Study On The Electrogenerated Chemiluminescence Biosensor Based On Ru(bpy)₃～(2+)-SiO₂ NPs DNA Probe

Deoxyribonucleic Acid (DNA) is known as inherited substance in life body. With the increasing knowledge about human diseases it is found that the mutations of DNA base sequence are responsible for numerous inherited human diseases. Inherited substance directly impact on living things through being translated into protein. Proteins are involved in every life process and life activities, which play important roles in all kinds of life functions. Abnormal expression of the protein always suggest many clinical diseases. In order to select genes or make early diagnosis and cure for disease, much effort is needed to detect the specific DNA sequences, DNA mutation and the proteins (especially low-abundance protein) related to diseases.Many detection techniques of DNA sequence and protein have been developed in recent years, but there is still a lot of dissatisfaction. The radioactive labels present many problems such as a potential hazard to analyst and environment. Non-radioactive labels such as fluorescent, chemiluminescent and biotin-avidin label probes also present many shortcomings such as low sensitivity or complex equipment or others. So it is necessary to develop another method for the more sensitive, easy-to-use, fast, inexpensive detection of specific DNA sequences and proteins to adapt to wide-scale genetic and protein testing requires.Electrogenerated chemiluminescence (ECL) is a technique that combines chemiluminescence (CL) and electrochemistry (EC). ECL introduces both the merits of CL and EC, such as high sensitivity and selectivity, rapid and convenient operation and relatively simple instrumentation system. The most important issue for making good use of ECL technology in biosensor field is to find better ECL labels or label methods. The lumiophore-doped SiO₂ nanoparticles will be a good ECL labels for their high sensitivity in analysis.This dissertation combines the principle of base-complementary, and the specific recognition of certain protein to design ECL biosensors based on SiO₂ NPs DNA probe with high sensitivity and unique selectivity. The dissertation is composed of four chapters as foliowings:Chapter 1: Introduced the ECL method, pay attention to the two most primary type of ECL reaction and their application in analytical chemistry field. Review the DNA biosensor and the aptamer biosensor, including their principles, characteristics andsummarized the methods for the immobilization of ssDNA onto electrode surface. Introduced the advantages ;and the application of the SiO₂ nanoparticles which are used as tag. At last pointed out the purpose of the dissertation. Chapter 2: Ru(bpy)₃²⁺-SiO₂ nanoparticles were prepared by water-in-oil(W/O) microemulsion method. A great deal of Ru(bpy)₃²⁺ was immobilized inside the nanoparticle, which could greatly enhance the ECL response and result in the increased sensitivity. Ru(bpy)₃²⁺-SiO₂ NPs labled DNA probe was hybridized with target DNA immobilized on the surface of PPy modified Pt electrode. The hybridization events were evaluated by ECL measurements in oxalic acid solution and only the complementary sequence could give strong ECL signals. A three-base mismatch sequence and a non-complementary sequence had almost negligible responses. The assay allows detection at levels as low as 1.0×10^-13mol/L of the target DNA.Chapter 3: Sensitive and .selective detection of thrombin via target protein-induced strand displacement of the ssDNA probe by △I_ecl is described. The hybridization between the ssDNA probe labeled by Ru(bpy)₃²⁺-doped SNPs and the aptamer self-assembled on a microfebricated thin film gold electrode was evaluated by ECL measurements. Then, the eleetarode incubated with the thrombin analyte, and the binding event between the throflabin analyte and the aptamer was monitored by ECL measurements again. The △I_ecl of the two events can be used to quantify the thrombin. The assay has excellent selectivity because the aptamer could bind thrombin with high specificity. The assay allows detection at levels as low as 1.0 fM of the thrombin due to a. large number of Ru(bpy)₃²⁺ molecules inside SNPs labeled on DNA probe.Chapter 4: Novel ECL protein biosensor in sandwich manner using the aptamers was developed. Two different aptamers (aptamer I and aptamer II), which recognize different positions of thrombin, were chosen to construct sandwich type sensing system for protein. Aptamer I was immobilized onto the gold electrode for capturing thrombin onto the electrode and aptamer II labled with Ru(bpy)₃²⁺- SiO₂ NPs was used for detection. The increase of the ECL signal generated by Ru(bpy)₃²⁺- SiO₂ NPs was observed in dependent manner of the concentration of thrombin added. The assay also has excellent selectivity and further more it has the better sensitivity. It could detect as low as 1.0 fM of the thrombin.