Combination Ab/Ag Paper-Based Lateral Flow Assay for COVID-19

Through our test, we aim to significantly increase testing capacity globally so that people, are not deprived of the resources necessary to combat COVID-19. Our hypothesis states that with our combination of antibody-antigen tests, we can provide researchers, labs, and hospitals a ‘truer’ indication of the severity of the virus outbreak so that the strain on the healthcare system can be reduced.

Problem Investigated:

As of the beginning of June, there have been over 3.5 million cases of infection and roughly 250,000 deaths due to COVID-19.  It is crucial to pinpoint the whereabouts of the virus in terms of rates and progression in individuals.  The only way to do this is via testing.  Yet this is an aspect that has failed, time and time again.  Developed countries despite having some of the strongest healthcare systems have the highest numbers of cases due to lack of effective and efficient testing and developing countries too face this very uncertainty due to the scarcity of cheap point of care testing kits.  With this lack of resources, even those working on the frontline, such as healthcare workers, have very little access to tests, let alone the general public. Hence, in order to reduce the high levels of uncertainty, to plan how to eventually lift lockdowns, and ultimately overcome this pandemic, developing effective and affordable testing kits is quintessential.

Our aims and hypothesis:

Through our test, we aim to significantly increase testing capacity globally and make testing more inclusive so that people, especially those in developing nations, are not deprived of the resources necessary to combat COVID-19.  Our hypothesis states that with our combination of antibody-antigen tests, we can provide researchers, labs, and hospitals a ‘truer’ value and indication of the severity and extent of the virus outbreak so that the strain on the healthcare system can be reduced.

Our solution:

We have designed a combination antibody/antigen (Ab/Ag) paper-based lateral flow assay that is sustainable, easily manufactured, and easy to utilize as a point-of-care (POC) device.  Integrating QR codes onto our tests, which would be connected to an app, we can connect researchers to non-personal data to track the virus and provide users a platform to assess their own progress.

Our prototype:

Diagnosis is the primary strategy towards the overarching goal of virus control and elimination, which would be effective ultimately everywhere to detect active cases, but mainly in developing countries and those with currently low numbers of cases as it may give those countries a chance to thoroughly control and prevent the outbreak from growing.  Testing for antibodies would be more effective in countries and regions where the virus has spread to a much wider vicinity.  Such tests can give an indication of historical infection and as many have debated, a possible sign of immunity, which can be used to slowly lift lockdown restrictions, however that is to be further explored.

Ultimately to get the fullest and the most accurate picture, both tests are needed simultaneously.

Our test is a double-sided combination Ab/Ag paper-based lateral flow assay, which on one side, can detect the presence of the COVID-19 virus itself, and on the other, both IgM and IgG antibodies from blood, urine, serum, plasma, saliva, and sputum samples [1].

FIGURE 1: A schematic representation of the appearance of our test.  Both sides of the paper will have this structure and appearance.

The standard method of diagnosis so far throughout this current pandemic has been through polymerase chain reaction(PCR) and serological tests.  PCR tests detect viral RNA, thus can only determine who has an active case of COVID-19.  Serological tests, on the other hand, can only reveal if an individual has previously been exposed to the virus and developed antibodies as a natural immune response.  Though these two forms of diagnostics respectively have their benefits such as high sensitivity and accuracy, they are constantly challenged by many quality assurance issues.  This includes but is not limited to: cross-reactivity of used antibodies in ELISA specifically, virus stability, reagent storage, equipment performance, and staff competency just to name a few [2].  Furthermore, with PCR and serology, there are high risks of false-positives from contamination and transcription errors, or false-negative results from malfunctioning equipment and degraded samples and reagents [2, 3].  Currently, these tests are administered separately, should an individual have the rare privilege to have access to both forms.

Dr James Gill, Locum GP & Honorary Clinical Lecturer at Warwick Medical school has stated, “The best test for early detection is combining the antibody test AND  the PCR swab taken from the patient.  Then we have a 98.6% detection rate within the first 5.5 days of infection.”

The timing of diagnosis has proven to be crucial in outbreak response in order to mitigate and track the virus not just for an entire country but for an individual’s health too.  Currently, PCR takes a few hours to run and prospective serology tests claim to show results in 15-30 minutes, however, on their own they do not normally benefit patients as immune responses can only be detected for a period of time after initial viral infection [1].  Studies have shown that within the first week of infection, PCR tests can detect covid-19 from nasal swabs with ~95% accuracy, in which gradually after that, the percentage decreases [4].  However at the moment of writing there seems to be a range of PCR detection rates according to many factors such as where the sample was taken from, lab and clinical settings and different protocols for different countries [5].

As stated before, both tests are clearly needed simultaneously therefore we believe our combination Ab/Ag test can possibly override the separate tests.  This has been proven possible in diagnosing HIV through 4th generation combination tests, which have overtaken the market.  This combination test is one of the most preferred and recommended methods of diagnosis for HIV, approved by authorities [6].

