Structure of lactate oxidase from Enterococcus hirae revealed new aspects of active site loop function: Product-inhibition mechanism and oxygen gatekeeper.

l-Lactate oxidase (LOx) is a flavin mononucleotide (FMN)-dependent triose phosphate isomerase (TIM) barrel fold enzyme that catalyzes the oxidation of l-lactate using oxygen as a primary electron acceptor. Although reductive half-reaction mechanism of LOx has been studied by structure-based kinetic studies, oxidative half-reaction and substrate/product-inhibition mechanisms were yet to be elucidated. In this study, the structure and enzymatic properties of wild-type and mutant LOxs from Enterococcus hirae (EhLOx) were investigated. EhLOx structure showed the common TIM-barrel fold with flexible loop region. Noteworthy observations were that the EhLOx crystal structures prepared by co-crystallization with product, pyruvate, revealed the complex structures with "d-lactate form ligand," which was covalently bonded with a Tyr211 side chain. This observation provided direct evidence to suggest the product-inhibition mode of EhLOx. Moreover, this structure also revealed a flip motion of Met207 side chain, which is located on the flexible loop region as well as Tyr211. Through a saturation mutagenesis study of Met207, one of the mutants Met207Leu showed the drastically decreased oxidase activity but maintained dye-mediated dehydrogenase activity. The structure analysis of EhLOx Met207Leu revealed the absence of flipping in the vicinity of FMN, unlike the wild-type Met207 side chain. Together with the simulation of the oxygen-accessible channel prediction, Met207 may play as an oxygen gatekeeper residue, which contributes oxygen uptake from external enzyme to FMN. Three clades of LOxs are proposed based on the difference of the Met207 position and they have different oxygen migration pathway from external enzyme to active center FMN.

Faradaic electrochemical impedance spectroscopy for enhanced analyte detection in diagnostics.

Electrochemical impedance spectroscopy (EIS) is a widely implementable technique that can be applied to many fields, ranging from disease detection to environmental monitoring. EIS as a biosensing tool allows detection of a broad range of target analytes in point-of-care (POC) and continuous applications. The technique is highly suitable for multimarker detection due to its ability to produce specific frequency responses depending on the target analyte and molecular recognition element (MRE) combination. EIS biosensor development has shown promising results for the medical industry in terms of diagnosis and prognosis for various biomarkers. EIS sensors offer a cost-efficient system and rapid detection times using minimal amounts of sample volumes, while simultaneously not disturbing the sample being studied due to low amplitude perturbations. These properties make the technique highly sensitive and specific. This paper presents a review of EIS biosensing advancements and introduces different detection techniques and MREs. Additionally, EIS's underlying theory and potential surface modification techniques are presented to further demonstrate the technique's ability to produce stable, specific, and sensitive biosensors.

Correction to: Project honeybee: Clinical applications for wearable biosensors.

The original version of this article unfortunately contained a mistake.

BODDEE BUDDEE: Evaluation of Different Foams and Thermoplastics to Develop a Biofidelic Manikin for Cardiopulmonary Resuscitation.

Cardiopulmonary resuscitation (CPR) is an emergency course of action developed to sustain oxygenated blood flow in persons suffering from cardiac arrest by manually compressing the heart in the chest and providing rescue ventilations. The best-selling CPR manikins, an integral part of training, are costly investments that lack biofidelic characteristics in appearance, feel, and response; as a result, the rescuer's learning experience suffers. The objective of the present study was to test the compressibility properties of different foams and thermoplastics in order to determine which material would most accurately imitate a human chest response. The results suggested that styrene-ethylene/butylene-styrene (SEBS) was the best choice, because its increasing stiffness under increasing compression was characteristic of a human chest cavity. Further testing must be done to determine the best composition of SEBS, analyze its response under cyclic compressions, and improve its durability.

A Comparison of Force Sensing for Applications in Prosthetic Haptic Feedback.

