Tyrosine phosphatase PTPN22: multifunctional regulator of immune signaling, development, and disease.

Inheritance of a coding variant of the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is associated with increased susceptibility to autoimmunity and infection. Efforts to elucidate the mechanisms by which the PTPN22-C1858T variant modulates disease risk revealed that PTPN22 performs a signaling function in multiple biochemical pathways and cell types. Capable of both enzymatic activity and adaptor functions, PTPN22 modulates signaling through antigen and innate immune receptors. PTPN22 plays roles in lymphocyte development and activation, establishment of tolerance, and innate immune cell-mediated host defense and immunoregulation. The disease-associated PTPN22-R620W variant protein is likely involved in multiple stages of the pathogenesis of autoimmunity. Establishment of a tolerant B cell repertoire is disrupted by PTPN22-R620W action during immature B cell selection, and PTPN22-R620W alters mature T cell responsiveness. However, after autoimmune attack has initiated tissue injury, PTPN22-R620W may foster inflammation through modulating the balance of myeloid cell-produced cytokines.

Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells.

Inflammation is a critical component of the immune response. However, acute or chronic inflammation can be highly destructive. Uncontrolled inflammation forms the basis for allergy, asthma and various autoimmune disorders. Here we identified a signaling pathway that was exclusively responsible for the production of inflammatory cytokines but not for cytotoxicity. Recognition of tumor cells expressing the NK cell-activatory ligands H60 or CD137L by mouse natural killer (NK) cells led to efficient cytotoxicity and the production of inflammatory cytokines. Both of those effector functions required the kinases Lck, Fyn and PI(3)K (subunits p85α and p110δ) and the signaling protein PLC-γ2. However, a complex of Fyn and the adaptor ADAP exclusively regulated the production of inflammatory cytokines but not cytotoxicity in NK cells. That unique function of ADAP required a Carma1-Bcl-10-MAP3K7 signaling axis. Our results have identified molecules that can be targeted to regulate inflammation without compromising NK cell cytotoxicity.

The autoimmunity-associated gene PTPN22 potentiates toll-like receptor-driven, type 1 interferon-dependent immunity.

Immune cells sense microbial products through Toll-like receptors (TLR), which trigger host defense responses including type 1 interferons (IFNs) secretion. A coding polymorphism in the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is a susceptibility allele for human autoimmune and infectious disease. We report that Ptpn22 selectively regulated type 1 IFN production after TLR engagement in myeloid cells. Ptpn22 promoted host antiviral responses and was critical for TLR agonist-induced, type 1 IFN-dependent suppression of inflammation in colitis and arthritis. PTPN22 directly associated with TNF receptor-associated factor 3 (TRAF3) and promotes TRAF3 lysine 63-linked ubiquitination. The disease-associated PTPN22W variant failed to promote TRAF3 ubiquitination, type 1 IFN upregulation, and type 1 IFN-dependent suppression of arthritis. The findings establish a candidate innate immune mechanism of action for a human autoimmunity "risk" gene in the regulation of host defense and inflammation.

Homeostatic mechanisms may shape the type and duration of oscillatory modulation.

Neural oscillations are observed ubiquitously in the mammalian brain, but their stability is known to be rather variable. Some oscillations are tonic and last for seconds or even minutes. Other oscillations appear as unstable bursts. Likewise, some oscillations rely on excitatory AMPAergic synapses, but others are GABAergic and inhibitory. Why this diversity exists is not clear. We hypothesized Ca2+-dependent homeostasis could be important in finding an explanation. We tested this hypothesis in a highly simplified model of hippocampal neurons. In this model homeostasis profoundly alters the modulatory effect of neural oscillations. Under homeostasis, tonic AMPAergic oscillations actually decrease excitability and desynchronize firing. Tonic oscillations that are synaptically GABAergic-like those in real hippocampus-don't provoke a homeostatic response, however. If our simple model is correct, homeostasis can explain why the theta rhythm in the hippocampus is synaptically inhibitory: GABA has little to no intrinsic homeostatic response and so can preserve the pyramidal cell's natural dynamic range. Based on these results we speculate that homeostasis may explain why AMPAergic oscillations in cortex, and in hippocampus, often appear as bursts. Bursts do not interact with the slow homeostatic time constant and so retain their normal excitatory effect.NEW & NOTEWORTHY The intricate interplay of neuromodulators, like acetylcholine, with homeostasis is well known. The interplay between oscillatory modulation and homeostasis is not. We studied oscillatory modulation and homeostasis for the first time using a simplified model of hippocampus. We report a paradoxical result: Ca-mediated homeostasis causes AMPAergic oscillations to become effectively inhibitory. This result, along with other new observations, means homeostasis might be just as complex and important for oscillations as it is for other neuromodulators.

