| Electrogenerated chemiluminescence (also called electrochemiluminescence, abbreviated as ECL) is the process where species generated at electrodes undergo electron-transfer reactions to form excited states that emit light. ECL method has many distinct advantages over fluorescence method because it does not involve a light source and avoids the attendant problems of scattered light and impurities luminescent. Moreover, the specificity of the ECL reaction associated with the ECL label and the coreactant species decreases problems with side reactions and is characterized by good spatial and temporal resolution. ECL has been widely used in many chemical and biochemical related applications over the past several years, which include immunoassay, DNA hybridization detection, food and water testing, as well as biowarfare agent or explosive material detection. Aptamers are nucleic acid (both DNA and RNA) receptors that bind to specific target molecules. Aptamers exhibit a strong and specific binding affinity towards their targets with a remarkable stability, simply synthesized via cost-effective and readily automated routes and so on. With regard to this, aptamers can be used as new recognition entities for specific sensing of a variety of analyte targets in biosensor. Some ECL aptamer-based biosensor about the combination of high sensitive ECL with aptamer have been attracted much attention in recent years. Up to date, all the ECL aptamer-based biosensor are broadly used thiolated SAMs on Au for the attachment of ruthenium complexes, this approach can only allow for the anodic ECL measurements conducted under potentials not positive than-0.80 V vs Ag/AgCl so that the thiol layer's damage can be avoided. Additionally, because gradual degradation of the SAMs in air or in a phosphate saline buffer (PBS) solution often occurs, the long-term storage stability of the ECL detector or the biosensor is a challenge.The aim of this thesis is to design reusable and long storage stability ECL biosensors in order to provide a new strategy in fabricating various ECL or electrochemical chemical sensors or biosensors. The research work in this thesis is financially supported by the National Natural Science Foundation of China (Grant No.20775046, No.20975065) and the innovation Funds for Graduate Programs of Shaanxi Normal University (No.2009CXS012).The thesis includes two parts. First part, chapter 1 is general introduction while second part consisting of four chapters, is a research report. In Chapter 1, general introduction to the principle of ECL, ECL aptamer-based biosensors including their principles and research development, and the purpose of this research work were presented. In Chapter 2, a double covalent coupling method for the fabrication of highly sensitive and reusable ECL chemical sensor for the detection of tertiary amines and ECL aptamer-based (ECL-AB) biosensor for the detection of cocaine was reported. The ECL sensors were constructed by covalent coupling of Ru(bpy)32+derivatives (Ru1,Ru(bpy)32+=tris(2,2'-bipyridyl)ruthenium(Ⅱ)) or cocaine aptamer-Ru1 to the surface of paraffin-impregnated graphite electrode (PIGE) that had been covalently modified with a monolayer of 4-aminobenzene sulfonic acid via electrochemical oxidations. ECL performance of the newly developed chemical sensors was evaluated using tri-n-propylamine (TPrA) and metoclopramide (MCP) as model analytes. The sensors exhibited excellent sensitivity, stability, and reproducibility with a detection limit of 30 nmol/L for TPrA and 2.0 nmol/L for MCP, and relative standard deviations (RSD) of 2.1% over 90 cyclic potential cycles (0 to 1.50 V vs Ag/AgCl) and 2.6% over 45 cycles (0.60 to+1.30 V vs Ag/AgCl) at 400 mV/s for 50 nmol/L TPrA and 200 nmol/L MCP, respectively. For the ECL-AB biosensor, it showed an extremely low detection limit of 10 pM for cocaine, and offered a good selectivity towards cocaine, heroin, and caffeine. This detection limit was about 4-6 orders of magnitude lower than that reported on the basis of AC voltammetry and optical aptamer-based cocaine biosensors. Additionally, the ECL-AB biosensor was highly reusable (RSD=2.8%, n=7) and possessed long-term storage stability (96.8% initial ECL recovery over 21 days storage). A binding constant of 4.6±0.3×109M-1 between cocaine and its aptamer was estimated using an ECL based Langmuir isotherm approach. Wide applications of the presently reported strategy in fabricating various chemical sensors or biosensors are expected.In Chapter 3, ECL-AB biosensors based on target-induced aptamer displacement were developed for the determination of ATP as a model system. "Signal on" and "Signal off" ECL-AB biosensors were fabricated by covalently coupling the partial complementary single strand DNA-ATP binding aptamer to the surface of PIGE modified with 4-ABSA through a cross-linking reaction amine-acyl chloride. In the absence of ATP, the "Signal off" ECL-AB biosensor led to a great ECL signal. In the presence of ATP, target-induced strand release of a 3'-end label Ru(bpy)32+ derivatives tagged aptamer from the aptamer capture DNA duplex bound on an electrode, led to decrease ECL signal. Therefore, this ECL-AB biosensor can be used to detect of ATP. With regard to the "Signal on" ECL-AB biosensor's principle is that the interaction between the target and the aptamer strand may induce the formation and subsequent dissociation of target-aptamer complex from an electrode surface, and consequently, the remaining DNA strand on the electrode surface can hybridize again with a ssDNA containing an ECL probe. The linear of "Signal on" and "Signal off" were in the range from 1.0×10-12 mol/L to 5.0×10-9mol/L and 1.0×10-10mol/L to 5.0×10-8mol/L with a detection limit were 1.0×10-12mol/L,1.0×10-10 mol/L, respectively.In Chapter 4, a simple ECL sensing platform for high specificity and sensitive sandwich assay for the detection of small molecule target ATP was demonstrated. A capture probe,5'-NH2-(CH2)6-ACC TGG GGG AGT AT-3'was covalently coupled to the surface of PIGE modified with r-aminobenzene sulfonic acid through a cross-linking reaction amine-acyl chloride. In the absence of ATP, a ECL probe 5'-TGC GGA GGA AGGT-(CH2)2-NH2-Rul-3'was weakly interaction between the capture probe, led to a small ECL signal. In the presence of ATP, ECL probe was strongly interaction between the capture probe, led to a great ECL signal.The ECL intensity is linear with the concentration of ATP in the range from 5.0×10-11mol/L to 5.0×10-8 mol/L and the detection limit is 3×10-11 mol/L (S/N=3).In Chapter 5, a sensitive ECL sensor for the determination of heroin was developed by employing ruthenium complex polymer as ECL signal and ionic liquid as binding reagent. ECL sensor was prepared by thoroughly mixing ruthenium complex polymer, graphite powder and ionic liquid, which show good electrochemical and ECL behaviors. The ECL intensity is linear with the concentration of heroin in the range from 2.0×10-9 mol/L to 2.0×10-5 mol/L and the detection limit is 8×10-10 mol/L. The ECL sensor exhibited a long-term stability, fine reproducibility with relative standard deviation less than 5% for 5.0×10-9 mol/L heroin in 60 continuous determinations. The developed method allows the detection of heroin in a serum sample with recovery in 94-101%. |
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