Unlike the traditional diagnostic methods like PCR, a paper-based platform provides a basis for point-of-care (POC) diagnosis in resource-limited settings, and removes the necessity for any heavy machinery or professional to administer the tests, whilst maintaining the high sensitivity and accuracy as diagnostic tests should.  In addition, they have numerous advantages including: affordability, sustainability, portability, disposability, and the ability to handle small volumes of unprocessed samples of biofluids such as blood, urine, saliva, sputum or plasma [1].  Paper is an inexpensive, lightweight, easy to functionalize and versatile material.  The most common kinds of paper used for POC devices are filter paper and chromatography paper [7].  Due to its 3D, porous, heterogeneous morphology, paper most often have the ability to store reagents and samples without the use of refrigeration, which would be especially useful in developing countries where there is often a lack of accessibility to electricity, and given the contagious nature of the samples, our paper-based assay should be able to protect users from even more exposure to other biohazards [1, 8].

FIGURE 2: A more in-depth representation as to how each side of the paper functions, and how the detection is carried out via bioconjugated AuNPs.  Top: Covid-19 detection.  Bottom: IgM and IgG detection.

How our test works:

Our test is a lateral flow assay thus functions quite similarly to pregnancy tests.  The user will put a sample of blood, urine, saliva, sputum, serum or plasma on to the sample pad.  Due to capillary force, the cellulose fibers of the paper allows for the biofluids to penetrate the hydrophilic matrix and flow up the test without external power [9].  As the sample rehydrates the conjugated pad, the target will bind to the bioconjugated gold nanoparticles on either side(AuNPs).  As studies have found that covid-19 enters host cells by using their spike glycoprotein (S-protein), on the top layer of the assay we have placed AuNP’s that are functionalised with a complementary antibody to that of the s-protein in order for only the virus to attach to it and aggregate the nanoparticles.  In addition, on the bottom layer, in order to only attach to the desired IgM and IgG antibodies, we functionalised the AuNP’s with dead or inactive covid-19 antigens.  The sample will continue to flow up the paper by capillary action.  As the sample reaches the detection zone and the control line,  there are capture reagents allocated in certain regions, overall forming a QR code image.  Should the test turn out to be positive, the conjugated AuNPs will bind to the capture reagents, causing a visible color change on the QR code to the naked eye, indicating positive for the biomarker detected.  Should the test be negative, no binding will be initiated, causing no color change.  We would expect our test to be able to show results within 5-10 minutes.  Once results have been processed, the user should scan the QR code, connected to our app, which would be able to recognize the different colored QR codes.  The app has an option by which the users would be able to report any symptoms should they have any, indicating their progress, and non-personal data such as how many are positive for COVID-19 can be sent easily to researchers, hospitals, and authorities.  Should the user report severe symptoms we will allow them to contact nearby hospitals, which will allow for hospitals to capacity plan a lot better, without further risk of being overwhelmed and will allow for those who really require intensive care, the opportunity to receive the help they need.

FIGURE 3: A depiction of the color changes of the QR code detection zone upon aggregation with different biomarkers. Top left: Red QR code contains unaggregated AuNPs indicating Negative results for both COVID-19 and IgM and IgG antibodies.  Top right: Deep blue QR code as a result of positive detection for COVID-19.  Bottom left: Light blue QR code as a result of positive for ONLY IgG antibodies.  Bottom centre: Light pink QR code as a result of positive for ONLY IgM antibodies.  Bottom right: Purple QR code as a result of positive for BOTH IgM and IgG antibodies.

In order to detect the presence of COVID-19 and IgG and IgM antibodies at the detection zone, we have decided to use gold nanoparticles (AuNPs) in order to efficiently provide results in a short time period.

Coronaviruses are enveloped positive single-stranded RNA (+ssRNA) viruses which are round, elliptical and often pleomorphic in their form [10].  They have a crown-like appearance under electron microscopes due to the spike glycoproteins (S-proteins) on the envelope, hence the name of coronavirus.  There are two main biorecognition strategies: directly detecting the pathogen, in this case, it would be through the viral RNA or detecting biomarkers, such as antigens like the S-proteins.  Traditionally, PCR directly detects nucleic acids, however, recent innovations in detecting biomarkers for many diseases and conditions have been proven to be faster and more robust [11].  In order to detect the virus, we wanted to use a particle or similar sized material which could detect the S-proteins.  In the case of COVID-19, the virus is 60-140nm in diameter, thus we found it most suitable to integrate nanotechnology and nanomaterials to be able to track the virus from the same scale, hence using APS.

