Patent Application
20250003967
The field of the invention relates generally the measurement and/or detection of viruses.
Severe acute respiratory syndrome coronavirus (SARS-CoV) was first identified in 2003 and spread into Southeast Asia and China. Roughly a decade later, another variant called Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged. Another decade later, we have witnessed the worldwide pandemic of SARS-CoV-2, causing the coronavirus disease 2019 (COVID-19). It is expected to originate from bats, although the cause of its appearance has not been identified (Zhou et al., 2020).
SARS-CoV-2 infection may be mild or asymptomatic for most healthy people. Individuals with pre-existing conditions, such as obesity, diabetes, cancer, heart and lung disease, can be seriously ill, requiring intensive care units (ICUs), and may die with SARS-CoV-2. In addition, such morbidity and mortality can also occur in healthy individuals (Fozouni et al., 2020). Millions have been impacted by the COVID-19 pandemic.
COVID-19 diagnostics have typically been carried out on the nasopharyngeal swabs and, recently, on the nasal swabs from the infected patients (Zenhausem et al., 2021). Since SARS-CoV-2 spreads through the air, like SARS-CoV and MERS-CoV, it may be too late to contain its spread by testing the infected individuals. Therefore, the best approach would be direct detection from the air.
Bioaerosol refers to particulate matter with life characteristics suspended in the air (Hinds, 1982). Bioaerosol sizes range from 0.02 to 30 μm. In SARS-CoV and SARS-CoV-2, water droplets are generated from the human mouth through exhaling, coughing, and sneezing. SARS-CoV-2, with a diameter of 0.1 μm, has been believed to spread through the droplets >5 μm (thus “droplets” but not “aerosols”). Therefore, 6-feet or 2-m physical distancing has emerged as a safety precaution (WHO, 2021). However, several reports have been made that SARS-CoV-2 can spread with droplets smaller than 5 μm (i.e., aerosols) (Vuorinen et al., 2020; Chia et al., 2020; Hwang et al., 2021; Chen et al., 2020).
While the best practice to prevent SARS-CoV-2 infection is a face mask (Zhang et al., 2020), it is imperfect in capturing all bioaerosols, especially when SARS-CoV-2 spreads through smaller droplets and aerosols. In addition, some people cannot wear face masks due to medical conditions, and some others are not willing to wear face masks. Therefore, the best practice might be direct detection from the air.
The number of viruses in such air samples tends to be very low, which cannot easily be detected conventionally. Air samplers are necessary to increase the number of captured viruses.
There are three widely adopted methods for collecting viruses (or bacteria) from the air: impaction, impinging, and filtration (Terzieva et al., 1996; Fronczek and Yoon, 2015). The impaction method is the most common, where aerosols are aerodynamically separated by size according to their diameter. In the impinging method, the air is sucked in and passed through a device containing a liquid buffer to collect viruses or bacteria. The filtration method is economical and straightforward, where bioaerosols are collected using a fibrous or membrane filter. An additional incubation for more than 24 hours may be required for all three methods due to the small number of viruses and bacteria in the air.
Very few papers have been published to detect the COVID-19 aerosols from the air directly. In the small number of such published works, droplets or aerosols were collected using the traditional air samplers for a long time. Additionally, conventional laboratory analyses were used, such as reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) (Piri et al., 2021; Su et al., 2020). These methods are not easy to be implemented in the field and cannot rapidly provide results.
In certain aspects, the invention encompasses a handheld, rapid, low-cost, smartphone-based paper microfluidic assay capable of directly detecting SARS-CoV-2 in the droplets or aerosols from the air, without needing an air sampler and the long collection time.
There is a strong need to develop a handheld, rapid, and extremely sensitive assay of coronaviruses such as SARS-CoV-2 amid the recent COVID-19 pandemic. The current gold standard is qRT-PCR from nasopharyngeal (NP) swabs, which requires laboratory and skilled personnel, and is time-consuming. Another emerging method is immunoassay from NP and other (nasal or buccal) swabs, including laboratory-based ELISA and field-based sandwich immunoassay strips (also known as rapid kits). As the immunoassays are not sensitive and have high limit of detection (high LOD), early-stage infections cannot be identified; SARS-CoV-2 is notoriously known for its early-stage propagation. In addition, they also suffer from false-positive results when used with complicated samples (NP or other swabs).
As such, there is a need to develop a device and a method for detecting and/or quantifying virus from aerosols and droplets. The present invention satisfies this need.
This background information is provided for the purpose of making information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the present invention.
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to certain embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, and alterations and modifications in the illustrated invention, and further applications of the principles of the invention as illustrated therein are herein contemplated as would normally occur to one skilled in the art to which the invention relates.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used).
