Gold nanoparticle-decorated fluorine-doped tin oxide substrate for sensitive label-free OIRD microarray chips.

Oblique incidence reflectance difference (OIRD) is an emerging technique enabling real-time and label-free detection of bio-affinity binding events on microarrays. The interfacial architecture of the microarray chip is critical to the performance of OIRD detection. In this work, a sensitive label-free OIRD microarray chip was developed by using gold nanoparticle-decorated fluorine-doped tin oxide (AuNPs-FTO) slides as a chip substrate. This AuNPs-FTO chip demonstrates a higher signal-to-noise ratio and improved sensitivity compared to that built on FTO glass, showing a detection limit of as low as 10 ng mL-1 for the model target, HRP-conjugated streptavidin. On-chip ELISA experiments and optical calculations suggest that the enhanced performance is not only due to the higher probe density enabling a high capture efficiency toward the target, but most importantly, the AuNP layer arouses optical interference to improve the intrinsic sensitivity of OIRD. This work provides an effective strategy for constructing OIRD-based microarray chips with enhanced sensitivity, and may help extend their practical applications in various fields.

Label-free OIRD detection of protein microarrays on high dielectric constant substrate with enhanced intrinsic sensitivity.

Oblique-incidence reflectivity difference (OIRD) is a dielectric constant-sensitive technique and exhibits intriguing applications in label-free and high-throughput detection of protein microarrays. With the outstanding advantage of being compatible with arbitrary substrates, however, the effect of the substrate, particularly its dielectric constant on the OIRD sensitivity has not been fully disclosed. In this paper, for the first time we investigated the dependence of OIRD sensitivity on the dielectric constant of the substrate under top-incident OIRD configuration by combining theoretical modeling and experimental evaluation. Optical modeling suggested that the higher dielectric constant substrate exhibits a higher intrinsic sensitivity. Experimentally, three substrates including glass, fluorine-doped tin oxide (FTO) and silicon (Si) with different dielectric constants were selected as microarray substrates and their detection performances were evaluated. In good agreement with the modeling, high dielectric constant Si-based microarray exhibited the highest sensitivity among three chips, reaching a detection limit of as low as 5 ng mL-1 with streptavidin as the model target. Quantification of captured targets on three chips with on-chip enzyme-linked immunosorbent assay (ELISA) further confirmed that the enhanced performance originates from the high dielectric constant enhanced intrinsic OIRD sensitivity. This work thus provides a new way to OIRD-based label-free microarrays with improved sensitivity.

Effects of sub-lethal doses of fentanyl on vital physiologic functions and withdrawal-like behaviors in adult goats.

Synthetic opioids like fentanyl have improved the standard of care for many patients in the clinical setting, but their abuse leads to tens of thousands of overdose deaths annually. The current opioid epidemic underscores a critical need for insights into the physiological effects of fentanyl on vital functions. High doses of opioids in small mammals cause opioid-induced respiratory depression (OIRD) leading to hypoventilation, hypoxemia, and hypercapnia. In addition, opioids can also increase the alveolar to arterial oxygen (A-a) gradient and airway dysfunction. However, little is known about the physiologic effects of sub-lethal doses of opioids in large mammals. Here we report the effects of a sub-lethal dose range of fentanyl (25-125 µg/kg; IV) on vital physiologic functions over 90 min (min) and withdrawal-like behaviors over the subsequent 4 h (h) in adult female goats (n = 13). Fentanyl induced decreases in breathing frequency in the first few min post-injection, but then led to a sustained increase in tidal volume, total ventilation, and blood pressure with a reduced heart rate for ≥90 min. These ventilatory changes resulted in time-dependent arterial hypocapnia and hypoxemia and an increased alveolar to arterial oxygen gradient ∼30 min post-injection indicative of impaired gas exchange in the lung. The predominant effects of fentanyl on breathing were stimulatory, underscored by an increased rate of rise of the diaphragm muscle activity and increased activation of upper airway, intercostal and abdominal muscles. Beginning 90 min post-injection we also quantified withdrawal-like behaviors over 4 h, demonstrating dose- and time-dependent increases in locomotor, biting, itching, and pawing behaviors. We conclude that fentanyl at sublethal doses induces multiple physiologic and behavior changes that emerge along different time courses suggesting multiple independent mechanisms underlying effects of opioids.

