High-affinity one-step aptamer selection using a non-fouling porous hydrogel.

Aptamers, commonly referred to as chemical antibodies, are used in a wide range of applications including drug delivery and biosensing. However, the process of aptamer selection poses a substantial challenge, as it requires numerous cycles of enrichment and involves issues with nonspecific binding. We present a simple, fast instrument-free method for aptamer enrichment and selection based on a diffusion-binding process in a three-dimensional non-fouling porous hydrogel with immobilized target proteins. Low-affinity aptamer candidates can be rapidly released from the hydrogel, whereas high-affinity candidates are restricted due to their strong binding to the immobilized protein targets. Consequently, a one-step enriched aptamer pool can strongly bind the protein targets. This enrichment is consistent across five proteins with isoelectric points in varying ranges. With thrombin as a representative model, the anti-thrombin aptamer identified from an enriched aptamer pool has been found to have a binding affinity that is comparable to those identified over ten cycles of selection using traditional methods.

A non-invasive wearable stress patch for real-time cortisol monitoring using a pseudoknot-assisted aptamer.

Stress is part of everyone's life and is exacerbated by traumatic events such as pandemics, disasters, violence, lifestyle changes, and health disorders. Chronic stress has many detrimental health effects and can even be life-threatening. Long-term stress monitoring outside of a hospital is often accomplished by measuring heart rate variability. While easy to measure, this digital biomarker has low specificity, greatly limiting its utility. To address this shortcoming, we report a non-invasive, wearable biomolecular sensor to monitor cortisol levels in sweat. Cortisol is a neuroendocrine hormone that regulates homeostasis as part of the stress pathway. Cortisol is detected using an electrochemical sensor functionalized with a pseudoknot-assisted aptamer and a flexible microfluidic sweat sampling system. The skin-worn microfluidic sampler provides rapid sweat collection while separating old and new sweat. The conformation-switching aptamer provides high specificity towards cortisol while being regenerable, allowing it to monitor temporal changes continuously. The aptamer was engineered to add a pseudoknot, restricting it to only two states, thus minimizing the background signal and enabling high sensitivity. An electrochemical pH sensor allows pH-corrected amperometric measurements. Device operation was demonstrated invitro with a broad linear dynamic range (1 pM - 1 µM) covering the physiological range and a sub-picomolar (0.2 pM) limit of detection in sweat. Real-time, on-body measurements were collected from human subjects using an induced stress protocol, demonstrating in-situ signal regeneration and the ability to detect dynamic cortisol fluctuations continuously for up to 90 min. The reported device has the potential to improve prognosis and enable personalized treatments.

Electrochemical Carbamazepine Aptasensor for Therapeutic Drug Monitoring at the Point of Care.

Monitoring the anti-epileptic drug carbamazepine (CBZ) is crucial for proper dosing, optimizing a patient's clinical outcome, and managing their medication regimen. Due to its narrow therapeutic window and concentration-related toxicity, CBZ is prescribed and monitored in a highly personalized manner. We report an electrochemical conformation-changing aptasensor with two assay formats: a 30 min assay for routine monitoring and a 5 min assay for rapid emergency testing. To enable "sample-to-answer" testing, a de novo CBZ aptamer (K d < 12 nM) with conformational switching due to a G-quadruplex motif was labeled with methylene blue and immobilized on a gold electrode. The electrode fabrication and detection conditions were optimized using electrochemical techniques and visualized by atomic force microscopy (AFM). The aptasensor performance, including reproducibility, stability, and interference, was characterized using electrochemical impedance spectroscopy and voltammetry techniques. The aptasensor exhibited a wide dynamic range in buffer (10 nM to 100 µM) with limits of detection of 1.25 and 1.82 nM for the 5 and 30 min assays, respectively. The clinical applicability is demonstrated by detecting CBZ in finger prick blood samples (<50 µL). The proposed assays provide a promising method to enable point-of-care monitoring for timely personalized CBZ dosing.

A 26.5 pArms Neurotransmitter Front-End With Class-AB Background Subtraction.

