Mitigating Medication Tampering and Diversion via Real-Time Intravenous Opioid Quantification.

Opioid tampering and diversion pose a serious problem for hospital patients with potentially life-threatening consequences. The ongoing opioid crisis has resulted in medications used for pain management and anesthesia, such as fentanyl and morphine, being stolen, substituted with a different substance, and abused. This work aims to mitigate tampering and diversion through analytical verification of the administered drug before it enters the patient. We present an electrochemical-based sensor and miniaturized wireless potentiostat that enable real-time intravenous (IV) monitoring of opioids, specifically fentanyl and morphine. The proposed system is connected to an IV drip system during surgery or post-operation recovery. Measurement results of two opioids are presented, including calibration curves and data on the sensor performance concerning pH, temperature, interference, reproducibility, and long-term stability. Finally, we demonstrate real-time fluidic measurements connected to a flow cell to simulate IV administration and a blind study classified using a machine-learning algorithm. The system achieves limits of detection (LODs) of 1.26 µg/mL and 2.75 µg/mL for fentanyl and morphine, respectively, while operating with >1-month battery lifetime due to an optimized ultra-low power 36 µA sleep mode.

A GMR enzymatic assay for quantifying nuclease and peptidase activity.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Plasmonic Sensing Studies of a Gas-Phase Cystic Fibrosis Marker in Moisture Laden Air.

A plasmonic sensing platform was developed as a noninvasive method to monitor gas-phase biomarkers related to cystic fibrosis (CF). The nanohole array (NHA) sensing platform is based on localized surface plasmon resonance (LSPR) and offers a rapid data acquisition capability. Among the numerous gas-phase biomarkers that can be used to assess the lung health of CF patients, acetaldehyde was selected for this investigation. Previous research with diverse types of sensing platforms, with materials ranging from metal oxides to 2-D materials, detected gas-phase acetaldehyde with the lowest detection limit at the µmol/mol (parts-per-million (ppm)) level. In contrast, this work presents a plasmonic sensing platform that can approach the nmol/mol (parts-per-billion (ppb)) level, which covers the required concentration range needed to monitor the status of lung infection and find pulmonary exacerbations. During the experimental measurements made by a spectrometer and by a smartphone, the sensing examination was initially performed in a dry air background and then with high relative humidity (RH) as an interferent, which is relevant to exhaled breath. At a room temperature of 23.1 °C, the lowest detection limit for the investigated plasmonic sensing platform under dry air and 72% RH conditions are 250 nmol/mol (ppb) and 1000 nmol/mol (ppb), respectively.

Development of a Soluble KIT Electrochemical Aptasensor for Cancer Theranostics.

An electrochemical sensor based on a conformation-changing aptamer is reported to detect soluble KIT, a cancer biomarker, in human serum. The sensor was fabricated with a ferrocene-labeled aptamer (Kd < 5 nM) conjugated to a gold electrode. Quantitative KIT detection was achieved using electrochemical impedance spectroscopy (EIS) and square-wave voltammetry (SWV). EIS was used to optimize experimental parameters such as the aptamer-to-spacer ratio, aptamer immobilization time, pH, and KIT incubation time, and the sensor surface was characterized using voltammetry. The assay specificity was demonstrated using interfering species and exhibited high specificity toward the target protein. The aptasensor showed a wide dynamic range, 10 pg/mL-100 ng/mL in buffer, with a 1.15 pg/mL limit of detection. The sensor also has a linear response to KIT spiked in human serum and successfully detected KIT in cancer-cell-conditioned media. The proposed aptasensor has applications as a continuous or intermittent approach for cancer therapy monitoring and diagnostics (theranostics).

An aptamer-based magnetic flow cytometer using matched filtering.

Facing unprecedented population-ageing, the management of noncommunicable diseases (NCDs) urgently needs a point-of-care (PoC) testing infrastructure. Magnetic flow cytometers are one such solution for rapid cancer cellular detection in a PoC setting. In this work, we report a giant magnetoresistive spin-valve (GMR SV) biosensor array with a multi-stripe sensor geometry and matched filtering to improve detection accuracy without compromising throughput. The carefully designed sensor geometry generates a characteristic signature when cells labeled with magnetic nanoparticles (MNPs) pass by thus enabling multi-parametric measurement like optical flow cytometers (FCMs). Enumeration and multi-parametric information were successfully measured across two decades of throughput (37 - 2730 cells/min). 10-µm polymer microspheres were used as a biomimetic model where MNPs and MNP-decorated polymer conjugates were flown over the GMR SV sensor array and detected with a signal-to-noise ratio (SNR) as low as 2.5 dB due to the processing gain afforded by the matched filtering. The performance was compared against optical observation, exhibiting a 92% detection efficiency. The system achieved a 95% counting accuracy for biomimetic models and 98% for aptamer-based pancreatic cancer cell detection. This system demonstrates the ability to perform reliable flow cytometry toward PoC diagnostics to benefit NCD control plans.

