University of Cambridge
Enzyme-Linked Electrochemical Detection of Urinary Sarcosine
Abstract
dc:description.abstractBiomarker-based diagnostics are crucial for the early detection of prostate cancer (PCa). One such biomarker that may be useful in PCa diagnostics is urinary sarcosine (N-methylglycine). However, its utility is confounded by several factors: the small differences in metabolite concentration that separate healthy (~ 0.2 µM) and sick (~ 5.0 µM) patient populations, the difficulties associated with detection of these low concentrations in complex matrix (i.e. urine), and the cumbersome methods that have typically been used for detection (i.e. HPLC). To address these problems, this thesis sought to develop a first-generation enzyme-linked electrochemical biosensor (EnzL-ECBS) to detect sarcosine at clinically relevant concentrations for PCa diagnosis in human urine. Combining a silica-immobilised chimeric monomeric sarcosine oxidase (mSOx) biorecognition element with a static platinum disc macroelectrode transducer created an easy-to-use, low-cost alternative for chronocoulometric sarcosine detection in static samples of PBS and artificial urine matrix (AUM). However, initial EnzL-ECBS designs suffered severe sensitivity limitations. Characterisation studies demonstrated that sarcosine detection was obstructed by the impact of several AUM components (uric, citric, lactic acids and CaCl₂) on biochemical and electrochemical transduction, along with the acidic pH (which could be induced or bolstered by AUM acids) and insensitivity of the initial macroelectrode design towards static H₂O₂ samples. Attempts to engineer kinetically enhanced biorecognition elements from different mSOx and glycine oxidase constructs failed to generate viable alternatives. However, kinetic enhancements to the initial mSOx-bound silica were artificially imbued by increasing the concentration and duration of its exposure to the sarcosine-containing sample. Exchanging the initial static macroelectrode cell design with either static microelectrodes or dynamic flow cell designs provided significant improvements to peroxide sensitivity in both PBS and AUM. Combining sample pretreatment methods, such as pH titration to pH ~ 8 and exposure to Amberlite™ exchange resins bolstered sarcosine detection by enhancing sarcosine transduction while simultaneously reducing the signature of background interferents in a statistically significant manner. These actions generated sensitivity to sarcosine at concentrations as low as 0.25 µM in PBS and AUM, aided in large part by a ~ 71% reduction in electrochemical interference of pretreated AUM samples compared to their untreated counterparts. While further research is needed, this work establishes a new platform for the detection of sarcosine at concentrations considered clinically relevant for prostate cancer detection in both PBS and AUM.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- McCarthy, John
- Advisors dc:contributor.advisor
-
- Hall, Elizabeth
- Higson, Seamus
Subjects
dc:subject × 19Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.108019
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/367424