An electroencephalogram biomarker of fentanyl drug effects.

Opioid drugs influence multiple brain circuits in parallel to produce analgesia as well as side effects, including respiratory depression. At present, we do not have real-time clinical biomarkers of these brain effects. Here, we describe the results of an experiment to characterize the electroencephalographic signatures of fentanyl in humans. We find that increasing concentrations of fentanyl induce a frontal theta band (4 to 8 Hz) signature distinct from slow-delta oscillations related to sleep and sedation. We also report that respiratory depression, quantified by decline in an index of instantaneous minute ventilation, occurs at ≈1700-fold lower concentrations than those that produce sedation as measured by reaction time. The electroencephalogram biomarker we describe could facilitate real-time monitoring of opioid drug effects and enable more precise and personalized opioid administration.

Current research in pathophysiology of opioid-induced respiratory depression, neonatal opioid withdrawal syndrome, and neonatal antidepressant exposure syndrome.

Respiratory depression (RD) is the primary cause of death due to opioids. Opioids bind to mu (µ)-opioid receptors (MORs) encoded by the MOR gene Oprm1, widely expressed in the central and peripheral nervous systems including centers that modulate breathing. Respiratory centers are located throughout the brainstem. Experiments with Oprm1-deleted knockout (KO) mice undertaken to determine which sites are necessary for the induction of opioid-induced respiratory depression (OIRD) showed that the pre-Bötzinger complex (preBötC) and the pontine Kölliker-Fuse nucleus (KF) contribute equally to OIRD but RD was not totally eliminated. Morphine showed a differential influence on preBötC and KF neurons - low doses attenuated RD following deletion of MORs from preBötC neurons and an increase in apneas after high doses whereas deletion of MORs from KF neurons but not the preBötC attenuated RD at both high and low doses. In other KO mice studies, morphine administration after deletion of Oprm1 from both the preBötC and the KF/PBN neurons, led to the conclusion that both respiratory centres contribute to OIRD but the preBötC predominates. MOR-mediated post-synaptic activation of GIRK potassium channels has been implicated as a cause of OIRD. A complementary mechanism in the preBötC involving KCNQ potassium channels independent of MOR signaling has been described. Recent experiments in rats showing that morphine depresses normal, but not gasping breathing, cast doubt on the belief that eupnea, sighs, and gasps, are under the control of preBötC neurons. Methadone, administered to alleviate symptoms of neonatal opioid withdrawal syndrome (NOWES), desensitized rats to OIRD. Protection lost between postnatal days 1 and 2 coincides with the preBötC becoming the dominant generator of respiratory rhythm. Neonatal antidepressant exposure syndrome (NADES) and serotonin toxicity (ST) show similarities including RD. Enzyme CYP2D6 involved in opioid detoxification is polymorphic. Individuals of different CYP2D6 genotype may show increased, decreased, or no enzyme activity, contributing to the variability of patient responses to different opioids and OIRD.

Epidural electrical stimulation of the cervical spinal cord opposes opioid-induced respiratory depression.

