Parametric study of 3D printed microneedle (MN) holders for interstitial fluid (ISF) extraction.

The need for novel, minimally invasive diagnostic, prognostic, and therapeutic biomedical devices has garnered increased interest in recent years. Microneedle (MN) technology has stood out as a promising new method for drug delivery, as well as extraction of interstitial fluid (ISF). ISF comprises a large portion of the extracellular fluid in living organisms yet remains inadequately characterized for clinical applications. Current MN research has focused on the fabrication of needles with different materials like silicone, carbon, and metals. However, little effort has been put forth into improving MN holders and patches that can be used with low cost MNs, which could effectively change how MNs are attached to the human body. Here, we describe different 3D-printed MN holders, printed using an MJP Pro 2500 3D printer, and compare the ISF extraction efficiencies in CD Hairless rats. We varied design parameters that may affect the skin-holder interface, such as throat thickness, tip curvature, and throat diameter. MN arrays, with insertion depths of 1500 µm, had extraction efficiencies of 0.44 ± 0.35, 0.85 ± 0.64, 0.32 ± 0.21, or 0.44 ± 0.46 µl/min when designed with flat, concave, convex, or bevel profile geometries, respectively. Our results suggest ISF extraction is influenced by MN holder design parameters and that a concave tip design is optimal for extracting ISF from animals. The future direction of this research aims to enable a paradigm in MN design that maximizes its efficiency and engineering performance in terms of volume, pressure, and wearability, thereby automatizing usage and reducing patient intervention to ultimately benefit remote telemedicine.

Developments in Transduction, Connectivity and AI/Machine Learning for Point-of-Care Testing.

We review some emerging trends in transduction, connectivity and data analytics for Point-of-Care Testing (POCT) of infectious and non-communicable diseases. The patient need for POCT is described along with developments in portable diagnostics, specifically in respect of Lab-on-chip and microfluidic systems. We describe some novel electrochemical and photonic systems and the use of mobile phones in terms of hardware components and device connectivity for POCT. Developments in data analytics that are applicable for POCT are described with an overview of data structures and recent AI/Machine learning trends. The most important methodologies of machine learning, including deep learning methods, are summarised. The potential value of trends within POCT systems for clinical diagnostics within Lower Middle Income Countries (LMICs) and the Least Developed Countries (LDCs) are highlighted.

Proteomic Characterization of Dermal Interstitial Fluid Extracted Using a Novel Microneedle-Assisted Technique.

As wearable fitness devices have gained commercial acceptance, interest in real-time monitoring of an individual's physiological status using noninvasive techniques has grown. Microneedles have been proposed as a minimally invasive technique for sampling the dermal interstitial fluid (ISF) for clinical monitoring and diagnosis, but little is known about its composition. In this study, a novel microneedle array was used to collect dermal ISF from three healthy human donors and compared with matching serum and plasma samples. Using a shotgun quantitative proteomic approach, 407 proteins were quantified with at least one unique peptide, and of those, 135 proteins were differently expressed at least 2-fold. Collectively, these proteins tended to originate from the cytoplasm, membrane bound vesicles, and extracellular vesicular exosomes. Proteomic analysis confirmed previously published work that indicates that ISF is highly similar to both plasma and serum. In this study, less than one percent of proteins were uniquely identified in ISF. Taken together, ISF could serve as a minimally invasive alternative for blood-derived fluids with potential for real-time monitoring applications.

Rapid detection of Ebola virus with a reagent-free, point-of-care biosensor.

Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 104 PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodology has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.

Feasibility of Interstitial Fluid Ketone Monitoring with Microneedles.

