Draft:Magnetic Modulation Biosensing

Magnetic Modulation Biosensing (MMB) system is an ultrasensitive, in vitro diagnostic, combines magnetic nanoparticles and fluorescent labeling to measure ultra-low biomarker s concentrations. The technology's defining characteristics are its extremely high limit of detection, sensitivity and dynamic range.

Background
Biomarkers, such as proteins, DNA fragments, or nucleic acids, are essential tool for detection of various diseases. Even minor changes in biomarkers concentrations may be an indication of first signs of a disease. Usually, biomarkers are present at relatively low concentrations. Therefore, in order to detect such low concentrations, highly sensitive medical instruments are required.

In many bioligical assays, the target protein is marked with a fluorescent marker. To see the fluorescent marker, one need to shine laser light on the solution. However, at low concentrations, only two or three molecules are positioned inside the detection volume and therefore the fluorescence signal is very weak which limits the minimum concentration of detection.

A novel high-sensitivity biomarker detection platform, named Magnetic Modulation Biosensing (MMB), has been developed in 2008 by Dr. Amos Danielli, a stuff member at the Faculty of Engineering in Bar-Ilan University. in 2018, the MMB platform was commercialized as MMB-1 system by MagBiosense Inc., a medical device company developing a diagnostic devices and disposables for a point-of-care (POC) applications.

Principles
In the MMB, the capture surfaces, magnetic beads, are conjugated to a fluorescently labeled target.

The MMB has 2 key elements:


 * Aggregation
 * Modulation

Aggregation
Using electromagnets the system aggregates the fluorescent-conjugated beads in order to increase the signal intensity.

Modulation
After the system has created a bead aggregate, using the electromagnets, it moves the aggregate back and forth, in and out laser area in order to separate the fluorescent signal from background nois e.

Detection process
A laser beam is reshaped using two plano-convex lenses, reflected by a dichroic mirror, and focused by an objective lens onto the sample. The sample is a magnetic bead aggregate inside a rectangular glass cell positioned between two electromagnets that cause the bead aggregates to move. The laser excites the fluorophores in the sample, and the emitted fluorescence is collected by the same objective lens, filtered by an emission filter, and detected by a CCD camera or by a photomultiplier tube (PMT). A precision pinhole blocks the scattered photons from reaching the PMT.

Qualifications
The MMB qualifications have been demonstrated using a commercially-available assay for detection and quantification of human Interleukin-8 over several orders of magnitude. The results have shown that MMB has a 6-log dynamic range available for detection and is capable of detecting 0.08 ng/L of IL-8 in blood plasma, a sensitivity comparable to the state of the art laboratory assays.

Zika
A 2018 study has shown that using in MMB for ZIKA virus asasy provides high sensitivity and specificity, and low cross-reactivity compare to ELISA-ZIKA assays. Zika virus is a member of the flavivirus in the Flaviviridae family. ZIKV is most commonly transmitted by mosquitos (ZIKV can also be spread through sexual transmission. Zika infection in pregnant women is a major concern), as it is linked to catastrophic fetal abnormalities. Therefore, the ability to accurately diagnose ZIKV, is important for both spouses and especially in pregnant women. ZIKV usually disappears from the blood a few days after the onset of symptoms, for this reason indirect serologic tests were developed, such as an enzyme-linked immunosorbent assay (ELISA) and plaque-reduction neutralization test that detects virus-specific neutralization antibodies. These methods are not limited to the period in which the virus is still in the blood stream, and they constitute the bulk of the workload of many virology laboratories. Current ZIKV serological diagnosis is designed to detect IgM and IgG antibodies for either envelope or NS1 proteins. The envelope-based ELISA tests are sensitive, but lack specificity and have high cross-reactivity. NS1 protein-based ELISA is more specific. However, low levels of IgG/IgM antibodies against the NS1 protein are produced, which can result in a higher rate of false negative results. MMB utilizes magnetic beads that are conjugated to NS1 protein, which specifically captures the Zika IgM/IgG antibodies. A second, fluorescently-labeled antibody is then added to form a “sandwich” assay with the analyte and the capture protein. The MMB Zika assays have shown 88%–97% sensitivity, much higher than the state-of-the-art EUROIMMUN ELISA assays (38%–74%). In addition, the specificity is 100%, and the cross-reactivity with West Nile and dengue viruses is minimal (0%–4%). Furthermore, the MMB assays detected Zika IgM antibodies as early as 5 days and as late as 180 days postsymptoms onset, significantly extending the number of days that the antibodies are detectable.

Dengue
Dengue virus (DENV) is a member of the flavivirus in the Flaviviridae family. There are 4 serotypes of the virus that cause dengue (DEN-1, DEN-2, DEN-3 and DEN-4). Dengue fever is transmitted to humans through the bites of infective female Aedes mosquitoes. The symptoms of first infection are clinically characterized by high fever, severe headache, pain behind the eyes, muscle and joint pain, nausea, vomiting, swollen lymph nodes and rash. After recovery, lifelong immunity to that serotype of dengue virus will develop. However, cross-immunity to the other three serotypes after recovery is only partial and temporary. Hence, infections with other serotypes of dengue virus are more likely to result in severe dengue. Severe dengue is a potentially deadly complication due to plasma leakage, fluid accumulation, respiratory distress, severe bleeding, or organ impairment.

DENV serological diagnosis is designed to detect IgM and IgG antibodies for either envelope or NS1 proteins. The envelope-based ELISA tests are sensitive, but lack specificity and have high cross-reactivity.. MMB utilizes magnetic beads that are conjugated to NS1 protein (DEN-1, DEN-2, DEN-3 and DEN-4), which specifically captures the Dengue IgM/IgG antibodies. A second, fluorescently-labeled antibody is added to form a “sandwich” assay with the analyte and the capture protein. The MMB DENV assays are in par with ELISA DENV assays.