Opioid overdose suppresses brainstem respiratory circuits, causes apnoea and may result in death. Epidural electrical stimulation (EES) at the cervical spinal cord facilitated motor activity in rodents and humans, and we hypothesized that EES of the cervical spinal cord could antagonize opioid-induced respiratory depression in humans. Eighteen patients requiring surgical access to the dorsal surface of the spinal cord between C2 and C7 received EES or sham stimulation for up to 90 s at 5 or 30 Hz during complete (OFF-State) or partial suppression (ON-State) of respiration induced by remifentanil. During the ON-State, 30 Hz EES at C4 and 5 Hz EES at C3/4 increased tidal volume and decreased the end-tidal carbon dioxide level compared to pre-stimulation control levels. EES of 5 Hz at C5 and C7 increased respiratory frequency compared to pre-stimulation control levels. In the OFF-State, 30 Hz cervical EES at C3/4 terminated apnoea and induced rhythmic breathing. In cadaveric tissue obtained from a brain bank, more neurons expressed both the neurokinin 1 receptor (NK1R) and somatostatin (SST) in the cervical spinal levels responsive to EES (C3/4, C6 and C7) compared to a region non-responsive to EES (C2). Thus, the capacity of cervical EES to oppose opioid depression of respiration may be mediated by NK1R+/SST+ neurons in the dorsal cervical spinal cord. This study provides proof of principle that cervical EES may provide a novel therapeutic approach to augment respiratory activity when the neural function of the central respiratory circuits is compromised by opioids or other pathological conditions. KEY POINTS: Epidural electrical stimulation (EES) using an implanted spinal cord stimulator (SCS) is an FDA-approved method to manage chronic pain. We tested the hypothesis that cervical EES facilitates respiration during administration of opioids in 18 human subjects who were treated with low-dose remifentanil that suppressed respiration (ON-State) or high-dose remifentanil that completely inhibited breathing (OFF-State) during the course of cervical surgery. Dorsal cervical EES of the spinal cord augmented the respiratory tidal volume or increased the respiratory frequency, and the response to EES varied as a function of the stimulation frequency (5 or 30 Hz) and the cervical level stimulated (C2-C7). Short, continuous cervical EES restored a cyclic breathing pattern (eupnoea) in the OFF-State, suggesting that cervical EES reversed the opioid-induced respiratory depression. These findings add to our understanding of respiratory pattern modulation and suggest a novel mechanism to oppose the respiratory depression caused by opioids.

Current and Future Perspective of Devices and Diagnostics for Opioid and OIRD.

OIRD (opioid-induced respiratory depression) remains a significant public health concern due to clinically indicated and illicit opioid use. Respiratory depression is the sine qua non of opioid toxicity, and early detection is critical for reversal using pharmacologic and non-pharmacologic interventions. In addition to respiratory monitoring devices such as pulse oximetry, capnography, and contactless monitoring systems, novel implantable sensors and detection systems such as optical detection and electrochemical detection techniques are being developed to identify the presence of opioids both in vivo and within the environment. These new technologies will not only monitor for signs and symptoms of OIRD but also serve as a mechanism to alert and assist first responders and lay rescuers. The current opioid epidemic brings to the forefront the need for additional accessible means of detection and diagnosis. Rigorous evaluation of safety, efficacy, and acceptability will be necessary for both new and established technologies to have an impact on morbidity and mortality associated with opioid toxicity. Here, we summarized existing and advanced technologies for opioid detection and OIRD management with a focus on recent advancements in wearable and implantable opioid detection. We expect that this review will serve as a complete informative reference for the researchers and healthcare professionals working on the subject and allied fields.

Postpartum Respiratory Depression.

Postpartum respiratory depression is a complex, multifactorial issue that encompasses a patient's baseline preexisting conditions, certain pregnancy-specific conditions or complications, as well as the iatrogenic element of various medications given in the peripartum period. In this review, we discuss many of these factors including obesity, sleep-disordered breathing, chronic lung disease, neuromuscular disorders, opioids, preeclampsia, peripartum cardiomyopathy, postpartum hemorrhage, amniotic fluid embolism, sepsis, acute respiratory distress syndrome (ARDS), and medications such as analgesics, sedatives, anesthetics, and magnesium. Current recommendations for screening, treatment, and prevention are also discussed.

Neural basis of opioid-induced respiratory depression and its rescue.

Opioid-induced respiratory depression (OIRD) causes death following an opioid overdose, yet the neurobiological mechanisms of this process are not well understood. Here, we show that neurons within the lateral parabrachial nucleus that express the µ-opioid receptor (PBL Oprm1 neurons) are involved in OIRD pathogenesis. PBL Oprm1 neuronal activity is tightly correlated with respiratory rate, and this correlation is abolished following morphine injection. Chemogenetic inactivation of PBL Oprm1 neurons mimics OIRD in mice, whereas their chemogenetic activation following morphine injection rescues respiratory rhythms to baseline levels. We identified several excitatory G protein-coupled receptors expressed by PBL Oprm1 neurons and show that agonists for these receptors restore breathing rates in mice experiencing OIRD. Thus, PBL Oprm1 neurons are critical for OIRD pathogenesis, providing a promising therapeutic target for treating OIRD in patients.

Neuronal mechanisms underlying opioid-induced respiratory depression: our current understanding.

