Enzyme Catalysis Causes Fluid Flow, Motility, and Directional Transport on Supported Lipid Bilayers.

The dynamic interplay between the composition of lipid membranes and the behavior of membrane-bound enzymes is critical to the understanding of cellular function and viability, and the design of membrane-based biosensing platforms. While there is a significant body of knowledge about how lipid composition and dynamics affect membrane-bound enzymes, little is known about how enzyme catalysis influences the motility and lateral transport on lipid membranes. Using enzyme-attached lipids in supported bilayers (SLBs), we provide direct evidence of catalysis-induced fluid flow that underlies the observed motility on SLBs. Additionally, by using active enzyme patches, we demonstrate the directional transport of tracer particles on SLBs. As expected, enhancing the membrane viscosity by incorporating cholesterol into the bilayer suppresses the overall movement. These are the first steps in understanding diffusion and transport on lipid membranes due to active, out-of-equilibrium processes that are the hallmark of living systems. In general, our study demonstrates how active enzymes can be used to control diffusion and transport in confined 2-D environments.

Interactions with amyloid beta peptide and acetylcholinesterase increase alkaline phosphatase activity.

Multiple studies have shown that the activity of alkaline phosphatase (AP) increases during Alzheimer's disease (AD). In this paper, using UV-Visible spectroscopy, we show that this increase in activity is due to its interaction with key components of AD such as amyloid ß peptide and acetylcholinesterase. Activity increase also occurs due to high concentrations of acetylcholine and choline. These conditions are present in AD or could occur due to drugs used for treating AD.

MEASURE: Multiplex Exhaled Breath Condensate - Scanning Using Rapid Electro-Analytics.

Exhaled breath condensate is an emerging source of inflammatory biomarkers suitable for the noninvasive detection of respiratory disorders. Current gold standard methods are highly invasive and pose challenges in sample collection during airway inflammation monitoring. Cytokine biomarkers are detectable in EBC at increased or decreased concentrations. IL-6, IL-1ß, IL-8, and hs-CRP are characteristic biomarkers identified in respiratory disorders. We have demonstrated the promising outcomes of a 16-plexed electrochemical platform - READ 2.0 for the multiplexed detection of characteristic biomarkers in EBC. The sensor demonstrates dynamic ranges of 1-243 pg/mL with a lower detection limit of 1 pg/mL for IL-6 and IL-1ß, while the detection range and limit of detection for IL-8 and hs-CRP is 1-150 pg/mL and 3 pg/mL, respectively. The detection accuracies for the biomarkers are in the range of ∼85 ± 15% to ∼100 ± 10%. The sensor shows a nonspecific response to similar cross-reacting biomarkers. Analytical validation of the sensor with ELISA as the standard reference generated a correlation of R2 > 0.96 and mean biases of 10.9, 3.5, 17.4, and 3.9 pg/mL between the two methods for IL-6, IL-1ß, IL-8, and hs-CRP, respectively. The precision of the sensor in detecting low biomarker concentrations yields a %CV of <7%. The variation in the sensor's response on repeat EBC sample measurements and within a 6 h duration is less than 10%. The READ 2.0 platform shows a promise that EBC-based biomarker detection can prove to be vital in predicting the severity and survival rates of respiratory disorders and serve as a reference point for monitoring EBC-based biomarkers.

EBC-SURE (exhaled breath condensate- scanning using rapid electro analytics): A non-faradaic and non-invasive electrochemical assay to screen for pro-inflammatory biomarkers in human breath condensate.

The innovation of this work lies in the trace detection of inflammatory biomarkers (IL-6, hs-CRP) in human exhaled breath condensate on the developed EBC-SURE platform as a point-of-care aid for respiratory disorder diagnosis. The unique design of the EBC-SURE leverages non-faradaic electrochemical impedance spectroscopy to capture target-specific biomolecular interactions for highly sensitive biomarker detection. For sensor calibration, EBC-SURE's performance is assessed to measure the response of the sensor to a known concentration by spike and recovery analysis with a recovery error of <20% and an extended dynamic range over 3-log orders. The lowest detection limits for IL-6 and hs-CRP detection in EBC were found to be 3.2 pg/mL and 4 pg/mL respectively. The intra-assay and inter-assay efficacy of EBC-SURE for its usage as a diagnostic device was established through repeatability and reproducibility (over 48 h s) performance testing. The percentage variations (<20%) met the Clinical and Laboratory Standards Institute standards (CLSI) indicating a highly stable performance for robust biomarker detection. EBC-SURE generated highly selective IL-6 and hs-CRP responses in the presence of other non-specific cytokines. Statistical validation methods- Correlation and Bland Altman analysis established the one-to-one agreement between EBC-SURE and the reference method. Correlation analysis generated a Pearson's R value of 0.99 for IL-6 and hs-CRP. Bland-Altman analysis indicated a good agreement between both the methods with all data points confined within the ±2SD limits. We have demonstrated EBC-SURE's ability in detecting inflammatory biomarkers in human breath condensate towards developing a non-invasive technology that can quantify biomarker levels associated with healthy and acute inflammatory conditions.