Clinical characteristics and organ system involvement in sarcoidosis: comparison of the University of Minnesota Cohort with other cohorts.

BACKGROUND: Sarcoidosis is a systemic granulomatous disease of unknown etiology. Clinical cohort studies of different populations are important to understand the high variability in clinical presentation and disease course of sarcoidosis. The aim of the study is to evaluate clinical characteristics, including organ involvement, pulmonary function tests, and laboratory parameters, in a sarcoidosis cohort at the University of Minnesota. We compare the organ system involvement of this cohort with other available cohorts. METHODS: We conducted a retrospective data collection and analysis of 187 subjects with biopsy-proven sarcoidosis seen at a tertiary center. Organ system involvement was determined using the WASOG sarcoidosis organ assessment instrument. Clinical phenotype groups were classified using the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis criteria. RESULTS: Mean subject age at diagnosis was 45.8 ± 12.4, with a higher proportion of males (55.1%), and a higher proportion of blacks (17.1%) compared to the racial distribution of Minnesota residents (5.95%). The majority (71.1%) of subjects required anti-inflammatory therapy for at least 1 month. Compared to the A Case Control Etiologic Study of Sarcoidosis cohort, there was a higher frequency of extra-thoracic lymph node (34.2% vs. 15.2%), eye (20.9% vs. 11.8%), liver (17.6% vs. 11.5%), spleen (20.9% vs. 6.7%), musculoskeletal (9.6% vs. 0.5%), and cardiac (10.7% vs. 2.3%) involvement in our cohort. A multisystem disease with at least five different organs involved was identified in 13.4% of subjects. A restrictive physiological pattern was observed in 21.6% of subjects, followed by an obstructive pattern in 17.3% and mixed obstructive and restrictive pattern in 2.2%. Almost half (49.2%) were Scadding stages II/III. Commonly employed disease activity markers, including soluble interleukin-2 receptor and angiotensin-converting enzyme, did not differ between treated and untreated groups. CONCLUSIONS: This cohort features a relatively high frequency of high-risk sarcoidosis phenotypes including cardiac and multiorgan disease. Commonly-utilized serum biomarkers do not identify subpopulations that require or do better with treatment. Findings from this study further highlight the high-variability nature of sarcoidosis and the need for a more reliable biomarker to predict and measure disease severity and outcomes for better clinical management of sarcoidosis patients.

Gene editing for inflammatory disorders.

Technology for precise and efficient genetic editing is constantly evolving and is now capable of human clinical applications. Autoimmune and inflammatory diseases are chronic, disabling, sometimes life-threatening, conditions that feature heritable components. Both primary genetic lesions and the inflammatory pathobiology underlying these diseases represent fertile soil for new therapies based on the capabilities of gene editing. The ability to orchestrate precise targeted modifications to the genome will likely enable cell-based therapies for inflammatory diseases such as monogenic autoinflammatory disease, acquired autoimmune disease and for regenerative medicine in the setting of an inflammatory environment. Here, we discuss recent advances in genome editing and their evolving applications in immunoinflammatory diseases. Strengths and limitations of older genetic modification tools are compared with CRISPR/Cas9, base editing, RNA editing, targeted activators and repressors of transcription and targeted epigenetic modifiers. Commonly employed delivery vehicles to target cells or tissues of interest with genetic modification machinery, including viral, non-viral and cellular vectors, are described. Finally, applications in animal and human models of inflammatory diseases are discussed. Use of chimeric autoantigen receptor T cells, correction of monogenic diseases with genetically edited haematopoietic stem and progenitor cells, engineering of induced pluripotent stem cells and ex vivo expansion and modification of regulatory T cells for a range of chronic inflammatory diseases are reviewed.

Nonsinusoidal Beta Oscillations Reflect Cortical Pathophysiology in Parkinson's Disease.