Giant magnetoresistive biosensors for real-time quantitative detection of protease activity.

Proteases are enzymes that cleave proteins and are crucial to physiological processes such as digestion, blood clotting, and wound healing. Unregulated protease activity is a biomarker of several human diseases. Synthetic peptides that are selectively hydrolyzed by a protease of interest can be used as reporter substrates of unregulated protease activity. We developed an activity-based protease sensor by immobilizing magnetic nanoparticles (MNPs) to the surface of a giant magnetoresistive spin-valve (GMR SV) sensor using peptides. Cleavage of these peptides by a protease releases the magnetic nanoparticles resulting in a time-dependent change in the local magnetic field. Using this approach, we detected a significant release of MNPs after 3.5 minutes incubation using just 4 nM of the cysteine protease, papain. In addition, we show that proteases in healthy human urine do not release the MNPs, however addition of 20 nM of papain to the urine samples resulted in a time-dependent change in magnetoresistance. This study lays the foundation for using GMR SV sensors as a platform for real-time, quantitative detection of protease activity in biological fluids.

Smartphone-Based pH Sensor for Home Monitoring of Pulmonary Exacerbations in Cystic Fibrosis.

Currently, Cystic Fibrosis (CF) patients lack the ability to track their lung health at home, relying instead on doctor checkups leading to delayed treatment and lung damage. By leveraging the ubiquity of the smartphone to lower costs and increase portability, a smartphone-based peripheral pH measurement device was designed to attach directly to the headphone port to harvest power and communicate with a smartphone application. This platform was tested using prepared pH buffers and sputum samples from CF patients. The system matches within ~0.03 pH of a benchtop pH meter while fully powering itself and communicating with a Samsung Galaxy S3 smartphone paired with either a glass or Iridium Oxide (IrOx) electrode. The IrOx electrodes were found to have 25% higher sensitivity than the glass probes at the expense of larger drift and matrix sensitivity that can be addressed with proper calibration. The smartphone-based platform has been demonstrated as a portable replacement for laboratory pH meters, and supports both highly robust glass probes and the sensitive and miniature IrOx electrodes with calibration. This tool can enable more frequent pH sputum tracking for CF patients to help detect the onset of pulmonary exacerbation to provide timely and appropriate treatment before serious damage occurs.

An Efficient Power Harvesting Mobile Phone-Based Electrochemical Biosensor for Point-of-Care Health Monitoring.

Cellular phone penetration has grown continually over the past two decades with the number of connected devices rapidly approaching the total world population. Leveraging the worldwide ubiquity and connectivity of these devices, we developed a mobile phone-based electrochemical biosensor platform for point-of-care (POC) diagnostics and wellness tracking. The platform consists of an inexpensive electronic module (< $20) containing a low-power potentiostat that interfaces with and efficiently harvests power from a wide variety of phones through the audio jack. Active impedance matching improves the harvesting efficiency to 79%. Excluding loses from supply rectification and regulation, the module consumes 6.9 mW peak power and can measure < 1 nA bidirectional current. The prototype was shown to operate within the available power budget set by mobile devices and produce data that matches well with that of an expensive laboratory grade instrument. We demonstrate that the platform can be used to track the concentration of secretory leukocyte protease inhibitor (SLPI), a biomarker for monitoring lung infections in cystic fibrosis patients, in its physiological range via an electrochemical sandwich assay on disposable screen-printed electrodes with a 1 nM limit of detection.

High-Resolution Analysis of Antibodies to Post-Translational Modifications Using Peptide Nanosensor Microarrays.

Autoantibodies are a hallmark of autoimmune diseases such as lupus and have the potential to be used as biomarkers for diverse diseases, including immunodeficiency, infectious disease, and cancer. More precise detection of antibodies to specific targets is needed to improve diagnosis of such diseases. Here, we report the development of reusable peptide microarrays, based on giant magnetoresistive (GMR) nanosensors optimized for sensitively detecting magnetic nanoparticle labels, for the detection of antibodies with a resolution of a single post-translationally modified amino acid. We have also developed a chemical regeneration scheme to perform multiplex assays with a high level of reproducibility, resulting in greatly reduced experimental costs. In addition, we show that peptides synthesized directly on the nanosensors are approximately two times more sensitive than directly spotted peptides. Reusable peptide nanosensor microarrays enable precise detection of autoantibodies with high resolution and sensitivity and show promise for investigating antibody-mediated immune responses to autoantigens, vaccines, and pathogen-derived antigens as well as other fundamental peptide-protein interactions.

