A wearable cardiac ultrasound imager.

Continuous imaging of cardiac functions is highly desirable for the assessment of long-term cardiovascular health, detection of acute cardiac dysfunction and clinical management of critically ill or surgical patients1-4. However, conventional non-invasive approaches to image the cardiac function cannot provide continuous measurements owing to device bulkiness5-11, and existing wearable cardiac devices can only capture signals on the skin12-16. Here we report a wearable ultrasonic device for continuous, real-time and direct cardiac function assessment. We introduce innovations in device design and material fabrication that improve the mechanical coupling between the device and human skin, allowing the left ventricle to be examined from different views during motion. We also develop a deep learning model that automatically extracts the left ventricular volume from the continuous image recording, yielding waveforms of key cardiac performance indices such as stroke volume, cardiac output and ejection fraction. This technology enables dynamic wearable monitoring of cardiac performance with substantially improved accuracy in various environments.

A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation.

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30 kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). In both soluble and condensed forms, N has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ∼1:1 ratio of N to oligonucleotide, LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here, assembly has a critical concentration of about 1 µM. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.

Wearable Electrochemical Glucose Sensors in Diabetes Management: A Comprehensive Review.

With the rising diabetic population, the demand for glucose sensing devices has also been on an increasing trend. Accordingly, the field of glucose biosensors for diabetes management has witnessed tremendous scientific and technological advancements since the introduction of the first enzymatic glucose biosensor in the 1960s. Among these, electrochemical biosensors hold considerable potential for tracking dynamic glucose profiles in real time. The recent evolution of wearable devices has opened opportunities to use alternative body fluids in a pain-free, noninvasive or minimally invasive manner. This review aims to present a comprehensive report about the status and promise of wearable electrochemical sensors for on-body glucose monitoring. We start by highlighting the importance of diabetes management and how sensors can contribute toward its effective monitoring. We then discuss the electrochemical glucose sensing mechanisms, evolution of such glucose sensors over time, different versions of wearable glucose biosensors targeting various biofluids, and multiplexed wearable sensors toward optimal diabetes management. Finally, we focus on the commercial aspects of wearable glucose biosensors, starting with existing continuous glucose monitors, followed by other emerging sensing technologies, and concluding with highlighting the key prospects toward personalized diabetes management in connection to an autonomous closed-loop artificial pancreas.

Structure of the Hepatitis B virus capsid quasi-6-fold with a trapped C-terminal domain reveals capsid movements associated with domain exit.

Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/ß complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impß as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T = 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0 Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impß-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.

Charge Detection Mass Spectrometry Reveals Favored Structures in the Assembly of Virus-Like Particles: Polymorphism in Norovirus GI.1.

The main capsid protein (CP) of norovirus, the leading cause of gastroenteritis, is expected to self-assemble into virus-like particles with the same structure as the wild-type virus, a capsid with 180 CPs in a T = 3 icosahedron. Using charge detection mass spectrometry (CD-MS), we find that the norovirus GI.1 variant is structurally promiscuous, forming a wide variety of well-defined structures, some that are icosahedral capsids and others that are not. The structures that are present evolve with time and vary with solution conditions. The presence of icosahedral T = 3 and T = 4 capsids (240 CPs) under some conditions was confirmed by cryo-electron microscopy (cryo-EM). The cryo-EM studies also confirmed the presence of an unexpected prolate geometry based on an elongated T = 4 capsid with 300 CPs. In addition, CD-MS measurements indicate the presence of well-defined peaks with masses corresponding to 420, 480, 600, and 700 CPs. The peak corresponding to 420 CPs is probably due to an icosahedral T = 7 capsid, but this could not be confirmed by cryo-EM. It is possible that the T = 7 particles are too fragile to survive vitrification. There are no mass peaks associated with the T = 9 and T = 12 icosahedra with 540 and 720 CPs. The larger structures with 480, 600, and 700 CPs are not icosahedral; however, their measured charges suggest that they are hollow shells. The use of CD-MS to monitor virus-like particles assembly may have important applications in vaccine development and quality control.

Decentralized ORP Measurements for Gut Redox Status Monitoring: Toward Personalized Gut Microbiota Balance.

