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Biosensor capable of directly probing bisphenol A
In recent years, the problems incurred with endocrine disrupting chemicals (EDCs) have aroused the public concerns. Bisphenol A, as one of the phenolic EDCs, has been widely used in the production of polycarbonate plastics, epoxy resins and flame retardants. Many evidences confirm that bisphenol A increases the incidence of infertility, genital tract abnormalities and breast cancer. Accordingly, there is an urgent need for developing an effective technology to remove and/or detect bisphenol A. For efficient removal/detection of bisphenol A, isolation of molecules directly and selectively cognitive of the target is expected to play a key role in overcoming major disadvantages of many other methods frequently relying on the use of linker or tagging molecules for target recognition. In this context, phage display is a powerful selection tool where a short peptide sequence displayed on the surface of a bacteriophage as a fusion with one of the viral coat proteins can be isolated from a vast library of similarly displayed peptides on the basis of its binding affinity to a target compound. Currently, Ph.D.-C7C library (one of filamentous M13 phage display libraries) is being used in search of bisphenol A affinity peptide moieties. Upon selection of bisphenol A binders, their relative affinity to bisphenol A and cross-binding affinity to other endocrine disruptors (especially bisphenol analogues) will be assessed, respectively. Finally, the peptide moieties will be further engineered to improve their binding affinity to bisphenol A and fused to protein docking system to design a recombinant protein exhibiting specific affinity to the target for development, which is expected to facilitate development of biosensors and biosorbent for detection and removal of endocrine disruptors in an economically viable way.
Screening of S-(−) verapamilbinding aptamer for its chiralseparation application
Aptamers are artificial nucleic acid ligands, specifically generated to recognize targetmolecules, such as amino acids, drugs, proteins or other molecules. Someaptamers can even discriminate optical isomers of target molecules such asL-arginine, L-tyrosinamide, and D-vasopressin. These artificial ligands have mostly been screened from combinatorial libraries of synthetic nucleic acidsby a method, known as systematic evolution of ligands by exponential enrichment (SELEX), comprising anin vitro iterative process of adsorption, recovery and amplification. Verapamil, a calcium entry blocker, has been usedin the treatment of hypertension, anginapectoris and cardiac arrhythmia. Verapamil formulations for drug administration usually consist of a blendof R-(+)and S-(−) enantiomers, pharmacokinetic and/or pharmacodynamic properties of which are radically different (i.e. S-(−) verapamil is 10–20-times more potentthat R-(+)-verapamil). In addition, an overdose injection of verapamil may cause serious side effects. Therefore, identification of aptamers specifically cognitive of S-(−)-verapamil would facilitate an efficient separation of verapamil enantiomers, rendering controlled administration of S-(−)-verapamil or close monitoring of drug concentration in order to avoidtoxic effects. In this project, we aim to develop a novel chromatographic SELEX method with the use of FPLC equipped with a monolith column in order to facilitate in vitro selection of aptamers with specific affinity to S-(−) verapamil from an 86-bp random single-stranded DNA pool. Pre-negative and negative screening were implemented with bare column and R-(−)-verapamilimmobilized column in the recycle mode to eliminate the non-specific binders priorto the main screening. The percentage of ssDNA eluted, measured for the fractionated DNApool following each round of main selection, increased with selection rounds, indicatingthe successful enrichment of aptamers with enhanced affinity to the target. Inthe future, an aptamer sequence exhibiting the highest affinity and specificityto S-(−)-verapamil will be immobilized on a chromatographic support to enable one-steppurification of the target enantiomer. The influence of various parameters (such as column temperature, eluent pH, and salt concentration) on the S-(−)- and R-(−)-verapamil retention will be investigated in order to find optimum separation condition and also to understand the binding mechanism between aptamer ligand and S-(−)-verapamil. It is expected that that DNA aptamers, specifically selected against an enantiomer, could soon become very attractive as new target-specific chiralselectors.
Targeted aptamer-based imaging, therapy and diagnosis of breast cancer
Breast cancer is the most common cancer in women and second most deadly. The conventional treatment involves a non-specific systemic delivery of therapeutic agent and this often results in unwarranted side effects which greatly decrease the patient’s quality of life. This work aims to identify aptamers which can specifically target the breast cancer cell surface protein to develop: 1) multifunctional magnetic nanoparticles for in vivo breast cancer detection and targeted therapy; 2) biosensor for the detection of tumor biomarker. Magnetic nanoparticles are deemed to be non-toxic and biodegradable and the magnetic properties will be harnessed for magnetic resonance imaging (MRI) of cancer cells and also for hyperthermia to induce cell apoptosis in the presence of a magnetic field. Human epidermal growth factor receptor 2 (HER2) is a transmembrane protein overexpressed in 20-30% of breast cancers. HER2 positive cancer is notorious for higher fatality and recurrence rates primarily due to strong drug resistance of HER2 positive cancer cells. Herceptin, a monoclonal antibody which binds to HER2 extracellular domain (ECD), is currently used in the treatment of HER2 positive cancer. Aptamer has advantages over monoclonal antibody in targeting HER2 in terms of lower cost, equal or greater target specificity, lower immunogenicity, more hydrophilic (hence less likely to cause particles aggregation in an aqueous environment once it is immobilized onto the particles), greater stability, and reproducible synthesis. The overall work scope of this project is as follows: 1) selection of aptamers with affinity to breast cancer cell surface protein HER2; 2) magnetic nanoparticle synthesis and functionalization with aptamer ligands; 3) assessment of cell targeting efficacy of functionalized nanoparticles in vitro and in vivo. Currently, we are characterizing the binding affinity and specificity of single-stranded DNAs, screened via CE-based SELEX method, against HER2 ECD and other target analogues.
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