The use of “or” means “and/or” unless stated otherwise.
The use of “a” or “an” herein means “one or more” unless stated otherwise or where the use of “one or more” is clearly inappropriate.
The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of” and/or “consisting of.”
As used herein, the term “about” refers to a 10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
The inventors designed a system that simulates a human cough using a simple sprayer in a controlled chamber. Droplets or aerosols are passively collected on paper microfluidic chips without any collector, pump, fan, or filter. Antibody-conjugated submicron fluorescent particle suspension is added to the paper microfluidic chip, inducing antibody-antigen binding and subsequent particle aggregation. A low-cost smartphone-based fluorescence microscope was fabricated, used to quantify the extent of this particle aggregation from the microscopic images (Chung et al., 2021), and confirmed the presence of SARS-CoV-2 from the air. The device and method can slow the spread of SARS-CoV-2 and other emerging respiratory viruses.
One aspect of the invention pertains to a method for detecting and/or quantifying coronavirus from aerosols and droplets, said method comprises (a) adding an anti-virus antibody conjugated fluorescent particle suspension to a paper microfluidic chip comprising coronavirus particles and (b) quantifying said coronavirus particles.
Another aspect of the invention pertains a kit for detecting an airborne virus, said kit comprises a paper microfluidic chip with a pore size of about 5 μm to 15 μm depending on the type of sample suspension and a suspension of antibody conjugated fluorescent particles, and optionally a smartphone-based fluorescence microscope.
A further aspect of the invention pertains to a kit for detecting airborne coronavirus, said kit comprises (a) a device for detecting and/or quantifying airborne coronavirus comprising a paper microfluidic chip, comprising one or more microfluidic channels, wherein said paper has a pore size of about 5 μm to 15 μm, and wherein said channels have a width about 2 mm to about 5 mm and/or a channel length between channel length of about 20 mm to about 50 mm and (b) one or more reagents (e.g., fluorescent particles conjugated with different antibodies) for carrying out detection and/or quantification of one or more coronaviruses.
A further aspect of the invention pertains to a method for detecting an airborne virus, said method comprises (a) fabricating a paper microfluidic chip with multiple channels on it for simultaneously conducting multiple assays; (b) conjugating an antibody to fluorescent particles to obtain an anti-virus conjugated fluorescent submicron particle suspension; (c) passively collecting aerosols or droplets on a paper microfluidic chip (e.g., by placing the chip in the environment where aerosols and droplets are generated and circulated); (d) adding said antibody conjugated fluorescent particle suspension to the paper microfluidic chip; (e) allowing the particles to aggregate and facilitating imaging of individual particles (e.g. by allowing particles and viruses spread spontaneously throughout the paper microfluidic channel via capillary action); (f) imaging the aggregation of antibody conjugated fluorescent particles; (g) removing the background noises and autofluorescence from paper substrate using an optimized threshold intensity and isolating only the fluorescent particles; (h) binarizing an entire image; (i) removing the smaller size of particles to isolate only the aggregated particles; and ( ) relating the total pixel area to the virus concentration to construct a standard curve and estimate the virus concentration from an unknown sample.
A further aspect of the invention pertains to a method for detecting and/or quantifying coronavirus from aerosols and droplets, said method comprises (a) collecting aerosols and/or droplets that contain coronavirus on a paper microfluidic chip; (b) adding an anti-virus antibody conjugated fluorescent particle suspension to the paper microfluidic chip; and (c) imaging the individual particles (e.g., by allowing particles and viruses spread spontaneously throughout the paper microfluidic channel via capillary action and allowing the particles to aggregate to facilitate imaging of individual particles).
In some embodiments, a method for detecting and/or quantifying coronavirus from aerosols and droplets, said method comprises (a) adding an anti-virus antibody conjugated fluorescent particle suspension to a paper microfluidic chip comprising coronavirus particles; (b) quantifying said coronavirus particles. In some embodiments, the virus may be present in a concentration ranging from 100 to 105 virions.
In some embodiments, said paper microfluidic chip comprises nitrocellulose paper, cellulose paper, or polymeric fiber filter. In some embodiments, said aerosols and/or droplets comprising coronavirus particles are passively collected. For instance, said aerosols and/or droplets comprising coronavirus particles may be passively collected using neither aerosol samplers (filter sampler, liquid sampler, impinger sampler, etc.) nor purification, concentration, and amplification. In further embodiments, said method involves a single virus copy level detection of said virus. In some embodiments, said virus is a SARS-CoV-2 virus.
