High throughput optical modulation biosensing for highly sensitive and rapid detection of biomarkers.

Rapid, highly sensitive, and high-throughput detection of biomarkers at low concentrations is invaluable for early diagnosis of various diseases. In many highly sensitive immunoassays, magnetic beads are used to capture fluorescently labeled target molecules. The target molecules are then quantified by detecting the fluorescent signal from individual beads, which is time consuming and requires a complicated and expensive detection system. Here, we demonstrate a high-throughput optical modulation biosensing (ht-OMB) system, which uses a small permanent magnet to aggregate the beads into a small detection volume and eliminates background noise by steering a laser beam in and out of the cluster of beads. Shortening the aggregation, acquisition, and well-to-well scanning transition times enables reading a 96-well plate within 10 min. Using the ht-OMB system to detect human Interleukin-8, we demonstrated a limit of detection of 0.14 ng/L and a 4-log dynamic range. Testing 94 RNA extracts from 36 confirmed RT-qPCR SARS-CoV-2-positive patients (Ct≤40) and 58 confirmed RT-qPCR SARS-CoV-2-negative individuals resulted in 100% sensitivity and 100% specificity.

Recent Advances in Rapid and Highly Sensitive Detection of Proteins and Specific DNA Sequences Using a Magnetic Modulation Biosensing System.

In early disease stages, biomolecules of interest exist in very low concentrations, presenting a significant challenge for analytical devices and methods. Here, we provide a comprehensive overview of an innovative optical biosensing technology, termed magnetic modulation biosensing (MMB), its biomedical applications, and its ongoing development. In MMB, magnetic beads are attached to fluorescently labeled target molecules. A controlled magnetic force aggregates the magnetic beads and transports them in and out of an excitation laser beam, generating a periodic fluorescent signal that is detected and demodulated. MMB applications include rapid and highly sensitive detection of specific nucleic acid sequences, antibodies, proteins, and protein interactions. Compared with other established analytical methodologies, MMB provides improved sensitivity, shorter processing time, and simpler protocols.

A Magnetic Modulation Biosensing-Based Molecular Assay for Rapid and Highly Sensitive Clinical Diagnosis of Coronavirus Disease 2019 (COVID-19).

Rapid and sensitive detection of human pathogens, such as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is an urgent and challenging task for clinical laboratories. Currently, the gold standard for SARS-CoV-2-specific RNA is based on quantitative RT-PCR (RT-qPCR), which relies on target amplification by Taq polymerase and uses a fluorescent resonance energy transfer-based hydrolysis probe. Although this method is accurate and specific, it is also time consuming. Here, a new molecular assay is described that combines a highly sensitive magnetic modulation biosensing (MMB) system, rapid thermal cycling, and a modified double-quenched hydrolysis probe. In vitro transcribed SARS-CoV-2 RNA targets spiked in PCR-grade water, were used to show that the calculated limit of detection of the MMB-based molecular assay was 1.6 copies per reaction. Testing 309 RNA extracts from 170 confirmed RT-qPCR SARS-CoV-2-negative individuals (30 of whom were positive for other respiratory viruses) and 139 RT-qPCR SARS-CoV-2-positive patients (CT ≤ 42) resulted in 97.8% sensitivity, 100% specificity, and 0% cross-reactivity. The total turnaround time of the MMB-based assay is 30 minutes, which is three to four times faster than a standard RT-qPCR. By adjusting the primers and the probe set, the platform can be easily adapted to detect most of the pathogens that are currently being diagnosed by RT-qPCR.

Optical modulation biosensing system for rapid detection of biological targets at low concentrations.

In many sensitive assays, target molecules are tagged using fluorescently labeled probes and captured using magnetic beads. Here, we introduce an optical modulation biosensing (OMB) system, which aggregates the beads into a small detection area and separates the signal from the background noise by manipulating the laser beam in and out of the cluster of beads. Using the OMB system to detect human interleukin-8, we demonstrated a limit of detection of 0.02 ng/L and a 4-log dynamic range. Using Zika-positive and healthy individuals' serum samples, we show that the OMB-based Zika IgG serological assay has 96% sensitivity and 100% specificity.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

A novel approach for simultaneous tibiofibular synostosis takedown and peroneus longus ligamentoplasty for posttraumatic tibiofibular synostosis: a case report and review of the literature.

INTRODUCTION: A singular procedure involving both a distal tibiofibular synostosis resection with syndesmosis repair by peroneus longus ligamentoplasty has not been reported in the English literature. We report a case of simultaneous distal tibiofibular synostosis resection and syndesmosis stabilization by peroneus longus ligamentoplasty for the treatment of symptomatic distal tibiofibular synostosis formation, following neglected syndesmosis injury. CASE PRESENTATION: A 42-year-old Caucasian man presented with ankle pain and painful range of motion 20 months following ankle trauma. Distal tibiofibular synostosis was identified, and our patient was successfully treated by simultaneous synostosis takedown and peroneus longus ligamentoplasty for distal tibiofibular syndesmosis repair. CONCLUSIONS: Our experience illustrates that in cases of painful posttraumatic distal tibiofibular synostosis, simultaneous synostosis resection with peroneus longus ligamentoplasty may show good clinical results. LEVEL OF EVIDENCE: 5.

