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.
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.
In fluorescence-based biosensing applications, to increase optical detection sensitivity, time-resolved measurements are extensively used. Magnetic modulation biosensing (MMB) is a novel, fast, and sensitive detection technology for various applications. While this technology provides high sensitivity detection of biomarkers, to date, only the time resolved signal was analyzed. Here, we use for the first time both time-resolved and spatial-resolved measurements and show that this combination drastically improves the sensitivity of an MMB-based assay.
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.
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.
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 novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose a global public health threat. Effective and rapid serological assays are needed to provide valuable information about acute and past viral infections. Using the receptor-binding domain of the SARS-CoV-2 spike protein 1 antigen and a highly sensitive detection technology, termed magnetic modulation biosensing (MMB), we demonstrate a quantitative and rapid SARS-CoV-2 IgG antibody test with high sensitivity and specificity compared with the gold standard ELISA test. The improved analytical and clinical sensitivity of the MMB-based assay can help clinical laboratories provide critical information in a timely manner and monitor the spread of the disease.
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.
One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical sensitivity of the assay. Here, we photobleached several widely used magnetic beads and reduced their autofluorescence to 1% of the initial value. The photobleached beads were stable over time and their surface functionality was retained. In a high sensitivity LX-200™ system using photobleached magnetic beads, human interleukin-8 was detected with a 3-fold improvement in analytical sensitivity and signal to noise ratio over results achievable with non-bleached beads.
Luminex 200 is a flow cytometer that is widely used to detect very small concentrations of biomarkers. For biomarkers detection, fluorescently labeled probes and magnetic beads are attached to target molecules. After several washing and separation steps to remove unbound fluorescent molecules, single magnetic beads pass one by one through a narrow channel and their fluorescent signal is measured. Previously, we presented a novel magnetic modulation biosensing (MMB) system, which provides very high sensitivity in biomarkers detection. Unlike the particle by particle approach of flow cytometry, the MMB system is based on aggregating the magnetic beads and manipulating them in and out of the excitation laser beam. The modulation separates the signal from the background noise without washing steps. Using the MMB platform, we were able to detect Interleukin-8 with a limit of detection of 0.04 ng/L. Here, we provide a head-to-head comparison of the MMB platform and the Luminex 200 instrument. Comparisons were performed by processing identical bead-based tests in both systems. Using Luminex Triple Dye Beads, we showed that the MMB has at least 2x better sensitivity than the Luminex 200. In addition, we used a cardiac Troponin I assay and showed that the MMB has 10x better sensitivity than the Luminex 200. While the Luminex 200 has multiplexing capabilities, the MMB system provides higher sensitivity and a washless protocol that facilitates the use of the platform in point of care applications.
Intravital microscopy techniques have become increasingly important in biomedical research because they can provide unique microscopic views of various biological or disease developmental processes in situ. Here we present an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system that visualizes internal organs with a much finer resolution than conventional acoustic-resolution photoacoustic endoscopy systems. By combining gradient index (GRIN) lens-based optical focusing and ultrasonic ring transducer-based acoustic focusing, we achieved a transverse resolution as fine as ~10 μm at an optical working distance of 6.5 mm. The OR-PAEM system’s high-resolution intravital imaging capability is demonstrated through animal experiments.
Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.
Photoacoustic microscopy (PAM) has been shown to be a valuable tool for quantifying hemoglobin oxygenation within
single vessels. Recently, optical-resolution PAM was developed to achieve higher resolution by reducing the laser beam
diameter, which increased the light intensity. As intensity increases, saturation of the optical absorption and multiphoton/
multi-step absorption can occur, which, together with the temperature dependence of thermal expansion, result in
a non-linear dependence of the photoacoustic signal on the excitation pulse fluence. For hemoglobin, the major absorber
in tissue for photoacoustic imaging, these non-linear phenomena have strong wavelength dependence. To enable
quantitative photoacoustic measurements at different wavelengths in the presence of nonlinearity, a careful wide range
analysis of the intensity-dependent absorption is required. Here, we built a photoacoustic spectrometer, using a tunable
nanosecond optical parametric oscillator that operates between 410 nm and 2400 nm as our light source. To reduce
uncertainty in our measurements due to inhomogeneous spatial distribution of the optical fluence, we used a flat-top
beam illumination and a flat transducer which was mounted in reflection mode, effectively reducing quantitative
measurements to a one dimensional problem. Intensity-dependent non-linear spectra of the photoacoustic signals of oxyand
deoxy-hemoglobin were obtained. These measurements have the potential to contribute significantly to quantitative
functional PAM.
