DNA INTERFACES FOR ELECTROCHEMICAL DETECTION OF NEURODEGENERATIVE REPEAT SEQUENCES

TITLE: DNA INTERFACES FOR ELECTROCHEMICAL DETECTION OF NEURODEGENERATIVE REPEAT SEQUENCES DNA repeat sequences in the human genome possess unique biophysical properties due to their sequence-directed structural flexibility. It has been assumed that unique helical flexibility of these sequences forms non-canonical structures inside the cell that disrupts transcription/translation functions and can lead to variety of fatal diseases such as neurodegenerative disorders. Neorodegenerative diseases are caused by certain types of mutations called repeat expansions. Expansion of certain trinucleotide repeat (TNR) sequences have been associated with almost two dozen neurodegenerative and neuromuscular diseases, such as CGG expansion with Fragile X, CAG repeats with Huntington’s disease (HD), CTG repeats with Myotonic Dystrophy, and GAA expansion with Friedreich’s Ataxia depending on their gene location and threshold length. Current diagnosis of repeat expansion disorders includes traditional techniques including two-dimensional gel electrophoresis and various polymerase chain reaction (PCR) based methods. However, these methods are complicated, high cost, frequently generate false negatives, and are time-consuming procedures. Therefore, to develop rapid and sensitive detection tools, a number of researchers have employed electrochemical strategies for detection of TNR sequences and their lengths using electroactive labels and enzyme-linked steps for signal amplification. However, an urgent need exists for further exploration in this area in order to develop label-free, low-cost, and simple biosensing platforms to detect such unique sequences. In this dissertation we investigated the properties of repeat sequences associated with neurodegenerative diseases to characterize and develop low-cost, label-free, and printable electrochemical platforms to detect TNR sequences. First, surface probe microscopy and electrochemical methods were employed for characterization of TNR sequences and showed that the properties of TNRs are sequence dependent rather than by flexibility rank as had been previously reported for them. Then, the interface properties of these repeat sequences were studied on the surface of two-dimensional materials such as graphene and molybdenum disulfide. Finally, we reported a label-free, low-cost, and simple inkjet printable platform to distinguish the CGG expansion associated with Fragile-X disease

PNA Microprobe for Label-Free Detection of Nucleic Acid Repeat Mutations

We present a PNA-based microprobe sensing platform to detect nucleic acid repeat mutations by electrochemical impedance spectroscopy. The microprobe platform discriminated Huntington’s disease-associated CAG repeats in cell-derived total RNA. This sensitive, label-free, and PCR-free detection strategy has the potential to detect a plethora of length mutation disorders

Sequence-Independent DNA Adsorption on Few-Layered Oxygen-Functionalized Graphene Electrodes: An Electrochemical Study for Biosensing Application

DNA is strongly adsorbed on oxidized graphene surfaces in the presence of divalent cations. Here, we studied the effect of DNA adsorption on electrochemical charge transfer at few-layered, oxygen-functionalized graphene (GOx) electrodes. DNA adsorption on the inkjet-printed GOx electrodes caused amplified current response from ferro/ferricyanide redox probe at concentration range 1 aM–10 nM in differential pulse voltammetry. We studied a number of variables that may affect the current response of the interface: sequence type, conformation, concentration, length, and ionic strength. Later, we showed a proof-of-concept DNA biosensing application, which is free from chemical immobilization of the probe and sensitive at attomolar concentration regime. We propose that GOx electrodes promise a low-cost solution to fabricate a highly sensitive platform for label-free and chemisorption-free DNA biosensing

Hand-Fabricated CNT/AgNPs Electrodes using Wax-on-Plastic Platforms for Electro-Immunosensing Application

