Cytokine-Detecting Biosensor: Covalently Functionalizing Surfaces with Aptamers for Reusable and Fast Detection of Small Proteins

This thesis outlines a modular method to covalently functionalized surfaces with aptamers to sense for small proteins. Small proteins, such as cytokines, are related to a variety of diseases such as Rheumatoid arthritis, cancer, Alzheimer’s, eczema, etc. Current methods for detecting these small proteins are antibody-based single-use systems requiring specialized skills, long incubation times, and expensive equipment. We propose a method of aptamer-based biosensing that would be reusable, more versatile, and offer simpler operations.

Aptamers are synthetic chains of nucleotides made in vitro selection (SELEX) through repeated purification and washing cycles that provide the ability to bind tightly to any target. Aptamers capture target molecules via hydrogen-bond interactions, which are weaker than the covalent bonds between aptamers and the sensor base surface. Using this characteristic, the biosensing layer can be reused by breaking the hydrogen-bonds with a high-concentration urea wash. Such a treatment will destroy antibody-based biosensors but can be sustained by aptamers.

Atomic-layer-deposition (ALD) of silica onto stable surfaces enables surface functionalization. The deposited silica allows silane chemistry functionalization, as well as passivates and protects underlying fragile 2D TMD surfaces. By immobilizing specific aptamers to a silica surface via silane chemistry, users can measure the mass change and/or fluorescence difference upon target attachment on the silica substrates.

Optical detection of proteins involves constructing a partial-complementary loopback-structured fluorescent dye-labeled aptamer. Upon aptamer binding with the higher binding-affinity target, the aptamer will deform and move the fluorescent dye to a different position. The deformation results in a fluorescence intensity change that could indicate a target protein binding.

A quartz crystal microbalance (QCM) is an acoustic sensor that can measure a material’s resonant vibration frequency. A material’s resonant frequency is correlated to its mass. By immobilizing aptamers onto QCM silica quartz crystal surfaces, the added mass of a target bounded by the aptamers can be sensed by a change in resonant frequency. With an increasing amount of target proteins, TNF Alpha (TNFa), interacting with the biosensor, a corresponding decrease in resonant vibration frequency is measured. Non-target cytokines, Interferon Gamma (IFNg), were introduced to the QCM to test the biosensing sensitivity. For the same concentration of IFNg and TNFa, only the TNFa interaction elicited a decrease in resonance vibration frequency. The proposed biosensor was able to obtain high selectivity and reusability.

Applications of functionalized TMD (ft-TMD) or general surfaces include target medicine, target therapy, acoustic wave, fluorescent, and electrochemical sensors. This thesis explores the design and sensing of the proposed reusable and target-generalizable sensing layer.