Electrochemical Biosensors for Point-of-Care Diagnostics in Whole Human Fluids

Electrochemical biosensors are sensing devices that selectively detect biomolecular analytes to generate quantitative signals using electroanalytical techniques. In general, electrochemical biosensing architectures consist of biorecognition elements (e.g., antibodies, enzymes, nuclei acids, receptors, etc.) that facilitate analyte detection due to their intrinsic biological selectivity. These biorecognition elements are immobilized on electrode transducers, and detection events can trigger electrochemical reactions/processes or changes to electrode properties such as conductance, resistance, capacitance, etc.. The transducer then translates these as electrical signals which can then be converted to a particular readout method. Due to the advantageous properties of electrochemical biosensors, i.e., rapid response times, high sensitivity and selectivity, portability, and ease of operation, they have attracted intense interest as promising tools for point-of-care (POC) applications over other biosensing devices. POC diagnostic testing refers to clinical analyses performed at the time and place of patient care and is a rapidly developing area of biomedical technology. The proliferation of POC technologies is highly desirable due to their potential to improve patient survival and health outcomes through early disease diagnosis and clinical intervention. Ideally, POC diagnostic devices should follow the ASSURED criteria: Affordable, Sensitive, Specific, User-friendly, Rapid/Robust, Equipment-free and Deliverable. Further, it is critical for these devices to detect bioanalytes directly in whole physiological media (e.g., blood/serum, urine, saliva, sweat, etc.) to minimize sample preparation steps and reduce testing length and complexity. These requirements are challenging to meet in practice and thus require careful design, construction, and testing for the development of effective POC electrochemical sensors.

The work described in this thesis summarizes the development of electrochemical biosensors POC applications. Specifically, we investigate multiple strategies to enable effective POC testing in whole human fluid samples. For instance, electrochemical biosensors are developed as affinity-based or biocatalytic sensing platforms based on their biorecognition element. We implemented approaches related to electrode surface chemistries that are robust against non-specific protein adsorption in a variety of complex fluids, e.g., whole blood, serum, cerebrospinal fluid (CSF). In addition, the role of electrode geometries and configurations, particularly those that enable signal amplification via redox cycling for enhanced sensing performance, is described. Both small and large bioanalytes, such as cytokines, and biometabolites, including cholesterol, glucose, lactate, are used as clinical biomarkers. Finally, the implementation of smartphone technology in sensor readout as well as comparison with commercial, FDA-approved POC devices is explored as an avenue to field deployment.

A label-free electrochemical immunosensor for the detection of interleukin-6 (IL-6) in human cerebrospinal fluid (CSF) and serum was developed for diagnostic and therapeutic monitoring. The IL-6 immunosensor was fabricated from gold interdigitated electrode arrays (IDEAs) modified with IL-6 antibodies for direct antigen recognition and capture. A rigorous surface analysis of the sensor architecture was conducted to ensure high structural fidelity and performance. Electrochemical characterization was conducted using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), and sensing was performed using differential pulse voltammetry (DPV). The DPV peak current was used to quantify IL-6 in buffer, CSF, and serum. The linear range of the sensor response as a function of the analyte concentration was obtained in each medium and detection limits, as low as 1.6 pg mL^-1 in buffer, were also determined. The IL-6 immunosensor was evaluated for selectivity over other common cytokines, including IL-10 and TNF-a, showing selectivities in the range 10x-100x against these potential interferents. EIS measurements showed that the resistance to charge transfer, R_CT, decreases upon IL-6 binding, an observation attributed to a structural change upon Ab-Ag binding that opens up the architecture so that the redox probe can more easily access the electrode surface.

A closed bipolar electrode (CBE)-based metabolite sensing platform was constructed for the detection of diagnostic metabolites in undiluted human blood. An electrode chemistry based on blood-compatible phosphorylcholine (PPC) and phenylbutyric acid (PBA) mixed layers, electroactive ferrocene (Fc) moieties, redox-active enzymes, and a diffusing mediating species was developed as a means of overcoming nonspecific-protein adsorption and amplifying sensing currents in human whole fluid, i.e., unprocessed, samples. Multiple electron mediation routes were identified as contributing to the overall sensing scheme, and results suggested that both surface-bound and diffusible mediating species complement each other in producing an enhanced electrochemical response. The sensing scheme was incorporated in the CBE blood metabolite sensor, where cholesterol, glucose, lactate levels in real blood samples were determined from a coupled redox-mediated color change captured by a smartphone camera and subsequent RGB analysis. Important sensor parameters such as the limits of detection, linear response range, selectivity, were evaluated for all blood metabolites. The CBE blood metabolite sensor was compared with commercial FDA-approved meters for the three blood metabolites, and the CBE device gave results in excellent agreement with the commercial devices, thereby validating the platform as a potential POC diagnostic device.

The work described in this thesis illustrates the importance of synergistically combining electrochemistry, device design, surface chemistry, and biology in biosensor development. Doing so has yielded practical and effective electrochemical biosensors to address POC determinations of health-critical biomarkers in human-derived whole fluids.