This paper presents an analog front-end (AFE) for fast-scan cyclic voltammetry (FSCV) with analog background subtraction using a pseudo-differential sensing scheme to cancel the large non-faradaic current before seeing the front-end. As a result, the AFE can be compact and low-power compared to conventional FSCV AFEs with dedicated digital back-ends to digitize and subtract the background from subsequent recordings. The reported AFE, fabricated in a 0.18- µ m CMOS process, consists of a class-AB common-mode rejection circuit, a low-input-impedance current conveyor, and a 1st-order current-mode delta-sigma (ΔΣ) modulator with an infinite impulse response quantizer. This AFE achieves an effective dynamic range of 83 dB with a state-of-the-art 39.2 pArms input-referred noise when loaded with a 1 nF input capacitance (26.5 pArms open-circuit) across a 5 kHz bandwidth while consuming an average power of 3.7 µW. This design was tested with carbon-fiber microelectrodes scanned at 300 V/s using flow-injection of dopamine, a key neurotransmitter.

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.

Cortisol Detection in Undiluted Human Serum Using a Sensitive Electrochemical Structure-Switching Aptamer over an Antifouling Nanocomposite Layer.

There is a strong and growing need to monitor stress biomarkers in vivo for real-time emotional and wellness assessment. Toward this, we report a reagent-free electrochemical aptasensor with a nanocomposite antifouling layer for sensitive and continuous detection of cortisol in human serum. A thiolated, methylene blue (MB)-tagged conformation-switching aptamer was immobilized over a gold nanowire (AuNW) nanocomposite to capture cortisol and generate a signal proportional to the cortisol concentration. The signal is recorded through differential pulse voltammetry (DPV) and chronoamperometry. The aptasensor exhibited a sensitive response with 0.51 and 0.68 nM detection limits in spiked buffer and undiluted serum samples, respectively. Interference from other structurally similar analogs, namely, epinephrine and cholic acid, was negligible (<10%). The developed nanocomposite-based aptasensor showed excellent stability in undiluted human serum, outperforming several other nanocomposite materials even after prolonged exposure. This work lays the foundation for new biosensor formats such as implantable and wearable sensors.

Dataset on optimization and development of a point-of-care glucometer-based SARS-CoV-2 detection assay using aptamers.

We present supplementary data for the published article, "Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer" [1]. The assay described is designed to be performed at home or in a clinic without expensive instrumentation or professional training. SARS-CoV-2 is detected by an aptamer-based assay that targets the Nucleocapsid (N) or Spike (S) antigens. Binding of the N or S protein to their respective aptamer results in the competitive release of a complementary antisense-invertase enzyme complex. The released enzyme then catalyzes the conversion of sucrose to glucose that is measured by an off-the-shelf glucometer. The data presented here describe the optimization of the assay parameters and their contribution to developing this aptamer-based assay to detect SARS-CoV-2. The assay performance was checked in a standard buffer, contrived samples, and patient samples validated with well-established scientific methods. The resulting dataset can be used to further develop glucometer-based assays for diagnosing other communicable and non-communicable diseases.

Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer.

Significant barriers to the diagnosis of latent and acute SARS-CoV-2 infection continue to hamper population-based screening efforts required to contain the COVID-19 pandemic in the absence of widely available antiviral therapeutics or vaccines. We report an aptamer-based SARS-CoV-2 salivary antigen assay employing only low-cost reagents ($3.20/test) and an off-the-shelf glucometer. The test was engineered around a glucometer as it is quantitative, easy to use, and the most prevalent piece of diagnostic equipment globally, making the test highly scalable with an infrastructure that is already in place. Furthermore, many glucometers connect to smartphones, providing an opportunity to integrate with contact tracing apps, medical providers, and electronic health records. In clinical testing, the developed assay detected SARS-CoV-2 infection in patient saliva across a range of viral loads - as benchmarked by RT-qPCR - within 1 h, with 100% sensitivity (positive percent agreement) and distinguished infected specimens from off-target antigens in uninfected controls with 100% specificity (negative percent agreement). We propose that this approach provides an inexpensive, rapid, and accurate diagnostic for distributed screening of SARS-CoV-2 infection at scale.

