Jeffrey T. La Belle

A methodology for rapid detection of Salmonella typhimurium using label-free electrochemical impedance spectroscopy.

A pathogen detection methodology based on Bayesian decision theory has been developed for rapid and reliable detection of Salmonella typhimurium. The methodology exploits principles from statistical signal processing along with impedance spectroscopy in order to analytically determine the existence of pathogens in the target solution. The proposed technique is validated using a cost-effective and portable immunosensor. This device uses label-free, electrochemical impedance spectroscopy for pathogen detection and has been demonstrated to reliably detect pre-infectious levels of pathogen in sample solutions. The detection process does not entail any pathogen enrichment procedures. The results using the proposed technique indicate a detection time of approximately 6min (5min for data acquisition, 1min for analysis) for pathogen concentrations in the order of 500CFU/ml. The detection methodology presented here has demonstrated high accuracy and can be generalized for the detection of other pathogens with healthcare, food, and environmental implications. Furthermore, the technique has a low computational complexity and uses a minimal data-set (only 30 data-samples) for data analysis. Hence, it is ideal for use in hand-held pathogen detectors.

Integrated explosive preconcentrator and electrochemical detection system for 2,4,6-trinitrotoluene (TNT) vapor

This article reports on an integrated explosive-preconcentration/electrochemical detection system for 2,4,6-trinitrotoluene (TNT) vapor. The challenges involved in such system integration are discussed. A hydrogel-coated screen-printed electrode is used for the detection of the thermally desorbed TNT from a preconcentration device using rapid square wave voltammetry. Optimization of the preconcentration system for desorption of TNT and subsequent electrochemical detection was conducted yielding a desorption temperature of 120 degrees C under a flow rate of 500 mL min(-1). Such conditions resulted in a characteristic electrochemical signal for TNT representing the multi-step reduction process. Quantitative measurements produced a linear signal dependence on TNT quantity exposed to the preconcentrator from 0.25 to 10 microg. Finally, the integrated device was successfully demonstrated using a sample of solid TNT located upstream of the preconcentrator.

A disposable tear glucose biosensor - Part 1: Design and concept testing

Background: Tear glucose has been suggested previously as a potential approach for the noninvasive estimation of blood glucose. While the topic remains unresolved, an overview of previous studies suggests the importance of a tear sampling approach and warrants new technology development. A concept device is presented that meets the needs of a tear glucose biosensor. Methods: Three approaches to chronoamperometric glucose sensing were evaluated, including glucose oxidase mediated by potassium ferricyanide or oxygen with a hydrogen peroxide catalyst, Prussian blue, and potassium ferricyanide-mediated glucose dehydrogenase. For tear sampling, calcium alginate, poly(2-hydroxyethyl methacrylate), and polyurethane foam were screened as an absorbent tear sampling material. A quantitative model based on the proposed function of concept device was created. Results: For glucose sensing, it was found that potassium ferricyanide with glucose dehydrogenase was ideal, featuring oxygen insensitivity, long-term stability, and a lower limit of detection of 2 μM glucose. Polyurethane foam possessed all of the required characteristics for tear sampling, including reproducible sampling from a hydrogel-simulated, eye surface (4.2 ± 0.5 μl; n = 8). It is estimated that 100 μM of glucose tear fluid would yield 135 nA (14.9% relative standard deviation). Conclusion: A novel concept device for tear glucose sampling was presented, and the key functions of this device were tested and used to model the performance of the final device. Based on these promising initial results, the device is achievable and within reach of current technical capabilities, setting the stage for prototype development.

A cytokine immunosensor for Multiple Sclerosis detection based upon label-free electrochemical impedance spectroscopy using electroplated printed circuit board electrodes.

A biosensor for the serum cytokine, Interleukin-12 (IL-12), based upon a label-free electrochemical impedance spectroscopy (EIS) monitoring approach is described. Overexpression of IL-12 has been correlated to the diagnosis of Multiple Sclerosis (MS). An immunosensor has been fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode and immobilizing anti-IL-12 monoclonal antibodies (MAb) onto the surface of the electrode. This approach yields a robust sensor that facilitates reproducible mass fabrication and easy alteration of the electrode shape. Results indicate that this novel PCB sensor can detect IL-12 at physiological levels, <100 fM with f-values of 0.05 (typically <0.0001) in a label-free and rapid manner. A linear (with respect to log concentration) detectable range was achieved. Detection in a complex biological solution is also explored; however, significant loss of dynamic range is noted in the 100% complex solution. The cost effective approach described here can be used potentially for diagnosis of diseases (like MS) with known biomarkers in body fluids and for monitoring physiological levels of biomolecules with healthcare, food, and environmental relevance.

