Patent Application
20220120692
The present disclosure relates in general to a method and system for analyte detection and monitoring, and more specifically to a method and system for detection, quantification and/or identification of an analyte such as a coronavirus, using analyte and reagent interactions detection and monitoring based on electrical and/or optical response to an electric field. More specific embodiments of the present disclosure relate in general to a method and system for virus detection and monitoring, and more specifically to a method and system for detection, quantification and/or identification of a coronavirus, using virus and protein-antibody interactions detection and monitoring based on electrical and/or optical response to an electric field.
In late December 2019, patients with an atypical pneumonia due to a novel coronavirus were reported in Wuhan, China. Since then, the novel coronavirus disease 2019 (COVID-19) has become a pandemic that has spread worldwide to virtually every country. This pandemic has caused massive social and economic disruptions in nearly every country and therefore global research and development efforts are being geared towards development of vaccines and therapeutics for the prevention and treatment of COVID-19, in order to normalize the situation.
Unfortunately, the complete clinical picture of COVID-19 is not yet fully known and most likely depends upon a number of factors, including virus characteristics. As of Jul. 7, 2020, more than 11,645,109 cases of COVID-19 infection had been confirmed worldwide with 538,780 deaths, revealing a case fatality rate (CFR) of 4.6%. Successful detection of SARS coronavirus 2 (SARS-CoV-2) plays an important role in stopping the spread.
The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19 enters the susceptible cells primarily via endocytosis using its spike (S) protein (3-5). The viral S protein is a homotrimer that protrudes from the virion surface (6) and is responsible for entry into susceptible cells by binding to the human angiotensin converting enzyme 2 (ACE2) protein (3-5). Once internalized, the virus starts to replicate within the cell (7). The nucleocapsid (N) protein of SARS-CoV-2, is the largest structural protein of the virus which coats its large genomic RNA and is responsible for creating its helical structure (8). Compared to the viral S protein, the N protein is much more conserved (-90%), is expressed at high levels during infection, and is highly immunogenic (8). This is for example described in the publication Y. Cong, M. Ulasli, H. Schepers, M. Mauthe, P. V'kovski, F. Kriegenburg, V. Thiel, C. A. M. de Haan, F. Reggiori, Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle. J. Virol. 94 (2019), doi:10.1128/JVI.01925-19.
Currently, oropharyngeal and nasopharyngeal swabs are primarily used for virus detection. However, it is not clear how many virus particles of SARS-CoV-2 are needed to trigger an infection. It has been anticipated that the corresponding dose to establish an infection in exposed people could be as little as 10 virus particles. Other studies suggest a relatively higher dose, ranging from a few hundred to thousands of particles. It has early been estimated and later confirmed in international medical reports that SARS-CoV-2 exhibits a higher rate of virus replication compared to SARS-CoV-1. The high level rate of virus replications of SARS-CoV-2 can quickly cause increased disease severity. Statistically, confirmed COVID-19 cases worldwide are 100 times higher than the confirmed cases of SARS and MERS. This is because, 1) SARS-CoV-2 replicates to much higher levels in the nose and mouth than SARS and MERS, and 2) this leads to very high levels of virus shedding in the environment by people who are either pre-symptomatic or asymptomatic. Thus, a huge percentage of infected people can transmit the virus without realizing that they are even infected.
Rapid detection methods independent of lab setting have been identified as one of the foremost priorities for promoting epidemic prevention and control. Currently, there are two main strategies for the detection of COVID-19. The first is a real time reverse transcriptase (RT) polymerase chain reaction (RT PCR)-based strategy that detects the viral nucleic acid in patient samples (presence of the viral RNA). The second strategy is an immunological assay that detects viral protein antigens or serum antibodies produced as a result of the body's immune response to the viral infection. The two strategies complement each other, with the qPCR strategy detecting the virus during its active phase, while the immunological assay identifies individuals who have developed antibodies to fight the disease.
Currently, the molecular technique of quantitative real time polymerase chain reaction (qRT PCR) is the gold standard for SARS-CoV-2 detection using samples from respiratory secretions. However, it is a time consuming and cumbersome procedure that takes long processing times over days for results. Several other molecular assays have been developed to detect SARS-CoV-2, such as enzyme-based assays like ELISAs, and rapid tests that aim to detect either antibodies against the virus or the viral antigen themselves. Nevertheless, most of these antigen-antibody-based assays have failed quality control due to their rapid development without proper testing and result in either false negative or false positive detection due to the long time it takes to develop serum responses to the viral infection (from days to two weeks). Although capacity for PCR testing as well as the quality of quick tests for coronavirus detection have been developed, the testing is inflexible and does not give a reliable quantification of virus present in a sample.
Another approach for virus detection that is applicable to the detection of coronavirus and protein-antibody interactions has been described for example in the co-pending patent application by the same applicant, U.S. patent application Ser. No. 17/379,496 for “Method and system for virus and protein-antibody interactions detection and monitoring based on optical light intensity and electrical parameters” based on U.S. 63/054,022 in the name of the United Arab Emirates University, inventors: Mahmoud F. Y. Al Ahmad, Tahir A. Rizvi, Farah Mustafa.
The advancement in the integration of bio sensing mechanisms and electrical characterization techniques, such as the approach mentioned above, have provided a powerful potential and huge impact in developing point of care systems. In principle biosensors are capable to transform biochemical information, such as analyte or particles concentrations into a corresponding electrical useful signal: current or voltage. Signal processing along with parametric modelling techniques have been developed for the purpose of deciding the outcome about a sample under test. Combining biosensor with wireless connectivity platforms and analysis capabilities have reduced the cost for employing such technology.
Examples of related art with different assay methods and analysing techniques are found in various publications. For example, the patent publication US2017362668A1 to Meso Scale Technologies with the title Co-binder assisted assay methods disclose methods for reducing cross-reactivity between species employed in multiplexed immunoassays.
Another example of related art is found in the patent publication US2021123883A1 to University of Utah Research Foundation with the title Whole virus quantum mechanical tunnelling current and electronic sensors. This publication discloses a field effect transistor (FET) biosensor for virus detection of a selected virus within a sample volume.
A further example of related is the patent publication WO2021081476A1 to University of Utah Research Foundation with the title Zero Power visible colorimetric pathogen sensors. This piece of related art shows a method in which a visibly perceived colorimetric pathogen sensor comprises a substrate and a molecular recognition group coupled to the substrate. The molecular recognition group can bind a target pathogen and when that occurs, the reflected light cab be altered thereby changing apparent color, thus indicating the detected target pathogen.
The article with the title An Analysis Review of Detection Coronavirus Disease 2019 (COVID-19) Based on Biosensor Application by Bakr Ahmed Taha et al. summarizes technologies for the detection of coronavirus disease 2019 (COVID-19) technologies with biosensors that operate using laser detection technology.
The related art patent publication WO2011060184 to Cermed Corporation with the title Cervical cancer screening by molecular detection of human papillomavirus-induced neoplasia further shows point-of-care tools for screening biological samples for markers associated with pathogenic microbial infections. This publication discloses a technology for screening cervical cells for the expression of proteins that occur because of human papillomavirus infection and progression to invasive cervical cancer.
Another related art patent publication WO2015116083 to Hewlett Packard Development with the title Microfluidic sensing device. This publication discloses a microfluidic sensing device that comprises a channel and an impedance sensor within the channel. A particle in a fluid passing the sensor is identified based on the sensed impedance characteristics.
On a general level there is a need for efficient methods and systems for detection, identification and quantification of analytes in specimen comprising cells, such as virus of any kind, coronavirus, bacteria, fungus, protein or other analyte.
A more specific and current need is the need for a cost effective method and system that yields fast, cheap and accurate results of detection, identification and quantification of a coronavirus such as SARS-CoV-2, which may be used to slow the spread of the virus and monitor the vaccine development process.
The present disclosure describes embodiments for a method and a system of detecting, identifying and quantifying an analyte in a specimen comprising one or more cells using detection and monitoring interactions between an analyte in the specimen and a reagent being prone to engage in a binding activity with such an analyte present in said one or more cells, using detection and monitoring of interactions based on electrical and/or optical response to an electric field. The analyte may be virus of any kind, coronavirus bacteria, fungus, protein or other analyte. The specimen may be taken from a living being for example in a bodily from a human patient, or from an object subject to possible analytes being present for example on a surface.
The present disclosure further describes more specific embodiments for a method and system for coronavirus detection and quantification using virus and protein-antibody interactions detection and monitoring based on electrical and/or optical response to an electric field.
Embodiments disclosed herein comprises a system and an electrical method that measures the viral nucleocapsid protein and anti-N antibody interactions to differentiate between SARS-CoV-2 negative and positive samples for example captured on nasal swab samples. Under electrical bias, the cell opens up by the formation of pores in the cell membrane. The antibodies penetrate the cell through these pores and binds with the N protein of SARS-CoV-2 virus that is expressed during infection. In exemplifying usage of embodiments disclosed herein, it has been found empirically that SARS-CoV-2 in patient nasal swab samples would be detected within five minutes if gaussian-like binding profile are detected, otherwise, the sample is considered negative. The calculated cost of such a test would be less than 3$, which is the cheapest ever one can have compared to existing tests.