CD300a Receptor Blocking Enhances Early Clearance of Leishmania donovani From Its Mammalian Host Through Modulation of Effector Functions of Phagocytic and Antigen Experienced T Cells.

The parasites of the genus Leishmania survive and proliferate in the host phagocytic cells by taking control over their microbicidal functions. The parasite also promotes differentiation of antigen-specific anti-inflammatory cytokines producing effector T cells, which eventually results in disease pathogenesis. The mechanisms that parasites employ to dominate host adaptive immunity are largely unknown. For the first time, we report that L. donovani, which causes visceral leishmaniasis in the Indian subcontinent, upregulates the expression of an immune inhibitory receptor i.e., CD300a on antigen presenting and phagocytic cells to dampen their effector functions. The blocking of CD300a signals in leishmania antigens activated macrophages and dendritic cells enhanced the production of nitric oxide, pro-inflammatory cytokines along with MHCI/II genes expression, and reduced parasitic uptake. Further, the abrogation of CD300a signals in Leishmania infected mice benefited antigen-experienced, i.e., CD4+CD44+ and CD8+CD44+ T cells to acquire more pro-inflammatory cytokines producing phenotypes and helped in the early clearance of parasites from their visceral organs. The CD300a receptor blocking also enhanced the conversion of CD4+ T effectors cells to their memory phenotypes i.e., CCR7high CD62Lhigh up to 1.6 and 1.9 fold after 14 and 21 days post-infection, respectively. These findings implicate that CD300a is an important determinant of host phagocytic cells functions and T cells differentiation against Leishmania antigens.

Development of an aptamer-based field effect transistor biosensor for quantitative detection of Plasmodium falciparum glutamate dehydrogenase in serum samples.

There has been a continuous strive to develop portable, stable, sensitive and low cost detection system for malaria to meet the demand of effective screening actions in developing countries where the disease is most endemic. Herein, we report an aptamer-based field effect transistor (aptaFET) biosensor, developed by using an extended gate field effect transistor with inter-digitated gold microelectrodes (IDµE) for the detection of the malaria biomarker Plasmodium falciparum glutamate dehydrogenase (PfGDH) in serum samples. A 90 mer long ssDNA aptamer (NG3) selective to PfGDH was used in the aptaFET to capture the target protein. The intrinsic surface net charge of the captured protein led to change in gate potential of the aptaFET device, which could be correlated to the concentration of the protein. This biosensor exhibited a sensitive response in broad dynamic range of 100 fM -10 nM with limits of detection of 16.7 pM and 48.6 pM in spiked buffer and serum samples, respectively. The high selectivity of the biosensor for PfGDH was verified by testing relevant analogous human and parasitic proteins on the device. Overall, the results validated the application potential of the developed aptaFET for diagnosis of both symptomatic and asymptomatic malaria.

Capacitive malaria aptasensor using Plasmodium falciparum glutamate dehydrogenase as target antigen in undiluted human serum.

A capacitive aptasensor for detecting the malaria biomarker, Plasmodium falciparum glutamate dehydrogenase (PfGDH), directly in human serum samples developed. A thiolated ssDNA aptamer (NG3) that binds specifically to PfGDH antigen with high affinity (Kd= 79 nM) was used to develop the aptasensor. The aptasensor produced capacitance response at an optimized frequency of 2 Hz in a non-Faradaic electrochemical impedance based signal transduction platform. The aptasensor exhibited a wide dynamic range of 100 fM-100 nM with a limits of detection of 0.77 pM in serum samples. The interference from other predominant malarial biomarkers, namely, Plasmodium falciparum -lactate dehydrogenase and -histidine rich protein-II on the aptasensor was negligible. This PfGDH aptasensor with highly sensitive and label free detection capability has great application potential for diagnosis of asymptotic malaria and monitoring the regression of malaria during treatment regime with antimalarial drugs.

Development of an Indicator Displacement Based Detection of Malaria Targeting HRP-II as Biomarker for Application in Point-of-Care Settings.