AuNPs are a leading class of metal nanostructures that are widely known for their versatile traits such as chemical inertness, water-solubility, high electron density, and strong optical absorption [12, 13].  They have such broad size and shape controllability, ranging from 1 to 800nm in size and have different morphological shapes from spheres, cubes, rods, dog bones, shells, crystals and even hollow structures[12].  Abundant in characteristics, AuNPs have in recent decades been applied in genomics, clinical chemistry, vaccine development, microorganisms control, cancer-cell imaging, and drug delivery, but have also been recognized to constitute ideal tools in virus detection [13, 14].  AuNPs as labeling agents and bioconjugates are easily visualized to the naked eye due to their intense colors, thus would contribute to the simplicity and portability of our prototype, making it an ideal POC platform, whilst maintaining high levels of sensitivity and accuracy.

To be able to visually and easily present test results, the QR code detection zone relies on the colorimetric changes of AuNP solutions upon aggregation, which can be mediated upon the recognition of COVID-19 antigens and IgM and IgG antibodies which are complementary to the bioconjugated AuNPs [15].

When aiming to use AuNPs in biomedicine as a diagnostic or therapeutic tool, it is necessary to rightly choose the targeting component such as a monoclonal antibody (mAb), and attach it on to the surface of the nanoparticle [16].  Sole AuNPs are ‘blind’ with respect to sensing COVID-19 or antibodies, so the bioconjugation of specific biomolecules to their surfaces is a crucial process.  There have been many successful examples of various AuNP bioconjugates being employed in colorimetric systems to detect well-known human viruses such as Dengue virus; Ebola virus; Hepatitis; HIV; Human Papillomavirus; Herpes; West Nile Virus and even SARS [3, 17].

As stated before, we believe our test would be able to present results within 5 minutes without compensating the accuracy and quality.  This contrasts previous attempts to employ efficiency to detect COVID-19 but with inaccuracy, as shown by the UK government using £3.5million to purchase faulty tests from China.  The technique of using bioconjugated AuNPs has exhibited very promising statistics when detecting SARS in the 2003 pandemic, with a sensitivity limit of 100fM, thus with SARS-CoV-2, we would expect it to show similar prospects [18].

Due to the need for easy and rapid access to data, for researchers, hospitals and authorities, and a platform to report symptoms and progress, we decided to integrate the use of smartphones and diagnosis through the detection zone for our test forming a QR code.  Upon aggregation of the AuNPs, the QR code on each side will change colors according to the sample contents [19].  As shown in figure 3, should the test be negative on each side, the QR codes will remain red.  Should the test show positive for COVID-19, the QR code on the antigen detection side will change into a deep blue color.  If positive for IgM antibodies the QR code will turn light pink, if only positive for IgG antibodies the QR will turn light blue, and if both are present the QR code will be purple.

According to Statista, the current number of smartphone users in the world today is 3.5 billion (45.04% of the current population) and these numbers are growing ever so fast in this day and age.  Aligning this to our aim of inclusivity in terms of availability to testing, we wanted to connect smartphone devices to the tests in a simple manner.  The first smartphone approaches for POC diagnostics have usually involved additional attachment parts, or very specific post-treatment of the used tests: Zangheri et al. described a lateral flow assay which required the use of an accessory lens [20], Mudanyali et Al. reported a smartphone-based platform with a 3D printed accessory with LED [21], You et Al. and Lee et Al. both used smartphone readers [22, 23].  These approaches all use customized add-ons and excessive resources, bringing down the people reached drastically.  In more recent attempts to make it easier to analyse or record data from lateral flow assays, Yang et Al. have innovated a way to integrate barcode on to the assay in order to be read by inexpensive barcode scanners similar to those used in supermarkets [9].  But yet again that lacks inclusivity as we don’t all have barcode scanners lying around hence QR codes seems to be the most effective.

Utilizing paper as the main material for our assay, functionalizing AuNPs for detection, and integrating QR codes as a mean of recording and analyzing data, we have considered the manufacturing process, how the tests will be utilized and the sustainability of our test, all in order to maximize the potential for our test to not be harmful to any party involved.  The materials for our test for COVID-19 can be easily synthesized, made, found, and disposed of, and show little to no toxicity in the case of AuNPs [13, 17].

Accessibility was a big motivation for us whilst designing the test.   Upon our research, we noticed that at the time of writing, out of the 50 countries with the lowest GDP per capita at nominal value in US dollars in 2019 according to the International Monetary Fund, 15 are in the lowest 50 nations in terms of numbers of cases and 13 are in the lowest 50 in terms of death from COVID-19.   But most of all, out of the 50 countries with the lowest recorded numbers of testing, there are 22 of those countries with the lowest GDP per capita.  With even more uncertainty as to how far and wide the disease has spread in these countries, it was our concern that we should not neglect them in our idea of how we should combat COVID-19.  Which raises the question: do these developing countries genuinely not have as many cases, or is it just due to a lack of testing, resources, and knowledge that has caused their data to appear the way that it does?  The only way to find out is through testing.  Testing as many people worldwide as possible.  And through our tests, we may find an answer.


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By Nara Ito

Nara Ito is a student from London, England. She is interested in neurology, immunology, and genetics

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