Embodiments disclosed herein comprises a method of detecting, identifying and quantifying an analyte in a specimen comprising one or more cells, the specimen being accommodated in a suspension medium, comprising: apportioning the suspended specimen into one or more test samples; optionally, adding a reagent to the one or more test samples, said reagent being prone to engage in a binding activity with an analyte present in a said one or more cells; applying an electric field with a first magnitude and with a second magnitude over said one or more test samples for a selected period of time, said second magnitude being higher than said first magnitude; measuring one or more electrical properties of said one or more test samples in response to said applied electric field for said first magnitude and for said second magnitude over said period of time; —identifying characteristics of said electrical properties responses; determining the presence, the identification and/or the quantity of the analyte based on the characteristics of said electrical properties responses to said first magnitude and to said second magnitude of the applied electric field.
In different embodiments of the method, the analyte is any of coronavirus, virus of any kind, bacteria, fungus, protein or other analyte.
Further embodiments of the method comprise:
In embodiments of the method:
Embodiments of the method, comprises:
Further embodiments of the method comprise providing two or more test samples with specimen and an added reagent, and for said two or more test samples, simultaneously: —measuring electrical properties responses for an applied low magnitude electric field over said test samples; —measuring electrical properties responses for an applied high magnitude electric field over said test samples; —analyze characteristics of said electrical properties responses for said two or more test samples; thereby processing two or more test samples in parallel for increased efficiency; said electrical properties responses preferably being capacitance versus applied voltage responses.
In embodiments of the method there is comprised measuring optical properties responses, preferably light intensities, of said two or more test samples for said applied low and high magnitude electric fields.
Embodiments of the method, comprises:
Embodiments described herein comprises a system for detecting, identifying and quantifying an analyte in a specimen comprising one or more cells, the system comprising: —a testing apparatus having a test sample holder arranged to house one or more test sample containers in a position in relation to one or more sets of electrodes configured to apply an electric field over a test sample in a test sample container and to conduct measurements of one or more electrical properties of a test sample in a test sample container.
In embodiments of the system the analyte is any of coronavirus, virus of any kind, bacteria, fungus, protein or other analyte.
Embodiments of the system further comprises a test sample container for a test sample of a specimen accommodated in a suspension medium, the test sample container being configured for applying an electric field over a test sample in the test sample container and/or configured to enable light passing through the test sample and the test sample container.
In embodiments of the system, the testing apparatus is configured to apply an electric field of a first, lower, magnitude and of a second, higher, magnitude over a said test sample in a said test sample container, wherein the applied electric field is induced by applying a DC voltage or a pulsating AC voltage over said test sample, said high magnitude electric field being generated by an applied voltage in the range of preferably 0.5 to 1 volt.
In other embodiments of the system the testing apparatus is configured to measure one or more parameters indicating electrical properties of a said test sample in a said test sample container for different magnitudes of applied electrical fields, for example a parameter indicating the capacitance of said test sample.
In embodiments of the system the test sample holder of the testing apparatus is further configured to hold the one or more test sample container in a position in relation to a light source to enable light to pass through the test sample and onto a light detector coupled to a device configured to measure parameters indicating optical properties of the test sample; and preferably said testing apparatus being configured to measuring optical properties responses of said one or more test samples in response to said applied electric of said first and second magnitudes.
Embodiments of system, further comprises: a processor provided with computer program code configured to determine the presence, the identification and/or the quantity of coronavirus based on the characteristics and/or on differences in the characteristics of said electrical properties responses to said first magnitude and to said second magnitude of the applied electric field; and/or a processor provided with computer program code configured to determine the presence, the identification and/or the quantity of coronavirus based on the characteristics and/or on the differences in characteristics of said optical properties responses to said first magnitude and to said second magnitude of the applied electric field.
Other aspects and embodiments of the invention will be apparent as will be shown in the detailed description of the invention.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The accompanying drawings illustrate non-limiting example embodiments of the invention.
Throughout the following description, specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure of embodiments. The following description of examples of the technology is not intended to be exhaustive or to limit the system to the precise forms of any example embodiment. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
As has been seen with the recent COVID-19 pandemic, viral outbreaks or pandemics can result in numerous global, social and economic problems. In particular, this outbreak has highlighted the need for rapid diagnosis and detection mechanisms that can play major role in stop spreading the virus. Although some studies show that anywhere from a few hundred to a few thousand COVID-19 viruses are required for a person to contact COVID-19, other studies have shown that even as few as 10 viral particles can result in viral infection. Embodiments disclosed herein comprise a technology that is scaled to detect down to few virions.
The technology with electrical detection of specific viral antigen binding to a specific antibody has the potential to accelerate the development of a new generation viral detectors. Techniques as described herein enable high sensitivity, specific detection and quantitative signature based on the binding characteristics. Moreover, the application of electrical conditions over a sample can be used to enable and accelerate such binding interactions that are required for efficient electrical detection. In a more general sense, the technique enables the control of chemical reactions using electric field gradients.
The need for rapid cost efficient detection of analytes is in fact universal for several different kinds of target analytes, such as virus of any kind, bacteria, fungus, protein or other analyte, in particular for supporting the health care sector around the world.
The typical structure of a biological cell is that a cell membrane encloses a nucleoid region with components such as a nucleus and/or other cell components suspended in cytoplasm. These components can interact in different manners with the environment of the cell. This is explained by means of an example detailing coronavirus characteristics.
The coronavirus SARS-CoV-2 appears as a spherical particle with external spike protein that displays crown-like appearance, as has been observed under an electron microscope. Corona virus (herein in short also called CoV) consist of positive-sense single-stranded RNA of approximately 27-32 kb (kilobase, unit equal to 100 base pairs bp of RNA or DNA). Its envelope is approximately 100 nm (nanometer) in diameter and the spikes are oligomers of the 180-200 kDa (kilodalton, atomic mass unit) S glycoprotein that can bind to the host cell. Thus, SARS-CoV-2 belongs to the betaCoVs category (betacoronavirus genus). It has round or elliptic and often pleomorphic form, and a diameter of approximately 60-140 nm (nanometer). Like other CoVs, it is sensitive to ultraviolet rays and heat. Ultraviolet (UV) is electromagnetic radiation with wavelength from 10 nm to 400 nm (750 THz). The spike protein appearances are much smaller much smaller in size than the particle itself with high aspect ratio, where the spike protein measures 1-5 nm and has an atomic mass of less than 200 kDa and has an arm of sub-nanometer dimension.
Several published reports suggest that SARS CoV-2 could be able to bind to the angiotensin-converting enzyme 2 receptor in humans. Nevertheless, the complete virus structure analysis and its future evolution, adaptation, still require further investigations.
The genomic characteristics of 2019-nCoV as well as similarities and differences to other coronaviruses has been discussed in the publication titled “Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding” Lu et al, 2020. Based on the genome sequences of 2019-nCoV it could be concluded that the outbreak was detected relatively rapidly after the first occurrences of infection in human patients. It has been found that the recombinant RBD protein bind strongly to human ACE2 (hACE2) and bat ACE2 (bACE2) receptors and it blocked the entry of SARS-CoV-2 and SARS-CoV into their respective hACE2-expressing cells, as discussed also in the publication titled “Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine”, Tai et al, 2020.
A coronavirus is composed of four structural proteins incorporating spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. S protein plays the most important roles in viral attachment, fusion and entry. The S protein mediates viral entry into host cells by first binding to a host receptor through the receptor-binding domain (RBD) in the 51 subunit and then fusing the viral and host membranes through the S2 subunit. Furthermore, it serves as a target for development of antibodies, entry inhibitors and vaccines.
SARS-CoV-2 particle diameter approximately ranges from 60 to 140 nm with distinctive spikes about 8 to 12 nm in length, as disclosed in the publication titled “A Novel Coronavirus from Patients with Pneumonia in China, 2019”, Zhu et al, 2020. In average the virus volume is of about 10-3 femtoliter with mass of 1 femtogram as disclosed in the publication titled “Science Forum: SARS-CoV-2 (COVID-19) by the numbers”, Bar-On et al, 2020. The virus concentration levels have been reported in several diagnoses as per follow: Nasopharynx 106-109 RNAs/swab; throat 104-108 RNAs/ swab, Stool 104-108 RNAS/g and Sputum 106-1011 RNAS/mL, the RNA counts can markedly overestimate infectious virions, which is discussed in the publications titled “Virological assessment of hospitalized cases of coronavirus disease 2019”, Wölfel et al. 2020, and another publication titled “Viral load of SARS-CoV-2 in clinical samples”, Pan et al, 2020.