Rapid and Sensitive Detection of Repetitive Nucleic Acid Sequences Using Magnetically Modulated Biosensors.

Repetitive DNA sequences are abundant in the genome of most biological species. These sequences are naturally "preamplified", which makes them a preferential target for a variety of biological assays. Current methods to detect specific DNA sequences are based on the quantitative polymerase chain reaction (PCR), which relies on target amplification by Taq polymerase and uses a fluorescent resonance energy transfer (FRET)-based probe. Here, to rapidly detect a repetitive DNA sequence, we combine a highly sensitive magnetic modulation biosensing (MMB) system and a modified double-quenched FRET-based probe. The high numbers of copies of the female-specific XhoI sequence of the domestic chicken (Gallus gallus), combined with the low background fluorescence levels of the modified double-quenched probe and the high sensitivity of the MMB system, allow us to determine the chick sex in ovo within 13 min, with 100% sensitivity and specificity. Compared to quantitative PCR, the presented assay is 4-9 times faster. More broadly, by specifically tailoring the primers and probe, the proposed assay can detect any target DNA sequence, either repetitive or nonrepetitive.

Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay.

We present a novel assay for rapid and highly sensitive detection of specific nucleic acid fragments in human serum. In a magnetic modulation biosensing (MMB) system, magnetic beads and fluorescently labeled probes are attached to the target analyte and form a "sandwich" complex. An alternating external magnetic field gradient condenses the magnetic beads (and hence the target molecules with the fluorescently labeled probes) to the detection volume and sets them in a periodic motion, in and out of a laser beam. A synchronous detection enables the removal of background signal from the oscillating target signal without complicated sample preparation. The high sensitivity of the MMB system, combined with the specificity of a sandwich hybridization assay, enables detection of DNA fragments without enzymatic signal amplification. Here, we demonstrate the sensitivity of the assay by directly detecting the EML4-ALK oncogenic translocation sequence spiked in human serum. The calculated limit of detection is 1.4 pM, which is approximately 150 times better than a conventional plate reader. In general, the MMB-assisted SHA can be implemented in many other applications for which enzymatic amplification, such as PCR, is not applicable and where rapid detection of specific nucleic acid targets is required.

Bilateral Common Peroneal Nerve Entrapment After Excessive Weight Loss: Case Report and Review of the Literature.

We report a case of excessive weight loss causing bilateral common peroneal nerve entrapment in a 60-year-old patient. The bilateral peroneal involvement suggested a systemic cause. Excessive weight loss during a relatively short period can cause changes in the tissues surrounding the common peroneal nerve and lead to its entrapment in the peroneal tunnel. Our patient underwent successful surgical decompression with significant improvement.

Improvement of hERG-ROMK index of spirocyclic ROMK inhibitors through scaffold optimization and incorporation of novel pharmacophores.

SAR in the previously described spirocyclic ROMK inhibitor series was further evolved from lead 4 by modification of the spirocyclic core and identification of novel right-side pharmacophores. In this process, it was discovered that the spiropyrrolidinone core with the carbonyl group α to the spirocenter was preferred for potent ROMK activity. Efforts aimed at decreasing hERG affinity within the series led to the discovery of multiple novel right-hand pharmacophores including 3-methoxythiadiazole, 2-methoxypyrimidine, and pyridazinone. The most promising candidate is pyridazinone analog 32 that showed an improved functional hERG/ROMK potency ratio and preclinical PK profile. In vivo evaluation of 32 demonstrated blood pressure lowering effects in the spontaneously hypertensive rat model.

Discovery of a Potent and Selective ROMK Inhibitor with Pharmacokinetic Properties Suitable for Preclinical Evaluation.

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

Comparison of human Ether-à-go-go related gene screening assays based on IonWorks Quattro and thallium flux.

In vitro screens using cellular preparations expressing human Ether-à-go-go related gene (hERG) potassium channels have become an intrinsic tool for evaluating cardiac liability of compounds during early preclinical stage development. Although hERG channel blocking effects are most reliably evaluated using the low-throughput, manual patch clamp technique, methods and technologies that yield hERG activity data in multiwell format are required to address increased throughput requirements. In most cases, multiwell approaches to measuring hERG activity involve achieving a reasonable balance between throughput and data fidelity. Here we compared two functional multiwell hERG assays: a fluorescence-based fluorometric imaging plate reader (FLIPR(®)) screen measuring thallium (Tl(+)) influx through hERG channels and an automated patch clamp assay using an IonWorks Quattro(®). Mean Z' values for FLIPR-Tl(+) and IonWorks Quattro assays were similar, 0.57 ± 0.09 (±SD; n = 10) versus 0.63 ± 0.11 (n = 12), respectively. IC50 determinations for a set of 17 reference compounds were used to evaluate potency shifts relative to conventional voltage clamp data. The reference compound set included members that are known to exert severe potency shifts in multiwell assays. Mean potency shift values for FLIPR-Tl(+) and IonWorks Quattro assays were 117- and 8-fold, respectively. On the basis of reduced potency shifts and low data variability, we conclude that the IonWorks Quattro screen was a better predictor of hERG activity in conventional whole-cell patch clamp than the Tl(+) influx assay.