Oscillations in neural activity play a critical role in neural computation and communication. There is intriguing new evidence that the nonsinusoidal features of the oscillatory waveforms may inform underlying physiological and pathophysiological characteristics. Time-domain waveform analysis approaches stand in contrast to traditional Fourier-based methods, which alter or destroy subtle waveform features. Recently, it has been shown that the waveform features of oscillatory beta (13-30 Hz) events, a prominent motor cortical oscillation, may reflect near-synchronous excitatory synaptic inputs onto cortical pyramidal neurons. Here we analyze data from invasive human primary motor cortex (M1) recordings from patients with Parkinson's disease (PD) implanted with a deep brain stimulator (DBS) to test the hypothesis that the beta waveform becomes less sharp with DBS, suggesting that M1 input synchrony may be decreased. We find that, in PD, M1 beta oscillations have sharp, asymmetric, nonsinusoidal features, specifically asymmetries in the ratio between the sharpness of the beta peaks compared with the troughs. This waveform feature is nearly perfectly correlated with beta-high gamma phase-amplitude coupling (r = 0.94), a neural index previously shown to track PD-related motor deficit. Our results suggest that the pathophysiological beta generator is altered by DBS, smoothing out the beta waveform. This has implications not only for the interpretation of the physiological mechanism by which DBS reduces PD-related motor symptoms, but more broadly for our analytic toolkit in general. That is, the often-overlooked time-domain features of oscillatory waveforms may carry critical physiological information about neural processes and dynamics.SIGNIFICANCE STATEMENT To better understand the neural basis of cognition and disease, we need to understand how groups of neurons interact to communicate with one another. For example, there is evidence that parkinsonian bradykinesia and rigidity may arise from an oversynchronization of afferents to the motor cortex, and that these symptoms are treatable using deep brain stimulation. Here we show that the waveform shape of beta (13-30 Hz) oscillations, which may reflect input synchrony onto the cortex, is altered by deep brain stimulation. This suggests that mechanistic inferences regarding physiological and pathophysiological neural communication may be made from the temporal dynamics of oscillatory waveform shape.

Inferring synaptic excitation/inhibition balance from field potentials.

Neural circuits sit in a dynamic balance between excitation (E) and inhibition (I). Fluctuations in E:I balance have been shown to influence neural computation, working memory, and information flow, while more drastic shifts and aberrant E:I patterns are implicated in numerous neurological and psychiatric disorders. Current methods for measuring E:I dynamics require invasive procedures that are difficult to perform in behaving animals, and nearly impossible in humans. This has limited the ability to examine the full impact that E:I shifts have in cognition and disease. In this study, we develop a computational model to show that E:I changes can be estimated from the power law exponent (slope) of the electrophysiological power spectrum. Predictions from the model are validated in published data from two species (rats and macaques). We find that reducing E:I ratio via the administration of general anesthetic in macaques results in steeper power spectra, tracking conscious state over time. This causal result is supported by inference from known anatomical E:I changes across the depth of rat hippocampus, as well as oscillatory theta-modulated dynamic shifts in E:I. Our results provide evidence that E:I ratio may be inferred from electrophysiological recordings at many spatial scales, ranging from the local field potential to surface electrocorticography. This simple method for estimating E:I ratio-one that can be applied retrospectively to existing data-removes a major hurdle in understanding a currently difficult to measure, yet fundamental, aspect of neural computation.

Autoimmune Variant PTPN22 C1858T Is Associated With Impaired Responses to Influenza Vaccination.

High-affinity-antibody production, T-cell activation, and interferon upregulation all contribute to protective immunity that occurs in humans following influenza immunization. Hematopoietic cell-specific PTPN22 encodes lymphoid phosphatase (Lyp), which regulates lymphocyte antigen receptor and pattern recognition receptor (PRR) signaling. A PTPN22 variant, R620W (LypW), predisposes to autoimmune and infectious diseases and confers altered signaling through antigen receptors and PRRs. We tested the hypothesis that LypW-bearing humans would have diminished immune response to trivalent influenza vaccine (TIV). LypW carriers exhibited decreased induction of influenza virus-specific CD4(+) T cells expressing effector cytokines and failed to increase antibody affinity following TIV receipt. No differences between LypW carriers and noncarriers were observed in virus-specific CD8(+) T-cell responses, early interferon transcriptional responses, or myeloid antigen-presenting cell costimulatory molecule upregulation. The association of LypW with defects in TIV-induced CD4(+) T-cell expansion and antibody affinity maturation suggests that LypW may predispose individuals to have a diminished capacity to generate protective immunity against influenza virus.

