New Methods For Detection Of Cancer Biomarkers And Carcinoma Cell Surface Glycans

Early diagnosis and therapeutics plays a crucial role in improving the survival rate of cancer patients. In the post-genomic era, discovery of potential cancer biomarkers due to the development of proteomics has held the promise of''individualized medicine,''bringing a new dimension to disease diagnosis, classification and prognosis. The emerging glycomics which deciphers altered glycosylation in oncogenic transformation, invasion and metastasis is quickly becoming a driving force for discovering new glycan-based cancer biomarkers and therapeutic targets. Thus, it is very important to develop analytical technologies with significant performance for potential biomarkers discovery and early cancer diagnosis and therapeutics.In this dissertation, by integrating analytical biochemistry, molecular biotechnology, nanotechnology and bioconjugate chemistry, a series of new methods have been developed for simple and ultrasensitive detection of cancer biomarkers and in situ evaluation of carcinoma cell surface glycans. This dissertation includes the following five parts:1. A cascade signal amplification strategy for sub-attomolar protein detection by rolling circle amplification and quantum dots taggingA cascade signal amplification strategy was proposed for detection of protein target at ultra-low concentration by combining rolling circle amplification (RCA) with oligonucleotide functionalized quantum dots (QDs), multiplex binding of biotin-strepavidin system and anodic stripping voltammetric detection. The RCA product containing tandem-repeat sequences serve as excellent template for periodic assembly of QDs, which present per protein recognition event to numerous quantum dot tags for electrochemical readout. Both the RCA and the multiplex binding system show remarkable amplification efficiency, very little nonspecific adsorption and low background signal. Using human vascular endothelial growth factor as a model protein, the designed strategy could quantitatively detect protein down to 16 molecules in a 100-μL sample with a linear calibration range from 1 aM to 1 pM, and be amenable to quantification of protein target in complex biological matrices.2. A facile scanometric strategy for ultrasensitive detection of protein using aptamer-initiated RCAThis work proposed a simple strategy for ultrasensitive detection of protein biomarker. This strategy contained aptamer-initiated rolling circle amplification, Au nanoparticle probe and simple scanometric readout. The method showed a dynamic range of five orders of magnitude and a detection limit down to 10 fM protein molecule, featuring high specificity and low matrix effect. By integrating multiple molecular biotechnologies, nanobiotechnology, immobilization chemistry and scanometric detection, this primary research opened new horizons for integrating different disciplines to develop pragmatic and simple technology with significant analytical performance. The proposed strategy would become a powerful tool for proteomics research and clinical diagnostics.3. Ultrasensitive scanometric detection of matrix metalloproteinases using a histidine tagged peptide–Au nanoparticle probeA simple scanometric approach was proposed for ultrasensitive assay of matrix metalloproteinases (MMPs) based on their discriminatory and proteolytic activity. This approach integrated a newly designed peptide-gold nanoparticle (AuNP) probe, a nitrilotriacetic acid modified chip and silver signal amplification. Using MMP-7 as a model of MMPs, the peptide-AuNP probe contained a six-histidine (His) tag and a MMP-7 specific peptide sequence. The His tag could trap a large number of AuNPs on the modified chip by Ni2+ induced affinity binding to obtain silver enhanced signal. In presence of MMP-7, the proteolysis of the peptide sequence led to the cleavage of His tag from AuNPs, and decreased the amount of AuNPs bound on the modified chip, which decreased the scanometric readout signal. The proposed method could conveniently quantify MMP-7 down to 1.2×10-17 moles in a 2.5-μL sample with a linear range from 0.1 to 100 ng mL-1. This protocol showed good reproducibility, stability, accuracy and low matrix effect. The designed strategy presented a useful platform for ultrasensitive detection of MMPs.4. Effective cell capture with tetrapeptide functionalized carbon nanotubes and dual signal amplification for cytosensing and evaluation of cell surface carbohydrateA novel electrochemical cytosenor was designed based on the specific recognition of integrin receptors on cell surface to arginine-glycine-aspartic acid-serine (RGDS) functionalized single-walled carbon nanotubes (SWNTs). The conjugated RGDS showed predominant ability to capture cells on electrode surface by the specific combination of RGD domains with integrin receptors. Using BGC-823 human gastric carcinoma cells (BGC cells) as model, the cell surface mannosyl groups could specifically bind with horseradish peroxidase labeled concanavalin A, producing an electrochemical cytosensor. Based on the dual signal amplification of SWNTs and enzymatic catalysis the cytosensor could response down to 620 cells mL-1 BGC cells with a linear calibration range from 1.0×103 to 1.0×107 cells mL-1, showing very high sensitivity. The dual signal amplification could be further used to evaluate the mannosyl groups on cell surface, and the mannosyl groups on single living intact BGC cell were detected to correspond to 5.3×107 molecules of mannose. 5. Electrochemical cytosensor array for simple dynamic analysis of carcinoma cell surface glycansA facile electrochemical cytosensor array was proposed for sensitive multiple analysis of intact cell surface glycome and effective monitoring of dynamic variation in cell surface glycan expression profile during drug treatment. This method utilized arginine-glycine-aspartic acid-serine functionalized single-walled carbon nanotubes modified screen-printed carbon electrode to capture cancer cells and maintain their biological activity, and horseradish peroxidase labeled lectins to recognize cell surface specific glycans. The proposed strategy showed very little nonspecific adsorption and background signal and conducted more sensitive profile of low abundance of glycans than flow cytometric analysis. The proposed method could monitor the dynamic change of glycome during drug treatment with high sensitivity and acceptable accuracy and reproducibility. It could be anticipated that this facile cytosensor array would become a powerful and pragmatic tool to decode cell surface glycome and discover potential glycan biomarkers and novel therapeutic targets.