The current study presents a comparison of two load sensor designs that can be applied toward haptic feedback sensing in upper limb prosthetics. A lab-standard capacitive load cell sensor is discussed, which is succeeded by the proposal of an electrochemical sensor. Experiments were conducted primarily as a proof-of-principle study to evaluate sensor characteristics for prosthetic applications. The aim is to address the need for minimally invasive, cost-effective prosthetic sensor technologies, as the investigated sensor designs conceptualize applications of average grip forces. Thus, force requirements for the sensors were determined to be 250-500 N per the average maximum grip strength of healthy adults. Comparable to a commercial gold-standard capacitive load cell design, a lab-standard load cell sensor was inexpensively manufactured using conductive foam. The lab-standard design was improved upon by employing electrochemical techniques and CP-9000, a thermoplastic elastomer material, to form an electrochemical sensor for enhanced sensitivity. Sustained loads ranging from 0.49 to 2.45 N resulted in average maximum current readouts of - 1.25 × 10-1 to - 4.25 × 10-1 for the lab-standard sensor, and - 5.95 µA to - 7.85 µA for the electrochemical sensor. The electrochemical sensor was reproducible and demonstrated the potential to discriminate between various loads. Force requirements were not reached; however, future studies will seek to increase the mechanical strength of the electrochemical sensor. As the initial electrochemical sensor design provides a potential method for low-cost computer-based prosthetics, thermoplastic elastomer materials with increased elastic and mechanical strength properties will be investigated.

Staggered Nitinol Wire Actuator Array for High Linear Displacement and Force-to-Mass Ratio.

We present the design and performance of a unique Nitinol (NiTi) actuator design for high linear displacement and force generation through joule heating. The device is comprised of a staggered linear array of NiTi in wire form that, as a shape memory alloy, can achieve linear displacement through material phase change when heated. This change allows the crystal lattice within the material to displace/adjust. The design results in strain levels of 20.4% that are comparable to those of biological muscles and provides potential for additional strain. Three- to seven-staggered NiTi wires are tested to demonstrate the different levels of strain that are achieved with a range of wires in a staggered array. In addition, we measure and compare force generated to the mass of each wire to show system force-to-mass ratio. The effective force to mass for the system is greater than 5500 combined with a seven-wire staggered array. The device shows that a lightweight, high-strain actuator can be developed, and our research demonstrates its potential use in prosthetic actuation.

Toward the Development of a Wearable Optical Respiratory Sensor for Real-Time Use.

Respiration rate is an important vital sign that can provide insight into a patient's status and health progression. This information is used from critical care to sports and human performance evaluation. The current state of the art has demonstrated effectiveness in monitoring respiration rate with the use of wearable sensors. However, their form factor, which refers to the embodiment of approach, size, and shape, makes it difficult to implement within a longterm monitoring setting. Problems relating to form factor, such as compliance, are a major issue in collecting useful and actionable data, because they directly impact comfort and ease of wear. We present a new approach based on an optical computer mouse sensor that can be rendered into a slim, wearable device without the need for a harness or shirt to hold the sensor in place. Its main objective is to achieve similar or better readings than those of the state of the art while reducing the overall size and thus, improve compliance by making it easier, more comfortable to wear. The principle of operation of the sensor allows for enhanced signal and computational noise reduction for movement artifacts. The sensor was tested to determine its limits of detection and was calibrated to expected distance of movement. Then, observations were made under normal breathing conditions, apnea, deep breathing, and hyperventilation covering a spectrum of 0 to 45 breathings per minute (BPM). The performance of the device was described by using the mean average error which was 0.37 and 0.83 under deep breathing and hyperventilation, respectively. Testing revealed that the device produces the best results when worn over the diaphragm and that its readings are comparable to the industry gold standard. The future version we are developing incorporates a slimmer, lighter design, Bluetooth data communication to remove leads and wires, adhesive electrodes and a reusable adhesive that is also waterproof.

Feasibility of Commercially Marketed Health Devices for Potential Clinical Application.

Concerns have been raised regarding the lack of validation on consumer-marketed health-monitoring devices. An investigation to characterize current health monitoring devices was carried out in the laboratory using widely accepted clinical and industry criteria. In total, 16 unique devices were examined. These devices were assessed according to their sensing modalities: step count, blood pressure, body temperature, electrocardiogram, blood oxygen saturation, and respiratory rate. Devices were tested at rest and immediately following exercise. Our results revealed that only four devices meet target requirements for accuracy. The AliveCor, a portable ECG monitor, accurately detected the heart rate for 87% of all recordings. To meet the target criterion for accuracy, the heart rate must be within ± 5 beats/minute or 10% of the standard measurement, whichever is lower. The Withings Pulse Ox, the Tinké, and the Santamedical SM-110 measured blood oxygen saturation with 2.1, 2.6, and 1.4 root-mean-square (rms) error, respectively. For blood oxygen saturation, the device should demonstrate rms error of < 3%. However, the Withings Pulse Ox and the Tinké failed to meet the accuracy criteria for their alternative biosensing capabilities: step count and respiratory rate, respectively. We conclude that the use of consumer-marketed health-monitoring devices for clinical or medical purposes should be undertaken with caution, especially in the absence of FDA or comparable clearance.

