Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events.

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.

Robust Visualization and Discrimination of Nanoparticles by Interferometric Imaging.

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.

Development of a miniaturized mechanoacoustic sensor for continuous, objective cough detection, characterization and physiologic monitoring in children with cystic fibrosis.

Cough is an important symptom in children with acute and chronic respiratory disease. Daily cough is common in Cystic Fibrosis (CF) and increased cough is a symptom of pulmonary exacerbation. To date, cough assessment is primarily subjective in clinical practice and research. Attempts to develop objective, automatic cough counting tools have faced reliability issues in noisy environments and practical barriers limiting long-term use. This single-center pilot study evaluated usability, acceptability and performance of a mechanoacoustic sensor (MAS), previously used for cough classification in adults, in 36 children with CF over brief and multi-day periods in four cohorts. Children whose health was at baseline and who had symptoms of pulmonary exacerbation were included. We trained, validated, and deployed custom deep learning algorithms for accurate cough detection and classification from other vocalization or artifacts with an overall area under the receiver-operator characteristic curve (AUROC) of 0.96 and average precision (AP) of 0.93. Child and parent feedback led to a redesign of the MAS towards a smaller, more discreet device acceptable for daily use in children. Additional improvements optimized power efficiency and data management. The MAS's ability to objectively measure cough and other physiologic signals across clinic, hospital, and home settings is demonstrated, particularly aided by an AUROC of 0.97 and AP of 0.96 for motion artifact rejection. Examples of cough frequency and physiologic parameter correlations with participant-reported outcomes and clinical measurements for individual patients are presented. The MAS is a promising tool in objective longitudinal evaluation of cough in children with CF.

Observational pilot study of multi-wavelength wearable light dosimetry for erythropoietic protoporphyria.

BACKGROUND: Erythropoietic protoporphyria (EPP) causes painful light sensitivity, limiting quality of life. Our objective was to develop and validate a wearable light exposure device and correlate measurements with light sensitivity in EPP to predict and prevent symptoms. METHODS: A wearable light dosimeter was developed to capture light doses of UVA, blue, and red wavelengths. A prospective observational pilot study was performed in which five EPP patients wore two light dosimeters for 3 weeks, one as a watch, and one as a shirt clip. RESULTS: Standard deviation (SD) increases from the mean in the daily blue light dose increased the odds ratio (OR) for symptom risk more than the self-reported outdoor time (OR 2.76 vs. 2.38) or other wavelengths, and a one SD increase from the mean in the daily blue light wristband device dose increased the OR for symptom risk more than the daily blue light shirt clip (OR 2.45 vs. 1.62). The area under the receiver operator curve for the blue light wristband dose was 0.78, suggesting 78% predictive accuracy. CONCLUSION: These data demonstrate that wearable blue light dosimetry worn as a wristband is a promising method for measuring light exposure and predicting and preventing symptoms in EPP.

An implantable device for wireless monitoring of diverse physio-behavioral characteristics in freely behaving small animals and interacting groups.

Comprehensive, continuous quantitative monitoring of intricately orchestrated physiological processes and behavioral states in living organisms can yield essential data for elucidating the function of neural circuits under healthy and diseased conditions, for defining the effects of potential drugs and treatments, and for tracking disease progression and recovery. Here, we report a wireless, battery-free implantable device and a set of associated algorithms that enable continuous, multiparametric physio-behavioral monitoring in freely behaving small animals and interacting groups. Through advanced analytics approaches applied to mechano-acoustic signals of diverse body processes, the device yields heart rate, respiratory rate, physical activity, temperature, and behavioral states. Demonstrations in pharmacological, locomotor, and acute and social stress tests and in optogenetic studies offer unique insights into the coordination of physio-behavioral characteristics associated with healthy and perturbed states. This technology has broad utility in neuroscience, physiology, behavior, and other areas that rely on studies of freely moving, small animal models.

Synchronized wearables for the detection of haemodynamic states via electrocardiography and multispectral photoplethysmography.

