Toward a Label-Free Electrochemical Impedance Immunosensor Design for Quantifying Cortisol in Tears.

Cortisol is a viable biomarker for monitoring physiological, occupational, and emotional stress and is normally present in tear fluid at approximately 40 nM, or higher as a result of stress. We present characterization and quantification of cortisol via several electrochemical methods versus the standard enzyme-linked immunosorbent assay, commonly known as ELISA. We also present a prototyped design of a disposable test strip and handheld sensor based on label-free electrochemical impedance spectroscopy to quantify cortisol levels in tear fluid within approximately 90 seconds. Electrochemical characterization of the cortisol molecule was conducted using cyclic voltammetry, amperometric i-t, and square wave voltammetry. Lower limits of detection for these techniques were not sufficient to quantify cortisol and phycological tear ranges: 0.1 M, 0.23 M, and 193 M for cyclic voltammetry, amperometric i-t, and square wave voltammetry, respectively. However, electrochemical impedance spectroscopy (EIS) was to be the best mode of cortisol quantification and comparison to ELISA technique (detection range of ~ 138 pM - 552 nM). The initial EIS biosensor obtained a lower limit of detection of 59.76 nM with an approximate 10% relative standard deviation. The cortisol assay and tear collection prototype presented here offer a highly reproducible and ultra-low level of detection with a label-free and rapid response.

Multi-Biomarker Detection Following Traumatic Brain Injury.

The Centers for Disease Control and Prevention estimates almost two million traumatic brain injuries (TBIs) occur annually in the U.S., resulting in nearly $80 billion of economic burden. Despite its prevalence, current TBI diagnosis methods mainly rely on cognitive assessments vulnerable to subjective interpretation, thus highlighting the critical need to develop effective unbiased diagnostic methods. The presented study aims to assess the feasibility of a rapid multianalyte TBI blood diagnostic. Specifically, two electrochemical impedance techniques were used to evaluate four biomarkers: glial fibrillary acidic protein, neuron specific enolase (NSE), S-100ß, and tumor necrosis factor-α. First, these biomarkers were characterized in purified solutions (detection limit, DL = 2-5 pg/mL), then verified in spiked whole blood and plasma solutions (90% whole blood DL = 14-67 pg/mL). Finally, detection of two of these biomarkers was validated in a controlled cortical impact model of TBI in rats, where a statistical difference between NSE and S-100ß concentrations differed several days postinjury (p = 0.02 and p = 0.06, respectively). A statistical difference between mild and moderate injury was found at the various time points. The proposed diagnostic method enabled preliminary quantification of TBI-relevant biomarkers in complex media without the use of expensive electrode coatings or membranes. Collectively, these data demonstrate the feasibility of using electrochemical impedance techniques to rapidly detect TBI biomarkers and lay the groundwork for development of a novel method for quantitative diagnostics of TBI.

Direct Measurement of a Biomarker's Native Optimal Frequency with Physical Adsorption Based Immobilization.

The optimal frequency (OF) of a biomarker in electrochemical impedance spectroscopy (EIS) is the frequency at which the EIS response best reflects the binding of the biomarker to its molecular recognition element. Commonly, biosensors rely on complicated immobilization chemistry to attach biological molecules to the sensor surface, making the direct study of a biomarker's native OF a challenge. Physical adsorption presents a simple immobilization strategy to study the native biomarker's OF, but its utility is often discouraged due to a loss in biological activity. To directly study a biomarker's native OF and investigate the potential of OF to overcome the limitations of physical adsorption, a combination of EIS and glutaraldehyde-mediated physical adsorption was explored. The experimental sensing platform was prepared by immobilizing either anti-lactoferrin (Lfn) IgG or anti-immunoglobulin E (IgE) onto screen printed carbon electrodes. After characterizing the native OFs of both biomarkers, investigation of the platform's specificity, stability, and performance in complex medium was found to be sufficient. Finally, a paper-based tear sampling component was integrated to transform the testing platform into a prototypical point-of-care dry eye diagnostic. The investigation of native OFs revealed a correlation between the native OFs (57.44 and 371.1 Hz for Lfn and IgE, respectively) and the molecular weight of the antibody-antigen complex. Impedance responses at the native OFs have enabled detection limits of 0.05 mg/mL and 40 ng/mL for Lfn and IgE, respectively, covering the clinically relevant ranges. The native OFs were found to be robust across various testing mediums and conditions.

Enzymatic Detection of Traumatic Brain Injury Related Biomarkers.

Electrochemical detection methods have been popular in the medical diagnostics field. Several well-known devices such as the self-monitoring blood glucose meter have relied on electrochemical techniques for their sensitivity, and ability to make direct measurements without optical labels. Currently, no point-of-care or handheld diagnostic tool exists to quantify the severity of a traumatic brain injury (TBI). We have shown that enzymatic detection of norepinephrine (NE), a biomarker which can indicate TBI severity, using impedance-based electrochemical techniques can achieve the required sensitivity, ∼100 pg/mL. Furthermore, the first steps have been taken to quantify NE in whole blood solutions and to optimize the technique for a handheld device.