Design of an Ultrasensitive Diagnostic Device for Point of Care Based on Thin Film Colour Interference
Creators: Vankka, Natalie Anne
Publisher: name: University of Alberta Library
Year: 2022

<p>Point-of-care diagnostic testing offers many benefits compared to standard laboratory testing for biomarkers or diseases, including reducing the time and cost required to obtain a medical diagnosis. Most available point-of-care diagnostics are complex, expensive, or limited to high concentration, low volume samples. The aim of this work was to develop a highly sensitive, stand-alone, point-of-care medical diagnostic device that can quickly detect low concentrations of analyte (~ 4 pM) in high volume samples based on thin film colour interference. The device functions by immobilizing an antigen on a porous anodic aluminum oxide surface, then exposing the surface to a high-volume fluid sample containing the corresponding antibody at a low concentration. Transitional or turbulent fluid flow (up to a Reynolds number of ~ 10,000) over the device surface ensures that the fluid will be adequately mixed, exposing as much of the fluid to the surface as possible, increasing the chance of an antibody from the sample binding to the antigen coated surface. Binding leads to the formation of an antigen-antibody complex which causes a visible colour shift on the device surface, due to an increase in the optical path length. Substrate materials were coated with thin films of tantalum and aluminum, then anodized at 4 V to form the desired anodic alumina surface prior to protein immobilization. Several substrate materials were investigated including polycarbonate membranes, resin polymer discs, quartz frits discs, high purity tantalum discs, and silicon wafers. Two anodization methods were investigated for the high purity tantalum discs and pore size, surface morphology, and protein adsorption were analyzed and compared between the two methods. Several designs were considered including a filter and syringe design, a stirred tank with an impeller, and an airlift bioreactor design containing a draft tube. The limits of detection for a prothrombin and anti-prothrombin antigen-antibody prototype were determined for a stationary 20 μL fluid sample, a stationary 500 mL fluid sample, and a well-mixed 100 mL or 500 mL fluid sample in the stirred tank and the draft tube apparatus, based on a visible colour shift after exposure to the sample for 20 minutes. The use of a stationary 500 mL sample was able to reduce the limit of detection for anti-prothrombin to 0.0025 mg/mL (16.7 nM), a twenty-fold improvement in sensitivity compared to the stationary 20 μL drop, which had a limit of detection of 0.05 mg/mL (0.33 μM). Introducing turbulence by mixing the solution with an impeller above the coated surface further improved the sensitivity, and the limit of detection for the stirred tank set up was found to be 6.25 × 10-4 mg/mL (4.2 nM) – an 80-fold improvement compared to the stationary 20 μL drop. The final design was similar to an airlift bioreactor with a draft tube and allowed for the fluid flow to be recirculated and directed towards the coated surface. The introduction of directed transitional and turbulent flow in a 500 mL sample allowed for an 8,000-fold improvement in sensitivity compared to the 20 μL drop, with a limit of detection of 6.25 × 10-6 mg/mL (41.7 pM). Using a conical shape draft tube allowed for an 80,000-fold improvement in sensitivity compared to the 20 μL drop, with a limit of detection of 6.25 × 10-7 mg/mL (4.2 pM). Overall, the diagnostic device was able to detect anti-prothrombin in a high-volume sample, quantified by a colour shift, with improved sensitivity compared to the low volume sample after 20 minutes. Introducing directed transitional or turbulent flow and mixing into the high-volume sample improved the device sensitivity by a factor of 80,000.</p>