Protein binding-dependent decreases in hERG channel blocker potency assessed by whole-cell voltage clamp in serum.

In vitro hERG blocking potency is measured in drug discovery as part of an integrated cardiovascular risk assessment. Typically, the concentrations producing 50% inhibition are measured in protein-free saline solutions and compared with calculated free therapeutic in vivo Cmax values to estimate a hERG safety multiple. The free/unbound fraction is believed responsible for activity. We tested the validity of this approach with 12 compounds by determining potencies in voltage clamp studies conducted in the absence and presence of 100% dialyzed fetal bovine serum (FBS). Bath drug concentrations in saline solutions were measured to account for loss of compounds due to solubility, stability, and/or adsorption. Protein binding in dialyzed FBS was measured to enable predictions of serum IC50s based on the unbound fraction and the saline IC50. For 11 of 12 compounds, the measured potency in the presence of dialyzed FBS was within 2-fold of the predicted potency. The predicted IC50 in dialyzed FBS for one highly bound compound, amiodarone, was 9-fold higher than the measured serum IC50. These data suggest that for highly bound compounds, direct measurement of IC50s in the presence of 100% serum may provide a more accurate estimate of in vivo potencies than the approach based on calculated serum shifts.

Additive effects of combined application of multiple hERG blockers.

Pro-arrhythmia by noncardiac drugs has become an important safety concern in the pharmaceutical industry. The most common underlying mechanism for induction of arrhythmias by noncardiac drugs is off-target block of the native cardiac repolarizing current, I Kr. The pore-forming subunit of I Kr is encoded by the human ether-a-go-go related gene (hERG), and in vitro measurements of hERG activity has become a standard component of drug safety evaluations. hERG/I Kr channels are blocked by a wide array of different chemical series; therefore, patients could be exposed to multiple blockers. There are few published studies addressing whether multiple blockers will exert independent actions on hERG channels. Whole cell patch clamp was used to evaluate the potential for cooperative effects when 2 hERG blocking agents were applied simultaneously. Cisapride, quinidine, fluvoxamine, and BeKm-1 were selected as representative agents binding to: (1) hydrophobic residues in the inner vestibule (cisapride and quinidine, the most frequent sites of interaction), (2) an extracellular segment near the pore (BeKm-1) or, (3) an unknown site (fluvoxamine). No synergistic blocking actions were seen. In some cases block was slightly less than additive. On balance, the results are consistent with additive and independent actions with simultaneous application of 2 hERG blockers.

Bicyclo[3.1.0]hexyl urea melanin concentrating hormone (MCH) receptor-1 antagonists: impacting hERG liability via aryl modifications.

Herein, we report the discovery of an effective strategy to modulate liabilities related to affinity of previously disclosed bicyclohexane MCHR-1 antagonists for the hERG channel. This paper describes one of several strategies incorporated to limit hERG binding via modifications of a terminal aryl group in an otherwise promising bicyclohexyl urea series.

Characterization of a hERG screen using the IonWorks HT: comparison to a hERG rubidium efflux screen.

The introduction of parallel patch clamp instruments offers the promise of moderate-throughput, high-fidelity voltage clamp for drug screening assays. One such device, the IonWorks HT (Molecular Devices, Sunnyvale, CA), was evaluated and compared to conventional human ethera- go-go-related gene (hERG) patch clamp data and an alternative functional screen based on rubidium flux. Data generated by the IonWorks HT and rubidium assays were compared to determine if either offered superior predictive value compared to conventional patch clamp. Concentration-effect curves for a panel of known hERG blockers were shifted to higher concentrations on the IonWorks HT compared to conventional voltage clamp determinations. The magnitude of the potency shifts was compound-specific and ranged from no shift (e.g., quinidine) to over 200-fold (astemizole). When the extreme value for astemizole was disregarded, the potency shift for 13 other known reference standards was 12-fold or less, with an average shift of fivefold. The same subset of compounds in the rubidium efflux assay exhibited an average potency shift of 12-fold. To provide a simulation of how the IonWorks HT assay might perform in a single concentration screening mode, a panel of test compounds was evaluated. The IonWorks HT screen did not outperform the rubidium efflux screen in predicting conventional voltage clamp measurements. The most likely explanation appears to rest with variable and compound-specific potency shifts in the IonWorks HT assay. The variable potency shifts make it difficult to select a screening concentration that meets the criterion of a high positive predictive value while avoiding false-positives.