Patent Application
20240277252
The outbreak of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in, as of Apr. 7, 2021, over 132 million cases worldwide including 1.96 million deaths. Given the long incubation period of COVID-19 (2-14 days) and its highly contagious nature, the early and large-scale screening of COVID-19 is of paramount importance to reduce viral transmission. Since the FDA EUA approval of real-time RT-PCR for molecular diagnosis of COVID-19, it has been utilized as the ‘gold standard’ for SARS-CoV-2 detection. However, RT-PCR suffers from long turnaround time due to complicated sample preparation and laboratory analysis which affects the diagnostic accuracy. In addition, the need for specialized instruments to perform the test and specialists to operate the equipment makes it un-reachable in resource-poor settings. Immunoassays to detect SARS-CoV-2 antigens are an alternative to PCR-based testing approaches, and they offer significant potential benefits. There are antigen tests available, but they often suffer from higher false positives and false negatives rates. Thus, the inaccurate tests are in fact detriment to disease spread and its management. Hence, there continues to be an urgent need for a rapid, cost-effective, point-of-care (POC) antigen detection tests that can directly detect SARS-CoV-2 immunogenic proteins with high sensitivity and specificity even before the onset of symptoms.
Nanomechanical sensors comprising an antibody-functionalized microcantilever and methods of using the same are described. The antibody-functionalized microcantilever may comprise a microcantilever, an antibody configured to bind a target antigen or an antigen configured to bind a target antibody, and a tether covalently tethering the antibody or the antigen to the microcantilever. The microcantilever has an effective surface density of tethered antibodies or antigens thereon, which allows for the detections of antigens or antibodies present in a sample. In some embodiments, the antibody-functionalized microcantilever comprises an antibody configured to bind a pathogenic antigen or an antibody indicative of an infection. The sensors described herein may be functionalized to detect a pulmonary or respiratory disease, such as SARS-CoV-2 infection. In some embodiments, the sensors comprise a field-effect transistor, such as a MOSFET, which allows for electrical detection of antibody-antigen binding.
Systems comprising the nanomechanical sensors are also described. The systems comprise a nanomechanical sensor and a detector for detecting a response of the nanomechanical sensor to the sample and generating a signal indicative of the response of the nanomechanical sensor to the sample. Suitably, the detector is configured to detect a response such as a bending moment. The systems described herein may comprise two or more different nanomechanical sensors, thereby allowing for multiplexed detection. The different sensors may be functionalized with different antibodies, different antigens, or both an antibody and antigen. In some embodiments, the system comprises a breath collector, thereby allowing for noninvasive sample collection.
Methods of detecting a target antigen or antibody in a sample are also provided. The method may comprise contacting a nanomechanical sensor with the sample and detecting for a response of the antibody-functionalized microcantilever to the sample. The methods described herein allow for rapid detection of the target. In some embodiments, the response to the target antigen or the target antibody is detected within 5 minutes of contacting the nanomechanical sensor with the sample. The methods also allow for sensitive detection. In some embodiments, the sample comprises less than 10 ng/ml of the target antigen or the target antibody.
Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.
Herein we report nanomechanical sensors, systems, and methods based on nanomechanical signal transduction of specific binding on top of microcantilevers that can readily and specifically detect targets in minutes. The Examples demonstrate rapid detection of SARS-CoV-2 nucleocapsid (N) and spike (S1) receptor binding domain (RBD) proteins of the virus at clinically relevant concentration by real-time monitoring of nanomechanical signaling induced by antibody-antigen binding. The Examples also demonstrate that results are available in less than 10 minutes between subject sampling and testing. Cross-corroborated with qRT-PCR, the extent of viral load in patient samples shows remarkable consistency with nanomechanical signaling. This allows for detection of weaker binding and lowered response, which may be commensurate with reduced binding interactions. Indeed, the Examples demonstrate weaker binding affinity of U.K. mutant variant against antibodies derived from the wild type antigens. Thus, integration and automation of microcantilever-based real-time diagnostic assay promises rapid, accurate and cost-effective detection of antigens, potentially mutant variants, and other specific-binding related phenomena.
Microcantilever-based sensor platforms offer low cost, high sensitivity, rapid response, and label-free detection system. The microcantilever sensor platforms work as a transducer for the translation of a biomolecular recognition phenomenon on its surface into a response signal. These microsensors may be integrated in an array inside a chip which makes them amenable for parallel detection of several analytes at a time.
One aspect of the invention provides for a nanomechanical sensor comprising an antibody-functionalized microcantilever. As used herein, “antibody-functionalized” means a microcantilever comprising a tethered antibody or tethered antigen where binding of an antigen in a sample to the tethered antibody or an antibody in a sample to the tethered antigen results in a detectable response. The Examples demonstrate the use of an antibody-functionalized microcantilever where an antibody is tethered to the microcantilever and a response is generated by binding of antigen in a sample to the tethered antibody, but the antigen and antibody may also be used.
The response may be a bending moment. A bending moment, means a reaction induced in the microcantilever when an external force or moment is applied to the microcantilever, causing the microcantilever to bend. In some instances, the response may be referred to as a deflection of the microcantilever.