Gut microbiome targeting has emerged as a new generation of personalized medicine and a potential wellness and disease driver. Specifically, the gut redox balance plays a key role in shaping the gut microbiota and its link with the host, immune system, and disease evolution. In this sense, precise and personalized nutrition has proven synergy and capability to modulate the gut microbiome environment through the formulation of dietary interventions, such as vitamin support. Accordingly, there are urgent demands for simple and effective analytical platforms for understanding the relationship between the tailored vitamin administration and the gut microbiota balance by rapid noninvasive on-the-spot oxidation/reduction potential monitoring for frequent and close surveillance of the gut redox status and targeting by personalized nutrition interventions. Herein, we present a disposable potentiometric sensor chip and a homemade multiwell potentiometric array to address the interplay of vitamin levels with the oxidation/reduction potential in human feces and saliva. The potentiometric ORP sensing platforms have been successfully validated and scaled up for the setup of a multiapplication prototype for cross-talk-free simple screening of many specimens. The interpersonal variability of the gut microbiota environment illustrates the potential of feces and saliva samples for noninvasive, frequent, and decentralized monitoring of the gut redox status to support timely human microbiota surveillance and guide precise dietary intervention toward restoring and promoting personalized gut redox balance.

Smart Materials for Microrobots.

Over the past 15 years, the field of microrobotics has exploded with many research groups from around the globe contributing to numerous innovations that have led to exciting new capabilities and important applications, ranging from in vivo drug delivery, to intracellular biosensing, environmental remediation, and nanoscale fabrication. Smart responsive materials have had a profound impact on the field of microrobotics and have imparted small-scale robots with new functionalities and distinct capabilities. We have identified four large categories where the majority of future efforts must be allocated to push the frontiers of microrobots and where smart materials can have a major impact on such future advances. These four areas are the propulsion and biocompatibility of microrobots, the cooperation between individual units and human operators, and finally, the intelligence of microrobots. In this Review, we look critically at the latest developments in these four categories and discuss how smart materials contribute to the progress in the exciting field of microrobotics and will set the stage for the next generation of intelligent and programmable microrobots.

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2.

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

Submersible voltammetric sensing probe for rapid and extended remote monitoring of opioids in community water systems.

The intensifying global opioid crisis, majorly attributed to fentanyl (FT) and its analogs, has necessitated the development of rapid and ultrasensitive remote/on-site FT sensing modalities. However, current approaches for tracking FT exposure through wastewater-based epidemiology (WBE) are unadaptable, time-consuming, and require trained professionals. Toward developing an extended in situ wastewater opioid monitoring system, we have developed a screen-printed electrochemical FT sensor and integrated it with a customized submersible remote sensing probe. The sensor composition and design have been optimized to address the challenges for extended in situ FT monitoring. Specifically, ZIF-8 metal-organic framework (MOF)-derived mesoporous carbon (MPC) nanoparticles (NPs) are incorporated in the screen-printed carbon electrode (SPCE) transducer to improve FT accumulation and its electrocatalytic oxidation. A rapid (10 s) and sensitive square wave voltammetric (SWV) FT detection down to 9.9 µgL-1 is thus achieved in aqueous buffer solution. A protective mixed-matrix membrane (MMM) has been optimized as the anti-fouling sensor coating to mitigate electrode passivation by FT oxidation products and enable long-term, intermittent FT monitoring. The unique MMM, comprising an insulating polyvinyl chloride (PVC) matrix and carboxyl-functionalized multi-walled carbon nanotubes (CNT-COOH) as semiconductive fillers, yielded highly stable FT sensor operation (> 95% normalized response) up to 10 h in domestic wastewater, and up to 4 h in untreated river water. This sensing platform enables wireless data acquisition on a smartphone via Bluetooth. Such effective remote operation of submersible opioid sensing probes could enable stricter surveillance of community water systems toward timely alerts, countermeasures, and legal enforcement.

Light-Switchable Biocatalytic Covalent-Organic Framework Nanomotors for Aqueous Contaminants Removal.

Self-propelled nanomotors represent a promising class of adaptable and versatile technologies with broad applications in the realms of biomedicine and environmental remediation. Herein, we report a biocatalytic nanomotor based on a covalent-organic framework (COF) that demonstrates intelligent and switchable motion triggered by a blue-to-red light switch. Consequently, when exposed to blue light, the nanomotor significantly enhances the removal of contaminants in aqueous solutions due to its elevated mobility. Conversely, it effectively deactivates its motion and contaminant removal upon exposure to red light. This study explores the heterogeneous assembly strategy of the COF-based nanomotor and its light-controlled propulsion performance and provides a novel strategy for the regulation of movement, offering valuable insights for the design and practical applications of nanomotors.

Ease and practicability of the 2017 classification of periodontal diseases and conditions: a study of dental electronic health records.