Diabetic ketoacidosis (DKA) is one of the most dangerous and costly complications of diabetes, accounting for approximately 50% of deaths in diabetic individuals under 24 years. This results in over 130,000 hospital admissions yearly and costs the USA over USD 2.4 billion annually. Earlier diagnosis, treatment, and management of DKA are of critical importance to achieving better patient outcomes and preventing prolonged hospital admissions. Diabetic patients undergoing stress from illness or injury may not recognize early ketosis and often present advanced ketoacidosis, requiring intensive care admission. We have recently developed a microneedle-based technology to extract dermal interstitial fluid (ISF) from both animals and humans, which could enable wearable sensors to rapidly detect ketosis. Metabolite concentrations in ISF may differ in urine and blood and could likely represent local metabolic conditions in the surrounding tissue. Development of a wearable ketone detector will require an understanding of ketone concentrations and kinetics in ISF. Here, we report data that is first of its kind, with regard to the ketone concentrations present in the dermal ISF of rats, their correlation to blood, and the possible impact on the development of a wearable ISF "early warning system" to prevent morbidity from DKA. We extracted ISF, using minimally invasive microneedle arrays, from control Sprague Dawley rats and 17 h fasted rats. ISF and blood ketone levels were measured using a common glucose/ketone meter and strips. Local tissue concentrations of glucose were similar to those of blood, with an average blood to ISF glucose ratio of 0.99 ± 0.15 mg/dL. ISF ketones (0.4 ± 0.3 mM) were significantly higher (p = 4.2 × 10-9), compared with blood ketones (0.0 ± 0.0 mM). Although the fasted animals had slightly higher ISF ketones (1.3 ± 1.1 mM) compared with blood ketones (1.0 ± 1.0 mM), the difference was not significant (p = 0.3). This suggests ISF could possibly be useful as a surrogate for blood when determining ketone levels within a clinical setting.

Microneedle Array Technique for the Longitudinal Extraction of Interstitial Fluid without Hair Removal.

Interstitial fluid (ISF) bathes the cells and tissues and is in constant exchange with blood. As an exchange medium for waste, nutrients, exosomes, and signaling molecules, ISF is recognized as a plentiful source of biomolecules. Many basic and pre-clinical small animal studies could benefit from an inexpensive and efficient technique that allows for the in vivo extraction of ISF for the subsequent quantification of molecules in the interstitial space. We have previously reported on a minimally invasive technique for the extraction of ISF using a 3D-printed microneedle array (MA) platform for comprehensive biomedical applications. Previously, hairless animal models were utilized, and euthanasia was performed immediately following the procedure. Here, we demonstrate the technique in Sprague Dawley rats, without the need for hair removal, over multiple extractions and weeks. As an example of this technique, we report simultaneous quantification of the heavy metals Copper (Cu), Lead (Pb), Lithium (Li), and Nickel (Ni) within the ISF, compared with whole blood. These results demonstrate the MA technique applicability to a broader range of species and studies and the reuse of animals, leading to a reduction in number of animals needed to successfully complete ISF extraction experiments.

Aerosol generation with various approaches to oxygenation in healthy volunteers in the emergency department.

OBJECTIVES: Health care workers experience an uncertain risk of aerosol exposure during patient oxygenation. To improve our understanding of these risks, we sought to measure aerosol production during various approaches to oxygenation in healthy volunteers in an emergency department. METHODS: This was a prospective study conducted in an empty patient room in an academic ED. The room was 10 ft. long x 10 ft. wide x 9 ft. tall (total volume 900 ft3) with positive pressure airflow (1 complete turnover of air every 10 minutes). Five oxygenation conditions were used: humidified high-flow nasal cannula (HFNC) at 3 flow rates [15, 30, and 60 liters per minute (LPM)], non-rebreather mask (NRB) at 1 flow rate (15 LPM), and closed-circuit continuous positive airway pressure (CPAP) using the ED ventilator; in all cases a simple procedural mask was used. The NRB and HFNC at 30 LPM maneuvers were also repeated without the procedural mask, and CPAP was applied both with and without a filter. Each subject then sequentially underwent 8 total oxygenation conditions, always in the same order. Each oxygenation condition was performed with the participant on a standard ED bed. Particles were measured by laser aerosol spectrometer, with the detector sampling port positioned directly over the center of the bed, 0.35 meters away and at a 45-degree angle from the subject's mouth. Each approach to oxygenation was performed for 10 minutes, followed by a 20-minute room washout (≈ 2 complete room air turnovers). Particle counts were summated for 2 size ranges (150-300 nm and 0.5-2.0 µm) and compared before, during, and after each of the 8 oxygenation conditions. RESULTS: Eight adult subjects were enrolled (mean age 42 years, body mass index 25). All subjects completed 8 oxygenation procedures (64 total). Mean particle counts per minute across all oxygenation procedures was 379 ± 112 (mean ± SD) for smaller aerosols (150-300 nm) and 9.3 ± 4.6 for larger aerosols (0.5-2.0 µm). HFNC exhibited a flow-dependent increase in particulate matter (PM) generation-at 60 LPM, HFNC had a substantial generation of small (55% increase) and large particles (70% increase) compared to 15 LPM. CPAP was associated with lowered small and large particle generation (≈ 10-15% below baseline for both sizes of PM). A patient mask limited particle generation with the NRB, where it was associated with a reduction in small and large particulates (average 40% and 20% lower, respectively). CONCLUSION: Among 3 standard oxygenation procedures, higher flow rates generally were associated with greater production of both small and large aerosols. A patient mask lowered aerosol counts in the NRB only. Protocol development for oxygenation application should consider these factors to increase health care worker safety.