The antibody-functionalized microcantilevers may be used to detect a target antigen or target antibody by detecting a response of the antibody-functionalized microcantilevers to a sample. Assaying samples for use in clinical and non-clinical settings is described below. It can be generally useful to quantitatively test a sample, such as a biofluid obtained from a subject. In some embodiments, the biofluid can include, without limitation, mucus, saliva, sputum, blood, serum, urine, cerebrospinal fluid, interstitial fluid, and other fluid samples. In some embodiments, the sample is obtained by nasopharyngeal swab. In another embodiment, the sample is obtained by an exhalation. The systems described herein generally operate by detecting the presence or absence of a target antigen or antibody. This may be accomplished by binding the target antigen or the antibody to the antibody-functionalized microcantilever such that a response can be detected with a detector.
Viral and antigen loads in breath tend to be lower compared to saliva and/or nasopharyngeal swabs. The average viral load in the patients' exhaled breath is of the order of 102-104 viral particles compared to nasopharyngeal swab samples (˜106-108) from the same patients. Thus, for successful breath sampling, the detection device must exhibit not only ultra-high sensitivity but also specificity for the antigens across other analytes as well as selectivity amidst other proteins and markers. The nanomechanical sensors described herein fulfill these stringent requirements and the Examples demonstrate that the sensors have excellent sensitivity for breath sampling, compatibility with multiplexing and are amenable to system integration that can be cost effective and widely deployable.
As used herein, “subject” or “patient” refers to mammals and non-mammals. A “mammal” may be any member of the class Mammalia including, but not limited to, humans, non-human primates (e.g., chimpanzees, other apes, and monkey species), farm animals (e.g., cattle, horses, sheep, goats, and swine), domestic animals (e.g., rabbits, dogs, and cats), or laboratory animals including rodents (e.g., rats, mice, and guinea pigs). Examples of non-mammals include, but are not limited to, birds, and the like. The term “subject” does not denote a particular age or sex. In one specific embodiment, a subject is a mammal, preferably a human. The subject may have or suffer from, or be suspected of having or suffering from, a disease, condition, or disorder.
In some embodiments, the subject may have or suffer from, or be suspected of having or suffering from, an infection by a pathogen. “Pathogen” means an organism that can produce disease in a subject. Examples of pathogens include, without limitation, viruses, bacteria, fungi, and parasites. Accordingly, the methods and systems described herein may be used to detect an antigen that originates from a pathogen or an antibody produced in response to the subject's infection by a pathogen. The Examples demonstrate detection of antigens that originate from a pathogenic source, e.g., SARS-CoV-2, but the antigens may originate from non-pathogenic sources, including without limitation self-antigens that originate from the subject.
In particular embodiments, the pathogen may be associated with a pulmonary or respiratory disease. Exemplary pathogens include enveloped, single-stranded positive-sense RNA virus of the family Coronaviridae and optionally of the genus Betacoronavirus. Exemplary viruses of the genus include SARS-CoV-2, SARS-CoV, and MERS-CoV. Examples demonstrate the detection of antigens originated from SARS-CoV-2 as well as antigens associated with influenza A, enterovirus, H1N1 (swine flu), and pneumonia. Antigens or antibodies from other sources may also be detected.
In some embodiments, the virus infecting the subject is SARS-CoV-2. SARS-CoV-2's RNA genome encodes many viral proteins, including four structural and 16 nonstructural proteins. An antibody to these viral proteins may be tethered to the microcantilever and binding of the viral protein with the antibody may be detected. In some embodiments, an antibody to a receptor binding domain (RBD) protein, such S1 or N, may be tethered to the microcantilever.
The advantages of the systems and devices of this disclosure are multifold and include, for example, the benefit of shortening the time between sample collection and analysis, thereby identifying subjects having particular test results and, if appropriate, providing medical treatment.
In some embodiments, the methods further comprise treating the subject. The terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.
Antibody-functionalized microcantilevers comprise a microcantilever, an antibody configured to bind a target antigen or an antigen configured to bind a target antibody, and a tether covalently tethering the antibody or the antigen to the microcantilever.
The antibody-functionalized microcantilevers comprise an effective surface density of tethered antibodies or tethered antigens. As used herein, “effective surface density” means an amount of tethered antibodies or tethered antigens that when in contact with sample comprising the target antigen or antibody is capable of inducing a detectable response. Antibody-functionalized microcantilevers may be prepared by contacting tether-functionalized microcantilevers with a precursor solution comprising antibodies or antigens for the desired target such that the antibodies or antigens can covalently bond to the tethers. In some embodiments, the precursor solution comprises between 10 μg/ml-100 μg/ml of the antibody or antigen to be bound.
In some embodiments, the effective surface density is capable of inducing a detectable response when the sample comprises a clinically relevant concentration of target antigen or antibody. As used herein, “clinically relevant concentration” of target antigen or antibody is a concentration of the target antigen or antibody indicative of a disease, disorder, or condition, such as an infection by SARS-CoV-2 or another pathogen.
In some embodiments, the effective surface density is capable of inducing a detectable response when the sample comprises at least 1 μg/ml, 100 ng/ml, 10 ng/ml, or at least 1 ng/ml of target antigen or antibody. In some embodiments, the effective surface density is capable of inducing a detectable response when the sample comprises less than 1 ng/ml of target antigen or antibody.