Enzyme aggregation and fragmentation induced by catalysis relevant species.

It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.

On-demand lactate monitoring towards assessing physiological responses in sedentary populations.

Identification of diseases in sedentary populations on a timely basis before reaching a critical stage is a continuing challenge faced by emergency care centers. Lactate is a key biomarker for monitoring restricted oxygen supply essential for assessing the physiological responses of the user for clinical diagnostics. The novelty of this work is the development of a non-invasive, mediator-free, stick and remove biosensor for the on-demand measurement of lactate in passive sweat targeted towards sedentary populations. The conformable interface of the biosensors with skin can be engineered to extract relevant biochemical signals and quantify the in situ sweat biomarker levels. In this work, we demonstrate a highly sensitive and specific on-demand biosensor with a fabricated hybrid nanotextured Au/ZnO electrode stack embedded within a flexible nanoporous material to capture the temporal dynamics of passive sweat lactate. The biosensor exhibits a lactate specific response in human sweat with a 1 mM lower limit of detection and a wide dynamic detection range of 1-100 mM (R2 = 0.98). The proposed biosensor has a sensitivity of 8.3% mM-1 while selectivity studies reveal negative interactions with non-specific molecules. The sensor stability studies showed an ∼30% degradation in the lactate biosensing response over a 4-day duration when stored at 4 °C. Non-faradaic electrochemical spectroscopy is employed as the detection modality to quantify the enzymatic catalysis of sweat lactate at the electrode-sweat interface. Spectroscopic characterization techniques such as XPS, ATR-FTIR, and zeta potential measurements confirm the enzymatic assay binding efficacy on a qualitative scale.

Next-Generation Continuous Metabolite Sensing toward Emerging Sensor Needs.

This article discusses the emergent biosensor technology focused on continuous biosensing of metabolites by non-invasive sampling of body fluids emphasized on physiological monitoring in mobility-constrained populations, resource-challenged settings, and harsh environments. The boom of innovative ideas and endless opportunities in healthcare technologies has transformed traditional medicine into a sustainable link between medical practitioners and patients to provide solutions for faster disease diagnosis. The future of healthcare is focused on empowering users to manage their own health. The confluence of big data and predictive analysis and the internet of things (IoT) technology have shown the potential of converting the abundant health profile data amassed from medical diagnosis of patients into useable information, whilst allowing caregivers to provide suitable treatment plans. The implementation of the IoT technology has opened up advanced approaches in real-time, continuous, remote monitoring of patients. Wearable, point-of-care biosensors are the future roadmap to providing direct, real-time information of health status to the user and medical professionals in this digitized era.

CLIP: Carbon Dioxide testing suitable for Low power microelectronics and IOT interfaces using Room temperature Ionic Liquid Platform.

Health and safety considerations of room occupants in enclosed spaces is crucial for building management which entails control and stringent monitoring of CO2 levels to maintain acceptable air quality standards and improve energy efficiency. Smart building management systems equipped with portable, low-power, non-invasive CO2 sensing techniques can predict room occupancy detection based on CO2 levels exhaled by humans. In this work, we have demonstrated the development and proof-of-feasibility working of an electrochemical RTIL- based sensor prototype for CO2 detection in exhaled human breath. The portability, small form factor, embedded RTIL sensing element, integrability with low-power microelectronic and IOT interfaces makes this CO2 sensor prototype a potential application for passive room occupancy monitoring. This prototype exhibits a wide dynamic range of 400-8000 ppm, a short response time of ~10 secs, and a reset time of ~6 secs in comparison to commercial standards. The calibration response of the prototype exhibits an R2 of 0.956. With RTIL as the sensing element, we have achieved a sensitivity of 29 pF/ppm towards CO2 at ambient environmental conditions and a three times greater selectivity towards CO2 in the presence of N2 and O2. CO2 detection is accomplished by quantifying the capacitance modulations arising within the electrical double layer from the RTIL- CO2 interactions through AC- based electrochemical impedance spectroscopy and DC- based chronoamperometry.

Point-of-use sweat biosensor to track the endocrine-inflammation relationship for chronic disease monitoring.

