C-MEMS-derived glassy carbon electrochemical biosensors for rapid detection of SARS-CoV-2 spike protein.

According to a World Health Organization (WHO) report, the world has experienced more than 766 million cases of positive SARS-CoV-2 infection and more than 6.9 million deaths due to COVID through May 2023. The WHO declared a pandemic due to the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, and the fight against this pandemic is not over yet. Important reasons for virus spread include the lack of detection kits, appropriate detection techniques, delay in detection, asymptomatic cases and failure in mass screening. In the last 3 years, several researchers and medical companies have introduced successful test kits to detect the infection of symptomatic patients in real time, which was necessary to monitor the spread. However, it is also important to have information on asymptomatic cases, which can be obtained by antibody testing for the SARS-CoV-2 virus. In this work, we developed a simple, advantageous immobilization procedure for rapidly detecting the SARS-CoV-2 spike protein. Carbon-MEMS-derived glassy carbon (GC) is used as the sensor electrode, and the detection is based on covalently linking the SARS-CoV-2 antibody to the GC surface. Glutaraldehyde was used as a cross-linker between the antibody and glassy carbon electrode (GCE). The binding was investigated using Fourier transform infrared spectroscopy (FTIR) characterization and cyclic voltammetric (CV) analysis. Electrochemical impedance spectroscopy (EIS) was utilized to measure the change in total impedance before and after incubation of the SARS-CoV-2 antibody with various concentrations of SARS-CoV-2 spike protein. The developed sensor can sense 1 fg/ml to 1 µg/ml SARS-CoV-2 spike protein. This detection is label-free, and the chances of false positives are minimal. The calculated LOD was ~31 copies of viral RNA/mL. The coefficient of variation (CV) number is calculated from EIS data at 100 Hz, which is found to be 0.398%. The developed sensor may be used for mass screening because it is cost-effective. A schematic representation of the SARS-CoV-2 spike protein sensing using surface functionalized glassy carbon electrode.

Design Guidelines for Thin Diaphragm-Based Microsystems through Comprehensive Numerical and Analytical Studies.

This paper presents comprehensive guidelines for the design and analysis of a thin diaphragm that is used in a variety of microsystems, including microphones and pressure sensors. It highlights the empirical relations that can be utilized for the design of thin diaphragm-based microsystems (TDMS). Design guidelines developed through a Finite Element Analysis (FEA) limit the iterative efforts to fabricate TDMS. These design guidelines are validated analytically, with the assumption that the material properties are isotropic, and the deviation from anisotropic material is calculated. In the FEA simulations, a large deflection theory is taken into account to incorporate nonlinearity, such that a critical dimensional ratio of a/h or 2r/h can be decided to have the linear response of a thin diaphragm. The observed differences of 12% in the deflection and 13% in the induced stresses from the analytical calculations are attributed to the anisotropic material consideration in the FEA model. It suggests that, up to a critical ratio (a/h or 2r/h), the thin diaphragm shows a linear relationship with a high sensitivity. The study also presents a few empirical relations to finalize the geometrical parameters of the thin diaphragm in terms of its edge length or radius and thickness. Utilizing the critical ratio calculated in the static FEA analysis, the basic conventional geometries are considered for harmonic analyses to understand the frequency response of the thin diaphragms, which is a primary sensing element for microphone applications and many more. This work provides a solution to microelectromechanical system (MEMS) developers for reducing cost and time while conceptualizing TDMS designs.

Part II: Impedance-based DNA biosensor for detection of isolated strains of phytopathogen Ralstonia solanacearum.