The cross section of a virion, i.e., a virus in the form of an independent particle when appearing not inside an infected cell or in the course of infecting a cell, is depicted in
The spike protein distribution across the virus particle surface can be explored with the help of electron microscopy images, as described in the publication titled “Brief Review on COVID-19: The 2020 Pandemic Caused by SARS-CoV-2”, Damian N. Valencia, 2020. With the help of image processing tools the spike protein variation in position and in in length have been extracted and their distribution versus spike protein counts are represented by
As illustrated in
As an example for a virion of 100 nm diameter, the distances between the spikes varies from 1 nm to 4.5 nm; and its length varies from 0.5 nm to 13 nm. These features have been extracted using imported SEM images relying on image pixel assessment. The number of spikes per virus circumference is approximate of 35 spikes, and of around more than 300 spikes per virus particle with complex randomly distributed pattern and not equal spaced.
In the illustration of
There are several antigens and elements in SARS-CoV2 virus that are active in binding and/or interaction, ACE2 is one example, nucleocapsid is another example and the technology of the present disclosure is applicable to other antigens and elements as well. The technology of embodiments disclosed herein can, as described herein, be applied on any virus, antigens, antibodies or elements.
For the sake of example, embodiments herein are explained with application of the technology on ACE2 and nucleocapsid. In terms of detection, one of the most prominent features of the SARS-CoV-2 virus, like other coronaviruses, is the spike protein (S) that protrudes out of the virus particle essentially like “spikes”; hence the name. The spike protein forms a trimer that is used by the virus to enter susceptible cells using the angiotensin-converting enzyme 2 (ACE2) protein as the cellular receptor, the same protein used by the SARS-CoV-1 virus that caused the first SARS epidemic in 2003 (
A reagent such as the ACE-2 as well as the nucleocapsid antibody NC, or any other possible antibody or other suitable reagent, can be introduced into the virus cell by electro-poration methods or any other type of method that can allow the cell content to go outside in the suspended media or allowing the antibodies to go inside the cells. In embodiments an electrical pulse is applied to the cells in the specimen of a test sample to stimulate interaction between a reagent and the virus cell. The cell permeability increases, due to pore formation in the cell membrane. The pores only stay open for the duration of the applied constant field or its time domain variation. The corresponding antibody penetrates the cell through these pores and binds with the antigen. In case of ACE-2 antibody it will bind with the Spike protein of SARS-CoV-2 virus. This Spike protein can be used as reliable markers for the indication of the presence of infection and of virus replication, since it causes the COVID-19 disease.
Detection, Quantification and Identification of Coronavirus and other Analytes
In embodiments, negative and positive specimen from COVID 19 patients and suspected patients are loaded into individual test samples. A regent, such as ACE-2 antibodies or NC antibodies also called anti-N ab. The selected reagents bind specifically to components of the SARS-CoV-2 virus. Under electrical bias, the virus cell opens up by formation of pores in the cell membrane. So, for example the anti-N antibody penetrates the cell through these pores and bind with the N protein (i.e., the nucleocapsid protein which herein is also called NC protein) that is expressed during infection. For the purpose of embodiments this binding event is used as a reliable marker for the presence of SARS-CoV-2 infection.
In accordance with the concepts described herein different embodiments are applied on different analytes, such as coronavirus, virus of any kind, bacteria, fungus, protein or other analyte. In embodiments of such a system including a method and an apparatus, of detecting, identifying and quantifying an analyte in a specimen comprising one or more cells, the specimen is accommodated in a suspension medium. The suspended specimen is preferably apportioned into one or more test samples. Optionally, a reagent is added to the one or more test samples, wherein said reagent is prone to engage in a binding activity with an analyte present in a said one or more cells. An electric field with a first magnitude and with a second magnitude is applied over said one or more test samples for a selected period of time, wherein the second magnitude is higher than said first magnitude. One or more electrical properties of said one or more test samples in response to said applied electric field are measured for said first magnitude and for said second magnitude over said period of time. Characteristics of said electrical properties responses are identified, and the presence, the identification and/or the quantity of the analyte are determined based on the characteristics of said electrical properties responses to said first magnitude and to said second magnitude of the applied electric field.
In exemplifying embodiments of such a system for detection, quantification and identification of coronavirus, a specimen is obtained from a patient using a nasopharyngeal swab. The swab is placed in a suspension medium such as a viral transport medium (VTM) solution or any other possible suspension liquid media, for example but not limited to blood, urine and saliva, and gently shaken to transfer the viruses from the swab to the suspension medium. The specimen accommodated in a suspension medium is put in a test sample container. A pure specimen test sample and/or a test sample with an added reagent are prepared and are for testing placed in a testing apparatus. An electric field, preferably a low magnitude and a high magnitude electric field is applied over the test sample in the testing apparatus. After or during the application of the electric field, electrical and/or optical responses are measured and recorded individually or simultaneously. The responses are processed individually or simultaneously to extract or determine one or more sets of parameters that in different embodiments are used for detection, quantification and identification of coronavirus. In different embodiments, determining the presence, the identification and/or the quantity of coronavirus is based on the characteristics and/or on the differences in characteristics of said electrical responses and/or said optical properties responses to said first magnitude and to said second magnitude of the applied electric field.
For the purpose of embodiments employing measurement of electrical as well as optical response parameter values, embodiment apparatuses would be configured as a combination of an arrangement comprising features configured for applying an electric field and measuring electrical response parameters as outlined in the example in
Thus, in a use case example of operating this experimental setup, the electrical analyzer is used to apply the required electrical condition over the test sample and to collect the corresponding electrical responses. For example, positive and negative specimen samples from patients may be extracted from the upper respiratory tract by nasopharyngeal swabs suspended in viral transport media (VTM) to form positive and negative test samples. In order to confirm and calibrate the test results of embodiments, specimen have been confirmed clinically for their virus status using real-time RT PCR identification of SARS-CoV-2 RNA, following all relevant guidelines and regulations. The individual specimen are loaded as test samples into test sample containers, for example in the form of 0.4 cm-gap micropulser electroporation cuvettes from Bio-Rad. This exemplifying test sample container cuvette incorporates aluminum electrodes plates with an area of 1 cm by 0.8 cm. Its outer dimensions are 12.5×12.5×45 mm with a path length of 10 mm and a functional volume between 50-1,500 μl. With a test sample container comprising electrode plates, the electrical analyzer may be coupled via cables 104, 105 directly to the test sample container or via electrodes 106,108 in contact with such electrode plates or positioned in close proximity to the test sample container.
In a general embodiment of detecting and quantifying coronavirus in a specimen from a patient, the specimen is accommodated in a suspension medium, such as the mentioned viral transport medium (VTM) or other suitable suspension liquid medium. Exemplifying embodiments show nasal swabs being used in the procedure of obtaining a specimen and preparing a test sample. However, the system and methods are not limited to that and in different embodiments any specimen taken from living beings such as humans or animals, or non-living entities such as object surfaces, wastewater, liquid substances or food.
The suspended specimen is apportioned into one or more test samples and a reagent, for example in the form of an antibody, is added to the one or more test samples. An electric field with a first magnitude and with a second magnitude is applied over said one or more test samples for a selected period of time, where said second magnitude is higher than said first magnitude. One or more electrical properties of said one or more test samples in response to said applied electric field are measured for said first magnitude and for said second magnitude over said period of time. Characteristics of said electrical properties responses are identified. The presence, the quantity and/or the identification of coronavirus is determined dependent on characteristics or on differences in characteristics of said electrical properties responses to said first magnitude and to said second magnitude of the applied electric field.
The reagent is in different embodiments ACE2 antibodies, NC antibodies, other suitable antibodies or a fluorescence-based reagent. In other embodiments, the reagent may be a protein devised for testing on protein-protein binding or other reaction, an antibody or an antigen devised for testing on antibody-antigen binding or reaction with embodiments of the method and system adapted to that. The reagent may in different embodiments be in the form of a single type or a mix of a plurality of types, for example a single type antibody or a mix of different type antibodies. For analytes in the form of virus in general, the reagent is for example the specific antibody to bind with the specific antigen and/or the specific nucleocapsid antibody to bind with the nucleocapsid protein specific antigen. In embodiments, testing on a test sample with pure specimen, i.e., having no reagent added, is comprised either as a stand-alone test or in combination with the testing of one or more test samples having a selected reagent added.
In order to apply the electrical field over the one or more test samples and to measure the electrical properties responses to the applied electrical field of the test samples, the one or more test samples in their test sample containers are placed in or on a test sample holder arranged to house the test sample containers in a position in relation to a selection of electrodes. Such an arrangement comprises electrodes that are configured to apply an electric field over the test samples as well as probe electrodes configured to conduct measurements of one or more electrical properties of the test sample. In different embodiments the electrodes have different configurations. For example, the electrodes for applying an electric field and the probe electrodes may be the same set of electrodes coupled in different circuits. In other examples, the electric field application electrodes and the probe electrodes may be arranged in different sets of electrodes. Some embodiments of test sample holders are adapted to house and test one test sample at a time. Other embodiments of test sample holders are adapted to house and test a plurality of test samples in parallel or serially, for example two, three or more test samples at a time. The test sample holder and electrode arrangement may in embodiments for handling a plurality of test sample containers be configured for applying an electric field and measure electrical and/or optical properties per individual test sample container, in parallel or consecutively in series. The test sample holder may in different embodiments for example be configured as a chamber, an adaptor, a container or a plate that holds the specimen in its test sample container in a defined position in a testing apparatus.
