Electrochemical Sensors Based On Nucleic Acid Probes For Detection Of Small Molecules And Myoglobin

Depletion of glutathione （GSH） is associated with a variety of human diseasesincluding diabetes, AIDS and neurodegenerative diseases. Cysteine has been provento act as a physiological regulator associated with Alzheimer’s, cardiovascular disease,etc. Myoglobin, a non-enzymatic cardiac protein, is used as one of the diagnosticmarkers for acute myocardial infarction （AMI）. Thus, it is very important to developsensitive and selective methods for the detection of these biomolecules. In this thesis,electrochemical sensors for detection of glutathione, cysteine and myoglobin wereintroduced based on nucleic acid probes and signal amplification. The main contentswere as followed:1. An electrochemical sensor was constructed for glutathione and cysteinedetection based on gold nanoparticle signal amplification strategies. Hg²⁺could formT-Hg²⁺-T structure with thymine in aqueous medium. In this system, the T-rich DNA1probes were immobilized on electrode surface as recognition element. Goldnanoparticles were modified by T-rich DNA2probes as signal amplifier. The cationicelectroactive complex [Ru（NH3）6]3+was bound to the DNA via electrostaticinteractions as signal molecules. The added GSH （or Cys） can selectively coordinatewith Hg²⁺via forming T-Hg²⁺-T structure, which resulted in the obviously reducedchronocoulometry signal, and then GSH （or Cys） could be detected according to thechange of chronocoulometry signal. The results showed that the electrochemicalsensor was highly sensitive, and it could detect as low as10pmol/L for GSH （or Cys）.In addition, the sensor demonstrated good selectivity.2. An electrochemical biosensor was constructed for myoglobin detection basedon gold nanoparticles signal amplification strategies. In this system, the myoglobinaptamer was immobilized on electrode surface as recognition element. The goldnanoparticles were modified by DNA probes as signal amplifier. The cationicelectroactive complex [Ru（NH₃）₆]³⁺was bound to the DNA via electrostaticinteractions as signal molecules. Myoglobin was quantitatively detected, and it coulddetect as low as50pmol/L for myoglobin, which was about1-3orders of magnitudelower than that of those electrochemical biosensors which used antibody asrecognition elements. More importantly, this new method demonstrated itseffectiveness for detection of myoglobin in human serum.3. An electrochemical biosensor was constructed for myoglobin based on “supersandwich” signal amplification. The myoglobin aptamer was immobilized onelectrode surface as recognition element and “supersandwich” structure has beenemployed as signal amplifier, which can bound to the cationic electroactive complex[Ru（NH₃）₆]³⁺ via electrostatic interactions as signal molecules. Myoglobin wasquantitatively detected, and it could detect as low as10pmol/L for myoglobin, whichwas about1order of magnitude lower than that of “non-supersandwich” method. Thesensor demonstrated good selectivity. More importantly, this new methoddemonstrated its effectiveness for detection of myoglobin in human serum. In addition,this assay has characteristics of enzyme-free and mild reaction conditions, and it is asimple, sensitive, selective and universal method.