AIM: The hypothalamic-pituitary-adrenal axis is involved in maintaining homeostasis by engaging with the parasympathetic nervous system. During the process of disease affliction, this relationship is disturbed and there is an imbalance driven response observed. MATERIALS & METHODS: By monitoring the two key components involved in these pathways, cortisol and TNF-α, the manifestations of chronic stress on the body's homeostasis can be evaluated in a comprehensive manner. This work highlights the development of an electrochemical detection system for the two biomarkers through human sweat. RESULTS: Limit of detection and dynamic ranges are 1 ng/ml, 1-200 ng/ml for cortisol and 1 pg/ml, 1-1000 pg/ml for TNF-α. CONCLUSION: This wearable system is designed to be a point of use, chronic disease self-monitoring and management platform.

A Rapid Response Electrochemical Biosensor for Detecting Thc In Saliva.

Marijuana is listed as a Schedule I substance under the American Controlled Substances Act of 1970. As more U.S. states and countries beyond the U.S. seek legalization, demands grow for identifying individuals driving under the influence (DUI) of marijuana. Currently no roadside DUI test exists for determining marijuana impairment, thus the merit lies in detecting the primary and the most sought psychoactive compound tetrahydrocannabinol (THC) in marijuana. Salivary THC levels are correlated to blood THC levels making it a non-invasive medium for rapid THC testing. Affinity biosensing is leveraged for THC biomarker detection through the chemical reaction between target THC and THC specific antibody to a measure signal output related to the concentration of the targeted biomarker. Here, we propose a novel, rapid, electrochemical biosensor for the detection of THC in saliva as a marijuana roadside DUI test with a lower detection limit of 100 pg/ml and a dynamic range of 100 pg/ml - 100 ng/ml in human saliva. The developed biosensor is the first of its kind to utilize affinity-based detection through impedimetric measurements with a rapid detection time of less than a minute. Fourier transform infrared spectroscopy analysis confirmed the successful immobilization of the THC immobilization assay on the biosensing platform. Zeta potential studies provided information regarding the stability and the electrochemical behavior of THC immunoassay in varying salivary pH buffers. We have demonstrated stable, dose dependent biosensing in varying salivary pH's. A binary classification system demonstrating a high general performance (AUC = 0.95) was employed to predict the presence of THC in human saliva. The biosensor on integration with low-power electronics and a portable saliva swab serves as a roadside DUI hand-held platform for rapid identification of THC in saliva samples obtained from human subjects.

Ultrasensitive and Rapid-Response Sensor for the Electrochemical Detection of Antibiotic Residues within Meat Samples.

Antimicrobial use in livestock has emerged as a pressing global issue because of the rise of antimicrobial-resistant bacteria. Regulatory authorities across the globe have taken steps to discourage the misuse of these antibiotics by banning or limiting the use of medically important antibiotics in food animals. However, to ensure that food animals are not being administered antibiotics inappropriately, there is a need for a reliable, raid-response biosensor that can detect the presence of these antibiotic residuals in meat products. We have developed an affinity-based electrochemical biosensor for the label-free detection of ceftiofur residues in meat samples. The sensor uses a self-assembled immunoassay to target the ceftiofur biomarker by employing electrochemical impedance spectroscopy to probe the interfacial capacitive changes as ceftiofur binds to the sensor surface. We have demonstrated a platform that can detect ceftiofur within 15 min of introducing the sample at concentrations down to 0.01 ng/mL in 1× phosphate-buffered saline and 10 ng/mL in 220 mg ground turkey meat samples.

Enzymatic Low Volume Passive Sweat Based Assays for Multi-Biomarker Detection.

Simultaneous detection of correlated multi-biomarkers on a single low-cost platform in ultra-low fluid volumes with robustness is in growing demand for the development of wearable diagnostics. A non-faradaic biosensor for the simultaneous detection of alcohol, glucose, and lactate utilizing low volumes (1⁻5 µL) of sweat is demonstrated. Biosensing is implemented using nanotextured ZnO films integrated on a flexible porous membrane to achieve enhanced sensor performance. The ZnO sensing region is functionalized with enzymes specific for the detection of alcohol, glucose, and lactate in the ranges encompassing their physiologically relevant levels. A non-faradaic chronoamperometry technique is used to measure the current changes associated with interactions of the target biomarkers with their specific enzyme. The specificity performance of the biosensing platform was established in the presence of cortisol as the non-specific molecule. Biosensing performance of the platform in a continuous mode performed over a 1.5-h duration showed a stable current response to cumulative lifestyle biomarker concentrations with capability to distinguish reliably between low, mid, and high concentration ranges of alcohol (0.1, 25, 100 mg/dL), glucose (0.1, 10, 50 mg/dL), and lactate (1, 50, 100 mM). The low detection limits and a broader dynamic range for the lifestyle biomarker detection are quantified in this research demonstrating its suitability for translation into a wearable device.