Minimally-invasive, microneedle-array extraction of interstitial fluid for comprehensive biomedical applications: transcriptomics, proteomics, metabolomics, exosome research, and biomarker identification.

Interstitial fluid (ISF) has recently garnered interest as a biological fluid that could be used as an alternate to blood for biomedical applications, diagnosis, and therapy. ISF extraction techniques are promising because they are less invasive and less painful than venipuncture. ISF is an alternative, incompletely characterized source of physiological data. Here, we describe a novel method of ISF extraction in rats, using microneedle arrays, which provides volumes of ISF that are sufficient for downstream analysis techniques such as proteomics, genomics, and extracellular vesicle purification and analysis. This method is potentially less invasive than previously reported techniques. The limited invasiveness and larger volumes of extracted ISF afforded by this microneedle-assisted ISF extraction method provide a technique that is less stressful and more humane to laboratory animals, while also allowing for a reduction in the numbers of animals needed to acquire sufficient volumes of ISF for biomedical analysis and application.

Extraction and biomolecular analysis of dermal interstitial fluid collected with hollow microneedles.

Dermal interstitial fluid (ISF) is an underutilized information-rich biofluid potentially useful in health status monitoring applications whose contents remain challenging to characterize. Here, we present a facile microneedle approach for dermal ISF extraction with minimal pain and no blistering for human subjects and rats. Extracted ISF volumes were sufficient for determining transcriptome, and proteome signatures. We noted similar profiles in ISF, serum, and plasma samples, suggesting that ISF can be a proxy for direct blood sampling. Dynamic changes in RNA-seq were recorded in ISF from induced hypoxia conditions. Finally, we report the first isolation and characterization, to our knowledge, of exosomes from dermal ISF. The ISF exosome concentration is 12-13 times more enriched when compared to plasma and serum and represents a previously unexplored biofluid for exosome isolation. This minimally invasive extraction approach can enable mechanistic studies of ISF and demonstrates the potential of ISF for real-time health monitoring applications.

Analysis of tear glucose concentration with electrospray ionization mass spectrometry.

We have developed a mass spectrometry-based method that allows one to accurately determine the glucose concentration of tear fluid. We used a 1 microL micro-capillary to collect tear fluid from the tear meniscus with minimal irritation of the eye. We analyzed the 1 muL volume of collected tear fluid with liquid-chromatography electrospray ionization mass spectrometry with the use of D-glucose-6,6-d2 as an internal standard. Repeated measurements and a recovery experiment on pooled, onion-induced tears showed that the analysis of the glucose in tears was precise (4% relative standard deviation) and provided 100% recovery. We found the tear glucose concentration of one fasting nondiabetic subject to be 13 to 51 microM while the onion-induced tear glucose concentration of a different nondiabetic subject to be 211 to 256 microM.

Tear glucose analysis for the noninvasive detection and monitoring of diabetes mellitus.

One approach to the noninvasive monitoring of blood glucose concentration is to monitor glucose concentrations in tear fluid. While several methods for sensing glucose in tear fluid have been proposed, controversy remains as to the precise concentrations of tear glucose in normal and diabetic subjects and as to whether tear fluid glucose concentrations correlate with blood glucose concentrations. This review covers the present understanding of the physiology of glucose transport in tears, the regulation of the aqueous tear fraction, and studies of tear glucose concentration over the last 80 years. The various tear collection methods employed greatly influence the measured tear glucose concentrations. Studies that involve mechanical irritation of the conjunctiva during sampling measure the highest tear glucose concentrations, while studies that avoid tear stimulation measure the lowest concentrations. Attempts to monitor tear glucose concentration in situ by using contact lens-based sensing devices are discussed, and new observations are presented of tear glucose concentration obtained by a method designed to avoid tear stimulation. These studies indicate the importance of the sampling method in determining tear glucose concentrations. On the basis of these results, we discuss the future of in vivo tear glucose sensing and outline the studies needed to resolve the remaining questions about the relationship between tear and blood glucose concentrations.