In some embodiments, said step (b) comprises imaging and counting aggregation of antibody-conjugated, fluorescent submicron particles is on said paper chip. In some embodiments, said step (b) comprising quantifying said coronavirus particles using a smartphone-based fluorescence microscope. In further embodiments, the volume of the antibody conjugated fluorescent particles is from 2 uL to 6 uL and wherein said suspension has a concentration from about 0.001% to about 0.04%. In further embodiments, said antibody is a polyclonal antibody or a monoclonal antibody. In some embodiments, the fluorescent particle is a fluorescent polystyrene particle.
In some embodiments, the method further comprises (1) fabricating a paper microfluidic chip with multiple channels on it (e.g., for simultaneously conducting multiple assays) and (2) conjugating an antibody to fluorescent particles to obtain an anti-virus antibody conjugated fluorescent submicron particle suspension, wherein said steps are performed prior to said steps (a)-(b).
In some embodiments, said method further comprises (i) imaging the aggregation of antibody conjugated fluorescent particles; (ii) removing the background noises and autofluorescence from paper substrate using an optimized threshold intensity and isolating only the fluorescent particles; (iii) binarizing an entire image; (iv) removing the smaller size of particles to isolate only the aggregated particles; and (v) relating the total pixel area to the virus concentration to construct a standard curve and estimate the virus concentration from an unknown sample, wherein said steps are performed after said steps (a)-(c).
In some embodiments, a method for detecting an airborne virus, said method comprises (a) fabricating a paper microfluidic chip with multiple channels on it for simultaneously conducting multiple assays; (b) conjugating an antibody to fluorescent particles to obtain an anti-virus conjugated fluorescent submicron particle suspension; (c) collecting (e.g., passively) aerosols or droplets on a paper microfluidic chip (e.g., by placing the chip in the environment where aerosols and droplets are generated and circulated); (d) adding said antibody conjugated fluorescent particle suspension to the paper microfluidic chip; (e) allowing the particles to aggregate and facilitating imaging of individual particles (e.g. by allowing particles and viruses spread spontaneously throughout the paper microfluidic channel via capillary action); (f) imaging the aggregation of antibody conjugated fluorescent particles; (g) removing the background noises and autofluorescence from paper substrate using an optimized threshold intensity and isolating only the fluorescent particles; (h) binarizing an entire image; (i) removing the smaller size of particles to isolate only the aggregated particles; and (j) relating the total pixel area to the virus concentration to construct a standard curve and estimate the virus concentration from an unknown sample.
In some embodiments, a method for detecting and/or quantifying coronavirus from aerosols and droplets, said method comprises (a) collecting aerosols and/or droplets that contain coronavirus on a paper microfluidic chip; (b) adding an anti-virus antibody conjugated fluorescent particle suspension to the paper microfluidic chip; (c) imaging the individual particles (e.g., by allowing particles and viruses spread spontaneously throughout the paper microfluidic channel via capillary action and allowing the particles to aggregate to facilitate imaging of individual particles).
In some embodiments, a kit for detecting an airborne virus, said kit comprises a paper microfluidic chip with a pore size of about 5 μm to 15 μm depending on the type of sample suspension, a suspension of antibody conjugated fluorescent particles, and optionally a smartphone-based fluorescence microscope.
In some embodiments, said kit further comprises a smartphone-based fluorescence microscope comprising a microscope attachment, a light source (such as LED), a battery to power said light source (such as LED) (e.g., a button battery), and an optical filter. In further embodiments, a microscope attachment, an LED, a battery to power LED (e.g., a button battery), and an optical filter are housed within a plastic enclosure to block ambient lighting. In some embodiments, said microscope attachment comprises a bandpass filter or acrylic films (also known as “filter cards”).
In some embodiments, a kit for detecting airborne coronavirus, said kit comprises (a) a device for detecting and/or quantifying airborne coronavirus comprising a paper microfluidic chip, comprising one or more microfluidic channels, wherein said paper has a pore size of about 5 μm to 15 μm, and wherein said channels have a width about 2 mm to about 5 mm and/or a channel length between channel length of about 20 mm to about 50 mm; and (b) one or more reagents (e.g., fluorescent particles conjugated with different antibodies) for carrying out detection and/or quantification of one or more coronaviruses.
In some embodiments, said kit further comprises a smartphone-based fluorescence microscope comprising a microscope attachment, a light source (such as LED), a battery to power said light source (such as LED) (e.g., a button battery), and an optical filter. In further embodiments, a microscope attachment, an LED, a battery to power LED (e.g., a button battery), and an optical filter are housed within a plastic enclosure to block ambient lighting. In some embodiments, said microscope attachment comprises a bandpass filter or acrylic films (also known as “filter cards”).