The microcantilever is composed of one or more materials that can respond to antibody-antigen binding and covalently bond with a tether. Suitably, the microcantilever may be composed of silicon coated with gold as in the Examples but other materials may also be used.
The microcantilever is also dimensioned to allow for a response in response to antibody-antigen binding. The microcantilever may also be dimensioned to have length from about 250 μm to 600 μm, and width of 50 μm to 100 μm with thickness of 1 μm to 2 μm. The Examples demonstrated the use of microcantilevers having a length of 500 μm, width of 80 μm in width, and thickness of 1.2 μm but other lengths, widths, and thickness may be used.
The cantilevers may be tipped or tipless depending on detection methodology. The Examples demonstrated the use of tipless cantilevers where detection was performed optically. However, tipped cantilevers may be used where detection is performed electrically. Example 1 demonstrates rapid and sensitive detection of SARS-CoV-2 antigen proteins in clinical samples using microcantilever platform through optical detection method for nanomechanical sensing. Example 2 demonstrated electrical detection based non-invasive method is disclosed for rapid detection of antigenic proteins through exhaled breath of the patients with a high degree of sensitivity and selectivity. The electronics readout of MOSFET-embedded microcantilever sensors was used to detect various respiratory pathogen deflection, including SARS-Cov-2.
The tethers covalently tether the antibody or antigen to the microcantilever. The tether may be a heterobifunctional molecule with a spacer between immobilization and conjugation functional moieties. The Examples demonstrate the use of a thiocarboxylic acid, where the thiol group is used to immobilize the tether to the microcantilever via gold-thiol bonding, the carboxylic acid moiety is used to conjugate the antibody to the tether, and an alkyl chain provides a spacing and conformation flexibility between the antibody and microcantilever. The immobilization group, conjugation group, and spacer may be appropriately selected based on the material of the microcantilever and antibody or antigen to be conjugated.
Conjugation is a chemical strategy for forming stable covalent linked between molecules, where one is a biomolecule such as the antibodies and antigens used herein. Conjugation chemistries often use carboxyl, amine, thiol, or bio-orthogonal groups that target functional groups that are not native in biomolecules. The Examples demonstrate the use of EDC-NHS chemistry, but other conjugation chemistry may also be used and are generally known in the art.
Another aspect of the invention provides for systems for the detection of a target antigen or target antibody in a sample. The system comprises any of the nanomechanical sensors described herein and a detector for detecting a response of the nanomechanical sensor to the sample and generating a signal indicative of the response of the nanomechanical sensor to the sample.
In some embodiments, the detector can detect a bending moment response. In some embodiments, the bending moment is detected by optically detecting a bending moment. In other embodiments, the bending moment is detected by electrically detecting a bending moment.
In some embodiments, the system is configured for multiplexed detection. Suitably, the system may comprise two or more antibody-functionalized cantilevers where different antibodies, antigens, or combinations or antibodies and antigens are tethered to different cantilevers. Where the system comprises two or more antibody-functionalize cantilevers, the detector may be configured to individually detect the response of each cantilever and generate a signal indicative of the response of each sensor.
In some embodiments, the different antibody-functionalized cantilevers are configured to detect antibodies or antigens that originate from the same source. In some embodiments, the system is configured to detect two or more antibodies, antigens, or a combination thereof that originate from the same pathogen. For example, the system may be configured to detect both the SARS-CoV-2 Spike (S1) protein and the SARS-CoV-2 Nucleocapsid (N) protein.
In other embodiments, the different antibody-functionalized cantilevers are configured to detect antibodies or antigens that originate from different sources. In some embodiments, the system is configured to detect two or more antibodies, antigens, or a combination thereof that originate from the different pathogens, including different species or different variants, or a pathogen and the subject for a control.
In some embodiments, the system further comprises a computing platform having a communication interface that receives one or more signals from the detector, and a computer in communication with the communication interface, wherein the computer comprises a computer processor and a computer readable medium comprising machine-executable code that, upon execution by the computer processor, implements a method for determining the response of the one or more nanomechanical sensors to the sample.
The computational platform is capable of characterizing and determining the response of one or more nanomechanical sensors to the sample. The computational platform may generally include various input/output (I/O) modules, one or more processing units, a memory, and a communication network.
In some implementations, the computational platform may be any general-purpose computing system or device, such as a personal computer, workstation, cellular phone, smartphone, laptop, tablet, or the like. In this regard, the computational platform may be a system designed to integrate a variety of software, hardware, capabilities, and functionalities. Alternatively, and by way of configurations and programming, the computational platform may be a special-purpose system or device.
The computational platform may operate autonomously or semi-autonomously based on user input, feedback, or instructions. In some implementations, the computational platform may operate as part of, or in collaboration with, various computers, systems, devices, machines, mainframes, networks, and servers. For instance, the computational platform may communicate with one or more servers or databases, by way of a wired or wireless connection.