Non-Contact Type Pulse Oximeter.

Heart rate and through-body blood perfusion are vital measurements in all stages of patient care, be it predictive, in the clinical setting, or outpatient monitoring. Irregular, underachieving, or overperforming heart rate is the main precursor of most cardiovascular diseases that have severe long-term complications. In addition to heart rate, the shape of the pulse waveforms can indicate the heart's valve health and electrophysiology health. The goal of the study was to design a noninvasive device for continuously measuring a patient's heart rate with clinical-grade accuracy along with the ability to indicate pulse waveforms for the patient and physician. An accurate, easy-to-use heart-rate measuring device prototype was developed that did not require the sensor to have direct skin contact to obtain measurements. The statistical analysis of the data gathered by the prototype compared to the data collected from the industry standard device indicated significant correlation. The two-sample T-test for the data recorded from the prototype and the data collected from the industry commercially available pulse oximeter showed a P-value of 0.521, which indicates that there was no significant difference between the prototype and the commercially available pulse oximeter when measuring heart rate.

Proof of Concept for a Universal Identification System for Medical Devices.

Medical devices need a unified way of accessing information that uniquely identifies them. This can provide traceability to specifications, lot numbers, recalls, and the like. Such a system would have applications for devices both in and out of the body. Common barcodes, such as a UPC code, can only be read in plain sight, when nothing comes between the scanner and the code. UPC coding is not suitable for all medical devices because some are implanted in the body or are otherwise inaccessible without invasive techniques. This article demonstrates a proof of concept for XRF coding on devices. Material codes were made and read externally by an XRF reader. The reading showed trace amounts of the chemicals that compose the medical device in the background signal. The energy levels of the chemicals were assigned values to build a readable code correlated with information about the medical device it is attached to. Attachment can be made during material synthesis, part or product manufacture, or even after final assembly. The technique demonstrated here is a promising concept for the future of medical device detection.

Development Toward a Triple-Marker Biosensor for Diagnosing Cardiovascular Disease.

Cardiovascular disease (CVD) is the leading cause of death in the United States and is responsible for 30% of all deaths globally. The diagnosis and management of CVD requires monitoring of multiple biomarkers, which comprehensively represents the state of the disease. However, many assays for cardiac biomarkers today are complicated and laborious to perform. Rapid and sensitive biosensors capable of giving accurate measurements of vital cardiac biomarkers without complex procedures are thus in high demand. In the work presented below, rapid, label-free biosensor prototypes for three Food and Drug Administration-approved biomarkers are reported: B-type natriuretic peptide (BNP), cardiac troponin I (cTnI), and C-reactive protein (CRP). The sensors were prepared by immobilizing each biomarker's antibody onto gold working electrodes with platinum counter and silver/silver chloride reference electrodes. The sensors were tested using electrochemical impedance spectroscopy (EIS), a femto-molar sensitive technique capable of label-free, multi-marker detection if a biomarker's optimal frequency (OF) can be identified. The OFs of BNP, cTnI, and CRP were found to be 1.74, 37.56, and 253.9 Hz, respectively. The performance of the BNP biosensor was also evaluated in blood and achieved clinically relevant detection limits of 100 pg/mL.

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.

Development of Electrochemical Methods to Enzymatically Detect Lactate and Glucose Using Imaginary Impedance for Enhanced Management of Glycemic Compromised Patients.

Lactate is an important biological marker that can provide valuable information for patients who have experienced a traumatic injury. Additionally, when coupled with glucose, the severity and likely prognosis of a traumatic injury can be determined. Because monitoring various markers proves useful in diagnosis and treatment of trauma patients, monitoring both glucose and lactate simultaneously may be especially useful for diabetic patients who have suffered a traumatic injury. Previously, using electrochemical impedance spectroscopy (EIS), a sensor capable of measuring two affinity-based biomarkers simultaneously was demonstrated using the biomarker's specific optimal frequency to develop a deconvolution algorithm, which allowed for the measurement of two biomarkers from a single signal. Herein, while developing an EIS lactate sensor, dual enzymatic biomarker detection of lactate and glucose via EIS was also attempted. Both biomarkers were validated individually with the lactate sensor being additionally validated on whole blood samples. The EIS lactate biosensor achieved a range of detection from 0 to 32 mM of lactate and the glucose sensor a range of 0-100 mg/dL of glucose, which are representative of the likely physiological ranges that trauma patients experience. However, the preliminary attempt of dual marker detection was unsuccessful due to suspected accumulation of reduced redox probe on the surface of the self-assembled monolayer (SAM). Individually, the optimal frequency of lactate was determined to be 69.75 Hz and that of glucose was determined to be 31.5 Hz. However, when combined onto one sensor, no discernable optimal frequency could be determined which again was suspected to be due to the accumulation of the reduced redox probe at the surface of the SAM.