SARS-CoV-2 Isolate USA-WA1/2020, was deposited by Dr. Natalie J. Thomburg at the U.S. Centers for Disease Control Prevention (CDC) and obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). SARS-CoV-2 was passaged on mycoplasma negative Vero cells (ATCC #CCL-81) at an MOI of 0.005 for 48 h. Supernatant and cell lysate were combined, subjected to a single freeze-thaw, and then centrifuged at 3000 RPM for 10 min to remove cell debris. Concentration ranged from 106 PFU/mL to 107 PFU/mL, corresponding to 2 to 6 ng/mL, respectively. Virus stock was poured into a 15-cm petri dish and irradiated in a Bio-Rad GS Gene Linker UV Chamber on the ‘sterilize’ setting twice for 90 s with a brief swirl in between. The virus's complete inactivation was confirmed by standard plaque assay or 50% tissue culture infectious dose (TCID50). All live virus manipulations were performed in a biosafety level 3 laboratory, and procedures were approved by the University of Arizona's Institutional Biosafety Committee.
Yellow-green fluorescent carboxylated polystyrene particles (CAYF500NM; Magsphere, Inc., Pasadena, CA, USA) were used for assaying SARS-CoV-2. According to the manufacturer, their diameter was 500 nm, the excitation peak was 488 nm (blue), and the emission peak was 509 nm. Rabbit polyclonal antibody to SARS-CoV-2 (40588-T30; SinoBiological, Inc., Wayne, PA, USA) was covalently conjugated to the particles via carbodiimide reaction. Detailed information and optimizations can be found in the previous works (Chung et al., 2019; Chung et al., 2021; Kim et al., 2021; Park and Yoon, 2015). The final particle concentration was 0.06 μg/μL, optimized for showing the maximum extent of particle aggregation (Chung et al., 2021; Kim et al., 2021). Particle stocks were stored in a refrigerator.
The paper microfluidic chip was designed, optimized, and wax-printed in the same way used in the previous work (Kim et al., 2021; Chung et al., 2021). There were four channels in each chip. Each channel was 21 mm long and 2.4 wide, with dumbbell-shaped squares at both ends (
A fish tank was purchased and used as an air chamber (Fish Tanks Direct, North Venice, FL, USA). A hole on one side was drilled, where a sprayer (Amber Spray Bottles; Maredash, Shenzhen, China) was inserted and sealed.
2.5. Direct air sampling on paper microfluidic chips and assay procedure UV-inactivated SARS-CoV-2 was spiked into the 10% v/v human saliva solutions (from human donors, confirmed negative for SARS-CoV-2) with varying concentrations and loaded to the sprayer. The final concentration was 600 pg/mL, comparable to that in human saliva from active COVID-19 patients (Zhu et al., 2020). Concentrations of 0, 200, 400, and 800 pg/mL were additionally used to demonstrate the quantification capability. A paper microfluidic chip was placed 6 in (15.2 cm; one-quarter point of the chamber length), 12 in (30.5 cm; one-half point), or 24 in (61 cm; full length) away from the sprayer nozzle. It was sprayed two times manually (simulating typical human coughs), consuming 3 mL, as well as five times (mimicking repetitive human coughs) consuming 7.5 mL. Spraying made the paper chips wet. After 15 minutes, the paper microfluidic chips were collected from the chamber. The chips were incubated at room temperature for additional 10 minutes, allowing liquid to be evaporated. 4 μL of antibody conjugated particle suspension was then pipetted to the center of each channel and incubated for another 5 minutes allowing the particles to interact with the viruses. The paper microfluidic chip was then imaged and analyzed with a smartphone-based fluorescence microscope. This procedure was illustrated in
A smartphone-based fluorescence microscope was designed and built following the method described in Chung et al. (Chung et al., 2021), with the modifications described below. A commercial microscope attachment to a smartphone was purchased and used (MicroFlip 100-250× High Power Pocket Microscope; Carson Optical, NY, USA). A 460 nm LED (WP7113QBC/G; Digi-Key Electronics, MN, USA) was used as a light source for the excitation of fluorescent particles. The excitation wavelength was slightly shorter than the peak excitation of the particles (488 nm) to avoid the overlap with the emission signal while providing sufficient excitation to the particles, as confirmed with the fluorescence images. A 9-V battery powered this LED. Acrylic films (#382 and #15; Color Filter Booklet; Edmund Optics, AZ, USA) were used as the low-cost excitation and emission filters placed between the microscope attachment and smartphone camera. A smartphone camera (Samsung Galaxy S20 FE 5G; Samsung Electronics America, Inc., NJ, USA) was used to image each paper channel and isolate only the aggregated particles, which would be discussed in the following section. All components (a microscope attachment, an LED, a 9-V battery, acrylic filter, and a smartphone) were mounted on a foldable stand and stage designed in SolidWorks and 3D-printed using Creality Ender-3 (Shenzhen Creality 3D Technology Co. Ltd.; Shenzhen, Guangdong, China) with PETG filament (Overture; Wilmington, DE, USA). This smartphone-based fluorescence microscope was illustrated in