The I/O modules of the computational platform may include various input elements, such as a mouse, keyboard, touchpad, touchscreen, buttons, microphone, and the like, for receiving various selections and operational instructions from a user. The I/O modules may also include various drives and receptacles, such as flash-drives, USB drives, CD/DVD drives, and other computer-readable medium receptacles, for receiving various data and information. To this end, I/O modules may also include a number of communication ports and modules capable of providing communication via Ethernet, Bluetooth, or Wi-Fi, to exchange data and information with various external computers, systems, devices, machines, mainframes, servers, networks, and the like. In addition, the I/O modules may also include various output elements, such as displays, screens, speakers, LCDs, and others.
The processing unit(s) may include any suitable hardware and components designed or capable of carrying out a variety of processing tasks, including steps implementing the present framework for response detection. To do so, the processing unit(s) may access or receive one or more signals generated by the detector. The signals may be stored or tabulated in the memory, in the storage server(s), in the database(s), or elsewhere. In addition, such information may be provided by a user via the I/O modules or selected based on user input.
In some configurations, the processing unit(s) may include a programmable processor or combination of programmable processors, such as central processing units (CPUs), graphics processing units (GPUs), and the like. In some implementations, the processing unit(s) may be configured to execute instructions stored in a non-transitory computer readable-media of the memory. The non-transitory computer-readable media may be included in the memory, it may be appreciated that instructions executable by the processing unit(s) may be additionally, or alternatively, stored in another data storage location having non-transitory computer-readable media.
In some configurations, the processing unit(s) may include one or more dedicated processing units or modules configured (e.g., hardwired, or pre-programmed) to carry out steps, in accordance with aspects of the present disclosure. Each solver module may be configured to perform a specific set of processing steps, or carry out a specific computation, and provide specific results
The processing unit(s) may also be configured to generate a report and provide it via the I/O modules. The report may be in any form and provide various information. For instance, the report may include various numerical values, text, graphs, maps, images, illustrations, and other renderings of information and data. In particular, the report may provide various information or properties generated by the processing unit(s) from the detector generated signals. The report may also include various metrics or indices for determining whether a target antigen or antibody is present in a sample. To this end, the report may be provided to a user, or directed via the communication network to an assembly line or various hardware, computers, or machines therein.
In some embodiments, the systems and devices of this disclosure can analyze samples at the point-of-need rather than in a laboratory. As used herein, the term “point of need” or “point of care” can be defined to mean a location on or near a site of patient care where medical or medically related services such as medical testing and/or treatment are provided, including but not limited to hospitals, emergency departments, intensive care units, primary care setting, medical centers, patient homes, physician offices, pharmacies, or sites of an emergency.
In summary, the Examples demonstrate microcantilever-based nanomechanical detection of SARS-CoV-2 in patient nasopharyngeal swab and breath samples with high degree of sensitivity, specificity, and selectivity in just a few minutes (“collection to signal” in less than 10 mins). The patient sample diagnostic selectivity was optimized for SARS-CoV-2 spike (S1) and nucleocapsid (N) proteins specific detection by microcantilever sensor platform. The sensitivity of microcantilever sensors is such to detect SARS-CoV-2 S1 and N proteins at concentrations as low as 1 ng/ml (33 pM), within 2-3 minutes of probe-target interaction. Furthermore, microcantilevers exhibit no measurable cross-reactivity with non-specific N/S1 proteins in different and varied combinations showing a high degree of specificity and patient sample data provide favorable selectivity. These results collectively demonstrate efficacy for highly sensitive, multiplexed, and rapid detection of antigens associated with SARS-CoV-2, responsible for the worldwide pandemic of COVID-19. The Examples also show that the spike protein (RBD) from mutant variant of SARS-CoV-2 (B.1.17) appears to exhibit lower binding affinity to its corresponding antibody derived from the wild-type strain. Accordingly, nanomechanical signal transduction allows for rapid and real-time assays based on specific binding on microcantilever arrays.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”
As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus≤10% of the particular term and “substantially” and “significantly” will mean plus or minus>10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
SARS-CoV-2 Chimeric monoclonal antibody for Spike (S1) protein (40150-D003), SARS-CoV-2 Chimeric monoclonal antibody for Nucleocapsid (N) protein (40588-R0004), SARS-CoV-2 Spike (RBD) protein (40150-V08B2), and nucleocapsid (N) protein (40588-V08B) were all purchased from Sino Biological Inc. Phosphate Buffer Saline (PBS), PBS-Tween 20 sachets, Bovine Serum Albumin (BSA), and 11-Mercaptoundecanoic acid (MUA) were purchased from Millipore-Sigma. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide and Sulfo-NHS were received from ThermoFisher Scientific. Nasopharyngeal swab specimens were collected from individuals presenting to Northwestern Memorial Hospital, Chicago. The spike protein (S1-RBD) from mutant UK variant B.1.1.7 was also purchased from Sino Biological Inc (40591-V08H12). The set-up used for the deflection experiments was Bruker Bioscope Resolve liquid imaging system. The tipless silicon cantilevers used in these experiments were acquired from Nanoworld Incorporation.