Skin dendritic cells induce follicular helper T cells and protective humoral immune responses.

BACKGROUND: The contribution of individual subsets of dendritic cells (DCs) to generation of adaptive immunity is central to understanding immune homeostasis and protective immune responses. OBJECTIVE: We sought to define functions for steady-state skin DCs. METHODS: We present an approach in which we restrict antigen presentation to individual DC subsets in the skin and monitor the effects on endogenous antigen-specific CD4(+) T- and B-cell responses. RESULTS: Presentation of foreign antigen by Langerhans cells (LC) in the absence of exogenous adjuvant led to a large expansion of T follicular helper (TFH) cells. This was accompanied by B-cell activation, germinal center formation, and protective antibody responses against influenza. The expansion of TFH cells and antibody responses could be elicited by both systemic and topical skin immunization. TFH cell induction was not restricted to LCs and occurred in response to antigen presentation by CD103(+) dermal DCs. CD103(+) DCs, despite inducing similar TFH responses as LCs, were less efficient in induction of germinal center B cells and humoral immune responses. We also found that skin DCs are sufficient to expand CXCR5(+) TFH cells through an IL-6- and IFN-α/ß receptor-independent mechanism, but B cells were required for sustained Bcl-6(+) expression. CONCLUSIONS: These data demonstrate that a major unappreciated function of skin DCs is their promotion of TFH cells and humoral immune responses that potentially represent an efficient approach for vaccination.

Immune complex-mediated autoimmunity in a patient With Smith-Magenis syndrome (del 17p11.2).

Smith-Magenis syndrome (SMS) is a sporadic congenital disorder involving multiple organ systems caused by chromosome 17p11.2 deletions. Smith-Magenis syndrome features craniofacial and skeletal anomalies, cognitive impairment, and neurobehavioral abnormalities. In addition, some SMS patients may exhibit hypogammaglobulinemia. We report the first case of SMS-associated autoimmunity in a woman who presented with adult onset of multiple autoimmune disorders, including systemic lupus erythematosus, antiphospholipid antibody syndrome, and autoimmune hepatitis. Molecular analysis using single-nucleotide polymorphism array confirmed a de novo 3.8-Mb deletion (breakpoints, chr17: 16,660,721-20,417,975), resulting in haploinsufficiency for TACI (transmembrane activator and CAML interactor). Our data are consistent with potential loss of function for the BAFF (B cell-activating factor) receptor TACI as a contributing factor to human autoimmune phenomena.

A distributed, high-channel-count, implanted bidirectional system for restoration of somatosensation and myoelectric control.

Objective. We intend to chronically restore somatosensation and provide high-fidelity myoelectric control for those with limb loss via a novel, distributed, high-channel-count, implanted system.Approach.We have developed the implanted Somatosensory Electrical Neurostimulation and Sensing (iSens®) system to support peripheral nerve stimulation through up to 64, 96, or 128 electrode contacts with myoelectric recording from 16, 8, or 0 bipolar sites, respectively. The rechargeable central device has Bluetooth® wireless telemetry to communicate to external devices and wired connections for up to four implanted satellite stimulation or recording devices. We characterized the stimulation, recording, battery runtime, and wireless performance and completed safety testing to support its use in human trials.Results.The stimulator operates as expected across a range of parameters and can schedule multiple asynchronous, interleaved pulse trains subject to total charge delivery limits. Recorded signals in saline show negligible stimulus artifact when 10 cm from a 1 mA stimulating source. The wireless telemetry range exceeds 1 m (direction and orientation dependent) in a saline torso phantom. The bandwidth supports 100 Hz bidirectional update rates of stimulation commands and data features or streaming select full bandwidth myoelectric signals. Preliminary first-in-human data validates the bench testing result.Significance.We developed, tested, and clinically implemented an advanced, modular, fully implanted peripheral stimulation and sensing system for somatosensory restoration and myoelectric control. The modularity in electrode type and number, including distributed sensing and stimulation, supports a wide variety of applications; iSens® is a flexible platform to bring peripheral neuromodulation applications to clinical reality. ClinicalTrials.gov ID NCT04430218.

Many hats: intratrial and reward level-dependent BOLD activity in the striatum and premotor cortex.

