Study On A New Method Of Electrochemical Luminescence Biosensing Based On Cutting Reaction

Prostate cancer (prostatic, cancer, PCa) is one of the most common male malignant tumor and has become the second most common cancer in men. The incidence of prostate cancer is rising trend in the global scope. Prostate specific antigen (PSA) is a30KDa serine protease which stem from normal prostate gland cells and prostate cancer cells. PSA screening procedures can provide the data for early diagnosis of prostate cancer patients, but also can provide a more adequate treatment for prostate cancer patients, so as to improve the expected survival time in patients with prostate cancer, which provides the possibility of reducing the rate of death from prostate cancer. To establish a new method for detecting malignant tumor biological marker is simple, rapid, sensitive and selective. It has very important significance for prevention and treatment of malignant tumor.Electrochemiluminescence (electrogenerated chemiluminescence, ECL) involves the generation of species at electrode surfaces that then undergo electron-transfer reactions to form excited states that emit light. ECL biosensors combine the advantages of both electrochemical and chemiluminescent biosensors, such as high sensitivity, ease of control and using of simple equipment and specific selectivity offered by the biological recognition elements, which has widely been used in pharmaceutical analysis, environmental analysis, bioanalysis, and clinical analysis.The aim of the present work is to develop highly sensitive and selective peptide based electrogenerated chemiluminescence biosensors for the detection of biomarker proteins. The peptides are taken as the molecular recognition substances while electrochemiluminescence reagents are signal substances. Establishing a new method which is suitable for detection of prostate specific antigen (PSA) with high sensitivity, high selectivity of electrochemical biosensor. The research work in this thesis is financially supported by the National Natural Science Foundation of China (Grant No.21027007and No.21275095).This thesis is composed of two parts:introduction and research reports. The first part is the summary, the second part is the research reports.The first part, namely the first chapter, a brief overview of electrogenerated electrochemiluminescence, peptide and prostate specific antigen; And then the trends in ECL biosensor were reviewed in detail; finally, the research background, research ideas, research purpose and research contents were presented.The second part is the research report, including the second chapter and the third chapter.Second chapter constructs the heterogeneous detection of prostate specific antigen electrogenerated electrochemiluminescence biosensor method with peptides as molecular recognition materials. The electrogenerated electrochemiluminescence signals is ruthenium bis(2,2’-bipyridine)(2,2’-bipyridine-4,4’-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy)2(dcbpy)NHS) with specific polypeptide PSA and the other end alkynyl modified lysine modification of the (C=C-(CH2)2-EEEHSSKLQKKK) as molecular recognition materials. Prostate specific antigen specific peptide on the selection of cutting based on established reaction, electrochemical detection of prostate specific antigen a new method of luminescent biosensor. The Rul-peptide by click reaction of modified on glassy carbon electrode surface, a light-emitting electrochemical sensor. When the sample solution contain the sensor into PSA, the electrode surface and its electrogenerated electrochemiluminescence probe specific polypeptides in glutamine (Q) and lysine (K) between the fracture, lysine with electrochemiluminescence signal substances from the electrode surface shedding. In TPrA solution, the sensor surface PSA specific action generates a light signal, the ECL intensity decreased with the increase of PSA concentration. The electrochemical detection of PSA chemiluminescence analysis was established. The experimental results show that the logarithmic reduction intensity electrochemical and PSA concentration showed a good linear relationship in1×10-10g/mL-1.0×10-8g/mL range, the detection limit is8×10-12ng/mL.Third chapter, A novel electrogenerated chemiluminescence peptide-based (ECL-PB) biosensor for highly sensitive and selective determination of prostate-specific antigen (PSA) was developed. A helix peptide (CHSSKLQK) was served as a molecular recognition element and ruthenium bis(2,2’-bipyridine)(2,2’-bipyridine-4,4’-dicarboxylic acid)-N-hydroxysuccinimide ester (Ru(bpy)2(dcbpy)NHS) was used as an ECL label. The helix peptide was labeled with the ECL label at NH2.containing lysine and utilized as ECL probe (Rul-peptide). The ECL-PB biosensor was fabricated by immobilizing the ECL probe onto a gold electrode surface via self-assembling technique through a thiol-containing cysteine incorporated at the end of the peptide. The principle of ECL measurement is based on the specific proteolytic cleavage event of Rul-peptide on the gold electrode surface in the presence of PSA, resulting in a decrease of ECL signal. The ECL intensity versus concentration of PSA was linear log in the range from0.10ng/mL to8.0ng/mL with a detection limit of0.038ng/mL. This work demonstrates that the direct transduction of peptide cleavage events into an ECL signal provides a simple and sensitive method for detecting target protein.