The magnitudes of applied electric field is selected dependent inter alia on the dimension and the materials of the test sample container holding a specimen and of the test sample holder of the testing apparatus. In embodiments the magnitude of the applied electric field may for example be caused by applying a voltage between 0 and about 3 volts over the test sample. In different embodiments the applied electric field may be caused by an applied voltage in a constant or pulsated form, DC or AC voltage. The applied electric field and/or the applied voltage inducing the electric field may range from negative to positive and may in embodiments be swept over negative to positive values as well as zeroes. Different embodiments comprise applying one, two or a further plurality of kinds of applied electric fields, i.e., fields with different magnitudes or caused by different magnitudes of applied voltage, such as low and high. Increasing the applied voltage over about 3 volts will result in essentially burning the suspension medium and destroy it with its content of cells with analytes such as coronavirus, virus of any kind, bacteria, fungus, protein or other analyte and/or reagents. Thus, an applied bias voltage should result in induction of a current across the suspension and if this current is high enough, it should have the potential to destroy the protein physiology and functionality, resulting in the loss of specific protein-protein interactions. So for example, with the analyte coronavirus it has been found that one volt bias, i.e., voltage applied over a test sample is preferable and an optimum value that promotes the entry of the anti-N antibody into cells without changing the configuration and physiology of the viral nucleocapsid protein, the cells, buffer, antibodies or other reagents. The applied voltage may in embodiments be in the range of −3 to 3 volts, for example for coronavirus or other analytes.
The applied electric field may be generated by a DC or AC voltage in any general form, for example as a pulsed electric field. The electric field may in further variants be applied as a plane wave.
The application of a high magnitude electric field induced by a high electric voltage applied over the test samples cause reagents to enter and/or interact, for example in binding events, with a coronavirus virion, which in accordance with embodiments are detected by measuring electrical properties responses and/or optical properties responses of the test samples. In further embodiments, the transport of reagents such as antibodies to the inside the cell or the transport of cell content to the outside of its membrane is carried out also through other methods of cell transport such as for example “Diffusion”. This can also be carried out by scratching the cells using the nano or micro-pored electrodes. In such embodiments the cell suspension can be inserted inside sandwiched flexible nanopore electrode-based system, then by exerting force across these two electrodes bringing them close to each other. The cell contents will in this procedure be transported out on the electrode surface and reagents such as antibodies can then be added to this content. These electrodes can be used to collect electrical responses before and after the addition of antibodies of the scratched cell suspensions. In this scenario, there is no need to apply a high magnitude electric field over the test sample.
The electrical properties of the test samples that are measured are different in different embodiments. For example, in embodiments the impedance with its magnitude and phase is measured and recorded over a selected period of time. For this purpose, the testing apparatus comprises an electrical analyzer or other measuring equipment configured to measure selected electrical properties of test samples placed in the testing apparatus. The measurements are in different embodiments, where a plurality of test samples are processed, performed in parallel or in sequence for two or more than two different electric fields. In different embodiments, measurement can be performed on a test sample before a reagent is added, i.e., on a pure specimen test sample, and after the reagent is added. The measurement may in embodiments be conducted for a non-zero applied electric field at different time steps, with such an applied electric field being generated by a DC or AC voltage in any general form, for example as pulsed electric field.
As mentioned, different sets of electrical measurements are conducted in different embodiments to extract parameters representing the electrical properties responses to applied electric field. For example, impedance related parameters with magnitude and phase versus an entity representing the applied electric field, and/or capacitances versus voltages applied over the test sample at operating frequency which can be ranged from 0 to infinity.
In further embodiments, responses of optical properties of the test sample to an applied electric field are measured and recorded, as an alternative to or in combination with measurement of electrical properties. In such embodiments, light passing through a test sample is detected by a suitable optical detector such as described in embodiments in the present disclosure. Based on the refractive index and dielectric constant relationship, and as the capacitance changes with the applied electric field, the optical responses will also change with the applied electric field. The optical properties response profile will also change due to the interaction/binding events due to protein-protein or antibody-antigen reactions occurring in a test sample. This is reflected in for example measured light intensities of one or more of transmitted light, absorbance, scattered light and/or reflected light, as described in embodiments disclosed herein. In embodiments, optical properties response parameters are correlated with electrical properties response parameters in order to further enhance the detection, identification and quantification of coronavirus. Smart chart approaches or relationships may be used to conduct or generate such correlation.
Thus, in embodiments, there are combinations of measurements of optical and electrical properties responses to one or more applied electric fields. In different measurement and/or test sample scenarios the optical and electrical response parameters are measured for single or multiple applied electrical fields at single or multiple operational frequencies, in testing schemes with parallel or sequential testing of different test samples.
The extracted or measured parameters representing the electrical properties or optical properties responses are in embodiments further processed to extract one or more further sets of parameters that are used to determine detection, identification and quantification of coronavirus. In embodiments, a list of reference parameters, also called reference coefficients, is defined or determined from corresponding measurements of test samples with specimen alone or test samples with specimen and added reagent, such as antibody or protein. Measured parameters representing a response after a selected time period, that for example may be sub seconds to sub days long, are then compared with the determined reference parameters. The measured parameters are in embodiments correlated with the reference parameters, for example by means of calibration curves or mathematical modelling, to determine the presence, the identification and/or the quantity of the analyte, for example coronavirus, dependent on differences in parameters from responses to different electric fields and/or different test samples. In further embodiments, these parameters are used to determine the infection stage in an infected human or animal patient.
The selected period of time may, as in different embodiments, be selected to last for intervals ranging from sub seconds to hours, or sub days or days.
Test scenarios are varied in different embodiments. For example, as applied on the analyte coronavirus, single test samples or sets of test samples comprising a test sample:
A more specific exemplifying embodiment comprises the employment of three test samples, here exemplified with the analyte coronavirus. The first test sample is purely the specimen. Reagents in the form of ACE-2 and NC antibodies are added to the second and third test samples respectively. The samples are placed in a test sample holder of a testing apparatus and the electrical properties of the test samples are measured. First electrical properties relating to impedance, for example magnitude and phase, of each of the test samples are measured and recorded under the application of 0 volts over the test samples, resulting in a low or a non-existent first magnitude electric field for a selected period of time, for example 400 S (seconds). Then the same electrical properties of each of the test samples are measured and recorded under the application of 1 volt, resulting in a high second magnitude electric field for the same period of time.
The presence, quantity and/or the identification of the analyte, here exemplified with coronavirus, in the test samples are deducted from the characteristics of the measured and recorded electrical responses to the different applied electric fields. So, for example, negative samples show a generally flat rising profile of electrical characteristics response, whereas positive samples exhibit non-linearities. The degree of nonlinearity and the differences between zero or low voltage profiles and high voltage profiles can then be correlated with a measure for the concentration of the virus. The quantification of the virus is in embodiments determined based on the concentration of the virus, or as in other embodiments based on a correlation of the virus quantity with the degree of nonlinearity and the differences between zero or low voltage profiles and high voltage profiles. Similarly, identification of virus present in the test samples are in embodiments based on parameters correlated to for example the characteristics of electrical properties responses.
On a more general level, a detection decision, i.e., determining whether there is an analyte, for example virus, or not present in a test sample is in embodiments carried out by comparing the discrepancy between the measured responses of one or more of electrical and/or optical parameters at different applied electric fields. This discrepancy can be measured in terms of nonlinearity or differences in measured parameters values. In embodiments algorithms are implemented in accordance with such methods to make the detection decision automatically using software or developed programs possibly involving artificial intelligence. The nonlinearity can be expressed in terms of extracted single or multi parameters and their response profiles over time variations be observed manually or automatically.
Determining the quantification is in embodiments carried out with the help of calibration curves that for example are generated with known concentration of analyte or analyte load, for example virus. In other embodiments, quantification is determined from the differences of capacitance versus voltage profiles by subtracting the capacitance per voltage values at an applied low magnitude electric field from the capacitance per voltage values at an applied high magnitude electric field, and as in embodiments processing this difference signal using semiconductor theory to extract single or multi parameters to determine the analyte load, for example virus load, and a quantification of the analyte, for example virus, in a test sample.
Determining identification of analytes, for example coronavirus, is in embodiments made based on single or multi figure of merits extracted from the single or multi parameters used in quantifications.
An embodiment of a more specific example of a method of detecting and quantifying coronavirus in a specimen from a patient, wherein the specimen being accommodated in a suspension medium, comprises: apportioning the suspended specimen into a first, a second and a third test sample; arranging said first test sample to contain purely the suspended specimen; adding ACE2 antibodies to said second test sample; adding NC antibodies to said third test sample; applying an electric field with a first magnitude and with a second magnitude over said first, second and third test samples for a selected period of time, said second magnitude being higher than said first magnitude; measuring one or more electrical properties of said test samples in response to said applied electric field for said first magnitude and for said second magnitude over said period of time; identifying characteristics of said electrical properties responses; determining the presence, the quantity and/or the identification of coronavirus dependent on differences in characteristics of said electrical properties responses to said first magnitude and to said second magnitude of the applied electric field.