Human functional magnetic resonance imaging (fMRI) studies, as well as lesion, drug, and single-cell recording studies in animals, suggest that the striatum plays a key role in associating sensory events with rewarding actions, both by facilitating reward processing and prediction (i.e., reinforcement learning) and by biasing and later updating action selection. Previous human neuroimaging research has failed to dissociate striatal activity associated with reward, stimulus, and response processing, and previous electrophysiological research in nonhuman animals has typically only examined single striatal subregions. Overcoming both these limitations, we isolated blood oxygen level-dependent (BOLD) signal associated with four intratrial processes (stimulus, preparation of response, response, and feedback) in a visuomotor learning task and examined activity associated with each within four striatal subregions (ventral striatum, putamen, head of the caudate nucleus, and body of the caudate) and the lateral premotor cortex. Overall, the striatum and lateral premotor cortex were recruited during all trial components, confirming their importance in all aspects of visuomotor learning. However, the caudate was most active at stimulus and feedback, whereas the putamen peaked in activity at response. Activation in the lateral premotor cortex was, surprisingly, strongest during stimulus and following response as feedback approached. Activity was additionally examined at three reward magnitudes. Reward magnitude affected neural activity only during stimulus in the caudate, putamen, and premotor cortex, whereas the ventral striatum showed reward sensitivity during both stimulus and feedback. Collectively, these results indicate that each striatal region makes a unique contribution to visuomotor learning through functions performed at different points within single trials.

Aperiodic Neural Activity is a Better Predictor of Schizophrenia than Neural Oscillations.

Diagnosis and symptom severity in schizophrenia are associated with irregularities across neural oscillatory frequency bands, including theta, alpha, beta, and gamma. However, electroencephalographic signals consist of both periodic and aperiodic activity characterized by the (1/fX) shape in the power spectrum. In this paper, we investigated oscillatory and aperiodic activity differences between patients with schizophrenia and healthy controls during a target detection task. Separation into periodic and aperiodic components revealed that the steepness of the power spectrum better-predicted group status than traditional band-limited oscillatory power in classification analysis. Aperiodic activity also outperformed the predictions made using participants' behavioral responses. Additionally, the differences in aperiodic activity were highly consistent across all electrodes. In sum, compared to oscillations the aperiodic activity appears to be a more accurate and more robust way to differentiate patients with schizophrenia from healthy controls.

Update: biomarkers for idiopathic inflammatory myopathies.

PURPOSE OF REVIEW: Establishing diagnoses and distinguishing active disease from chronic injury remain significant clinical challenges in idiopathic inflammatory myopathies (IIM). Recent 'discovery' approaches utilizing novel genomic and proteomic techniques have revealed candidate molecular biomarkers to augment clinical and classical histological data. RECENT FINDINGS: Whole blood and serum Type 1 interferons (IFN-1) and IFN-1 inducible genes are gaining traction as disease biomarkers in IIM. IFNß is emerging as a disease activity marker specifically for dermatomyositis. Recently, molecules associated with innate immune-cell function, including TLR-3, high mobility group box (HMGB)-1, B7 Homolog 1, S100A4, and resistin have been detected in tissues of dermatomyositis patients. Serum Interleukin-17 (IL-17) and IL-23 correlate with active disease in early IIM. Antibodies recognizing the Survival Motor Neuron complex have been newly identified in a subset of patients with polymyositis. Protein aggregates are potential disease activity sensors for inclusion body myositis. Skin and lung harbor potential biomarkers for IIM. SUMMARY: Recent advances in understanding the pathogenesis of IIM have led to discovery of molecules that are candidate biomarkers of disease activity. Type 1 interferon and myeloid-cell signatures are leading candidate markers for use in IIM activity monitoring.

Quantifying Rheumatoid Arthritis Disease Activity Using a Multimodal Sensing Knee Brace.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory syndrome that features painful and destructive joint disease. Aggressive disease-modifying treatment can result in reduced symptoms and protection from irreversible joint damage; however, assessment of treatment efficacy is currently based largely on subjective measures of patient and physician impressions. In this work, we address this compelling need to provide an accurate and quantitative capability for monitoring joint health in patients with RA. METHODS: Joint acoustic emissions (JAEs), electrical bioimpedance (EBI), and kinematics were measured noninvasively from 11 patients with RA over the course of three weeks using a custom multimodal sensing brace, resulting in 49 visits with JAE recordings and 43 with EBI recordings. Features derived from all sensing modalities were fed into a linear discriminant analysis (LDA) model to predict disease activity according to the validated disease activity index (the DAS28-ESR). Erythrocyte sedimentation rate (ESR) was predicted using ridge regression and classified into a high or low class using LDA. RESULTS: DAS28-ESR level was predicted with an area under the receiver operating characteristic curve (AUC) of 0.82. With JAEs alone, we were able to track intrasubject differences in the disease activity score as well as classify ESR level with an AUC of 0.93. The majority of patients reported both an interest and ability to use the brace at home for longitudinal monitoring. CONCLUSION: This work demonstrates the ability to detect RA disease activity using noninvasive sensing. SIGNIFICANCE: This system has the potential to improve RA disease activity monitoring by giving treating clinicians objective data that can be acquired independent of a face-to-face clinic visit.