BACKGROUND: A new classification for Periodontal and Peri-implant Diseases and Conditions was introduced in the 2017 World Workshop. In the past the 1999 Armitage Classification was commonly used in practice. This study aimed to assess the ease and practicability of retroactively diagnosing a subset of patients formerly diagnosed using the 1999 AAP/CDC classification with the 2017 AAP/EFP disease classification. METHODS: A random subset of 10% of all patients referred over a 7-year period (2011-2018) to the Post-Doctoral Periodontics Clinic at Columbia University College of Dental Medicine were reviewed by accessing the Electronic Health Records (EHRs) on axiUm. Patients diagnosed with periodontal disease based on the 1999 AAP/CDC classification (including chronic and aggressive Periodontitis) were reclassified using the 2017 classification (stage: I, II, III and grade: A, B, C). RESULTS: A sample of 336 patient records were examined. 132 were diagnosed with gingivitis, and 204 with periodontitis. Of these 204 patients, 68 (33.3%) were diagnosed with aggressive and 136 (66.7%) with chronic periodontitis. Patients diagnosed with aggressive periodontitis, 10% were reclassified as stage II, 47% as stage III, and 43% as stage IV periodontitis, and 100% were reclassified as grade C. Among patients with chronic periodontitis, 7% were reclassified as stage I, 65% as stage II, 21% as stage III, and 7% as stage IV; 11% of these were reclassified as grade A, 63% grade B, and 26% grade C. CONCLUSIONS: The majority of those originally diagnosed with aggressive (90%) and chronic (80%) periodontitis were reclassified as either molar/incisor pattern stage III grade C or stage IV grade C periodontitis, and stage II or III periodontitis, respectively. The study demonstrated that it is practical to retroactively reassign a diagnosis according to the new 2017 classification using available information included in dental EHRs.

Biosensor Strip for Rapid On-site Assessment of Levodopa Pharmacokinetics along with Motor Performance in Parkinson's Disease.

While levodopa (L-Dopa) is the primary treatment for alleviating Parkinson's disease (PD), its efficacy is hindered by challenges such as a short half-life and inconsistent plasma levels. As PD progresses, the rising need for increased and more frequent L-Dopa doses coupled with symptom fluctuations and dyskinesias underscores the urgency for improved comprehension of the interplay between L-Dopa levels and PD motor symptoms. Addressing this critical need, we present a decentralized testing method using a disposable biosensor strip and a universal slope (U-slope) calibration-free approach. This enables reliable, rapid, simple, and cost-effective decentralized L-Dopa measurements from capillary blood. A pilot study with PD persons demonstrates the ability to monitor real-time L-Dopa pharmacokinetics from fingerstick blood after oral L-Dopa-Carbidopa (C-Dopa) tablet administration. Correlating capillary blood L-Dopa levels with PD motor scores revealed a well-defined inverse correlation with temporal motor fluctuations. We compared the resulting dynamic capillary blood L-Dopa levels with plasma L-Dopa levels using the traditional but clinically impractical high-performance liquid chromatography technique. By providing timely feedback on a proper L-Dopa dosing regimen in a decentralized and rapid fashion, this new biosensing platform will facilitate tailored optimal L-Dopa dosing, towards improving symptom management and enhancing health-related quality of life.

Region-Specific Hepatitis B Virus Genome Exposure from Nucleocapsid Modulated by Capsid Linker Sequence and Inhibitor: Implications for Uncoating.

Hepatitis B virus (HBV) contains a partially double-stranded, relaxed circular (RC) DNA genome synthesized within a nucleocapsid (NC) in the host cell cytoplasm. The release of RC DNA from the NC, in an ill-defined process called uncoating, to the nucleus is required for its conversion to the covalently closed circular (CCC) DNA, the viral episome serving as the transcriptional template for all viral RNAs necessary for replication and, thus, essential for establishing and sustaining viral infection. In efforts to better understand uncoating, we analyzed HBV core (HBc) mutants that show various levels of nuclear CCC DNA but little to no cytoplasmic RC DNA. We found that RC DNA could be synthesized by these mutants outside the cell, but in contrast to the wild type (wt), the mutant NCs were unable to protect RC DNA from digestion by the endogenous nuclease(s) in cellular lysates or exogenous DNase. Subcellular fractionation suggested that the major RC DNA-degrading activity was membrane associated. Digestion with sequence-specific and nonspecific DNases revealed the exposure of specific regions of RC DNA from the mutant NC. Similarly, treatment of wt NCs with a core inhibitor known to increase CCC DNA by affecting uncoating also led to region-specific exposure of RC DNA. Furthermore, a subpopulation of untreated wild type (wt) mature NCs showed site-specific exposure of RC DNA as well. Competition between RC DNA degradation and its conversion to CCC DNA during NC uncoating thus likely plays an important role in the establishment and persistence of HBV infection and has implications for the development of capsid-targeted antivirals. IMPORTANCE Disassembly of the hepatitis B virus (HBV) nucleocapsid (NC) to release its genomic DNA, in an ill-understood process called uncoating, is required to form the viral nuclear episome in the host cell nucleus, a viral DNA essential for establishing and sustaining HBV infection. The elimination of the HBV nuclear episome remains the holy grail for the development of an HBV cure. We report here that the HBV genomic DNA is exposed in a region-specific manner during uncoating, which is enhanced by mutations of the capsid protein and a capsid-targeted antiviral compound. The exposure of the viral genome can result in its rapid degradation or, alternatively, can enhance the formation of the nuclear episome, thus having a major impact on HBV infection and persistence. These results are thus important for understanding fundamental mechanisms of HBV replication and persistence and for the ongoing pursuit of an HBV cure.