Opioid-induced respiratory depression (OIRD) represents the primary cause of death associated with therapeutic and recreational opioid use. Within the United States, the rate of death from opioid abuse since the early 1990s has grown disproportionally, prompting the classification as a nationwide "epidemic." Since this time, we have begun to unravel many fundamental cellular and systems-level mechanisms associated with opioid-related death. However, factors such as individual vulnerability, neuromodulatory compensation, and redundancy of opioid effects across central and peripheral nervous systems have created a barrier to a concise, integrative view of OIRD. Within this review, we bring together multiple perspectives in the field of OIRD to create an overarching viewpoint of what we know, and where we view this essential topic of research going forward into the future.

Opioid-Induced Respiratory Depression in Pediatric Palliative Care Patients with Severe Neurological Impairment-A Scoping Literature Review and Case Reports.

Pediatric Palliative Care (PPC) addresses children, adolescents, and young adults with a broad spectrum of underlying diseases. A substantial proportion of these patients have irreversible conditions accompanied by Severe Neurological Impairment (SNI). For the treatment of pain and dyspnea, strong opioids are widely used in PPC. Nonetheless, there is considerable uncertainty regarding the opioid-related side effects in pediatric patients with SNI, particularly concerning Opioid-Induced Respiratory Depression (OIRD). Research on pain and OIRD in pediatric patients with SNI is limited. Using scoping review methodology, we performed a systematic literature search for OIRD in pediatric patients with SNI. Out of n = 521 identified articles, n = 6 studies were included in the review. Most studies examined the effects of short-term intravenous opioid therapy. The incidence of OIRD varied between 0.13% and 4.6%; besides SNI, comorbidities, and polypharmacy were the most relevant risk factors. Additionally, three clinical cases of OIRD in PPC patients receiving oral or transdermal opioids are presented and discussed. The case reports indicate that the risk factors identified in the scoping review also apply to adolescents and young adults with SNI receiving low-dose oral or transdermal opioid therapy. However, the risk of OIRD should never be a barrier to adequate symptom relief. We recommend careful consideration and systematic observation of opioid therapy in this population of patients.

Novel Oral Nanoparticle Formulation of Sustained Release Naloxone with Mild Withdrawal Symptoms in Mice.

Chronic use of opioids can lead to tolerance, dependence, abuse, and addiction. This in turn can result in dose escalation and opioid overdose. Opioid overdose can be fatal due to severe opioid-induced respiratory depression (OIRD). Naloxone, a nonspecific antagonist of the mu-opioid receptors, is used for the reversal of OIRD. However, one of the major challenges of using naloxone is its short elimination half-life, which is significantly shorter compared to many opioid analgesics. Thus, renarcotization and rapid return to full respiratory depression might occur, specifically in individuals who have taken large doses or long-acting opioid formulations. Additionally, because of the very low oral bioavailability of naloxone, an oral formulation is not currently available. This study examines in mice a novel oral formulation of naloxone based on polymer nanoparticles (NP-naloxone). A single dose of 1 or 5 mg/kg NP-naloxone was highly effective at inhibiting the activating effects of repeated administration of 10 mg/kg morphine for at least up to 24 h. Onset of action was approximately 5 min. Reversal of morphine-induced locomotion was already detected within 1 min and a full effect of returning to baseline activity levels was observed within 5 min. Importantly, at 1 mg/kg, NP-naloxone precipitated very minimal withdrawal behaviors. At the 5 mg/kg dose, NP-naloxone precipitated approximately 40% of the jumping withdrawal behaviors of injectable naloxone. Thus, this study demonstrates that orally administered naloxone based on polymer nanoparticles has high potential to be developed to circumvent OIRD and withdrawal symptoms.

Label-Free Sensing on Microarrays.