Cardiovascular health is typically monitored by measuring blood pressure. Here we describe a wireless on-skin system consisting of synchronized sensors for chest electrocardiography and peripheral multispectral photoplethysmography for the continuous monitoring of metrics related to vascular resistance, cardiac output and blood-pressure regulation. We used data from the sensors to train a support-vector-machine model for the classification of haemodynamic states (resulting from exposure to heat or cold, physical exercise, breath holding, performing the Valsalva manoeuvre or from vasopressor administration during post-operative hypotension) that independently affect blood pressure, cardiac output and vascular resistance. The model classified the haemodynamic states on the basis of an unseen subset of sensor data for 10 healthy individuals, 20 patients with hypertension undergoing haemodynamic stimuli and 15 patients recovering from cardiac surgery, with an average precision of 0.878 and an overall area under the receiver operating characteristic curve of 0.958. The multinodal sensor system may provide clinically actionable insights into haemodynamic states for use in the management of cardiovascular disease.

Wireless broadband acousto-mechanical sensing system for continuous physiological monitoring.

The human body generates various forms of subtle, broadband acousto-mechanical signals that contain information on cardiorespiratory and gastrointestinal health with potential application for continuous physiological monitoring. Existing device options, ranging from digital stethoscopes to inertial measurement units, offer useful capabilities but have disadvantages such as restricted measurement locations that prevent continuous, longitudinal tracking and that constrain their use to controlled environments. Here we present a wireless, broadband acousto-mechanical sensing network that circumvents these limitations and provides information on processes including slow movements within the body, digestive activity, respiratory sounds and cardiac cycles, all with clinical grade accuracy and independent of artifacts from ambient sounds. This system can also perform spatiotemporal mapping of the dynamics of gastrointestinal processes and airflow into and out of the lungs. To demonstrate the capabilities of this system we used it to monitor constrained respiratory airflow and intestinal motility in neonates in the neonatal intensive care unit (n = 15), and to assess regional lung function in patients undergoing thoracic surgery (n = 55). This broadband acousto-mechanical sensing system holds the potential to help mitigate cardiorespiratory instability and manage disease progression in patients through continuous monitoring of physiological signals, in both the clinical and nonclinical setting.

A wireless and battery-less implant for multimodal closed-loop neuromodulation in small animals.

Fully implantable wireless systems for the recording and modulation of neural circuits that do not require physical tethers or batteries allow for studies that demand the use of unconstrained and freely behaving animals in isolation or in social groups. Moreover, feedback-control algorithms that can be executed within such devices without the need for remote computing eliminate virtual tethers and any associated latencies. Here we report a wireless and battery-less technology of this type, implanted subdermally along the back of freely moving small animals, for the autonomous recording of electroencephalograms, electromyograms and body temperature, and for closed-loop neuromodulation via optogenetics and pharmacology. The device incorporates a system-on-a-chip with Bluetooth Low Energy for data transmission and a compressed deep-learning module for autonomous operation, that offers neurorecording capabilities matching those of gold-standard wired systems. We also show the use of the implant in studies of sleep-wake regulation and for the programmable closed-loop pharmacological suppression of epileptic seizures via feedback from electroencephalography. The technology can support a broader range of applications in neuroscience and in biomedical research with small animals.

Real-Time UV Measurement With a Sun Protection System for Warning Young Adults About Sunburn: Prospective Cohort Study.