A first exemplifying embodiment procedure comprises:
1. Loading a specimen of a COVID sample as a test sample into a test sample container and placing the test sample container in the test sample holder of a testing apparatus.
2. Applying a first magnitude electric field induced by applying a first voltage over the test sample container for a selected period of time, the first magnitude electric field preferably being a low magnitude electric field induced by a low voltage for example in the range of −0.5 to 0.5 volts. A first low magnitude electric field induced by a voltage of 0 volts, i.e., no actual electric field applied over the test sample is employed in exemplifying embodiments.
3. Measuring electrical properties responses in the form of Capacitance per applied low voltage at a frequency (f0).
4. Applying a second magnitude electric field induced by applying a second voltage over the test sample container for a selected time period, the second magnitude electric field preferably being a high magnitude electric field being induced by a high voltage for example ranging from 0.5 to 3 volts.
5. Re-measuring the electrical properties responses in the form of Capacitance per applied voltage at the same frequency (f0).
6. Optionally measuring optical properties responses in the form of light intensities for the test sample simultaneously with the measuring of the electrical properties responses.
7. Determining, based on the electrical properties response parameters and/or optionally also based on the optical properties response parameters, detection, quantification and/or identification of coronavirus in the test sample.
A second exemplifying embodiment procedure devised for parallel processing of two samples comprises:
1. Loading a specimen of a COVID sample as a first test sample into a first test sample container (container 1) and placing the first test sample container in the test sample holder of a testing apparatus being configured for testing a plurality of test sample containers.
2. Loading a specimen of the COVID sample as a second test sample into a second test sample container (container 2) and placing the second test sample container in the test sample holder of said testing apparatus.
3. Applying a first magnitude electric field induced by applying a first voltage over the first test sample container (container 1) for a selected period of time, the first magnitude electric field preferably being a low magnitude electric field induced by a low voltage for example in the range of −0.5 to 0.5 volts. A first low magnitude electric field induced by a voltage of 0 volts, i.e., no actual electric field applied over the test sample is employed in exemplifying embodiments.
4. Measuring electrical properties responses of the first test sample container (container 1) in the form of Capacitance per applied low voltage at a frequency (f0).
5. Applying a second magnitude electric field induced by applying a second voltage over the second test sample (container 2) for a selected time period, the second magnitude electric field preferably being a high magnitude electric field being induced by a high voltage for example ranging from 0.5 to 3 volts.
6. Measuring the electrical properties responses of the second test sample container (container 2) in the form of Capacitance per applied voltage at the same frequency (f0).
7. Optionally measuring optical properties responses of the first test sample (container 1) in the form of light intensities simultaneously with the measuring of the electrical properties responses of said first test sample (container 1).
8. Optionally measuring optical properties responses of the second test sample (container 2) in the form of light intensities simultaneously with the measuring of the electrical properties responses of said second test sample (container 2).
7. Determining, based on the electrical properties response parameters and/or optionally also based on the optical properties response parameters, detection, quantification and/or identification of coronavirus in the first test sample (container 1) and the second test sample (container 2).
A third exemplifying embodiment procedure devised for parallel processing of a plurality of samples simultaneously for the purpose of reducing testing time comprises:
1. Loading a specimen of a COVID sample as a first test sample into a first test sample container (container 1) and placing the first test sample container in the test sample holder of a testing apparatus being configured for testing a plurality of test sample containers.
2. Loading a specimen of the COVID sample as a second test sample into a second test sample container (container 2) and placing the second test sample container in the test sample holder of said testing apparatus.
3. Loading a specimen of the COVID sample with an added reagent for example in the form of antibodies, that may be a single type antibodies or a mix of antibodies as a third test sample into a third test sample container (container 3) and placing the third test sample container in the test sample holder of said testing apparatus.
4. Loading a specimen of the COVID sample with an added reagent for example in the form of antibodies, that may be a single type antibodies or a mix of antibodies as a fourth test sample into a fourth test sample container (container 4) and placing the fourth test sample container in the test sample holder of said testing apparatus.
5. Applying a first magnitude electric field induced by applying a first voltage over the first test sample container (container 1) for a selected period of time, the first magnitude electric field preferably being a low magnitude electric field induced by a low voltage for example in the range of −0.5 to 0.5 volts. A first low magnitude electric field induced by a voltage of 0 volts, i.e., no actual electric field applied over the test sample is employed in exemplifying embodiments.
6. Measuring electrical properties responses of the first test sample container (container 1) in the form of Capacitance per applied low voltage at a frequency (f0).
7. Applying a second magnitude electric field induced by applying a second voltage over the second test sample (container 2) for a selected time period, the second magnitude electric field preferably being a high magnitude electric field being induced by a high voltage for example ranging from 0.5 to 3 volts.
8. Measuring the electrical properties responses of the second test sample container (container 2) in the form of Capacitance per applied voltage at the same frequency (f0).
9. Applying a first magnitude electric field induced by applying a first voltage over the third test sample container (container 3) for a selected period of time, preferably a low voltage in similarity to point 5.
10. Measuring electrical properties responses of the third test sample container (container 3) in the form of Capacitance per applied low voltage at a frequency (f0).
11. Applying a second magnitude electric field induced by applying a second voltage over the fourth test sample (container 4) for a selected time period, preferably a high voltage in similarity to point 7.
12. Measuring electrical properties responses of the fourth test sample container (container 4) in the form of Capacitance per applied high voltage at a frequency (f0).
13. Optionally measuring optical properties responses of the first test sample (container 1) in the form of light intensities simultaneously with the measuring of the electrical properties responses of said first test sample (container 1).
14. Optionally measuring optical properties responses of the second test sample (container 2) in the form of light intensities simultaneously with the measuring of the electrical properties responses of said second test sample (container 2).
15. Optionally measuring optical properties responses of the third test sample (container 3) in the form of light intensities simultaneously with the measuring of the electrical properties responses of said third test sample (container 3).
16. Optionally measuring optical properties responses of the fourth test sample (container 4) in the form of light intensities simultaneously with the measuring of the electrical properties responses of said fourth test sample (container 4).
17. Determining, based on the electrical properties response parameters and/or optionally also based on the optical properties response parameters, detection, quantification and/or identification of coronavirus in the first test sample (container 1) and the second test sample (container 2).
In a fourth exemplifying embodiment procedure the example procedure of the second exemplifying embodiments as described above is generalized to a plurality of 2 to N test samples in 2 to test sample containers, where N is an integer 3 and upwards.
Examples of electrical properties response characteristics of embodiments are shown in
In a first set of examples,
The diagram of
The diagram of
Comparing the plotted capacitance responses for the negative test samples in
In another set of examples,
The diagram of
The diagram of
Comparing the plotted capacitance responses for the negative test samples in
In yet another set of examples,
Further examples of response profiles in embodiments comprising measurement of optical response characteristics are illustrated in
In
Similarly, in
In an embodiment of a measurement approach, as e.g., illustrated in
In the example of normalized curves of light intensities shown in the diagram of
To illustrate an analysis of the behavior of the samples further, the slopes of the corresponding sample normalized profiles were extracted and have been summarized in Table 1 from ten negative COVID-19(−) and ten positive COVID-19(+) individuals. The table shows the rate of change in the normalized measured light intensity of nasal swab samples from ten negative COVID-19 (−) and ten positive COVID (+) samples.
In
Embodiments configured for higher accuracy detection, comprises applying the following Figure of Merit (FOM) in accordance with the relationship:
where SA and S are the samples with reagent, e.g., antibody and the same sample alone, i.e., specimen without any added reagent, respectively.
The FOM represents the relative variation of the sample with and without antibody at two different voltages. The corresponding extracted FOM is listed in the last column of Table 1. As illustrated in
The optical response profiles of the respective test samples have their individual characteristics, and in embodiments such optical response profiles are further processed to be used in determining the detection, quantification and identification of coronavirus.
In a further example, with AC voltage applied over test samples, capacitance versus time responses appear as illustrated in
In a measurement series in one embodiment, a testing apparatus as described above was used first to measure the viral transport medium (VTM) with and without the anti-N antibodies. The corresponding measured electrical current versus time as well as capacitance versus time with the application of one volt electrical bias over the test sample is shown in
As illustrated in
For further understanding this, the corresponding electrical capacitance profiles have been measured as illustrated in
In a measurement series in another embodiment, similarly a testing apparatus as described above was used to measure negative and positive test samples with and without the anti-N antibodies. The measured capacitance versus time responses as well as detected binding events for negative and positive test samples are shown in
Turning to the measured capacitances profiles displayed in
Specimen taken with nasal swabs from individuals that were known to be free from rhinovirus infection were used as negative controls.
