, using signal amplification with Au NPs and [Ru(NH3)6]3+ as redox electroactive indicators. The properties of the avidin/4-ATP/Au NPs/Au, the characteristics of the immobilization and hybridization of DNA were studied by cyclic voltammetry (CV), difference pulse voltammetry (DPV) and electrochemical impedance spectroscopy(EIS). The dynamic detection range of the sequence-specific DNA was from 2.0×10-9 to 1.4×10-11mol·L-1 and the detection limit was 9.5×10-12 mol L-1. The DNA sensor not only showed low detection limit, but also exhibited excellent selectivity against two-base mismatched DNA.