Electrochemical Detection of Fertility Hormones.

Fertility hormone levels are constantly changing, but it is crucial for a woman to be able to monitor her fertility levels if she is interested in conceiving. Women and physicians often have a difficult time determining ovulation windows due to fluctuating menstrual cycles and inaccurate interpretations of hormone levels. Current methods of fertility monitoring include physical or vaginal exams, laparoscopy, ultrasound scans, as well as evaluation of hormone levels. A rapid, at-home fertility monitoring tool can help alleviate the apprehensiveness associated with routine screenings and give women the privacy desired when trying to conceive. Herein, we discuss the development of an electrochemical biosensor for quantification of three fertility hormones: beta-estradiol, progesterone, and FSH. Each biomarker's MRE was immobilized onto a gold disk electrode through the use of self-assembled monolayer linking chemistry. Using electrochemical impedance spectroscopy (EIS), the biomarker concentration was correlated to impedance magnitude. An optimal binding frequency was identified for each biomarker, permitting simplistic hardware requirements and investigation into multimarker detection. Analytes were tested in both purified solutions and 1%-90% whole blood. Each biomarker exhibited a unique imaginary impedance peak and optimal binding frequency. The determination was made by assessing the response parameters including the linear fit correlation across the physiological hormone ranges. The existence of unique optimal frequencies permits for simultaneous detection of multiple hormones in a single test. Additionally, the identified frequency was robust across purified and complex solutions. Response characteristics were negatively impacted by the introduction of blood-based contaminants. However, the introduction of Nafion membranes, similar to ones used in commercial glucose sensors, is both feasible and beneficial.

An Experimental Platform for Characterizing Cancer Biomarkers with Capabilities in Noninvasive and Continuous Screening.

Early detection is crucial to the proper and effective treatment of two metastatic cancers, prostate cancer and small cell lung cancer. Currently, preventative screenings for these conditions are restricted to high-risk populations and extremely expensive. The discovery of clinically indicative biomarkers has been revolutionary in advancing screening and diagnostic capabilities. Prostate-specific antigen (PSA), an extracellular secreted protein of the prostate gland, and neuron-specific enolase (NSE), an enzyme of neuronal origin, have reported reputable specificity for prostate cancer and small cell lung cancer (SCLC). Current efforts are underway to develop a rapid, label-free means of measuring both PSA and NSE levels in a clinical environment for early screening applications of highly metastatic cancers. Electrochemical impedance spectroscopy (EIS) and impedance time (Z-t) are rapid, sensitive electrochemical techniques previously validated in the detection of several clinically relevant biomarkers, including cardiovascular disease and diabetes mellitus. Herein, we determine the optimal frequencies of PSA (81.38 Hz) and NSE (14.36 Hz) using EIS that are robust across analytical platforms and in the presence of potentially interfering species. The reported empirical evidence supports the prevalence of electrostatic interactions in electrochemical systems and provides alternative theoretical support of previous findings. Finally, Z-t was implemented for its utility in continuous monitoring applications and to lay the foundation for future improvements to continuous sensor platforms.

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.

Project honeybee: Clinical applications for wearable biosensors.