Development of a novel single sensor multiplexed marker assay

There is an increasing desire to measure multiple analytes simultaneously for disease management and detection. However, in the case of invasive devices, it would be better to obtain one small sample and immediately be able to detect the analytes rapidly, as in the case of self-monitoring blood glucose, without the need for additional steps, arrays, or reagents. Electrochemical impedance spectroscopy is used to measure the interaction between ultralow levels of analyte and molecular recognition element in a label-free and rapid manner. Gold nanoparticles were attached to antibodies against interleukin-12 and tumor necrosis factor-α, typical inflammatory markers found with near overlapping responses, on an impedance spectroscopy based biosensor. Cross-reactivity and specificity of tuned antibodies were verified using ELISA. Impedance frequency was quantified by concentration gradients of marker against the device. The natural impedance frequency for interleukin-12 (5.00 Hz) was tuned to a lower frequency four Hertz away from one another for better signal processing. This was accomplished without significantly altering the lower limits of detection (<4 pg ml(-1) and ∼60 pg ml(-1) for interleukin-12 and tumor necrosis factor-α, respectively), no cross-reactivity and specificity as determined by ELISAs. With modeling the nanoscale effects and further development, a larger tuning will be possible for making a better multiplexed sensor. Although interleukin-12 and TNF-α equivalent circuit calculations were modeled here, a sensor with the potential to measure multiple markers at once might offer a solution on the sensor front for simplified management of conditions such as diabetes, where both glucose and hemoglobin A1c values could be obtained.

Mesoporous carbon amperometric glucose sensors using inexpensive, commercial methacrylate-based binders

Two ordered, soft-templated mesoporous carbon powders with cubic and hexagonal framework structure and four different commercial, low cost methacrylate-based polymer binders with widely varying physical properties are investigated as screen printed electrodes for glucose sensors using glucose oxidase and ferricyanide as the mediator. Both the chemistry and concentration of the binder in the electrode formulation can significantly impact the performance. Poly(hydroxybutyl methacrylate) as the binder provides hydrophilicity to enable transport of species in the aqueous phase to the carbon surface, but yet is sufficiently hydrophobic to provide mechanical robustness to the sensor. The current from the mesoporous carbon electrodes can be more than an order of magnitude greater than for a commercial printed carbon electrode (Zensor) with improved sensitivity for model glucose solutions. Even when applying these sensors to rabbit whole blood, the performance of these glucose sensors compares favorably to a standard commercial glucose meter with the lower detection limit of the mesoporous electrode being approximately 20mgdL(-1) despite the lack of a separation membrane to prevent non-specific events; these results suggest that the small pore sizes and high surface areas associated with ordered mesoporous carbons may effectively decrease some non-specific inferences for electrochemical sensing.