Implementation of small-group reflection rounds at an emergency medicine residency program.

Few residency curricular interventions have focused on improving well-being and promoting humanism. We describe the implementation of a novel curriculum based on small-group reflection rounds--the Emergency Medicine Reflection Rounds (EMRR)--at a 4-year US emergency medicine (EM) residency. During the inaugural year (2010-2011), nine residents volunteered to take part in 1-hour monthly sessions with faculty facilitators. Residents were provided with a confidential environment to discuss difficult ethical and interpersonal encounters from their clinical experiences. Ongoing feedback from participants was solicited, culminating with a four-question survey in which all respondents remarked that the EMRR contributed to improving their own well-being and agreed that it provided an important forum for residents to discuss difficult issues in a safe environment. In this article, we describe our innovation as an example of a wellness initiative that has promoted reflective practice and fostered cooperative learning around the communication, professional, and ethical challenges inherent in EM practice. Our EMRR model may be useful to other EM residences looking to supplement their wellness curriculum.

Progress in developing polymerized crystalline colloidal array sensors for point-of-care detection of myocardial ischemia.

The difficulty of rapid, definitive diagnosis of myocardial ischemia leads to unnecessary hospital admissions and treatment delays. Previously, decreased metal binding affinity in human serum was investigated as a marker for myocardial ischemia. Polymerized Crystalline Colloidal Array (PCCA) sensors for Ni2+ may be useful in developing a point-of-care test to determine metal binding affinity in plasma and to help rule out myocardial ischemia. PCCA sensors for Ni2+, with 5-amino-8-hydroxyquinoline as a chelating agent, were tested in aqueous solutions and diluted human plasma. The peak wavelength diffracted by the sensors was monitored by reflectance spectrometry and correlated with Ni2+ concentration. The PCCA sensors show a linear response to aqueous Ni2+ concentrations between 0.2 and 1.0 mmol L(-1), and can detect changes in free Ni2+ concentration of <60 micromol L(-1). The sensors respond at physiologic pH and can be reversibly dehydrated. The PCCA sensors developed here can report on free Ni2+ concentration in the presence of human plasma. These sensors can be used to detect a decrease in the Ni2+ affinity of plasma proteins, which may indicate recent myocardial ischemia. PCCA sensors offer a practical approach to rapid, point-of-care detection of a proposed biochemical signature of myocardial ischemia.

Mass spectral determination of fasting tear glucose concentrations in nondiabetic volunteers.

BACKGROUND: There is considerable disagreement regarding the concentration of glucose in tears and its relationship to the concentration in blood. Improved sampling and analysis methods may resolve these discrepancies and possibly provide a basis for in situ tear glucose sensors. METHODS: We used liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) to determine glucose in 1-muL tear fluid samples obtained from 25 fasting study participants. Tear fluid was collected with microcapillaries and a slitlamp microscope. RESULTS: The median (range) of fasting tear glucose concentrations was 28 (7-161) micromol/L or 0.50 (0.13-2.90) mg/dL. The SD of tear glucose measurements for individuals varied linearly with the mean tear glucose concentration and was approximately half of the mean. We found no significant difference in tear glucose concentrations between contact lens users and nonusers (P = 0.715). We observed significant correlations between fasting blood and tear glucose concentrations (R = 0.50, P = 0.01). CONCLUSIONS: Our tear fluid collection and analysis method enables reliable measurement of equilibrium, fasting tear glucose concentrations. These concentrations are lower than those previously reported for nondiabetic persons. Larger population studies are required to determine correlations between blood and tear glucose concentrations and to determine the utility of contact lens-based sensors for the monitoring of diabetes. Our methods are applicable for study of other tear fluid analytes and may prove useful for monitoring other disease states.