Abstract
The use of molecular sensors for the screening of target analytes is an attractive concept for point-of-care diagnostics, environmental and food safety surveillance, security and counterterrorism, product safety/quality compliance monitoring, and many other real-world applications. They supplement advanced instrumental analytical and bioanalytical measurements with their ease of use, rapid responses, high portability and low operational cost characteristics. Among the targets of these sensors, proteases are essential to a wide variety of physiological processes for normal cellular and tissue functioning such as regulation of intracellular signal transduction and protein/peptide transportation, digestion, reproduction, innate immune responses, wound healing, etc. Their potentials as biomarkers for disease diagnostics and as drug targets for therapies are being increasingly recognized in recent years. However, traditional assays for proteases by gel zymography and antibody-based immunoassays are usually slow, tedious and costly.In this thesis, the development and validation of a new signal amplification approach for the biosensing of proteases that offers rapid colorimetric responses readily detectable by naked eye is reported. This analyte-triggered mutual emancipation of linker-immobilized enzymes (AMELIE) strategy for biosensing enables the generation of multiple signaling events from a single sensing event via a pair of bound enzymes immobilized by linkers that are substrates to one another. One of the linkers, the “response-linker” contains the specific peptide sequence that can be selectively proteolyzed by the target protease analyte. The other linker, the “looping-linker”, is composed of the substrate for the enzyme immobilized by the response-linkers. It is responsible for the controlled release of the other member of the dual-enzyme pair that can also cleave the response-linkers, spawning a self-sustaining, autocatalytic loop of enzymatic reactions until the emancipation of all the bound enzymes. Spatial separation of these bound enzymes prevents the mutual cleavage reactions from occurring in the absence of target analyte which upsets many signal amplification mechanisms in chemo-/biosensing.
To demonstrate this concept, in Chapter 2, a biosensor for the detection of collagenase, a zinc-dependent matrix metalloproteinases was constructed. The signal amplification autocatalytic-loop was composed of alginate lyase and collagenase immobilized by a peptide sequence specific for the target MMP as the response-linker, and alginate as the looping-linker, respectively. This AMELIE biosensing afforded a detection sensitivity of 2.5 pg mL-1 with a wide linear range up to 4 orders of magnitude. This performance enables the detection of the extracellular metalloproteinases in the culture medium containing as low as 103 CFU mL-1 of Escherichia coli. Chapter 3 elaborates the kinetics of such an amplification autocatalytic loop mechanism based on solid-phase Michaelis-Menten equation. The kinetic parameters Km and kcat measured and utilized to build a kinetic model to simulate the performances of the AMELIE biosensor. To further extend the application of the AMELIE system, in Chapter 4, another pair of response-linker and looping-linker, thrombin and TEV protease specific peptide sequences were adopted to a high sensitivity and selectivity biosensor for thrombin.
In summary, the novel AMELIE approach is capable to detect proteases with high sensitivity and specificity. This new mechanism provides substantial signal amplification in chemosensing and biosensing. Moreover, by incorporation of suitable peptide sequence into the response-linker, or substituting it with other substrates, this AMELIE approach is prospective in the detection of other designated peptidases, hydrolases, lipases and nucleases.
| Date of Award | 19 Sept 2022 |
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| Original language | English |
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| Supervisor | Yun Wah LAM (Supervisor) |