Nanosensor dosimetry of mouse blood proteins after exposure to ionizing radiation.

Giant magnetoresistive (GMR) nanosensors provide a novel approach for measuring protein concentrations in blood for medical diagnosis. Using an in vivo mouse radiation model, we developed protocols for measuring Flt3 ligand (Flt3lg) and serum amyloid A1 (Saa1) in small amounts of blood collected during the first week after X-ray exposures of sham, 0.1, 1, 2, 3, or 6 Gy. Flt3lg concentrations showed excellent dose discrimination at ≥ 1 Gy in the time window of 1 to 7 days after exposure except 1 Gy at day 7. Saa1 dose response was limited to the first two days after exposure. A multiplex assay with both proteins showed improved dose classification accuracy. Our magneto-nanosensor assay demonstrates the dose and time responses, low-dose sensitivity, small volume requirements, and rapid speed that have important advantages in radiation triage biodosimetry.

A 256 pixel magnetoresistive biosensor microarray in 0.18µm CMOS.

Magnetic nanotechnologies have shown significant potential in several areas of nanomedicine such as imaging, therapeutics, and early disease detection. Giant magnetoresistive spin-valve (GMR SV) sensors coupled with magnetic nanotags (MNTs) possess great promise as ultra-sensitive biosensors for diagnostics. We report an integrated sensor interface for an array of 256 GMR SV biosensors designed in 0.18 µm CMOS. Arranged like an imager, each of the 16 column level readout channels contains an analog front- end and a compact ΣΔ modulator (0.054 mm2) with 84 dB of dynamic range and an input referred noise of 49 nT/√Hz. Performance is demonstrated through detection of an ovarian cancer biomarker, secretory leukocyte peptidase inhibitor (SLPI), spiked at concentrations as low as 10 fM. This system is designed as a replacement for optical protein microarrays while also providing real-time kinetics monitoring.

Quantification of protein interactions and solution transport using high-density GMR sensor arrays.

Monitoring the kinetics of protein interactions on a high-density sensor array is vital to drug development and proteomic analysis. Label-free kinetic assays based on surface plasmon resonance are the current gold standard, but they have poor detection limits, suffer from non-specific binding, and are not amenable to high-throughput analyses. Here, we show that magnetically responsive nanosensors that have been scaled to over 100,000 sensors per cm² can be used to measure the binding kinetics of various proteins with high spatial and temporal resolution. We present an analytical model that describes the binding of magnetically labelled antibodies to proteins that are immobilized on the sensor surface. This model is able to quantify the kinetics of antibody-antigen binding at sensitivities as low as 20 zeptomoles of solute.

Matrix-insensitive protein assays push the limits of biosensors in medicine.

Advances in biosensor technologies for in vitro diagnostics have the potential to transform the practice of medicine. Despite considerable work in the biosensor field, there is still no general sensing platform that can be ubiquitously applied to detect the constellation of biomolecules in diverse clinical samples (for example, serum, urine, cell lysates or saliva) with high sensitivity and large linear dynamic range. A major limitation confounding other technologies is signal distortion that occurs in various matrices due to heterogeneity in ionic strength, pH, temperature and autofluorescence. Here we present a magnetic nanosensor technology that is matrix insensitive yet still capable of rapid, multiplex protein detection with resolution down to attomolar concentrations and extensive linear dynamic range. The matrix insensitivity of our platform to various media demonstrates that our magnetic nanosensor technology can be directly applied to a variety of settings such as molecular biology, clinical diagnostics and biodefense.

Multiplex protein assays based on real-time magnetic nanotag sensing.

Magnetic nanotags (MNTs) are a promising alternative to fluorescent labels in biomolecular detection assays, because minute quantities of MNTs can be detected with inexpensive giant magnetoresistive (GMR) sensors, such as spin valve (SV) sensors. However, translating this promise into easy to use and multilplexed protein assays, which are highly sought after in molecular diagnostics such as cancer diagnosis and treatment monitoring, has been challenging. Here, we demonstrate multiplex protein detection of potential cancer markers at subpicomolar concentration levels and with a dynamic range of more than four decades. With the addition of nanotag amplification, the analytic sensitivity extends into the low fM concentration range. The multianalyte ability, sensitivity, scalability, and ease of use of the MNT-based protein assay technology make it a strong contender for versatile and portable molecular diagnostics in both research and clinical settings.