Preparation Of Photoelectrochemical Biosensor Based On Ruthenium Complex

The photoelectrochemical biosensor is a newly developed and promising analytical method for biological assay. The use of electronic detection makes the photoelectrochemical instrument simple and low-cost. At the same time, due to its separate source for excitation and detection, the sensitivity of a photoelectronchemistry based analytical method can potentially match that of the electrochemiluminescence. Photoelectrochemical biosensor was developed for the detection of various biomolecule in this thesis. ATP, DNA and Tumer cell could be detected sensitively and selectively using this technique. It provided a newly developed method for clinic diagnoses of many diseases at early stage. The major contents of this paper are as follows:1. A novel photoelectrochemical biosensor for sensitive detection of small molecules was developed based on the recognition interaction between aptamer and target molecule-ATP. Firstly, NH2-functionalized complementary DNA was coupled to carboxyl-modified MBs. After hybridization with aptamer, photoelectrochemically active species Ru(bpy)2dppz2+ can intercalate into the double helix of double-stranded DNA. When target molecule ATP was added, the double helixes were opened and photoelectrochemically active species Ru(bpy)2dppz2+ was entered intosolution. The supernatant was then subjected to photoelectrochemical measurement. The sensitivity of the developed biosensor was investigated . A linear range from 6.0×10-7 to 1.0×10-5 M was achieved and the detection limit was 1.3×10-7 M.2. A photoelectrochemical sensing strategy for highly sensitive detection of small molecules was developed based on the recognition interaction between aptamer and target molecule-ATP. The specificity of bio-barcoding and the recognition of aptamer have been integrated in photoelectrochemistry for the first time. The developed photoelectrochemical sensor was used in the detection of ATP and under the optimal conditions, a detection limit of 3.2×10-9 M was achieved. With its simplicity, selectivity,sensitivity and practicality in cell extract assay, this strategyshows great promise in the photoelectrochemical detection not only for the ATP, but also for other bio-active molecules.3. We report a strategy for the transduction of DNA hybridization into a readily detectable photoelectrochemical signal by means of a conformational change analogous to the E-DNA approach. The strategy involves a photosensitizer ruthenium complex tagged DNA stem–loop structure that self assembles onto a nanogold modified ITO electrode by means of gold-thiol chemistry. In the presence of target DNA, a stem-loop of the photoelectrochemical probe on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the tag moving away from the electrode surface, which in turn decreased the photocurrent. Under the optimal condition, a detection limit of 3.7×10-11 M was achieved. In order to improve the sensitivity of the photoelectrochemical biosensor, an amplified detection method based on isothermal strand displacement polymerization reaction was employed. With the multiple rounds of isothermal strand replication, which led to strand displacement and consecutive signal amplification, a detection limit of 9.4×10 -14 M target DNA was achieved. At the same time, the developed biosensor showed good specificity, and single mismatched target DNA was effectively discriminated from complementary target DNA. Inspite of its sensitivity and specificity, this convenient method can expand the application scope of photoelectrochemistry and reveal a good prospect of this platform for biological sample analysis.4. Signal-on type of photoelectrochemical biosensor was developed based on conformational change of probe DNA. To improve the sensitivity of the developed biosensor, polymerase and nick enzyme was introduced. Under the optimal experimental conditions, a detection limit of 5.0×10-17 M for target DNA was achieved, and a detection limit of 54 cells/mL for target Romas cell was achieved. At the same time, the developed biosensor showed good specificity for target DNA and target Romas cell respectively. Considering its sensitivity and specificity, this convenient method can expand the application scope of photoelectrochemistry and reveal a good prospect for biological sample analysis.