Mutations at the Alphavirus E1'-E2 Interdimer Interface Have Host-Specific Phenotypes.

Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane, and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at the following two interfaces: (i) the intradimer interactions between E2 and E1 of the same heterodimer and (ii) the interdimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1'). We hypothesized that the interdimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (chikungunya piggyback [CPB]) where we replaced six interdimeric residues in the E2 protein of Sindbis virus (wild-type [WT] SINV) with those from the E2 protein from chikungunya virus and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to the capsid protein and contained defects in particle morphology compared with virus derived from mosquito cells. Using cryo-electron microscopy (cryo-EM), we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the interdimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design. IMPORTANCE Alphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as emerging pathogens, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This finding is important because it identifies a region in the spike that could be a target for antiviral drug design.

Nanoparticle-modified microrobots for in vivo antibiotic delivery to treat acute bacterial pneumonia.

Bioinspired microrobots capable of actively moving in biological fluids have attracted considerable attention for biomedical applications because of their unique dynamic features that are otherwise difficult to achieve by their static counterparts. Here we use click chemistry to attach antibiotic-loaded neutrophil membrane-coated polymeric nanoparticles to natural microalgae, thus creating hybrid microrobots for the active delivery of antibiotics in the lungs in vivo. The microrobots show fast speed (>110 µm s-1) in simulated lung fluid and uniform distribution into deep lung tissues, low clearance by alveolar macrophages and superb tissue retention time (>2 days) after intratracheal administration to test animals. In a mouse model of acute Pseudomonas aeruginosa pneumonia, the microrobots effectively reduce bacterial burden and substantially lessen animal mortality, with negligible toxicity. Overall, these findings highlight the attractive functions of algae-nanoparticle hybrid microrobots for the active in vivo delivery of therapeutics to the lungs in intensive care unit settings.

Locking the Ultrasound-Induced Active Conformation of Metalloenzymes in Metal-Organic Frameworks.

Enhancing the enzymatic activity inside metal-organic frameworks (MOFs) is a critical challenge in chemical technology and bio-technology, which, if addressed, will broaden their scope in energy, food, environmental, and pharmaceutical industries. Here, we report a simple yet versatile and effective strategy to optimize biocatalytic activity by using MOFs to rapidly "lock" the ultrasound (US)-activated but more fragile conformation of metalloenzymes. The results demonstrate that up to 5.3-fold and 9.3-fold biocatalytic activity enhancement of the free and MOF-immobilized enzymes could be achieved compared to those without US pretreatment, respectively. Using horseradish peroxidase as a model, molecular dynamics simulation demonstrates that the improved activity of the enzyme is driven by an opened gate conformation of the heme active site, which allows more efficient substrate binding to the enzyme. The intact heme active site is confirmed by solid-state UV-vis and electron paramagnetic resonance, while the US-induced enzyme conformation change is confirmed by circular dichroism spectroscopy and Fourier-transform infrared spectroscopy. In addition, the improved activity of the biocomposites does not compromise their stability upon heating or exposure to organic solvent and a digestion cocktail. This rapid locking and immobilization strategy of the US-induced active enzyme conformation in MOFs gives rise to new possibilities for the exploitation of highly efficient biocatalysts for diverse applications.

Using Cell Membranes as Recognition Layers to Construct Ultrasensitive and Selective Bioelectronic Affinity Sensors.