Preparation and Functionalization of the Microcantilever Surface: Prior to use, the gold coated cantilevers were plasma cleaned prior to antibody immobilization to remove any organic residues. The anti-SARS-CoV-2 antibodies (for S1 and N proteins) were covalently attached onto the gold coated cantilever surface through EDC-NHS chemistry inside a glass plate containing tiny wells (See
Experimental Detection Method: Specific biomolecular binding results in lateral stress that imposes a bending moment on the free end of the microcantilever. The microcantilever bends upwards or downwards in response to a tensile or compressive surface stress, respectively. This deflection can be monitored real-time with exquisite sensitivity, similar to AFM in operation. The receptor-coated microcantilever is typically positioned next to a passivated reference cantilever for differential signal of deflection as well as added redundancy, duplication ad multiplexing for improved specificity. The sensitivity in these measurements is governed by ability to measure the slightest deflections quantitatively and reproducibly.
The tipless gold coated silicon cantilevers used in these experiments (500 μm long, 100 μm wide and 1 μm in thickness) were acquired from Nanoworld Incorporation (See
where L is the effective length of the cantilever, t is the thickness, and E/(1-ν) is the ratio between the Young's modulus E (130 GPa) and Poisson ratio ν of Si (0.28). The force constant of these cantilevers was around 0.03 N/m. Because a cantilever deflection depends on geometry, all the cantilevers used in these measurements were exactly similar in their geometry. The cantilever positive (upward) or negative (downward) bending due to change in surface stress as a result of antibody-antigen interaction is directly related to the quantitative measurements for the bindings. The deflection experiments were performed inside a microfluidic reaction chamber sized approximately 2 mm in diameter. The biofunctionalized microcantilevers was brought in close proximity to a microfluidics chamber through stepper motor which contains antigen solution (3 μl approx.) The deflection experiments were performed for different concentrations of SARS-CoV-2 spike (S1) and nucleocapsid proteins (N), in addition to actual patient samples. As the cantilever starts bending due to surface stress, vertical deflection of the cantilever was measured.
The micro cantilever based optical detection system was employed to detect SARS-CoV-2 S1 proteins. Apart from being a major transmembrane structural protein, spike protein is also highly immunogenic and possesses diverse amino acid sequences among the corona virus family making it an important target to develop a specific diagnostic platform for SARS-CoV-2 Microcantilevers biofunctionalized with anti-S1 protein antibody (20 μg/ml) were used to quantify the detection signal using different concentrations of S1 protein (1 μg/ml, 100 ng/ml, 10 ng/ml, and 1.0 ng/ml, respectively). The real-time deflection signal was observed for 15 minutes, as demonstrated in
The deflection signal (Δz) exhibited descending trends with decreasing antigen concentrations, i.e., 1 μg/ml (79.20+/−3-4 nm), 100 ng/ml (58.20+/−3-4 nm), 10 ng/ml (37.70+/−3-4 nm), and 1 ng/ml (22.10+/−3-4 nm), respectively, demonstrating highly specific binding between anti-S1 antibody and S1 protein. As illustrated in the
The cantilever response towards the SARS-CoV-2 Nucleocapsid (N) protein was evaluated. It is one of the 5 major non-structural proteins of coronavirus which is also highly immunogenic and has more stable and conserved amino acid sequences among the coronaviruses. This led us to study N protein mediated cantilever deflection and a comparison with S1 protein. Microcantilevers biofunctionalized with anti-N protein antibody (20 μg/ml) were used for the deflection measurements (
As shown in
To establish the specificity and to greatly increase the efficacy of our detection method to specific markers, ross-validation experiments to check the cross-reactivity with non-specific proteins were performed. First, the cantilever coated with anti-S1 antibody was allowed to react with SARS-CoV-2 N protein (1 μg/ml) and data was recorded.
After successfully demonstrating the specificity in SARS-CoV-2 S1 and N protein detection, the cantilever sensor was then employed for a control experiment also. Here, the cantilever was not conjugated with any antibody (anti-S1/anti-N) which was further allowed to interact with its corresponding antigen protein. In the absence of any antibody-antigen interaction, there was no significant bending of the microcantilever. The control experiment indicates that an antibody conjugation is necessary on the cantilever surface for a specific binding with its complementary antigen to trigger an antibody-antigen interaction.
Having successfully detected S1 and N proteins in isolation, the selectivity of our microcantilever system for detecting SARS-CoV-2 in patient specimens was evaluated. As per standard protocol, the nasopharyngeal swabs were stored in viral transport medium (VTM) and transported to the pathology laboratory. Prior to testing, the samples underwent a viral heat inactivation procedure by incubating them in a water bath at 65° C. for 30 minutes. The positive samples used for the study had a spectrum of Ct values consistent with high (low Ct value) and low (high Ct value) viral load. The information regarding patient sample collection and processing is further described below. The deflection measurements were carried out with respect to the different Ct values from 9 different patient samples. As seen in
In addition, the deflection signal from samples from subjects without SARS-CoV-2 infection (negative samples) was evaluated. As shown in
The different Ct values and the corresponding cantilever deflection from SARS-CoV-2 positive and negative individuals are given below in Table 2.