In Part I, we demonstrated the complete development of a label-free, ultra-low sample volume requiring DNA-based biosensor to detect Ralstonia solanacearum, an aerobic non-spore-forming, Gram-negative, plant pathogenic bacterium, using non-faradaic electrochemical impedance spectroscopy (nf-EIS). We also presented the sensor's sensitivity, specificity, and electrochemical stability. In this article, we highlight the specificity study of the developed DNA-based impedimetric biosensor to detect various strains of R. solanacearum. We have collected seven isolates of R. solanacearum isolated from locally infected host plants (eggplant, potato, tomato, chilli, and ginger) from different parts of Goa, India. The pathogenicity of these isolates was tested on the eggplant, and the pathogen was confirmed by microbiological plating and polymerase chain reaction (PCR). We further report the insight into the DNA hybridization on the surface of Interdigitated Electrodes (IDEs) and the expansion of the Randles model for more accurate analysis. The interpretation of the sensor specificity is clearly demonstrated by the capacitance change observed at the electrode-electrolyte interface.

Part I: Non-faradaic electrochemical impedance-based DNA biosensor for detecting phytopathogen - Ralstonia solanacearum.

Herein, we report for the first time the development of a label-free, non-faradaic, and highly sensitive DNA-based impedimetric sensor using micro-sized gold interdigitated electrodes (IDE) to detect a soil-borne agricultural pathogen Ralstonia solanacearum. A universal 30 oligomer single-stranded DNA (ssDNA) probe lpxC4 having specificity towards R. solanacearum is successfully immobilized on the surface of IDE along with mercaptohexanol. The electrochemical stability of the developed sensor surface is determined using open circuit potential measurements. The DNA probe immobilization protocol is validated using the changes configured on the surface of IDE by contact angle and ATR-FTIR analysis. The DNA target hybridization is detected using non-faradaic electrochemical impedance spectroscopy measurement with an ultra-low sample volume of 10 µL. The non-faradaic approach is verified by studying redox behavior using cyclic voltammetry. We investigate the hybridization of the surface-immobilized label-free probe with the complementary DNA targets obtained from infected eggplant saplings and cross-reactive studies with mismatched DNA strands. Our impedimetric sensor can detect target concentrations as low as 0.1 ng/µL. This standardization and detection of DNA hybridization serves as part I of the work and paves the way for further study in the detection of pathogenic field samples.

PSA detection using label free graphene FET with coplanar electrodes based microfluidic point of care diagnostic device.

Affordable point-of-care (PoC) diagnostic devices enable detection of prostate specific antigen (PSA) in resource limited settings. Despite the advancements in PoC systems, most of the reported methods for PSA detection have unsatisfactory detection limits and are based on labelled assays, requiring multiple reagent flow steps which increases both expenses and inconvenience. Circumventing these constraints, we report here the development and validation of a label free, affordable dielectrophoresis (DEP) based graphene field effect transistor (FET) sensor implemented using coplanar electrodes and integrated uniquely with a compact disc based microfluidic platform along with electronics readout for the estimation of PSA at the point of care. Design of coplanar gate electrode which has not been explored earlier is not a straightforward approach. In fact, it has been observed that there is a non-monotonic dependence of the capture of PSA molecules in the channel region of the FET with varying widths and spacings of the gate electrode. The graphene FET based PoC device with optimized coplanar gate electrode is the only label free analytical system for PSA detection requiring simple operation and achieving a detection limit of 1 pg/ml in serum with a wide dynamic range upto 4 ng/ml and appreciable selectivity against potential interferents like bovine serum albumin (BSA) and human immunoglobulin G (IgG). Further, it has been validated satisfactorily with commercially available existing systems using human serum samples. Moreover, the proposed sensing system lowers the detection limit by three orders of magnitude compared to a recent study on label free PoC device on other cancer biomarkers.

Synthesis of colloidal silver nanoparticle clusters and their application in ascorbic acid detection by SERS.