Microarrays of biological molecules such as DNAs, proteins, carbohydrates, and small molecules provide a high-throughput platform for screening tens of thousands of biomolecular interactions simultaneously, facilitating the functional characterization of these biomolecules in areas of genomics, proteomics, glycomics, and cytomics. Routinely, analysis of binding reactions between solution-phased probes and surface-immobilized targets involves some kinds of fluorescence-based detection methods. Even though these methods have advantages of high sensitivity and wide dynamic range, labeling probes and/or targets inevitably changes their innate properties and in turn affects probe-target interactions in often uncharacterized ways. Therefore, in recent years, various label-free sensing technologies have been developed for characterizing biomolecular interactions in microarray format. These biosensors, to a certain extent, take the place of fluorescent methods by providing a comparable sensitivity as well as retaining the conformational and functional integrality of biomolecules to be investigated. More importantly, some of these biosensors are capable of real-time monitoring probe-target interactions, providing the binding affinities of these reactions. Using label-free biosensors in microarrays has become a current trend in developing high-throughput screening platforms for drug discoveries and applications in all areas of "-omics." This article is aimed to provide principles and recent developments in label-free sensing technologies applicable to microarrays, with special attentions being paid to surface plasmon resonance microscopy and oblique-incidence reflectivity difference microscopy.

Evaluation of Kinetics Using Label-Free Optical Biosensors.

Optical biosensors provide a platform for qualitatively and quantitatively analyzing various biomolecular interactions. In addition to advantages such as label-free and high-throughput detection, these devices are also capable of measuring real-time binding curves in response to changes in optical properties of biomolecules. These kinetic data may be fitted to models to extract binding affinities such as association rates, dissociation rates, and equilibrium dissociation constants. In these biosensors, one of the binding pair is usually immobilized on a solid substrate for capturing the other. Due to the nature of these surface-based methods, mass transport effects and immobilization heterogenetity may cause problems when fitting the kinetic curves with the simple one-to-one Langmuir model. Here real-time binding curves of various antibody-antigen reactions were obtained by using an ellipsometry-based biosensor, and the results were fitted to the simple one-to-one model as well as a more sophisticated approach. The results show that the one-to-two model fitted much better to the curves than the one-to-one model. The two-site model may be explained by assuming two immobilization configurations on the surface. In summary, in fitting real-time curves obtained from optical biosensors, more sophisticated models are usually required to take surface-related issues, such as immobilization heterogenetity and mass transport effects within targets, into account.

Real-time, label-free characterization of oligosaccharide-binding proteins using carbohydrate microarrays and an ellipsometry-based biosensor.

Carbohydrates present on cell surfaces mediate cell behavior through interactions with other biomolecules. Due to their structural complexity, diversity, and heterogeneity, it is difficult to fully characterize a variety of carbohydrates and their binding partners. As a result, novel technologies for glycomics applications have been developed, including carbohydrate microarrays and label-free detection methods. In this paper, we report using the combination of oligosaccharide microarrays and the label-free oblique-incidence reflectivity difference (OI-RD) microscopy for real-time characterization of oligosaccharide binding proteins. Aminated human milk oligosaccharides were immobilized on epoxy-coated glass substrates as microarrays for reactions with Family 1 of solute binding proteins from Bifidobacterium longum subsp. infantis (B. infantis). Binding affinities of these protein-oligosaccharide interactions showed preferences of Family 1 of solute binding proteins to host glycans, which helps in characterizing the complex process of human milk oligosaccharides foraging by B. infantis.

Use of Microarrays as a High-Throughput Platform for Label-Free Biosensing.

In recent years, various label-free biosensing technologies have been developed for studying the real-time kinetics of diverse biomolecular interactions. These biosensors partially take the place of fluorescence-based methods by providing a comparable sensitivity as well as retaining the conformational and functional integrality of biomolecules to be investigated. However, to completely eliminate the need of fluorescence, throughput is the next big consideration. Microarrays provide a high-throughput platform for screening tens of thousands of biomolecular interactions simultaneously, and many compatible fluorescent scanners have been commercially available. The combination of microarrays and label-free biosensors will be of great interest to researchers in related fields. Microarrays are fabricated by spotting, imprinting, or directly synthesizing biomolecules on solid supports such as glasses, silicon wafers, and other functionalized substrates, and they have been applied to detect DNAs, proteins, toxins, and so on in surface plasmon resonance (SPR) imaging systems and oblique-incidence reflectivity difference (OI-RD) microscopes. Current challenges include increasing sensitivity, reducing sampling time, improving surface chemistry, identifying captured molecules, and minimizing reagent consumption. Future research directions are to improve the instruments themselves, modify the microarray surface for more efficient analyte capture, and combine the systems with mass spectrometry and microfluidics.

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach.

Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the Kon and Koff values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.