Conventional sandwich immunosensors rely on antibody recognition layers to selectively capture and detect target antigen analytes. However, the fabrication of these traditional affinity sensors is typically associated with lengthy and multistep surface modifications of electrodes and faces the challenge of nonspecific adsorption from complex sample matrices. Here, we report on a unique design of bioelectronic affinity sensors by using natural cell membranes as recognition layers for protein detection and prevention of biofouling. Specifically, we employ the human macrophage (MΦ) membrane together with the human red blood cell (RBC) membrane to coat electrochemical transducers through a one-step process. The natural protein receptors on the MΦ membrane are used to capture target antigens, while the RBC membrane effectively prevents nonspecific surface binding. In an attempt to detect tumor necrosis factor alpha (TNF-α) cytokine using the bioelectronic affinity sensor, it demonstrates a remarkable limit of detection of 150 pM. This new sensor design integrates natural cell membranes and electronic transduction, which offers synergistic functionalities toward a broad range of biosensing applications.

Screen-Printed Technologies Combined with Flow Analysis Techniques: Moving from Benchtop to Everywhere.

Screen-printed electrodes (SPEs) coupled with flow systems have been reported in recent decades for an ever-growing number of applications in modern electroanalysis, aiming for portable methodologies. The information acquired through this combination can be attractive for future users with basic knowledge, especially due to the increased measurement throughput, reduction in reagent consumption and minimal waste generation. The trends and possibilities of this set rely on the synergistic behavior that maximizes both SPE and flow analyses characteristics, allowing mass production and automation. This overview addresses an in-depth update about the scope of samples, target analytes, and analytical throughput (injections per hour, limits of detection, linear range, etc.) obtained by coupling injection techniques (FIA, SIA, and BIA) with SPE-based electrochemical detection.

Concept of the "Universal Slope": Toward Substantially Shorter Decentralized Insulin Immunoassays.

Decentralized sensing of analytes in remote locations is today a reality. However, the number of measurable analytes remains limited, mainly due to the requirement for time-consuming successive standard additions calibration used to address matrix effects and resulting in greatly delayed results, along with more complex and costly operation. This is particularly challenging in commonly used immunoassays of key biomarkers that typically require from 60 to 90 min for quantitation based on two standard additions, hence hindering their implementation for rapid and routine diagnostic applications, such as decentralized point-of-care (POC) insulin testing. In this work we have developed and demonstrated the theoretical framework for establishing a universal slope for direct calibration-free POC insulin immunoassays in serum samples using an electrochemical biosensor (developed originally for extended calibration by standard additions). The universal slope is presented as an averaged slope constant, relying on 68 standard additions-based insulin determinations in human sera. This new quantitative analysis approach offers reliable sample measurement without successive standard additions, leading to a dramatically simplified and faster assay (30 min vs 90 min when using 2 standard additions) and greatly reduced costs, without compromising the analytical performance while significantly reducing the analyses costs. The substantial improvements associated with the new universal slope concept have been demonstrated successfully for calibration-free measurements of serum insulin in 30 samples from individuals with type 1 diabetes using meticulous statistical analysis, supporting the prospects of applying this immunoassay protocol to routine decentralized POC insulin testing.

Microneedle Aptamer-Based Sensors for Continuous, Real-Time Therapeutic Drug Monitoring.

The ability to continuously monitor the concentration of specific molecules in the body is a long-sought goal of biomedical research. For this purpose, interstitial fluid (ISF) was proposed as the ideal target biofluid because its composition can rapidly equilibrate with that of systemic blood, allowing the assessment of molecular concentrations that reflect full-body physiology. In the past, continuous monitoring in ISF was enabled by microneedle sensor arrays. Yet, benchmark microneedle sensors can only detect molecules that undergo redox reactions, which limits the ability to sense metabolites, biomarkers, and therapeutics that are not redox-active. To overcome this barrier, here, we expand the scope of these devices by demonstrating the first use of microneedle-supported electrochemical, aptamer-based (E-AB) sensors. This platform achieves molecular recognition based on affinity interactions, vastly expanding the scope of molecules that can be sensed. We report the fabrication of microneedle E-AB sensor arrays and a method to regenerate them for multiple uses. In addition, we demonstrate continuous molecular measurements using these sensors in flow systems in vitro using single and multiplexed microneedle array configurations. Translation of the platform to in vivo measurements is possible as we demonstrate with a first E-AB measurement in the ISF of a rodent. The encouraging results reported in this work should serve as the basis for future translation of microneedle E-AB sensor arrays to biomedical research in preclinical animal models.