Rapid, highly sensitive, and high-throughput detection of biomarkers at low concentrations is invaluable for the early diagnosis of various diseases. In many sensitive immunoassays, the protocol is time-consuming and requires a complicated and expensive detection system. Previously, we presented a high-throughput optical modulation biosensing (ht-OMB) system, which enables reading a 96-well plate within 10 minutes. In ht-OMB, to aggregate and immobilize the magnetic beads to one spot, a single cylindrical permanent magnet with a sharp tip is positioned under a 96-well plate. To reduce washing and separation steps, the laser beam is manipulated relative to the fixed magnetic beads. Recently, MagBiosense Inc., which commercializes the ht-OMB technology, provided us with a fully automated OMBi detection system. Here, we show the use of the OMBi system for highly sensitive serological (clinical anti-Zika, anti-DENV, and anti-West Nile IgG) and molecular (SARS-CoV-2) assays. Using the OMBi, to detect 336 RNA extracts from 70 confirmed RT-qPCR SARS-CoV-2-positive patients (Ct≤40) and 236 confirmed RT-qPCR SARS-CoV-2-negative individuals, resulted in 100% specificity and 96% sensitivity.
The COVID-19 pandemic has emphasized the inability of diagnostic laboratories' testing capacity to keep up with the surging demand. The primary reasons were the lack of reagents (e.g., viral transport media and nucleic acid extraction kits) and the low throughput of the gold-standard molecular detection method (RT-qPCR). While the reagent shortages were eventually resolved, the limited throughput of the RT-qPCR remains a bottleneck for high-throughput testing applications even today.
Here, we introduce a rapid saliva-based extraction-free molecular assay, which utilizes a non-invasive saliva sampling and extraction-free sample preparation, a fast endpoint RT-PCR and a high-throughput optical modulation biosensing (ht-OMBi) detection platform. We blindly tested 364 paired nasopharyngeal swabs and saliva samples from suspected SARS-CoV-2 cases in Israel. Compared with the gold standard swab-based RT-qPCR, the presented assay's sensitivity and specificity are 90.7% and 95.3%, respectively, but is achieved with only 50 min. sample-to-result turnaround time (~60% faster than the regular RT-qPCR), allowing high throughput and considerable savings of the reagents and disposables.
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.
Rapid, highly sensitive, and high-throughput detection of biomarkers at low concentrations is invaluable for early diagnosis of various diseases. In many sensitive immunoassays the protocol is time consuming and requires a complicated and expensive detection system. Here, we demonstrate a high-throughput optical modulation biosensing (ht-OMB) system, which enables reading a 96-well plate within 10 minutes. Using the 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 (C_t≤40) and 58 confirmed RT-qPCR SARS-CoV-2-negative individuals resulted in 100% sensitivity and 100% specificity.
KEYWORDS: Molecules, Luminescence, Magnetism, Target detection, Signal detection, Signal to noise ratio, Modulation, Diagnostics, Polymers, In vitro testing
The COVID-19 pandemic demands fast, sensitive, and specific diagnostic tools for virus surveillance and containment. Current methods for diagnosing the COVID-19 are based on direct detection of either viral antigens or viral ribonucleic acids (RNA) in swab samples. Antigen-targeting tests are simple, have fast turnaround times, and allow rapid testing. Unfortunately, compared with viral RNA-targeting tests, their sensitivity is low, especially during the initial stages of the disease, which limits their adoption and implementation. Direct detection of SARS-CoV-2 RNA using reversetranscription quantitative polymerase chain reaction (RT-qPCR) is sensitive and specific, making it a golden standard in SARS-CoV-2 detection. However, it had not seen a significant update since its introduction three decades ago. It has a long turnaround time, requires a high number of amplification cycles, and a complicated and expensive detection system for real-time monitoring of the signal. While insignificant for research applications, these limitations present severe problems for mass testing required to contain the disease. Here, we introduce a diagnostic platform for rapid and highly sensitive clinical diagnosis of COVID-19. Based on the biochemical principles of the RT-PCR, it utilizes the endpoint detection by the magnetic modulation biosensing (MMB) system, allowing the detection of as little as two copies of SARS-CoV-2 in ~30 minutes. Testing 309 RNA samples from verified SARS-CoV-2 carriers and healthy subjects resulted in 97.8% sensitivity, 100% specificity, and 0% crossreactivity. This level of performance is on par with the gold standard (RT-qPCR) but requires 1/3 of the time. The platform can be easily adapted to detect almost any other pathogen of choice.
The outbreak of the coronavirus disease emphasized the need for fast and sensitive inhibitor screening tools for the identification of new drug candidates. In SARS-CoV-2, one of the initial steps in the infection cycle is the adherence of the receptor-binding domain (RBD) of the spike protein 1 (S1) to the host cell by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, inhibition of S1-ACE2 interaction may block the entry of the virus to the host cell, and thus may limit the spread of the virus in the body. We demonstrate a rapid and quantitative method for the detection and classification of different types of molecules as inhibitors or non-inhibitors of the S1-ACE2 interaction using magnetically modulated biosensors (MMB). In the MMB-based assay, magnetic beads are attached to the S1 protein and the ACE2 receptor is fluorescently labeled. Thus, only when the proteins interact, the fluorescent molecule is connected to the magnetic bead. To increase the sensitivity of fluorescence detection, the complex of magnetic beads and attached fluorescent molecules are aggregated by two opposing electromagnets and are moved from side to side in a periodic motion in and out of a laser beam, emitting a flashing signal that is collected by a digital camera. When an inhibitor interferes with the interaction, the signal is reduced. The MMB-based assay is much faster and has minimal non-specific binding than the commonly used ELISA. It can be adjusted to other interactions, and therefore can be utilized as a global tool for inhibitor screening.
Detection of biomarkers at low concentrations is essential for early diagnosis of numerous diseases. In many sensitive assays, the target molecules are tagged using fluorescently labeled probes and captured using magnetic beads. Current devices rely on quantifying the target molecules by detecting the fluorescent signal from individual beads. Here, we demonstrate a high-throughput optical modulation biosensing (ht-OMB) system 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, values which are on par with the most sensitive devices, but are achieved without their bulk and laborious protocols.
Detection of biomarkers at low concentrations is essential for early diagnosis of numerous diseases. In many sensitive assays, the target molecules are tagged using fluorescently labeled probes and captured using magnetic beads. Current devices rely on quantifying the target molecules by detecting the fluorescent signal from individual beads. Here, we propose a compact fluorescence-based magnetically aggregated biosensors (MAB) system. Using the device to detect human Interleukin-8, we demonstrated a 0.1 ng/L limit of detection and a 4-log dynamic range, performance which is on par with the most sensitive devices, but is achieved without their bulk and cost.
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