A novel label free spectrophotometric detection of malarial biomarker HRP-II following an indicator displacement assay has been developed. The assay is based on competitive displacement of murexide dye from its complex with Ni2+ by HRP-II present in serum samples. The binding constant (Kd) discerned for the dye and HRP-II to Ni2+ were 1.4 × 10-6 M-1 and 6.8 × 10-9 M-1, respectively. The progress of the reaction could be monitored from the change of color from orange (∼λ482 nm) to pink (∼λ515 nm) with the concomitant increase in HRP-II concentration in the mixture. A linear response (R2 = 0.995) curve was generated by plotting the ratio of absorbance (λ515 nm/λ482 nm) against the HRP-II concentrations. The method offers to detect HRP-II as low as 1 pM without any interference from some common salts and the major protein, HSA, present in the blood serum. The detection method was reproduced in a microfluidic paper based analytical device (µPAD), fabricated by printing hydrophobic alkyl ketene dimer on a chromatographic paper to create hydrophilic microchannels, test zone, and sample application zone. The device offers to use a maximum sample volume of 20 ± 0.06 µL and detects HRP-II within 5 min with LOD of 30 ± 9.6 nM in a dynamic range of 10 to 100 nM. The method has thus immense potential to develop as rapid, selective, simple, portable, and inexpensive malarial diagnostic device for point-of-care and low resource setting applications.

Molecular identification of Aedes aegypti mosquitoes from Pilani region of Rajasthan, India.

BACKGROUND & OBJECTIVES: Aedes aegypti is the most important vector of dengue virus infection in humans worldwide. Accurate identification and colonization are the essential requirements to understand vector biology as well as its diseases transmission potential. In this study, we have used molecular approaches for the identification of Ae. aegypti mosquitoes that were collected from the Pilani region of Rajasthan, India Methods: Field collected mosquito larvae were colonized under laboratory conditions. Conserved genetic markers, ITS-2 and mtCOI were used for amplification through species-specific primers to identify the mosquito species/ strain. Sequencing result of this strain was phylogenetically compared with other global strains through MEGA software. RESULTS: A comprehensive multiple sequence alignment and phylogenetic analysis revealed that COI gene of Ae. aegypti has extremely low genetic variability with one of the Indian isolate from Thirumala, Andhra Pradesh region (GenBank: HM807262.1). However, in context of different geographical locations, it indicated close similarity with Thailand's strain and high variability when compared with Madagascar strain. On the other hand, ITS-2 illustrated highest identity with Ae. aegypti of Saudi Arabia (GenBank: JX423807.1) whereas, high divergence was observed from Mayotte, France strain (GenBank: KF135506). INTERPRETATION & CONCLUSION: The findings suggest that this isolate from Rajasthan is similar to other Asian continent strains possibly due to the same origin. Understanding the vectorial capacity of these geographically distributed mosquito strains will enhance our knowledge to improve existing vector surveillance and control programme.

Human serum albumin-stabilized gold nanoclusters act as an electron transfer bridge supporting specific electrocatalysis of bilirubin useful for biosensing applications.

Human serum albumin (HSA)-stabilized Au18 nanoclusters (AuNCs) were synthesized and chemically immobilized on an Indium tin oxide (ITO) plate. The assembly process was characterized by advanced electrochemical and spectroscopic techniques. The bare ITO electrode generated three irreversible oxidation peaks, whereas the HSA-AuNC-modified electrode produced a pair of redox peaks for bilirubin at a formal potential of 0.27V (vs. Ag/AgCl). However, the native HSA protein immobilized on the ITO electrode failed to produce any redox peak for bilirubin. The results indicate that the AuNCs present in HSA act as electron transfer bridge between bilirubin and the ITO plate. Docking studies of AuNC with HSA revealed that the best docked structure of the nanocluster is located around the vicinity of the bilirubin binding site, with an orientation that allows specific oxidation. When the HSA-AuNC-modified electrode was employed for the detection of bilirubin using chronoamperometry at 0.3V (vs. Ag/AgCl), a steady-state current response against bilirubin in the range of 0.2µM to 7µM, with a sensitivity of 0.34µAµM(-1) and limit of detection of 86.32nM at S/N 3, was obtained. The bioelectrode was successfully applied to measure the bilirubin content in spiked serum samples. The results indicate the feasibility of using HSA-AuNC as a biorecognition element for the detection of serum bilirubin levels using an electrochemical technique.