Binding Study for Mutant Strain from U.K. Variant (B.1.1.7): Potential Variation in Specificity
Since new SARS-CoV-2 variants are emerging rapidly, following specific detection of SARS-CoV-2 in patient samples, we further extended the experiments to study the binding affinity of spike protein (S1) from mutant U.K. variant (B.1.1.7) with wild type antibodies from the original strain (wild type). This variant carries different mutations in its RBD region (H69del, V70del, Y144del, N501Y, A570D, D614G, P681H). It is of particularly interest whether the mutations in receptor binding domain (RBD) of spike protein affect the binding with its complementary antibody from the original strain and whether it shows the same sensitivity and specificity as their wild type counterpart. This is obviously crucial for designing therapeutics and vaccines as well as assessing the antigenic implications of viral evolution, among others. Four different concentrations of S1 protein (1 μg/ml, 100 ng/ml, 10 ng/ml, and 1 ng/ml) were used to make a comparison with our data from the original strain. Spike (RBD) protein from the original strain (1 μg/ml) was used as a control. Cantilever functionalization and antibody grafting were carried out in a similar fashion as mentioned earlier in this work. As seen in
These experimental findings demonstrate that the mutations in the RBD region of spike protein have an impact on its binding affinity with its wild type receptor molecule (antibody) on the cantilever surface and thus the specificity.
The Examples demonstrate specific and selective detection of both N and S1 (RBD) proteins of the virus at a concentration of less than 1 ng/ml (33 pM). A site-directed antibody immobilization technique was adopted to attach antibodies for S1 and N proteins on gold coated microcantilever using EDC-NHS chemistry. This method facilitates a specific bio-molecular binding between ligands and receptors on the surface of a microcantilever beam resulting in physical bending of the beam by some tens of nanometers. The Examples demonstrate that the assay exhibited an excellent sensitivity and specificity based on cantilever deflection measurements. The specific-biomolecular interaction results in lateral stress that induces a torque on the microcantilever resulting in deflection of the cantilever beam. This reflects the interplay between the strain energy increase of the cantilever and the free energy reduction of the interaction. It provides a unique approach for investigating the connection between the nanomechanics and the chemistry of antibody-antigen interaction at very low concentration. This qualifies the technology as a rapid method to detect the SARS-CoV-2 and other pathogens that reveals disease risk and its progression. This approach provides a label free signature for specific binding events and is readily amenable to not only massively parallel and multiplexed sensing but fully compatible microfluidics with all the advantages of the commercially successful microelectronics platform.
Prior to deflection measurements, microcantilevers were analyzed to study the optimum surface coverage for maximum sensitivity using anti-SARS-CoV-2 antibodies. Four different concentrations (i.e., 10 μg/ml, 20 μg/ml, 50 μg/ml, and 100 μg/ml) of anti-S1 (RBD) antibodies were grafted on the cantilever surface using EDC-NHS chemistry. Deflection measurements using spike proteins concentration of 1 ng/ml showed that the cantilever with antibody concentration of 20 μg/ml, 50 μg/ml and 100 μg/ml demonstrated almost the same degree of deflection (
The tipless gold-coated microcantilevers used for this study was 500 μm in length, 80 μm in width and 1.2 μm thick. Because a cantilever deflection also depends on geometry, all the cantilevers used in these measurements were exactly similar in their geometry.
Nasopharyngeal swab specimens were collected from individuals presenting to Northwestern Memorial Hospital. Per standard protocol, the nasopharyngeal swabs were placed in 1.5 to 3 mL of viral transport medium (VTM) and transported to the pathology laboratory. Prior to testing, the samples underwent a viral heat inactivation procedure by incubating them in a water bath at 65° C. for 30 minutes. Next, the samples underwent testing for the presence of the SARS-CoV-2 RNA genome by qRT-PCR (Cepheid). Positive samples were defined by their cycle threshold number (Ct) less than 45. After clinical testing was completed, positive and negative samples were stored at 4° C. for 7 days before the diagnostic testing. Patient information was removed from the sample container prior to use for research purpose. The positive samples used for the study had a spectrum of Ct values consistent with high (low Ct value) and low (high Ct value) viral load. All patient samples were typically processed prior optical detection method for microcantilever deflection monitoring. The samples were first centrifuged at 5000 rpm for 1 minute to remove mucous and other interferants. The samples were then mixed with the extraction buffer (lysis buffer) for 10 minutes and then used for the measurements. In parallel, gold-coated cantilevers were functionalized through EDC-NHS process and anchored with 50 μg/ml anti-N protein antibody.
Materials: SARS-CoV-2 Chimeric monoclonal antibody for Spike (S1) protein (40150-D003), SARS-CoV-2 Chimeric monoclonal antibody for Nucleocapsid (N) protein (40588-R0004), SARS-CoV-2 Spike (RBD) protein (40150-V08B2), and nucleocapsid (N) protein (40588-V08B) were all purchased from Sino Biological Inc. Phosphate Buffer Saline (PBS), PBS-Tween 20 sachets, Bovine Serum Albumin (BSA), and 11-Mercaptoundecanoic acid (MUA) were purchased from Millipore-Sigma. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide and Sulfo-NHS were received from Thermo Fisher Scientific. For Influenza A virus detection, Mouse monoclonal antibody to Influenza A virus Nucleoprotein (ab128193) was used. Rabbit polyclonal antibody to H1N1 virus Nucleocapsid (ab104870) was used for swine flu (H1N1) detection. Rabbit polyclonal to Mycoplasma pneumonia antibody (ab53600) was used for the detection of Pneumonia. Mouse polyclonal to Enterovirus 71 (ab169442) was used for Enterovirus detection. All the antibodies for the detection of Influenza A, H1N1, Enterovirus and Pneumonia were procured from Abcam. Delta variants detection was carried out using in-house developed highly specific rabbit monoclonal antibodies against delta variants.