Please provide an abstract of 150 to 250 words. The abstract should not contain any undefined abbreviations or unspecified references. The Project Honeybee Observational Clinical Trials were 12-month studies designed to validate the use of commercially available ambulatory medical devices costing $50-$300 for clinical applications. Each trial had a patient population of about 15-30 subjects with a broad range of disease types including heart failure, diabetes, sepsis, and Parkinson's disease. Over 30 supported proposals were funded in the 4-year period, as well as the creation of a database of all commercially available devices. Each year a call for proposals was published within ASU and Mayo Clinic Arizona. Proposals were selected for funding by a committee of ASU faculty from engineering, nursing, and exercise physiology departments. The progress of each research trial was monitored through monthly colloquia with the nursing, biomedical engineering, computer science, and nutrition graduate research assistants, to discuss the challenges and opportunities arising with each research trial. PIs were required to report on study progress 6 months into the trial period and 3 months following the conclusion of the 12-month project. The project was very successful in meeting our goals of testing consumer wearable devices on patients for a variety of conditions across a variety of clinical settings in the greater Phoenix community. The following clinical sites participated in one or more of these clinical trials: Adelante Healthcare, Arizona Arrhythmia Consultants, Arizona Cardiology Group, Banner University Medical Center, Barrow Neurological Institute, Honor Health, Mayo Clinic, and St Joseph's Hospital. A total of 12 ASU faculty and 39 clinicians participated.

Facilitating Earlier Diagnosis of Cardiovascular Disease through Point-of-Care Biosensors: A Review.

Cardiovascular disease (CVD) accounts for 30% of all global deaths and is predicted to dominate in the coming years, despite vast improvements in medical technology. Current clinical methods of assessing an individual's cardiovascular health include blood tests to monitor relevant biomarker levels as well as varying imaging modalities such as electrocardiograms, computed tomography, and angiograms to assess vasculature. As informative as these tools are, they each require lengthy scheduling, preparation, and highly trained personnel to interpret the results before any information is accessible to patients, often leading to delayed treatment, which can be fatal. A point-of-care (POC) sensor platform is thus paramount in rapid and early diagnosis of CVD. Among the many POC detection platforms, including established optical and mechanical methods, electrochemical-based detection mechanisms have become increasingly desirable because of their superior sensitivity, low cost, and label-free detection. Specifically, electrochemical impedance spectroscopy (EIS) has demonstrated remarkable abilities in low-level (femtomolar) detection of several clinically useful biomarkers and has been reported in CVD diagnostic applications. In this review, we provide an in-depth overview of prevalent CVD diseases and clinically relevant proteomic biomarkers for assessing them. Subsequently, we discuss the ongoing development of POC sensors for CVD, highlighting the current clinical gold standard, potential alternative modalities, and electrochemical methodologies previously successful in quantifying specific biomarkers approved by the Food and Drug Administration (FDA). A discussion of EIS highlighting the attributes and capabilities of novel analysis algorithms is included to showcase the possibility of simultaneous dual-marker detection.

A Disposable Tear Glucose Biosensor-Part 5: Improvements in Reagents and Tear Sampling Component.

A tear glucose (TG) sensor with an integrated tear sampler can provide a noninvasive method for calibrating the continuous TG contact lens and monitoring glucose. Expanding from previous work, an improved TG sensor that implements dried reagents, genetically modified glucose dehydrogenase (GDH), and a tear sampler was developed and compared against the TG sensor prepared with commercial GDH. It was found that neither sensor was affected by the tear interferents: ascorbic acid, acetaminophen, and uric acid. The sensor prepared with commercial GDH generated higher current. This suggests that using enzymes with lower Km may be advantageous when operating in low glucose environments like tears. The improved TG sensor also demonstrated the potential of integrating Schirmer's test strip as a tear sampler for self-monitoring of TG.

The electrochemical behavior of a FAD dependent glucose dehydrogenase with direct electron transfer subunit by immobilization on self-assembled monolayers.

Continuous glucose monitoring (CGM) is a vital technology for diabetes patients by providing tight glycemic control. Currently, many commercially available CGM sensors use glucose oxidase (GOD) as sensor element, but this enzyme is not able to transfer electrons directly to the electrode without oxygen or an electronic mediator. We previously reported a mutated FAD dependent glucose dehydrogenase complex (FADGDH) capable of direct electron transfer (DET) via an electron transfer subunit without involving oxygen or a mediator. In this study, we investigated the electrochemical response of DET by controlling the immobilization of DET-FADGDH using 3 types of self-assembled monolayers (SAMs) with varying lengths. With the employment of DET-FADGDH and SAM, high current densities were achieved without being affected by interfering substances such as acetaminophen and ascorbic acid. Additionally, the current generated from DET-FADGDH electrodes decreased with increasing length of SAM, suggesting that the DET ability can be affected by the distance between the enzyme and the electrode. These results indicate the feasibility of controlling the immobilization state of the enzymes on the electrode surface.