CLASP (Continuous lifestyle awareness through sweat platform): A novel sensor for simultaneous detection of alcohol and glucose from passive perspired sweat.

Wearable- IOT based low- cost platforms can enable dynamic lifestyle monitoring through enabling promising and exciting opportunities for wellness and chronic- disease management in personalized environments. Diabetic and pre- diabetic populations can modulate their alcohol intake by tracking their glycemic content continuously to prevent health risks through these platforms. We demonstrate the first technological proof of a combinatorial biosensor for continuous, dynamic monitoring of alcohol and glucose in ultra- low volumes (1-5 µL) of passive perspired sweat towards developing a wearable- IOT based platform. Non-invasive biosensing in sweat is achieved by a unique gold- zinc oxide (ZnO) thin film electrode stack fabricated on a flexible substrate suitable for wearable applications. The active ZnO sensing region is immobilized with enzyme complexes specific for the detection of alcohol and glucose through non- faradaic electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA). Biomolecular interactions occurring at the electrode- sweat interface are represented by the impedance and capacitive current changes in response to charge modulations arising in the double layer. We also report the detection of alcohol concentrations of 0.01-100 mg/dl and glucose concentrations of 0.01-50 mg/dl present in synthetic sweat and perspired human sweat. The limit of detection obtained for alcohol and glucose was found to be 0.1 mg/dl in perspired human sweat. Cross- reactivity studies revealed that glucose and alcohol did not show any signal response to cross- reactive molecules. Furthermore, the stable temporal response of the combinatorial biosensor on continuous exposure to passive perspired human sweat spiked with alcohol and glucose over a 120-min duration was demonstrated.

Simultaneous lancet-free monitoring of alcohol and glucose from low-volumes of perspired human sweat.

A lancet-free, label-free biosensor for simultaneous detection of sweat glucose and alcohol was demonstrated using zinc oxide thin films integrated into a nanoporous flexible electrode system. Sensing was achieved from perspired human sweat at low volumes (1-3 µL), comparable to ambient conditions without external stimulation. Zinc oxide thin film electrodes were surface functionalized with alcohol oxidase enzyme and with glucose oxidase enzyme towards developing an affinity biosensor specific to the physiological relevant range of alcohol comprising of 0-2 drinks (0-50 mg/dl) and physiologically relevant range of glucose ranging from hypo- to hyper-glycaemia (50-130 mg/dl) in perspired human sweat. Sensing was achieved by measuring impedance changes associated with alcohol and glucose binding onto the sensor interface using electrochemical impedance spectroscopy with a dynamic range from 0.01-200 mg/dl and a limit of detection of 0.01 mg/dl for alcohol in human sweat. Sensor calibration in synthetic sweat containing interferents (25-200 mg/dl) and comparison using regression and Bland-Altman analysis of sweat sensor performance was done with BACtrack®. Combinatorial detection of glucose and ethanol in perspired human sweat and comparison of sweat sensor performance with Accu-Chek® blood glucose monitoring system that we expect would be relevant for pre-diabetics and diabetics for monitoring their glucose levels and alcohol consumption.

Differential electronic detector to monitor apoptosis using dielectrophoresis-induced translation of flowing cells (dielectrophoresis cytometry).

The instrument described here is an all-electronic dielectrophoresis (DEP) cytometer sensitive to changes in polarizability of single cells. The important novel feature of this work is the differential electrode array that allows independent detection and actuation of single cells within a short section ([Formula: see text]) of the microfluidic channel. DEP actuation modifies the altitude of the cells flowing between two altitude detection sites in proportion to cell polarizability; changes in altitude smaller than 0.25 µm can be detected electronically. Analysis of individual experimental signatures allows us to make a simple connection between the Clausius-Mossotti factor (CMF) and the amount of vertical cell deflection during actuation. This results in an all-electronic, label-free differential detector that monitors changes in physiological properties of the living cells and can be fully automated and miniaturized in order to be used in various online and offline probes and point-of-care medical applications. High sensitivity of the DEP cytometer facilitates observations of delicate changes in cell polarization that occur at the onset of apoptosis. We illustrate the application of this concept on a population of Chinese hamster ovary (CHO) cells that were followed in their rapid transition from a healthy viable to an early apoptotic state. DEP cytometer viability estimates closely match an Annexin V assay (an early apoptosis marker) on the same population of cells.