|Author:||Chau, Li Yin|
|Title:||Development of new enzyme mimetics for highly sensitive DNA and protein detection|
|Advisors:||Lee, Ming Hung Thomas (BME)|
|Subject:||Hong Kong Polytechnic University -- Dissertations|
DNA -- Biotechnology
Proteins -- Biotechnology
|Department:||Department of Biomedical Engineering|
|Pages:||xxix, 194 pages : color illustrations|
|Abstract:||Biomolecule detection is a powerful tool for medical diagnostics. For many biomarkers, however, their abundance levels could be below the detection threshold of many labeling reagents, particularly in the early stage of the diseases. As a general route to improving the detection sensitivity, enzymes are widely applied as the amplifying agents in various platforms. These natural enzymes, however, have the drawbacks of laborious, time-consuming, and expensive production. To address these issues, recent focus has been directed towards the exploration of synthetic materials with intrinsic enzyme-like activities. Compared with their natural counterparts, enzyme mimetics have the advantages of simple and low-cost production as well as high stability. However, the present enzyme mimetics-based platforms still have severe limitations. For deoxyribonucleic acid (DNA) detection, although nanomaterial-based enzyme-mimetic labels were demonstrated superior catalytic performance, limit attempt has been made to explore their applicability for DNA detection using simple and cost-effective salt-induced aggregation approach. Moreover, a great potential has been shown to couple enzyme-mimetic labels with DNA amplification techniques, so that ultrasensitive DNA detection with simple colorimetric readout can be achieved. However, due to the lack of effective detection strategies, only few related work was reported. For small molecule and protein detection, compared with conventional sandwich-like enzyme-linked immunosorbent assay (ELISA) format, target-recycling amplification has been shown the advantages of cost effectiveness, ease of handling, and homogeneous assay format. However, the use of nuclease mimetics for triggering target-recycling in aptamer-bound graphene oxide (GO) platforms remains unexplored. To provide possible solutions for these challenges, this work compromises three individual studies.|
In the first study, we developed a simple and sensitive colorimetric assay for specific DNA sequence by using peroxidase mimetics of platinum nanoparticles on reduced graphene oxide (PtNPs/rGO). PtNPs/rGO possessed the combined advantages of PtNPs which had superior peroxidase-like activity and rGO which showed preferential binding with single-stranded DNA and the resulting stabilization effect on salt-induced aggregation. For DNA detection, the linear range and limit of detection of this assay platform were 0.5-10 nM and 0.4 nM, respectively. Moreover, this platform featured high specificity that 3-base-mismatched sequence could be distinguished with the naked eye and 1-base-mismatched sequence with absorbance measurement. Furthermore, the applicability for real sample detection was demonstrated with PCR product analysis. In the second study, we developed a simple and ultrasensitive colorimetric detection platform for loop-mediated isothermal amplification (LAMP) reaction using PtNPs/rGO. PtNPs/rGO possessed a pH-dependent peroxidase-like activity, with the threshold for turning on/off found between pH 6 and 7. On the other hand, by reducing the buffering capacity of LAMP reaction mixture, LAMP amplification resulted in a decrease of solution pH from ~8.5 to below 6. Taken together, a new platform for colorimetric detection of LAMP reaction using pH-sensitive enzyme mimetics was developed. Notably, a detection limit down to attomolar range was achieved by using this new platform. In the last study, we discovered that redox-active metal ions together with ascorbic acid and hydrogen peroxide (H2O2) served as nuclease mimetics, which was capable of triggering target regeneration in aptamer-bound GO platform. We developed an amplified assay for insulin based on Cu2+-mediated target-recycling. With the nuclease mimetics, the detection limit was improved by almost 150-fold compared with the unamplified method. Furthermore, we demonstrated the capability of target-recycling amplification with different aptamer-target systems or redox-active metal ions.
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