Functionalization of the MOSFET chips: MOSFET-embedded microcantilever chips were prepared for antibody functionalization by first blocking (passivating) the SiN sides of the cantilevers to reduce the incidence of non-specific binding during antibody and antigen exposure. Chip surfaces were cleaned by washing sequentially with acetone, isopropanol, and methanol. Chips were than briefly treated with piranha (10 seconds) followed by sequential washing with de-ionized water and ethanol.
Electronic measurements of the transistor characteristics were carried out using a Keithley 4200 semiconductor characterization system. The observed changes in the drain current at fixed gate bias and sweeping the drain voltage demonstrate the modulation of channel current with surface stress (generated due to biomolecular interactions) due to microcantilever bending. The change in drain current of the transistor results from the modulation of channel mobility because of surface stress, which increases the channel resistance. The mobility change may also arise from the changes in the interface charge densities, generation of trap-states, band structure alteration and generation of shallow defects due to localized bending stress. MOSFET transistors were passivated with silicon nitride thin coating and electrical contacts were isolated for the binding experiments in the fluidic environment.
The MOSFET microcantilever devices were first immersed in 10 mM ethanolic solution of 11-Mercaptoundecanoic acid (MUA) to attach Carboxylic moieties on the cantilever surface and then rinsed through Ethanol solution. The grafted —COOH groups were activated by putting the devices in solution mixture of 5 mM Carbodiimide EDC and 5 mM Sulfo-NHS in DI water for an hour followed by rinsing with DI water. The devices were then covalently immobilized by SARS-CoV-2 antibodies (20 μg/ml) against nucleocapsid protein by incubating overnight in an antibody solution prepared in PBS and 0.05% BSA (pH=7.4). Functionalized chips were washed repeatedly with PBS-Tween 20 solution and stored in PBS until further analysis. The detailed chemical functionalization process of MOSFET devices has been shown in
Breath sampling and measurements: Breath detection was carried out by using an in-house built breath collection chamber/analyzer (
Nasopharyngeal swab specimens were freshly collected from individuals presenting to Northwestern Memorial Hospital and transported to the pathology laboratory. Prior to testing, the samples underwent a viral heat inactivation procedure by incubating them in a water bath at 65° C. for 30 minutes. Next, the samples underwent testing for the presence of the SARS-CoV-2 RNA genome by qRT-PCR (Cepheid). Positive samples were defined by their cycle threshold number (Ct) less than 45. After clinical testing was completed, positive and negative samples were stored at 4° C. for 7 days before the diagnostic testing. Patient information was removed from the sample container prior to use for research purpose. The positive samples used for the study had a spectrum of Ct values consistent with high (low Ct value) and low (high Ct value) viral load. All patient samples were typically processed prior optical detection method for microcantilever deflection monitoring. The samples were first centrifuged at 5000 rpm for 1 minute to remove mucous and other interferants. The samples were then mixed with the extraction buffer (lysis buffer) for 10 minutes and then used for the measurements.
Prior to breath based detection, we optimized microcantilever measurements using patients' nasopharyngeal swab samples pre-analyzed by qRT-PCR. These measurements helped establish MOSFET drain current sensitivity, optimized affinity coating coverage and associated parameters. We then confirmed the notable consistency between MOSFET drain current change (due to microcantilever deflection) and the Ct values (i.e., the number of PCR cycles that are needed for confirmation of viral genome presence). This combination of high sensitivity coupled to very high sensitivity to viral load prompted us to implement and subsequently validate breath sampling for Covid diagnosis.
We first conducted optimized MOSFET detection of SARS-CoV-2 through patients' nasopharyngeal swab specimens collected freshly from individuals admitted to Northwestern Memorial Hospital, Chicago. Prior to testing, typical sampling and handling procedures consistent with other test methods were employed for both COVID positive and healthy individuals (negative samples). For example, it followed a viral heat inactivation procedure for patients' samples by incubating them in a water bath at 65° C. for 30 minutes. The positive samples used for the study had a spectrum of Ct values consistent with high viral load (low Ct value to achieve detection) and low viral load (high Ct value). The drain current measurements were carried out with respect to the differing Ct values from different COVID positive patient samples (n=19) along with negative patient samples. As mentioned earlier, the nasopharyngeal swab samples were collected from the patients and Ct values were pre-determined with qRT-PCR. In the first set of measurements (
The MOSFET drain current measurements were performed by using two different sets of MOSFET devices coated with antibodies for SARS-CoV-2 N and S1 proteins to compare the current change. As seen from the
Following the SARS-CoV-2 detection using patients' swab samples, another study was conducted on the patients who further volunteered for breath detection. Here samples were collected from 10 positive and 10 negative patients with different Ct values and current measurement was conducted. As shown in
After successfully demonstrating MOSFET electronic detection of viral antigens with great sensitivity and requisite specificity, we explored whether the sensitivity could be adequate as a step towards breath-based detection. The MOSFET possesses sufficient signal to noise ratio (S/N ratio) that it may be possible to detect even lower concentration of viral antigens through exhaled breath as we show next.