As illustrated in
The response profile of the positive test sample with added antibodies, Positive+ Anti-N Ab also exhibits stable and smooth behavior however with distinctly different characteristics. This response profile can be split into three regions: the first region extending from 0 to 165 seconds, the next region extending from 165 to 290 seconds and the third region extending from 290 to the end of the depicted time interval, here 350 seconds. The first and third regions both exhibit linear behavior. The middle region exhibits nonlinear behavior and is marked by the binding region as illustrated in
The binding event profile can be extracted by detrending the positive profiles and consider its first region as its baseline. The binding event profile is depicted in
As explained above, under electrical bias i.e., with an electric field induced by an electric voltage over the cell, the cell opens up by the formation of pores in the cell membrane. The pores only stay open for the duration of the applied constant field or its time domain variation. The anti-N antibodies penetrate the cell through these pores and binds with the N protein of SARS-CoV-2 virus that is expressed during infection. The application of electric fields will modify the orientation of molecules and alter the ion transport rates, they can modify the quantum states of molecules, which lead to shifts in the energy levels. The infected cell physiology with and without the application of electric field is illustrated in
As revealed from the corresponding binding measured light intensity profiles, such interactions exhibit Gaussian-like peaks. In embodiments, Brownian motion is fitted with Gaussian function. The fitting parameters of the distributions provide a plurality of features of the binding interactions. In embodiments, this is used to provide a quantitative signature or characterization of a specific antigen binding to a specific antibody such as intrinsic specificity and binding rate.
On a general level, embodiments comprise a system and a method to detect and possibly monitor the possible binding or interactions between proteins and proteins, proteins and antibody, viruses and proteins, viruses and antibodies, cells and proteins, cells and antibodies. Also, virus-cell, or cell-cell interactions, or any interactions between any part of protein, antibody, virus, cell with any part of protein, antibody, virus, cell of the same or different kind.
Embodiments comprises a method of opto-electrical detection of the presence of possible bindings or interactions between analytes in a sample, comprising: exposing a sample to light from a light source; detecting light passing through the sample; applying an electrical field over the sample; determining the values of a selection of light scattering parameters for the light passing through the sample in response to the electrical field; and/or
Further embodiments comprises a system of opto-electrical detection of the presence of possible bindings or interactions between analytes in a sample, comprising: a light source configured to emit or transfer natural or manmade light and to expose a sample with said light; an electric field device configured to apply a biasing electric field over the sample; a light detector configured to detect light passing through a said light exposed sample; an electric parameter detector configured to detect electric parameters. Such embodiments further comprise a processing device having code portions configured direct the processor to: determine the values of a selection of one or more light scattering parameters of the detected light passing through a said light exposed sample; determine the values of a selection of electrical parameters, for example electrical scattering parameters, in response to the electrical field, and to determine the presence of bindings or interactions between analytes in the sample based on the values of the determined light scattering parameters and the determined values of the electrical parameters, for example electrical scattering parameters.
An underlying mechanism for the concept of embodiments is that when two proteins bind or a molecular interaction takes place between them, due to this interaction, a possible form of kinetic energy is produced, perhaps emitted as a cloud. This kinetic energy can cause disturbances, such as altering the Brownian motion of the molecules which can change or alter light distribution on the surface of the sample or inside the sample. The generation of a cloud-like response due to the biochemical interaction should also disturb the light intensity distribution and accordingly the measured light intensities.
Further, the linear electro-optic effect is the change in the index of refraction that is proportional to the magnitude of an externally applied electric field. As mentioned herein. the electro-optic effect may also be non-linear, for example over a wider range. Hence, the measured light intensity varies with DC electric voltage. The DC bias will also change the current and capacitance as they depend on the applied voltage. This relationship/ dependency may for example be converted to a chart that could be used for detection enhancement and further identification of the sample under test. For a specific virus type, the corresponding capacitance and light intensity at zero bias could also be used for further detection purposes.
In method embodiments, the presence of bindings or interactions between analytes in the sample is determined is determined based on a determined characteristic of the detected light scattering parameters, and/or electrical scattering parameters for specific values of the electric parameters. In system embodiments, the processing device comprises code portions configured to determine the presence of bindings or interactions in the sample based on determined characteristic of the detected light scattering parameters, and/or electrical scattering parameters, for specific values of the electric parameters.
Method embodiments comprises determining the presence of bindings or interactions between analytes in the sample by comparing the discrepancy between the measured responses at different applied electrical fields. In system embodiments, for this purpose the processing device comprises code portions configured to determine the presence of bindings or interactions between analytes in the sample by comparing the discrepancy between the measured responses at different applied electrical fields. Further method and system embodiments are configured to determine the presence of bindings or interactions between analytes in the sample by comparing the discrepancy between the measured responses at different applied electrical fields; and/or wherein the measured light intensity varies with DC electric voltage and/or values for the capacitance, for example, can be extracted from scattering parameters or impedance measurements or from determined relations between the parameters; and/or to determine information about the viral load and/or identifying the viral stage infection based on the voltage dependency. In embodiments of system, the processing device comprises code portions configured account for these possible features.
The emitted or transferred, manmade or natural, light from the light source may comprise the whole spectrum of wavelengths.
In such method and system embodiments the light detector is one or more of: an image-based system such as a camera, a spectrometer or any light-based detector, sensor or device that is capable to detect light intensity dependent or derivable parameter values. The method and system are in embodiments configured to detect light intensity and to determine light scattering parameters based on one or more of absorbance, reflectance, transmittance or any other type of light scattering, corresponding intensity or other parameter that is extractable or collectable by means of a light detector.
In method and system embodiments a selection of one or more light scattering parameters, for example intensities, are measured and possibly recorded over a time-period and/or mapped to a time domain. In system embodiments the processing device comprises code portions configured to measure and possibly record a selection of one or more light scattering parameters over a time-period and/or to map said parameters to a time domain.
In method and system embodiments the sample is loaded into a transparent holder or container allowing light to pass through the material without appreciable scattering of light and being configured for holding a sample, for example on or more of a plate, box, tube or any type of transparent paper or other transparent material. For this purpose, system embodiments, may further comprise a transparent holder or container allowing light to pass through the material without appreciable scattering of light and being configured for holding a sample, for example on or more of a plate, box, tube or any type of transparent paper or other transparent material.
A sample may, as in method embodiments, be tested in place in close connection with or in the proximity of the taking of the sample or be stored and/or transported for testing spatially and/or temporally remote from the taking of the sample. For this purpose, in system embodiments, the transparent holder or container is configured to store and/or transport a sample.
In method and system embodiments, the light source, a sample and the light detector are aligned such that measurement of light passing through the sample is enabled, for example by alignment in a straight line or other possible such alignment.
The electric field device comprises, in method and system embodiments, two electrodes configured or configurable at respective sides of a sample and being coupled or couplable to an electric energy source.
In method and system embodiments, the sample is added to a holder or container aligned with the light source and the light detector such that measurement of light passing through the sample is enabled; and measured responses in the form of the determined values of said selection of optical or electrical parameters, for example electrical scattering parameters, in response to the electrical field are collected to determine the presence of bindings or interactions between analytes in the sample. In system embodiments, the processing device comprises code portions configured to collect measured responses in the form of the determined values of said selection of optical or electrical parameters, for example electrical scattering parameters, in response to the electrical field for a sample that is added to a holder or container aligned with the light source and the light detector such that measurement of light passing through the sample is enabled.
Such method embodiments comprises steps, and in such system embodiments the processing device comprises code portions, configured to collect measured responses comprising one or more of: collecting directly measured light intensity; and/or collecting directly measured light intensity and establishing direct relationship with time; and/or processing direct measured data to extract parameters or a set of parameters and correlating said parameters with time to establish relationship with the presence of bindings or interactions between analytes in the sample.
Method and system embodiments further comprise determining the type of binding and/or interaction between analytes in the sample based on a relationship between the values of determined light scattering parameters and/or of determined electrical scattering parameters, and time, for example by direct judgement or by further processing. For this purpose, in system embodiments the processing device comprises code portions configured to determine the type of binding and/or interaction between analytes in the sample based on a relationship between the values of the determined light scattering parameters and time, and/or of the electrical scattering parameters and time.
In method and system embodiments, a mathematical relation for the determined parameters represents the process of bindings or interactions between analytes in the sample. For this purpose, in system embodiments, the processing device comprises code portions configured to apply a mathematical relation for the determined parameters to represent the process of bindings or interactions between analytes in the sample.
In examples of such method and system embodiments, with a mathematical relation between determined light scattering parameters, and/or electrical scattering parameters, and time for measured responses at selected applied electrical fields, binding and/or interaction between analytes is determined to be present if the relations shows nonlinearity and not to be present if the relation shows constant behaviour. In system embodiments, for this purpose, the processing device comprises code portions configured to, for a mathematical relation between determined light scattering parameters, and/or electrical scattering parameters, and time for measured responses at selected applied electrical fields, determine that binding and/or interaction between analytes is present if the relations show non nonlinearity and not present if the relation shows constant behaviour. In case of linear relationship that can be represented with a slope, the two kind of elements or more than two elements under investigation, their individual intensities should be measured and considered accordingly, in method and corresponding system embodiments. Further, in such embodiments the non-linearity may be expressed in terms of extracted single or multi-parameters, the non-linearity may be observed manually or automatically and the variation of response profiles of the one or more parameters may be detected over time. In corresponding system embodiments, the processing device comprises code portions configured to express the non-linearity in terms of extracted single or multi-parameters, to enable the non-linearity to be observed manually or automatically and to detect the variation of response profiles of the one or more parameters over time. Further method embodiments and corresponding system embodiments comprises or are configured to determining a mathematical relation for the light scattering parameters, and/or electrical scattering parameters, and values of the electric parameters to represent the presence and/or the process of one or more of said bindings or interactions between analytes in the sample based on the value of light scattering parameters for the detected light passing through the sample.