Alcohol oxidase protein mediated in-situ synthesized and stabilized gold nanoparticles for developing amperometric alcohol biosensor.

A simple one step method for the alcohol oxidases (AOx) protein mediated synthesis of gold nano-particles (AuNPs) in alkaline (pH 8.5) condition with simultaneous stabilization of the nanoparticles on the AOx protein surface under native environment has been developed. The formation of the AOx conjugated AuNPs was confirmed by advanced analytical and spectroscopic techniques. The significant increase in zeta potential (ζ) value of -57mV for the synthesized AOx-AuNPs conjugate from the AOx (pI 4.5) protein (ζ, -30mV) implied good stability of the in-situ synthesized nano-conjugate. The AOx-AuNPs conjugate showed steady stability in alkaline (upto pH 8.5) and NaCl (up to 10(-1)M) solutions. The efficiency (Kcat/Km) of the AuNP conjugated AOx was increased by 18% from the free enzyme confirming the activating role of the surface stabilized AuNPs for the enzyme. The AuNPs-AOx conjugate was encapsulated with polyaniline (PANI) synthesized by oxidative polymerization of aniline using H2O2 generated in-situ from the AOx catalysed oxidation of alcohol. The PANI encapsulated AuNPs-AOx assembly was stabilized on a glassy carbon electrode (GCE) by chitosan-Nafion mixture and then utilized the fabricated bioelectrode for detection of alcohol amperometrically using H2O2 as redox indicator at +0.6V. The constructed biosensor showed high operational stability (6.3% loss after 25 measurements), wide linear detection range of 10µM-4.7mM (R(2)=0.9731), high sensitivity of 68.3±0.35µAmM(-1) and low detection limit of 7±0.027µM for ethanol. The fabricated bioelectrode was successfully used for the selective determination of alcohol in beverage samples.

Thermodynamic properties of a magnetically modulated graphene monolayer.

The effect of magnetic modulation on thermodynamic properties of a graphene monolayer in the presence of a constant perpendicular magnetic field is reported here. One-dimensional spatial electric or magnetic modulation lifts the degeneracy of the Landau levels and converts into bands and their bandwidth oscillates with magnetic field, leading to Weiss-type oscillations in the thermodynamic properties. The effect of magnetic modulation on the thermodynamic properties of a graphene sheet is studied and then compared with electrically modulated graphene and magnetically modulated conventional two-dimensional electron gas (2DEG). We observe Weiss-type and de Haas-van Alphen oscillations at low and high magnetic fields, respectively. There is a definite phase difference in Weiss-type oscillations in thermodynamic quantities of magnetically modulated graphene compared to electrically modulated graphene. On the other hand, the phase remains the same and the amplitude of the oscillation is large when compared with the magnetically modulated two-dimensional electron gas (2DEG). Explicit asymptotic expressions of the density of states and the Helmholtz free energy are provided to understand the phase and amplitude of the Weiss-type oscillations qualitatively. We also study thermodynamic properties when both electric and magnetic modulations are present. The Weiss-type oscillations still exist when the modulations are out-of-phase.

Use of diode laser in oral submucous fibrosis with trismus: prospective clinical study.

INTRODUCTION: Oral submucous fibrosis (OSMF) in later stages invariably leads to trismus due to retromolar fibrosis and buccal mucosa involvement. Medical treatment has limited role once trismus is established. Various surgical methods have been used with varying success to relieve trismus. We used diode laser to relieve trismus. All patients were diagnosed to have OSMF with trismus. The results are quite encouraging. STUDY DESIGN: Prospective clinical study. This study involved 8 patients between the years 2002 and 2006. OBJECTIVE: To evaluate the efficacy of laser to reduce trismus in OSMF METHODS: Laser with follow-up physiotherapy CONCLUSION: Diode laser gave good result in all our patients. Diode laser is a less expensive and alternative method in group III and group IVA cases in whom bilateral temporalis myotomy and coronoidectomy are considered to be the only solution. This technique has less morbidity and is suitable for Asian population as it requires less hospital stay and less followup as compared to other surgical methods.