Abstract

Antibiotic resistance poses a significant global health challenge, particularly in urinary tract infections (UTIs), where 92% of cases exhibit resistance to at least one antibiotic, causing over 260,000 deaths annually. The emergence of antibiotic-resistant bacteria and growing awareness of the adverse effects of unnecessary antibiotic use highlight the urgent need for better UTI diagnostics. Current state-of-the-art methods are either expensive and time-consuming, or they lack specific details about the nature of the antibiotic and only determine inflammation. This hinders timely and effective treatment decisions regarding whether an antibiotic is actually necessary. Given this backdrop, there is a demand for a complementary method that is affordable, user-friendly, and easily accessible for point-of-care (POC) analysis. Recent developments have shown that prominent uropathogens release specific small molecules during UTIs, which could serve as valuable diagnostic markers. In response to this challenge, microelectrode arrays offer a potential solution, providing a cost-effective, rapid, and label-free approach to monitor multiple binding events and thus provide multiplex sensing of metabolites. This study introduces a diblock copolymer-based microelectrode array platform designed for the quantitative analysis of such small molecules. We demonstrate the ability to site-selectively functionalize this stable, polymer-coated, high-density array with aptamers using the Cu(II)-mediated Chan-Lam coupling reaction. Initial work with borate ester-based polymer surfaces revealed challenges with selectivity due to significant background reactions. These studies revealed significant background fluorescence across the microelectrode array, indicating that the lack of selectivity was not due to a loss in confinement of the desired reaction. Further investigation showed that the background reactions occurred with the reactive borate ester surface even in the absence of the copper reagent used for the Chan-Lam coupling reaction. To overcome this problem, a dual-surface strategy was developed, primarily utilizing a less reactive arylbromide-based polymer that could be selectively converted to the reactive borate ester only at desired electrode sites. This approach significantly enhanced precise aptamer immobilization, reducing background reactions to below 3% and generating stable electrochemical signals for small molecule detection. In simple terms, non-targeted sites on the array lacked borate esters, eliminating borate ester-related background reactions. Further exploration into the reaction mechanism confirmed that the acetylene-functionalized aptamer itself contributes to confinement by forming dimers in the solution above the array, rapidly consuming any Cu(II) reagent that diffuses from the targeted electrodes. Utilizing this optimized platform, three distinct aptamers were successfully placed on a single array that was then used to demonstrate the quantitative and selective electrochemical detection of their respective target metabolites. The polymer-coated microelectrodes were compatible with the multiplex detection of small molecules. Other studies showed that the array-based approach was compatible with the use of human urine samples and demonstrated that the polymer functionalized array was stable for 50 weeks. The array was stable through multiple uses during this “year-long” period, a scenario that clearly illustrated the compatibility of the arrays for a point-of-care application. Taken together, these findings pave the way for constructing robust devices for multiplexed POC detection of metabolites, thereby improving the performance and accuracy of diagnostic tools for the identification of infectious pathogens.

Committee Chair

Kevin Moeller

Committee Members

John-Stephen Taylor; Jeffrey Henderson; Timothy Wencewicz; Vladimir Birman

Degree

Doctor of Philosophy (PhD)

Author's Department

Chemistry

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-6-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0002-9307-3780

Download

Included in

Chemistry Commons

Share

COinS