BACKGROUND: Melanoma is attributable to predisposing phenotypical factors, such as skin that easily sunburns and unprotected exposure to carcinogenic UV radiation. Reducing the proportion of young adults who get sunburned may reduce the incidence of melanoma, a deadly form of skin cancer. Advances in technology have enabled the delivery of real-time UV light exposure and content-relevant health interventions. OBJECTIVE: This study aims to examine the feasibility of young adults performing the following tasks daily: wearing a UV dosimeter, receiving text messages and real-time UV-B doses on their smartphone, and responding to daily web-based surveys about sunburn and sun protection. METHODS: Young adults aged 18-39 years (n=42) were recruited in the United States in June 2020 via social media. Participants received the UV Guard sun protection system, which consisted of a UV dosimeter and a smartphone app. During 3 consecutive periods, intervention intensity increased as follows: real-time UV-B dose; UV-B dose and daily behavioral facilitation text messages; and UV-B dose, goal setting, and daily text messages to support self-efficacy and self-regulation. Data were self-reported through daily web-based surveys for 28 days, and UV-B doses were transmitted to cloud-based storage. RESULTS: Patients' median age was 22 years (IQR 20, 29), and all patients had sun-sensitive skin. Sunburns were experienced during the study by fewer subjects (n=18) than those in the preceding 28 days (n=30). In July and August, the face was the most commonly sunburned area among 13 body locations; 52% (22/42) of sunburns occurred before the study and 45% (19/42) occurred during the study. The mean daily UV-B dose decreased during the 3 periods; however, this was not statistically significant. Young adults were most often exercising outdoors from 2 to 6 PM, walking from 10 AM to 6 PM, and relaxing from noon to 2 PM. Sunburn was most often experienced during exercise (odds ratio [OR] 5.65, 95% CI 1.60-6.10) and relaxation (OR 3.69, 95% CI 1.03-4.67) relative to those that did not exercise or relax in each category. The self-reported exit survey indicated that participants felt that they spent less time outdoors this summer compared to the last summer because of the COVID-19 pandemic and work. In addition, 38% (16/42) of the participants changed their use of sun protection based on their app-reported UV exposure, and 48% (20/42) shifted the time they went outside to periods with less-intense UV exposure. A total of 79% (33/42) of the participants were willing to continue using the UV Guard system outside of a research setting. CONCLUSIONS: In this proof-of-concept research, young adults demonstrated that they used the UV Guard system; however, optimization was needed. Although some sun protection behaviors changed, sunburn was not prevented in all participants, especially during outdoor exercise. TRIAL REGISTRATION: ClinicalTrials.gov NCT03344796; http://clinicaltrials.gov/ct2/show/NCT03344796.

Real-Time Capture and Visualization of Individual Viruses in Complex Media.

Label-free imaging of individual viruses and nanoparticles directly in complex solutions is important for virology research and biosensing applications. A successful visualization technique should be rapid, sensitive, and inexpensive, while needing minimal sample preparation or user expertise. Current approaches typically require fluorescent labeling or the use of an electron microscope, which are expensive and time-consuming to use. We have developed an imaging technique for real-time, sensitive, and label-free visualization of viruses and nanoparticles directly in complex solutions such as serum. By combining the advantages of a single-particle reflectance imaging sensor, with microfluidics, we perform real-time digital detection of individual 100 nm vesicular stomatitis viruses as they bind to an antibody microarray. Using this approach, we have shown capture and visualization of a recombinant vesicular stomatitis virus Ebola model (rVSV-ZEBOV) at 100 PFU/mL in undiluted fetal bovine serum in less than 30 min.

A Portable, Pressure Driven, Room Temperature Nucleic Acid Extraction and Storage System for Point of Care Molecular Diagnostics.

Many new and exciting portable HIV viral load testing technologies are emerging for use in global medicine. While the potential to provide fast, isothermal, and quantitative molecular diagnostic information to clinicians in the field will soon be a reality, many of these technologies lack a robust front end for sample clean up and nucleic acid preparation. Such a technology would enable many different downstream molecular assays. Here, we present a portable system for centrifuge-free room temperature nucleic acid extraction from small volumes of whole blood (70 µL), using only thermally stable reagents compatible with storage and transport in low resource settings. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of simulated samples demonstrate a lower limit of detection of 1000 copies/ml, with the ability to detect differences in viral load across four orders of magnitude. The system can also be used to store extracted RNA on detachable cartridges for up to one week at ambient temperature, and can be operated using only hand generated air pressure.

An interferometric reflectance imaging sensor for point of care viral diagnostics.

The use of in vitro diagnostic devices is transitioning from the laboratory to the primary care setting to address early disease detection needs. Time critical viral diagnoses are often made without support due to the experimental time required in today's standard tests. Available rapid point of care (POC) viral tests are less reliable, requiring a follow-on confirmatory test before conclusions can be drawn. The development of a reliable POC viral test for the primary care setting would decrease the time for diagnosis leading to a lower chance of transmission and improve recovery. The single particle interferometric reflectance imaging sensor (SP-IRIS) has been shown to be a sensitive and specific-detection platform in serum and whole blood. This paper presents a step towards a POC viral assay through a SP-IRIS prototype with automated data acquisition and analysis and a simple, easy-to-use software interface. Decreasing operation complexity highlights the potential of SP-IRIS as a sensitive and specific POC diagnostic tool. With the integration of a microfluidic cartridge, this automated instrument will allow an untrained user to run a sample-to-answer viral assay in the POC setting.