Method and system embodiments further comprise the use of and configuration with a chronoamperometry or a chronopotentiometry measurement method used to detect and/or monitor the presence and/or the process of one or more of said bindings or interactions.
In method and system embodiments the measured value, for example either current or voltage over time, can be used to explore the interaction. When an interaction occurs, the measured profile over time will increase or decrease in trend, depending upon the nature of the test samples, resulting in either an exponential growth or a decay behaviour. Furthermore, current voltage or capacitance voltage, impedance, or any other possible electrical-based measurements can also be considered. In method embodiments, the presence and/or the process of one or more of said bindings or interactions is determined, detected and/or monitored based on measurements of an electrical parameter, for example an electrical scattering parameter, for example a parameter based on one or more of the voltage, the current, the capacitance and/or the impedance over time in relation to a said applied electric field. For this purpose, in system embodiments, the processing device comprises code portions configured to determine, detect and/or monitor the presence and/or the process of one or more of said bindings or interactions based on measurements of an electrical parameter, for example one or more of the voltage, the current, the capacitance and/or the impedance over time in relation to a said applied electric field.
In further embodiments of method and system, low and high frequency scattering parameters or propagating waves, or any other possible form of measurements can also be considered. For this purpose, method and system embodiments, further comprise or are configured to measure and process parameter values based on low and high frequency scattering parameters or propagating waves.
In method embodiments, and in corresponding system embodiments comprising configured code portions, a characteristic of the determined light scattering parameters, and/or electrical scattering parameters, for specific values of the electric parameters is determined to represent the response of the determined light scattering parameters and/or electrical scattering parameters, due to the possible binding or interaction between one or more analytes in a sample, such as between proteins and proteins, proteins and antibody, viruses and proteins, viruses and antibodies, cells and proteins, cells and antibodies as well as virus-cell, or cell-cell interactions, or any interactions between any part of protein, antibody, virus, cell with any part of protein, antibody, virus, cell of the same or different kind.
Method and system embodiments are configured and/or used to monitor a vaccine or drug development process.
Method and system embodiments described herein may be configured for virus detection. A method embodiment is for this purpose used for virus detection and comprises collecting and processing electrical and optical responses individually or simultaneously to extract a set of parameters for detection, quantification and identification of virus. In a system embodiment for this purpose, the processing device comprises code portions configured to collecting and processing electrical and optical responses individually or simultaneously to extract a set of parameters for detection, quantification and identification of virus. In variants, such method and system embodiments comprise, or are configured to enable that, a virus cell is contacted by a selected antibody in a sample. Variants of such method and system embodiments are further adapted and used for detection of one or more of virus from the group SARS-CoV-2, SARS, MERS, influenza, respiratory syncytial virus (RSV), adenoviruses, or any other respiratory virus.
Further embodiments of the method and the system adapted for virus detection, further comprises or are configured to applying an electrical pulse over or through a sample containing analytes from a test subject and selected antibodies, thereby enabling electro-insertion of said antibodies into any virus cell present in the sample.
In method and system embodiments adapted for virus detection, a virus cell in a sample is suspended in an aqueous transport medium of a nasopharyngeal swab along with anti-N antibodies (anti-N).
In further method and system embodiment adapted for virus detection, the method comprises steps, and the system the processing device comprises code portions, configured to determine a characteristic of the light scattering parameters, and/or electrical scattering parameters, for specific values of the electric parameters and to measure the viral nucleocapsid protein and anti-N antibody interactions in a sample to differentiate between SARS-CoV-2 negative and positive nasal swab samples.
Embodiments of the method and the system adapted for virus detection, further comprises a sample strip configured for collecting a sample of analytes from a test object, the sample strip comprising a portion coated with antibodies. In variants, the sample strip is coated with an antibody configured to bind or interact with of one or more of virus from the group SARS-CoV-2, SARS, MERS, influenza, respiratory syncytial virus (RSV), adenoviruses, or other respiratory virus.
In further method and system embodiments adapted for virus detection, the sample strip comprises an electrode coated thereon and configured to conduct current measurements directly.
Other method and system embodiments for opto-electric virus detection, further comprises a device for nasal sampling configured such that a sampling strip is attachable at a section of the nasal sampling device that is configured to be insertable in the nose of a human test object, the nasal sampling device comprising two electrodes configured to apply an electric field over a sample of analytes collected on a said sampling strip. In such system embodiments the nasal sampling device may further comprise an integrated light source and detector configured to expose the sample to light and to detect light passing through the sample.
Such integrated method and system embodiments are, in variants, configured such that when the presence of a binding or interaction between a virus and an antibody is detected in a sample from a patient, an indication signal is presented confirming that patient is infected with the tested virus. The indication signal for infection may for example be in the form of light, sound, text or image presented via a corresponding presentation device.
In embodiments of method and system, a sample is taken from bodily tissue or fluid, for example blood, breath, urine, nasal swabs, stool, in the system embodiments with a strip configured for this purpose.
These method and system embodiments are further described and explained by illustrating examples below.
Embodiments described herein, comprises a light-based method to detect SARS-CoV-2 and potentially disrupt its binding ability with its receptor, rendering the virus non-infectious by combining optical detection with electric current. The measured intensity of light is used to determine information about different cellular parameters in a sample under study with method embodiments. Light scattering and electrical scattering is correlated with cell/virus size and reflect the complexity of their exterior or interior structures. When light passes through a cell, the intensity is associated with the DNA/RNA content of the cells. The nuclei size, cell shape and the refractive index variation of cells contribute to light intensity in the cell. It is worth adding that the measured optical spectrum of light passing through a sample consists of many features that in embodiments is used to reveal and determine important information about the sample under test. Usually when the cell/tissue changes from a normal state to infectious, metabolic alterations and genetic modifications occur. This causes dramatic changes in their physiological, biochemical and morphological characteristics. Indeed, the metabolic differences between infected and normal cells leads to several variations in cellular parameters, such as their size. In this text, embodiments with an optical label-free detection method incorporating a smartphone light source and a portable mini spectrometer for SARS-CoV-2 detection are described. The detection of parameters reflecting light interactions with control and viral protein solutions in accordance with embodiments enables a quick decision regarding whether a sample under test is positive or negative, thus enabling SARS-CoV-2 detection in a rapid and label-free manner.
An experimental setup utilized to illustrate an exemplary embodiment presented in
The graph in
An experimental setup in accordance with this embodiment may be used to characterize the two spike proteins subunits, S1 and S2 that are encoded by all coronaviruses and allow virus entry into susceptible cells, as illustrated in
The graphs in
Embodiments comprise calibrating a system for opto-electric virus detection in order to correct for errors that may occur when samples are loaded into the sample holder, and the angle and position of a container for a sample for example in the form of a microcentrifuge tube changes which may affect the results obtained. To ensure that the results are reproducible, the measurements for the same samples may, as in embodiments, be conducted over different days. In such embodiments the setup may be standardized for example on each day since the position of the mobile phone, spectrometer and/or samples may vary. To overcome this caveat and have more consistence measurements without constant standardization, advantage may be made of the ability of the spectrometer to provide light intensity measurements over time. Hence, after placing the microcentrifuge tube into the holder, the measurement mode may be started and the corresponding “blank” recorded. Then the sample would be added after for example ˜1.5 seconds, while keeping the measurement mode on.
In an example of conducting a test on the proof-of-principle of these embodiments for experimental design, initially a mixing experiment was conducted at a light wavelength of 623 nm. Towards this end, 250 μL of S1 protein solution was tested at the same maximum concentration at 5,000 copies/mL followed by addition of same amount of S2.
Table 2 below lists the extracted parameters at specific time points. The relative change in light intensity per light path length is a constructed parameter that should correlate with the loaded mass (concentration) of the protein in a suspension.
In equation (1): mi, mf and l are the initial mass of the buffer, the mass of the final suspension composite, and the light path length, respectively. α is the decay factor, unique for each control buffer. Its unit is in mm and could be correlated with the material absorptivity. ΔI is the relative changes in light intensity expressed as follows in equation (2):
In equation (2): I and Ib are the instantaneous measured light intensity of the suspension and the corresponding blank, respectively.
In method and system embodiments, relations for example those described herein, above and/or below, are applied to determine concentration of virus or virus load by serial dilution of antibody and simultaneous measurements using one or more microfluidic channels filled with a virus sample in solution. A microfluidic sensing device comprised in embodiments comprises one or more microfluidic channels. In the case with a plurality of microfluidic channels, the channels are in preferred embodiments arranged in parallel.