West Nile
West Nile virus (WNV) is a member of the flavivirus genus in the Flaviviridae family, and it is capable of causing severe neurological disease and death. The virus is transferred to humans and animals through bites of over 150 species of mosquitoes. Humans can also be infected by blood transfusion or organ transplantation. Symptoms for West Nile fever (WNF) may include fever, headache, weakness, joint and muscle pain, conjunctivitis, rash, nausea, and diarrhea. Approximately 80% of infections are asymptomatic and 20% will show mild infection symptoms. Less than 1% will develop a fatal neurological disease, namely West Nile Neuro invasive Disease (WNND), such as meningitis, encephalitis, or acute flaccid paralysisץ

The MMB WNV assays are on par with ELISA WNV assays.

Detection of repetitive oligonucleotide sequences
In 2019, two studies on using in MMB for detection of repetitive oligoncleotide sequences were published. Modern biomedical research extensively relies on detection and quantification of specific nucleic acid targets. The most widely used technique is based on Polymerase Chain Reaction (PCR), which allows amplification of the target molecule and its subsequent detection by gel electrophoresis or fluorescence (e.g., quantitative PCR). To detect rapidly a repetitive DNA sequence in MMB, two alternatives to the PCR method were examined:


 * 1) Partial PCR - using a double quenched fluorescent resonance energy transfer (FRET) based probe.
 * 2) Non PCR - using a sandwich hybridization assay (SHA).

In the first technique, combination of a modified double-quenched FRET-based probe with the MMB in allows to detect chick sex in ova within 13 min, with 100% sensitivity and specificity.

In the second technique sandwich hybridization assay (SHA), the presence of the target DNA/RNA sequence is recognized by two oligonucleotide probes, complementary to specific sites on the target molecule. The specificity of a sandwich hybridization assay (SHA) and the high sensitivity of a magnetic modulation biosensing (MMB), have enabled detection of DNA fragments without enzymatic signal amplification. The calculated limit of detection (LoD) was 685 fM.

Identification of protein-protein interactions
A 2019 study has shown the using in MMB for identification of protein-protein interactions. Investigating protein-protein interactions (PPIs) is essential in basic and clinical research. In the magnetic modulation biosensing (MMB) system, one protein of interest is attached to a magnetic bead and another protein is coupled to a fluorescent molecule. Thus, when the two proteins interact, the fluorescent molecule is connected to the magnetic bead. Using recombinant and native proteins,, the interaction between erythropoietin and its receptor was identified. In addition, the MMB demonstrated for the first time the direct interaction of a identified and characterized toxin antitoxin pair in Pseudomonas aeruginosa.

Diagnosis of Coronavirus Disease 2019 (COVID-19)
The outbreak of the coronavirus disease 2019 (COVID-19) emphasized the need for fast, sensitive, and specific diagnostic tools for virus surveillance. The diagnosis of the acute phase of the COVID-19 is based on direct detection of either viral antigens or viral RNA in nasopharyngeal, oropharyngeal, or mid turbinate swab samples. Antigen-targeting tests are simple to use, have fast turnaround times, and allow rapid testing for point-of-care applications. However, 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 severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA is based on amplification of the specific viral RNA sequences by quantitative RT-PCR (RT-qPCR), which theoretically allows for the detection of as little as a single copy of a target RNA. Overall, the RT phase, real-time monitoring, and the high number of amplification cycles are time consuming, and therefore the turnaround time of a standard RT-qPCR system is 90 to 120 minutes, which hinders its use for rapid screening. In reverse transcription loop-mediated isothermal amplification, nicking and extension amplification reaction and RT-PCR–based assays, the products of the reaction are visualized by either detection of increased fluorescence or a simple color change. To reach a detectable fluorescent signal or a color change and retain high sensitivity, long reaction times are used.

A 2021 study showed that using magnetic modulation biosensing (MMB) system, rapid thermal cycling, and a modified double-quenched hydrolysis probe minimizes the turnaround time while maintaining high sensitivity and specificity. 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.

SARS-CoV-2 Serological tests
in 2022, a study on using in MMB for SARS-CoV-2 Serological tests was published. The gold standard for the diagnosis of coronavirus disease 2019 (COVID-19) uses the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to directly detect the virus’ ribonucleic acid (RNA) in nasopharyngeal swab samples. While RT-qPCR is accurate, its diagnostic efficacy is limited to a few weeks after the infection. Serological assays to detect antiviral antibodies against SARS-CoV-2, namely immunoglobulin M (IgM), immunoglobulin G (IgG), and immunoglobulin A (IgA), are not limited to the first weeks of the disease and they indicate previous or recent SARS-CoV-2 infection, irrespective of whether the individual had a severe, mild, or even asymptomatic infection. IgA and IgM antibodies typically rise several days after SARS-CoV-2 infection, remain high for ~7–14 days, and indicate an acute viral infection. IgG antibodies are usually detected ~14 days after infection, remain detectable for several months, and indicate past viral infection. The study presents a rapid, highly sensitive, and specific anti-SARS-CoV-2 IgG serological assay based on the MMB technology. Compared with the gold standard ELISA test, the MMB-based assay demonstrates an ~3.8–6-fold better limit of detection. In clinical tests, the MMB-based assay showed similar sensitivity (93% vs. 92%) and specificity (98% vs. 99%) as the ELISA test but managed to correctly detect 14 positive samples (56%) in a group of 25 RT-qPCR SARS-CoV-2-positive samples taken from a large cohort study and had been falsely identified as negatives using the ELISA test. In addition, it detected an increase in IgG antibody concentrations in vaccinated individuals much earlier with a much faster turnaround time (45 vs. 245 minutes).