A Disposable Tear Glucose Biosensor—Part 3: Assessment of Enzymatic Specificity

Background: A concept for a tear glucose sensor based on amperometric measurement of enzymatic oxidation of glucose was previously presented, using glucose dehydrogenase flavin adenine dinucleotide (GDH-FAD) as the enzyme. Glucose dehydrogenase flavin adenine dinucleotide is further characterized in this article and evaluated for suitability in glucose-sensing applications in purified tear-like saline, with specific attention to the effect of interfering substances only. These interferents are specifically saccharides that could interact with the enzymatic activity seen in the sensors performance. Methods: Bench top amperometric glucose assays were performed using an assay solution of GDH-FAD and ferricyanide redox mediator with samples of glucose, mannose, lactose, maltose, galactose, fructose, sucrose, and xylose at varying concentrations to evaluate specificity, linear dynamic range, signal size, and signal-to-noise ratio. A comparison study was done by substituting an equivalent activity unit concentration of glucose oxidase (GOx) for GDH-FAD. Results: Glucose dehydrogenase flavin adenine dinucleotide was found to be more sensitive than GOx, producing larger oxidation currents than GOx on an identical glucose concentration gradient, and GDH-FAD exhibited larger slope response (-5.65 × 10−7 versus −3.11 × 10−7 A/mM), signal-to-noise ratio (18.04 versus 2.62), and linear dynamic range (0–30 versus 0–10 mM), and lower background signal (−7.12 versus −261.63 nA) than GOx under the same assay conditions. GDH-FAD responds equally to glucose and xylose but is otherwise specific for glucose. Conclusion: Glucose dehydrogenase flavin adenine dinucleotide compares favorably with GOx in many sensor-relevant attributes and may enable measurement of glucose concentrations both higher and lower than those measurable by GOx. GDH-FAD is a viable enzyme to use in the proposed amperometric tear glucose sensor system and perhaps also in detecting extreme hypoglycemia or hyperglycemia in blood.

A Disposable Tear Glucose Biosensor—Part 2: System Integration and Model Validation

Background: We presented a concept for a tear glucose sensor system in an article by Bishop and colleagues in this issue of Journal of Diabetes Science and Technology. A unique solution to collect tear fluid and measure glucose was developed. Individual components were selected, tested, and optimized, and system error modeling was performed. Further data on prototype testing are now provided. Methods: An integrated fluidics portion of the prototype was designed, cast, and tested. A sensor was created using screen-printed sensors integrated with a silicone rubber fluidics system and absorbent polyurethane foam. A simulated eye surface was prepared using fluid-saturated poly(2-hydroxyethyl methacrylate) sheets, and the disposable prototype was tested for both reproducibility at 0, 200, and 400 μM glucose (n = 7) and dynamic range of glucose detection from 0 to 1000 μM glucose. Results: From the replicated runs, an established relative standard deviation of 15.8% was calculated at 200 μM and a lower limit of detection was calculated at 43.4 μM. A linear dynamic range was demonstrated from 0 to 1000 μM with an R 2 of 99.56%. The previously developed model predicted a 14.9% variation. This compares to the observed variance of 15.8% measured at 200 μM glucose. Conclusion: With the newly designed fluidics component, an integrated tear glucose prototype was assembled and tested. Testing of this integrated prototype demonstrated a satisfactory lower limit of detection for measuring glucose concentration in tears and was reproducible across a physiological sampling range. The next step in the device design process will be initial animal studies to evaluate the current prototype for factors such as eye irritation, ease of use, and correlation with blood glucose.

Reliable, rapid and simple voltammetric detection of urea nitrate explosive.

A highly selective and rapid electrochemical assay of the improvised explosive urea nitrate (UN) is reported. The method involves a short ( approximately 10 s) acid-catalyzed reaction of UN with 4-nitrotoluene (NT) followed by a rapid ( approximately 2 s) square-wave voltammetric (SWV) detection of the 2,4-dinitrotoluene (DNT) product. The new protocol offers great promise for a reliable field detection of UN, with significant advantages of speed, sensitivity, portability, simplicity, and cost.

A label-free, rapid multimarker protein impedance-based immunosensor

A multiplexing, multimarker protein immunosensor based upon electrochemical impedance spectroscopy was designed, fabricated, and analyzed. The antibody based molecular recognition element sensor was demonstrated using five inflammatory cytokines and receptors (Figure 1). These pro- and anti-inflammatory markers are used in cellular signaling and could be useful in early detection of diseases including Multiple Sclerosis, cancer, or pathogenic infection. The disposable strip of five sensors was characterized over four orders of magnitude of target protein concentration. The assay takes only 90 seconds from sample introduction to impedance output in a label-free manner. The goal of this work is to develop a platform technology that can be made reproducible, scalable, and efficient for high throughput screening for proteomic markers of disease. These sensors detect the markers at the respective physiological ranges found in vivo, albeit in purified fashion. Electrochemical Impedance Spectroscopy (AC impedance) was chosen as the appropriate (label-free and rapid) approach resulting in signals that are discernible using a simple, rapid strip sensor platform that is readily scalable to accommodate future high throughput screening. This scalability could allow for the detection of 100s of proteins, or the antibody immunosignatures themselves.