pH is a tightly regulated indicator of metabolic activity. In mammalian systems, imbalance of pH regulation may result
from or result in serious illness. Even though the regulation system of pH is very robust, tissue pH can be altered in many
diseases such as cancer, osteoporosis and diabetes mellitus. Traditional high-resolution optical imaging techniques, such
as confocal microscopy, routinely image pH in cells and tissues using pH sensitive fluorescent dyes, which change their
fluorescence properties with the surrounding pH. Since strong optical scattering in biological tissue blurs images at
greater depths, high-resolution pH imaging is limited to penetration depths of 1mm. Here, we report photoacoustic
microscopy (PAM) of commercially available pH-sensitive fluorescent dye in tissue phantoms. Using both opticalresolution
photoacoustic microscopy (OR-PAM), and acoustic resolution photoacoustic microscopy (AR-PAM), we
explored the possibility of recovering the pH values in tissue phantoms. In this paper, we demonstrate that PAM was
capable of recovering pH values up to a depth of 2 mm, greater than possible with other forms of optical microscopy.
pH is a tightly regulated indicator of metabolic activity. In mammalian systems, an imbalance of pH regulation may result from or result in serious illness. In this paper, we report photoacoustic microscopy (PAM) of a commercially available pH-sensitive fluorescent dye (SNARF-5F carboxylic acid) in tissue phantoms. We demonstrated that PAM is capable of pH imaging in absolute values at tissue depths of up to 2.0 mm, greater than possible with other forms of optical microscopy.
Functional photoacoustic microscopy is a valuable tool in quantifying hemoglobin oxygenation within single vessels. In
several functional studies with this tool, quantitative sO2 measurements were taken both in vitro and in vivo. Although in
vitro measurements of sO2 showed high agreement with expected values from premade samples, in practice, in vivo
measurements were less accurate. The reported values of 70%-100% sO2 in the arteries present large deviations from the
expected range of 95-100%. Several factors, such as fluence wavelength dependence, optical wavelength range, and
transducer central frequency have been suggested and investigated in order to understand these discrepancies. Despite
additional knowledge of systematic errors arising from such factors, measuring the absolute value of sO2 in vivo remains
a challenge. All previous studies assumed linear dependence of the photoacoustic signal on absorption and used the
linear least squares model. However, several factors, such as wavelength calibration errors, photodiode-wavelength
dependence, and intensity dependent non-linearity, all of which may have a significant effect on the final calculation,
have not been investigated. Here we evaluate both in vitro and in vivo the effects on sO2 measurements of photodiode
wavelength dependence, laser wavelength accuracy, and intensity dependent absorption of oxygenated and
deoxygenated hemoglobin. We show that these factors may contribute significantly to the deviations in sO2 calculations
in vivo.
Magnetic modulation biosensing (MMB) system rapidly and homogeneously detected coding sequences of the nonstructural
Ibaraki virus protein 3 (NS3) complementary DNA (cDNA). A novel fluorescent resonance energy transfer
(FRET)-based probe discriminated the target DNA from the control. When the target sequence is detected, the FRETbased
probe is cleaved using Taq-polymerase activity and upon excitation with a laser beam fluorescent light is
produced. The biotinylated probes are attached to streptavidin-coupled superparamagnetic beads and are maneuvered
into oscillatory motion by applying an alternating magnetic field gradient. The beads are condensed into the detection
area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is
demodulated using synchronous detection. Condensation of the beads from the entire volume increases the signal while
modulation separates the signal from the background noise of the non-magnetized solution. 1.9 picomolar of the Ibaraki
virus NS3 cDNA was detected in homogeneous solution within 18 minutes without separation or washing steps. In this
paper we will review the magnetic modulation system and present its capability in specific DNA sequences detection.
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