“Breathalyzer” Test for COVID-19: Non-Invasive Detection of SARS-CoV-2 Antigens with Breath Sampling
The nasopharyngeal swab results demonstrate fast, sensitive, and specific antigen detection by the MOSFET-embedded microcantilever assay. Given the sensitivity of our microcantilever detection scheme down to just 100+ viral particles, it opens prospects for detecting via breath sampling. The analysis of antigen-based analytes in exhaled breath samples would represent potentially an entirely new paradigm in diagnostics because it is a noninvasive, may be sensitive to early-stage diagnosis and potentially inexpensive way to detect respiratory threat exposure. The major benefits of breath analysis with respect to its clinical application are its non-invasive nature, acceptable for most vulnerable patients, ease of use and rapid. Analysis from breath requires very short time and avoid any complex sample preparation procedures used in other methods.
We implemented our MOSFET-embedded cantilever-based diagnostics system for detecting COVID-19 via breath since MOSFET with swab samples demonstrated a great sensitivity which may be amenable to breath sampling as well. This integrated technology promises to provide a turn-key non-invasive diagnostics system for rapid COVID-19 detection via breath. These MOSFET chips were used to distinguish different level of pathogenicity (viral load) among the patients volunteered for the breath-based SARS-CoV-2 detection study.
Similar experiments were performed for detection of SARS-CoV-2 using patients' breath (as shown in
These results strongly demonstrate high sensitivity of the non-invasive detection method, and we are able to detect to the level the RT-PCR can do. All the patients underwent standard RT-PCR detection first to determine the level of the viral infection (Ct values determination). Here, patient with the lowest Ct value (13.24) exhibited the minimum drain current (bottom curve line), whereas the exhaled breath coming from the patient with the highest Ct value (36.14) shows the maximum drain current (black curve line).
Since Covid-19 and other respiratory disease symptoms often overlap and can be mistaken for one or the other, we attempted to demonstrate that breath sampling can provide sufficient discrimination across a broader swathe of pathogen detection. The MOSFET/cantilever technology is amenable to not only massively parallel and multiplexed sensing but fully compatible with microfluidics with all the advantages of the commercially versatile microelectronics platform.
In this study, array of ten silicon based MOSFET-embedded direct readout cantilevers were used to detect different respiratory disease pathogen biomarkers through exhaled breath from different patients. Here, subjects suffering from COVID-19 symptoms (Delta variant), Influenza A, HIN1 (Swine flu), Enterovirus mediated disease, and Pneumonia volunteered for this study along with some healthy patients (control). The arrays were prior bio-functionalized using different capillaries (channels) filled with individual antibody solution against these viral antigens. The antibody coated MOSFET chips were then placed inside the breath-based measurement setup and direct differential readout was carried out to minimize any false positives. Differential measurements of sensor versus reference microcantilever responses are very important to eliminate false positives as bending signal can be influenced by temperature variations as well as non-specific reactions/binding.
The current modulation data mediated through antibody-antigen based interaction were collected for different subjects under study. As seen from the figure that SARS-CoV-2 variant (Delta) yielded the maximum change in the drain current (70-78 μA) among different patients volunteered indicating typically higher viral load compared to other subjects under study. All the measurements were performed simultaneously. The results in
Table 5. Shows the details about the COVID patients volunteered for swab and breath-based study. In total, 68 COVID positive patients and 32 COVID negative patients were tested for breath, nasopharyngeal swab, and serum samples (serological testing,). The serological testing data have been presented in
The results discussed here indicate the detection capabilities of the integrated MOSFET device as a potential platform for a non-invasive, ultra-rapid (breath to signal≤3 min), and highly selective breath-based antigen test for SARS-CoV-2 and other related respiratory pathogens. These results provide impetus for building towards miniature and turnkey breath sampling “portable” system that is integrated with a mobile phone device.
We demonstrate MOSFET based electrical detection of SARS-CoV-2 in patient through ‘breath’ as a sampling method with high degree of sensitivity, specificity, and selectivity in just a few minutes (“breath to signal” in less than 3 mins). In addition, we also report multiplexed detection capabilities by detecting series of respiratory virus simultaneously along with SARS-CoV-2 Delta variant. We have conducted additional correlative study with swab and serological patient's samples to demonstrate the high sensitivity of our diagnostics system and compatibility with other sample collection methods.
We believe, the non-invasive rapid detection method through exhaled breath will not only reduce unnecessary viral exposure but also availability to vulnerable and underserved population and children's going back to school. The ability to detect the infection before the symptoms appears because of its high sensitivity will significantly reduce the spread of this contagious disease especially when the active variants are moving around.
The present application claims priority to U.S. Provisional Patent Application No. 63/202,713, filed Jun. 22, 2021, the entire contents of which are hereby incorporated by reference.
This invention was made with government support under HL119810 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/34607 | 6/22/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63202713 | Jun 2021 | US |