The MATLAB (Me MathWorks, Inc.; Natick, MA, USA) script used in this study was based on the previous work (Chung et al., 2019; Kim et al., 2021). Fluorescence images were acquired from five different locations of each channel since the field-of-view (FOV) was small and could not represent the overall particle aggregation behavior over the length of the channel. Each image was split into three color channels (red, green, and blue). The green channel images were used to capture the green fluorescence (peaked at 509 nm) of the particles. The color histogram of the fluorescence signal was investigated to optimize the intensity threshold value (Park et al., 2014). The pixels with intensities lower than 60 (out of 255) were eliminated to remove background noise (McCracken et al., 2016), which was optimized through a series of experiments using only the fluorescent particles on the paper. (For the benchtop fluorescence microscope analysis, the threshold value was 200). The images were then binarized (black and white). Next, size thresholding was applied to isolate only the aggregated particles. Only the spheres with 10 to 400 pixels (the same size thresholding was considered and used for the benchtop fluorescence microscope analysis); all other spheres were considered noise or artifacts (especially dust).
Two computer case cooling fans (NFD1293259B-2F; Y.S. Tech., Garden Grove, CA, USA, and FD08025, Masscool, Walnut, CA, USA) were installed in the chamber, as shown in
3.1. Confirmation of Particle Aggregation with SARS-CoV-2
Two types of antibodies were used to assay SARS-CoV-2 from the air samples. One was rabbit monoclonal antibody, and the other was rabbit polyclonal antibody, both to SARS-CoV-2. Experiments were initially conducted by sequentially pipette-adding the SARS-CoV-2 solution (0 and 600 pg/mL in 10% saliva) and the antibody-particle suspension on each paper microfluidic channel to demonstrate the initial feasibility. A benchtop fluorescence microscope was used for this initial investigation, and the results were shown in
3.2. Droplet/Aerosol Assay with a Benchtop Fluorescence Microscope
Droplets and aerosols containing SARS-CoV-2 were generated from a sprayer and passively collided with the paper microfluidic chips, as shown in
It was first investigated whether the sensor system works with a benchtop fluorescence microscope.
3.3. Droplet/Aerosol Assay with a Smartphone-Based Fluorescence Microscope
Experiments were repeated using a smartphone-based fluorescence microscope (
To demonstrate the quantification capability, additional experiments were conducted with varying concentrations (0, 200, 400, 600, and 800 pg/mL) with two-time spraying at a 6-inch distance. The result was shown in
Additional experiments were performed to see if this method could be applied to the environment that can better represent the air-conditioned rooms. As shown in
The assay results were summarized in
The SARS-CoV-2 from the sprayed droplets/aerosols directly on a paper microfluidic chip was collected without using any sampler device. Neither pumps nor fans were necessary (besides the fans used to simulate air conditioning) to collect these droplets, and the collection was entirely passive. Assays were conducted directly on the paper microfluidic chip without the need for sample collection, transfer, dilution, and purification. Antibody conjugated particle suspension was pipette-added to the channel, allowing particle aggregation induced by antibody-virus binding. A smartphone-based fluorescence microscope captured fluorescent images, and a MATLAB script isolated and quantified particle aggregation. Despite the small number of viruses captured on each channel, they could be detected with only two sprays (presumably equivalent to two coughs) at a 6-inch (15.2 cm) distance. Successful capture and detection were also demonstrated with the air circulated through the chamber. The method required a paper microfluidic chip (neither pre-loading nor immobilization was necessary), antibody-particle suspension, and a smartphone-based fluorescence microscope (requiring low-cost and off-the-shelf components, such as an LED, a 9-V battery, acrylic film, and a microscope attachment. The total cost of parts and supplies for a smartphone-based fluorescence microscope was US$46.60 (excluding a smartphone), as shown in
The following is list of exemplary embodiments of the invention:
A number of patents and publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.
This application claims the benefit of U.S. Provisional Application No. 63/281,605, filed on Nov. 19, 2021, entitled Direct Capture and Smartphone Quantification of Airborne SARS-CoV-2 on a Paper Microfluidic Chip. The entirety of the foregoing is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/50506 | 11/18/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63281605 | Nov 2021 | US |