Abstract Fabrication of inexpensive and flexible electronic and electrochemical sensors is in high demand for a wide range of biochemical and biomedical applications. We explore hand fabrication of CNT modified AgNPs electrodes using wax-on-plastic platforms and their application in electrochemical immunosensing. Wax patterns were printed on polyethylene terephthalate-based substrates to laydown templates for the electrodes. Hand painting was employed to fabricate a silver conductive layer using AgNPs ink applied in the hydrophilic regions of the substrate surrounded by wax. CNT was drop cast on top of the working electrodes to improve their electrochemical signal. The device layers were characterized by scanning electron microscopy. The electrochemical performance of the hand fabricated AgNPs and CNT/AgNPs electrodes was tested using cyclic voltammetry, differential pulse voltammetry, and amperometry. The electrochemical response of CNT/AgNPs electrodes was relatively faster, higher, and more selective than unmodified AgNPs sensing electrodes. Finally, the hand-painted CNT/AgNPs electrodes were applied to detect carcinoembryonic antigen (CEA) by measuring the end-product of immunoassay performed on magnetic particles. The detection limit for CEA was found to be 0.46 ng/mL

A system for bioelectronic delivery of treatment directed toward wound healing.

The development of wearable bioelectronic systems is a promising approach for optimal delivery of therapeutic treatments. These systems can provide continuous delivery of ions, charged biomolecules, and an electric field for various medical applications. However, rapid prototyping of wearable bioelectronic systems for controlled delivery of specific treatments with a scalable fabrication process is challenging. We present a wearable bioelectronic system comprised of a polydimethylsiloxane (PDMS) device cast in customizable 3D printed molds and a printed circuit board (PCB), which employs commercially available engineering components and tools throughout design and fabrication. The system, featuring solution-filled reservoirs, embedded electrodes, and hydrogel-filled capillary tubing, is assembled modularly. The PDMS and PCB both contain matching through-holes designed to hold metallic contact posts coated with silver epoxy, allowing for mechanical and electrical integration. This assembly scheme allows us to interchange subsystem components, such as various PCB designs and reservoir solutions. We present three PCB designs: a wired version and two battery-powered versions with and without onboard memory. The wired design uses an external voltage controller for device actuation. The battery-powered PCB design uses a microcontroller unit to enable pre-programmed applied voltages and deep sleep mode to prolong battery run time. Finally, the battery-powered PCB with onboard memory is developed to record delivered currents, which enables us to verify treatment dose delivered. To demonstrate the functionality of the platform, the devices are used to deliver H[Formula: see text] in vivo using mouse models and fluoxetine ex vivo using a simulated wound environment. Immunohistochemistry staining shows an improvement of 35.86% in the M1/M2 ratio of H[Formula: see text]-treated wounds compared with control wounds, indicating the potential of the platform to improve wound healing

A system for bioelectronic delivery of treatment directed toward wound healing

Abstract The development of wearable bioelectronic systems is a promising approach for optimal delivery of therapeutic treatments. These systems can provide continuous delivery of ions, charged biomolecules, and an electric field for various medical applications. However, rapid prototyping of wearable bioelectronic systems for controlled delivery of specific treatments with a scalable fabrication process is challenging. We present a wearable bioelectronic system comprised of a polydimethylsiloxane (PDMS) device cast in customizable 3D printed molds and a printed circuit board (PCB), which employs commercially available engineering components and tools throughout design and fabrication. The system, featuring solution-filled reservoirs, embedded electrodes, and hydrogel-filled capillary tubing, is assembled modularly. The PDMS and PCB both contain matching through-holes designed to hold metallic contact posts coated with silver epoxy, allowing for mechanical and electrical integration. This assembly scheme allows us to interchange subsystem components, such as various PCB designs and reservoir solutions. We present three PCB designs: a wired version and two battery-powered versions with and without onboard memory. The wired design uses an external voltage controller for device actuation. The battery-powered PCB design uses a microcontroller unit to enable pre-programmed applied voltages and deep sleep mode to prolong battery run time. Finally, the battery-powered PCB with onboard memory is developed to record delivered currents, which enables us to verify treatment dose delivered. To demonstrate the functionality of the platform, the devices are used to deliver H $$^+$$ + in vivo using mouse models and fluoxetine ex vivo using a simulated wound environment. Immunohistochemistry staining shows an improvement of 35.86% in the M1/M2 ratio of H $$^+$$ + —treated wounds compared with control wounds, indicating the potential of the platform to improve wound healing