Dissociating hippocampal and basal ganglia contributions to category learning using stimulus novelty and subjective judgments.

We identified factors leading to hippocampal and basal ganglia recruitment during categorization learning. Subjects alternated between blocks of a standard trial and error category learning task and a subjective judgment task. In the subjective judgments task subjects categorized the stimulus and then instead of receiving feedback they indicated the basis of their response using 4 options: Remember: Conscious episodic memory of previous trials. Know-Automatic: Automatic, rapid response accompanied by conscious awareness of category membership. Know-Intuition: A "gut feeling" without fully conscious knowledge of category membership. Guess: Guessing. In addition, new stimuli were introduced throughout the experiment to examine effects of novelty. Categorization overall recruited both the basal ganglia and posterior hippocampus. However, basal ganglia activity was found during Know judgments (both Automatic and Intuition), whereas posterior hippocampus activity was found during Remember judgments. Granger causality mapping indicated interactions between the basal ganglia and hippocampus, with the putamen exerting directed influence on the posterior hippocampus, which in turn exerted directed influence on the posterior caudate nucleus. We also found a region of anterior hippocampus that showed decreased activity relative to baseline during categorization overall, and showed a strong novelty effect. Our results indicate that subjective measures may be effective in dissociating basal ganglia from hippocampal dependent learning, and that the basal ganglia are involved in both conscious and unconscious learning. They also indicate a dissociation within the hippocampus, in which the anterior regions are sensitive to novelty, and the posterior regions are involved in memory based categorization learning.

BTI1, an azoreductase with pH-dependent substrate specificity.

The group II azoreductase BTI1 utilizes NADPH to directly cleave azo bonds in water-soluble azo dyes, including quenchers of fluorescence. Unexpectedly, optimal reduction was dye specific, ranging from a pH of <5.5 for Janus green B, to pH 6.0 for methyl red, methyl orange, and BHQ-10, to pH >8.3 for flame orange.

Pregnancy and rheumatoid arthritis: insights into the immunology of fetal tolerance and control of autoimmunity.

It has long been recognized that symptoms and signs of rheumatoid arthritis (RA) frequently improve spontaneously during pregnancy, only to flare postpartum. Although the mechanisms behind this phenomenon remain poorly understood, there is growing interest in the immunologic changes that occur during healthy pregnancy as a possible explanation. Because the maternal immune system must adapt during pregnancy to accept the semi-allogeneic fetus, it has been hypothesized that these natural changes induced by pregnancy on maternal immune regulatory cells may have the additional benefit of controlling the immunopathology driving disease activity in RA. Here, we review our current understanding on the effects of pregnancy on RA and highlight some of the recent literature related to advancing our understanding on the immunology of pregnancy as well as the immunologic changes in RA during pregnancy.

Single-step label-free nanowell immunoassay accurately quantifies serum stress hormones within minutes.

A non-faradaic label-free cortisol sensing platform is presented using a nanowell array design, in which the two probe electrodes are integrated within the nanowell structure. Rapid and low volume (≤5 µl) sensing was realized through functionalizing nanoscale volume wells with antibodies and monitoring the real-time binding events. A 28-well plate biochip was built on a glass substrate by sequential deposition, patterning, and etching steps to create a stack nanowell array sensor with an electrode gap of 40 nm. Sensor response for cortisol concentrations between 1 and 15 µg/dl in buffer solution was recorded, and a limit of detection of 0.5 µg/dl was achieved. Last, 65 human serum samples were collected to compare the response from human serum samples with results from the standard enzyme-linked immunosorbent assay (ELISA). These results confirm that nanowell array sensors could be a promising platform for point-of-care testing, where real-time, laboratory-quality diagnostic results are essential.