Ascorbic acid (vitamin C) has an essential role in the human body mainly due to its antioxidant function. In this work, metallic silver nanoparticle (AgNP) colloids were used in SERS experiments to detect ascorbic acid in aqueous solution. The AgNPs were synthesized by a green method using potato starch as reducing and stabilizing agent, and water as the solvent. The optical properties of the yellowish as-synthesized silver colloids were characterized by UV-vis spectroscopy, in which besides a typical band at 410 nm related to the localized surface plasmon resonance of the silver nanoparticles, a shoulder band around 500 nm, due to silver nanoparticle cluster formation, is presented when relatively higher concentrations of starch are used in the synthesis. These starch-capped silver nanoparticles show an intrinsic Raman peak at 1386 cm-1 assigned to deformation modes of the starch structure. The increase of the intensity of the SERS peak at 1386 cm-1 with an increase in the concentration of the ascorbic acid is related to a decrease of the gap between dimers and trimers of the silver nanoparticle clusters produced by the presence of ascorbic acid in the colloid. The limit of detection of this technique for ascorbic acid is 0.02 mM with a measurement concentration range of 0.02-10 mM, which is relevant for the application of this method for detecting ascorbic acid in biological specimen.

Glassy carbon microneedles-new transdermal drug delivery device derived from a scalable C-MEMS process.

Because carbon is the basic element of all life forms and has been successfully applied as a material for medical applications, it is desirable to investigate carbon for drug delivery applications, as well. In this work, we report the fabrication of a hollow carbon microneedle array with flow channels using a conventional carbon-microelectromechanical system (C-MEMS) process. This process utilizes the scalable and irreversible step of pyrolysis, where prepatterned SU-8 microneedles (precursor) are converted to glassy carbon structures in an inert atmosphere at high temperature (900 °C) while retaining their original shape upon shrinkage. Once converted to glassy carbon, the microneedles inherit the unique properties of hardness, biocompatibility, and thermal and chemical resistance associated with this material. A comparative study of hardness and Young's modulus for carbon microneedles and SU-8 microneedles was performed to evaluate the increased strength of the microneedles induced by the C-MEMS process steps. Structural shrinkage of the carbon microneedles upon pyrolysis was observed and estimated. Material characterizations including energy-dispersive X-ray spectroscopy (EDX) and Raman spectroscopy were carried out to estimate the atomic percentage of carbon in the microneedle structure and its crystalline nature, respectively. Our investigations confirm that the microneedles are glassy in nature. Compression and bending tests were also performed to determine the maximum forces that the carbon microneedles can withstand, and it was found that these forces were approximately two orders of magnitude higher than the resistive forces presented by skin. A microneedle array was inserted into mouse skin multiple times and was successfully removed without the breakage of any microneedles.

Fabrication of 3D Carbon Microelectromechanical Systems (C-MEMS).

A wide range of carbon sources are available in nature, with a variety of micro-/nanostructure configurations. Here, a novel technique to fabricate long and hollow glassy carbon microfibers derived from human hairs is introduced. The long and hollow carbon structures were made by the pyrolysis of human hair at 900 °C in a N2 atmosphere. The morphology and chemical composition of natural and pyrolyzed human hairs were investigated using scanning electron microscopy (SEM) and electron-dispersive X-ray spectroscopy (EDX), respectively, to estimate the physical and chemical changes due to pyrolysis. Raman spectroscopy was used to confirm the glassy nature of the carbon microstructures. Pyrolyzed hair carbon was introduced to modify screen-printed carbon electrodes ; the modified electrodes were then applied to the electrochemical sensing of dopamine and ascorbic acid. Sensing performance of the modified sensors was improved as compared to the unmodified sensors. To obtain the desired carbon structure design, carbon micro-/nanoelectromechanical system (C-MEMS/C-NEMS) technology was developed. The most common C-MEMS/C-NEMS fabrication process consists of two steps: (i) the patterning of a carbon-rich base material, such as a photosensitive polymer, using photolithography; and (ii) carbonization through the pyrolysis of the patterned polymer in an oxygen-free environment. The C-MEMS/NEMS process has been widely used to develop microelectronic devices for various applications, including in micro-batteries, supercapacitors, glucose sensors, gas sensors, fuel cells, and triboelectric nanogenerators. Here, recent developments of a high-aspect ratio solid and hollow carbon microstructures with SU8 photoresists are discussed. The structural shrinkage during pyrolysis was investigated using confocal microscopy and SEM. Raman spectroscopy was used to confirm the crystallinity of the structure, and the atomic percentage of the elements present in the material before and after pyrolysis was measured using EDX.