A light source may as in some embodiments integrated with the microfluidic sensing device, or the microfluidic sensing device may as in other embodiments be configured to receive light from an external light source. Both variant embodiments are configured such that light passes through the sample in each of the one or more microchannels, or groups of microchannels.
Embodiments of such microfluidic sensing devices further comprise an electric field device configured to apply an electric field over individual microchannels or groups of microchannels. Such an electrical field device may be integrated in the microstructure of the microfluidic device or be configured as a device external to the microstructure of the microfluidic device. The electrical field device may comprise electrodes and/or electrode connectors configured for individual channels, groups of channels or the whole microstructure.
Embodiments of such a microfluidic sensing device comprises a light sensor and/or an electrical sensor. Such sensors may be integrated in the said one or more channels or in the microstructure surrounding the channels, and said sensors are configured such that they can measure light and/or electrical parameters, e.g., scattering parameters, for each individual microchannel or groups of microchannels.
Mathematical models based on such said relations are used to extract a measurement value for the concentration of virus or virus load in the sample, thus reflecting the concentration of virus or virus load of test subject or patient from which the sample is taken. Quantification of the measurement value is for example carried out by means of calibration values e.g., represented in curves or tables. Such calibration values can for example be generated by calibrating parameter values determined in accordance with embodiments herein with known values for virus concentration or virus load. In embodiments, a quantification of concentration of virus concentration or virus load is determined from the differences of capacitance voltage profiles, and by processing a difference signal using semiconductor theory to extract single parameters or a collection of multiple parameters to determine said virus concentration or virus load.
Variants of these microfluidic channel embodiments, as with other embodiments herein, are configured to use for for the detection of for SARS-CoV-2 diagnosis, whereas other variants are configured, dependent on selection of the antibody used, for detection of any respiratory virus such as influenza, respiratory syncytial virus (RSV), adenoviruses, or other coronaviruses like SARS and MERS.
Method embodiments for these purposes are configured such that: a said sample is placed and distributed in one or more microfluidic channels; measurement of said light scattering parameters and/or said electrical parameters is conducted for the sample content in each of said microfluidic channels; and virus concentration and/or virus load is determined based on said measured parameters.
In further variants of such embodiments said sample is diluted such that there is stepwise increasing dilution of antibody content in said one or more microfluidic channels and said measurement of parameters is conducted for said dilution steps.
In method embodiments said sample is placed and distributed in a plurality of parallel microfluidic channels; the antibody content is serially diluted in said plurality of parallel channels, simultaneously measuring said parameters in said plurality of channels; and said virus concentration and/or virus load is determined based on said measured parameters.
The method of the preceding claim 30, wherein the virus concentration and/or virus load is based on a mathematical relationship between said parameters and virus concentration and/or virus load, said mathematical relationship for example being calibrated against known virus concentration or virus load.
System embodiments for the above purposes, comprise: a microfluidic sensing device with one or more one or more microfluidic channels configured for placing and distributing a said sample in one or more of said microfluidic channels; one or more sensors configured for measurement of said light scattering parameters and/or said electrical parameters for the sample content in each of said microfluidic channels; and code portions, in said processing device, configured to determine virus concentration and/or virus load based on said measured parameters.
Further variants of system embodiments are configured for dilution of said sample such that there is stepwise increasing dilution of antibody content in said one or more microfluidic channels and said measurement of parameters is conducted for said dilution steps.
In further system embodiments: said microfluidic sensing device is configured with a plurality of parallel microfluidic channels for placing and distributing said sample; said microfluidic sensing device is configured for serially diluting the antibody content in said plurality of parallel channels, simultaneously measuring said parameters in said plurality of channels; and said system is configured to determine said virus concentration and/or virus load based on said measured parameters.
In some such system embodiments, said code portions are configured such that the virus concentration and/or virus load is based on a mathematical relationship between said parameters and virus concentration and/or virus load, said mathematical relationship for example being calibrated against known virus concentration or virus load.
These embodiments with microfluidic channels may be applied in conjunction with other embodiments described herein or independently.
Above it is described by means of exemplifying embodiments how to detect proteins in solution using light. Further, as in embodiments described herein, light intensity is used to characterize the binding interactions of the spike protein with the viral receptor ACE2. As an exemplifying demonstration of this mechanism, two different variants of the S1 subunit of the spike protein, S1A(S1X) and S1B(S1Y), were tested. One of these variants could bind ACE2 with a much stronger affinity than the other one. The S1 subunit of the spike protein S1A and S1B were tested along with a non-specific control protein, bovine serum albumin (BSA) that should not bind to ACE2. These proteins were selected to demonstrate the detection of the binding process with ACE2 over time. Examples of measurement results are shown in
The measurement process in this example started with the blank, and after 200 seconds, 250 μL of ACE2 protein suspension was tested, as depicted in the graph of
The corresponding individual profiles of ACE2, S1A (S1X), S1B (S1Y) and BSA are depicted in
To explore the interaction and binding characteristics, by example between ACE2 and S1A (S1X) in more detail, the measurement time between the two proteins was extended over one hour the results of which are plotted in
In embodiments the following coefficients in equation (3) have the values: a, b, c, d and e are 20215.00393±6.54838, −0.15559±9.83118×10−4, 1.70666×10−5±5.093×10−8, −4.41996×10−10±1.09328×10−12 and 3.46019×10−15±8.30214×10−18, respectively. These coefficients can be used in embodiments to detect and identify the kind of analyte that binds with the ACE2 receptors. With other antigens and elements, the coefficients would have values characterised by the specific antigens or elements.
In further embodiments, the described optical system is used to detect protein-protein interactions. Using proteins that are well known to interact with each other in such measurements confirm the results described above. In an example, this can be done by testing the molecular interactions between an antigen and an antibody which is similar to the interaction between the spike protein and its receptor. In such an example, two proteins were tested along with their specific antibodies: the first protein was the receptor binding domain RBD of SARS-CoV-2 spike protein and its antibody and the other was the nucleocapsid protein NCP of SARS-CoV-2 and its antibody. Similar to the procedure described above, the two proteins were tested individually in an optical assay as described above followed by addition of their corresponding antibodies that were mixed and their interactions.
Embodiments makes use of the effect of direct current DC biasing on the ability of two proteins to bind specifically. An example to illustrate this was carried out by subjecting the nucleocapsid protein (NCP or NC protein) solution to DC voltage bias.
The breakdown field depends on the electrical characteristics of both the buffer and the analyte such as proteins, viruses, etc. In further exemplifying application of embodiments, the suspension of protein was subjected to 3 V for 1 minute and then the antibody to nucleocapsid NC was added to the NC protein solution. The corresponding measured response is shown in
Embodiments are configured for virus detection on samples on paperbased nitrocellulose membrane. The nitrocellulose membrane is a popular matrix that is frequently used due to its high protein-binding affinity with a pore size of 0.25-0.45 μm in paper-based diagnostics. Protein molecules usually bind to the nitrocellulose membranes through hydrophobic interactions. Due to the ease of their handling, cheap cost, and the presence of hydrophobic interactions between them and the suspended proteins, we tested whether the binding between the SARS-CoV-2 spike protein and antibody could be detected optically when both were added to each other on the nitrocellulose membrane. Using embodiments illustrated with the experimental setup described in
As these illustrating protein-antibody interactions took place within a time domain, the measurements are further illustrated using the well-known technique of chronoamperometry to detect this interaction electrically. Chronoamperome try is an electrochemical technique in which an electric potential is applied between two electrodes to measure the resulting current at the surface that is created from faradaic processes over time. The corresponding chronoamperometry measurements were conducted accordingly as plotted in
The applied potential in this example was of 0.5 mVolts, the minimum voltage that can be applied without causing any harm to the protein and antibody; therefore, it should not have affected the interaction process. As shown in
As described and illustrated in the above examples, different embodiments are configured as follows.
Specific examples of device and method have been described herein for purposes of illustration. These are only examples. The technology provided herein can be applied to device and method other than the examples described above. Many alterations, modifications, additions, omissions and permutations are possible within the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and/or acts with equivalent features, elements and/or acts; mixing and matching of features, elements and/or acts from different embodiments; combining features, elements and/or acts from embodiments as described herein with features, elements and/or acts of other technology; and/or omitting combining features, elements and/or acts from described embodiments.
This application is a non-provisional application claiming priority to U.S. Provisional Application No. 63/107,191 and filed on Oct. 29, 2020, the entire contents of which are incorporated herein by reference. This application is also a continuation-in-part and claims priority to U.S. Non-Provisional application Ser. No. 17/379,496 filed on Jul. 19, 2021, which claims priority to U.S. Provisional Application No. 63/054,022 filed on Jul. 20, 2020, the entire contents of both are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63107191 | Oct 2020 | US | |
| 63054022 | Jul 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17379496 | Jul 2021 | US |
| Child | 17514078 | US |
