Detection Of Golgi Protein 73 And Trinucleotide Polymorphism For Diagnosis Of Biliary Tract Cancer

Objective Biliary tract cancer(BTC) is a malignant tumor frequently associated with extremely high mortality due to the lack of effective methods for early diagnosis. In order to improve the diagnosis of BTC, novel methods are developed in this thesis for the highly sensitive detection of two BTC-relevant biomacromolecules.First, Golgi protein 73(GP73), a serum protein with much more elevated level in BTC than in physiological condition, is detected by a designed electrochemical immunosensor. Second, since the length polymorphism of trinucleotide repeats in BTC-relevant genes such as androgen genes has recently been proved to be strongly correlated with BTC, we have developed a novel fluorescence method to evaluate the length of this type of repeat via the interaction of doxorubicin(Dox) with structured DNA, and the resulted fluorescence quenching. Both methods have been employed in the analysis of biologically complex or even clinical samples, so as to facilitate the diagnosis of BTC, as well as to validate the clinical significance of the two biomacromolecules.Methods 1. Two functional nanomaterials, quantum dots(QDs) and graphene oxide(GO), are employed to develop an immunosensor for GP73. First, taking advantage of the good conductivity of electrochemically reduced GO, a highly conductive electrode surface layer is constructed to present the antibody of GP73. After the capturing of GP73 by the surface-immobilized antibody, aleuria aurantia lectin(AAL) capped QDs may bind oligosaccharide moieties on the captured GP73 via specific lectin-oligosaccharide interaction. So the surface-bound QDs can then generate amplified signal readout through anodic strip. The obtained signal response is positively correlated to GP73 abundance.2. Trinucleotide repeats(TNRs) rich in cytosines(C) and guanines(G) are apt to form the stable “hairpin” secondary structure. On the other hand, Dox is prone to intercalate into the double-stranded stem of the structured trinucleotide repeats, leading to the fluorescence quenching of Dox. Owing to this phenomenon, the fluorescence intensity of Dox is reversely proportional to TNRs length, so the length polymorphism of TNRs can be studied.Results 1. The proposed method show satisfactory analytical performance in terms of sensitivity, specificity and stability. Under the optimized conditions, the method has a linear range from 20 to5000 pM with a limit of detection as low as 12 pM. Furthermore, in the detection of serum in BTC patients before and after surgery,there is a significant decrease in GP73 levels, indicating effectiveness of the treatment. 2. The proposed method can determine the length of trinucleotide repeats in the range of 10-35 repeats, and the trinucleotide length polymorphism for PCR products has also been realized.Conclusions 1. We have developed a sensitive electrochemical immunosensor for GP73 based on GO and QDs. This method can detect serum GP73 with high sensitivity, showing promising prospect in BTC diagnosis and prognosis. 2. A fast TNR assay thatcan complete in less than 30 min is established based on quantitative fluorescence quenching of Dox by structured DNA. Without introducing chemical modifications, our method is simple,cost-effective, convenient, sensitive, and has greatly simplified relevant researches on length polymorphism of(CAG)n trinucleotide repeats.