Perspective Chapter: Microfluidic Technologies for On-Site Detection and Quantification of Infectious Diseases – The Experience with SARS-CoV-2/COVID-19

Abstract

Over the last 2 years, the economic and infrastructural damage incurred by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has exposed several limitations in the world’s preparedness for a pandemic-level virus. Conventional diagnostic techniques that were key in minimizing the potential transmission of SARS-CoV-2 were limited in their overall effectiveness as on-site diagnostic devices due to systematic inefficiencies. The most prevalent of said inefficiencies include their large turnaround times, operational costs, the need for laboratory equipment, and skilled personnel to conduct the test. This left many people in the early stages of the pandemic without the means to test themselves readily and reliably while minimizing further transmission. This unmet demand created a vacuum in the healthcare system, as well as in industry, that drove innovation in several types of diagnostic platforms, including microfluidic and non-microfluidic devices. In this chapter, we will explore how integrated microfluidic technologies have facilitated the improvements of previously existing diagnostic platforms for fast and accurate on-site detection of infectious diseases.

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a type of highly infectious pathogen that is unique compared to many other viruses seen in recent history [1]. The virulent nature of SARS-CoV-2 has allowed it to set the stage for the current global pandemic that continues to plague our society, known as coronavirus disease 2019 (COVID-19) [1]. Over the last 2 years, COVID-19 has drastically altered the way in which humans have lived and is expected to continue with no foreseeable end. As a result of the pandemic, people, businesses, and governments alike have been forced to adapt to continuously changing situations. The changes in people’s daily routines, workplace habits, and social interactions over the past 2 years, only account for a fraction of the seemingly permanent COVID-19-induced changes [2]. Otherwise known as the COVID-19 experience, these adaptations and changes to our society are not limited to the experiences of average citizens. SARS-CoV-2 has also fundamentally altered how institutions such as the healthcare industry and governments respond to potentially pandemic-level infectious diseases, both in terms of diagnostic capabilities and social practices [2].

Unlike many other viruses in the same coronavirus family, SARS-CoV-2 was found to be sufficiently infectious and complex that it was not readily or accurately diagnosed in the early stages of the pandemic. SARS-CoV-2 almost always initially presents itself as a mild case of the common cold or seasonal flu virus [1]. These near-asymptomatic early-stage primary infections, coupled with the limitations in early diagnostic testing, have led to some of the highest rates of lethality and long-lasting symptoms seen in the last century [3]. As of March 2022, it has been reported that COVID-19 has been officially linked to over 6,000,000 deaths worldwide. At one time, it was the second-leading cause of global deaths in both children and adults [3]. SARS-CoV-2’s unique characteristic of presenting seemingly mild or asymptomatic cases continues to be one of the most difficult aspects of managing the spread and treatment of infected patients. In one study, it has been reported that roughly 50% of all new SARS-CoV-2 infections were estimated to have originated from asymptomatic individuals [4], which included both individuals who transmitted prior to the development of symptoms (42%) and individuals who never developed symptoms (8%) [4]. Studies such as these reinforced the unnerving truth that transmission between primary infections and secondary infections was just as likely to originate from a seemingly asymptomatic individual as a symptomatic individual. In the early stages of the pandemic, this inability to discern between infected and uninfected COVID-19 patients forced researchers and healthcare professionals around the world to collaborate and assess the potential for the initial epidemic of COVID-19 to evolve into a pandemic. To do so, researchers and healthcare professionals evaluated the risk of reaching a pandemic status through epidemiological studies, with various empirical techniques [5]. One of the best-established techniques for assessing risk is the reproduction number (R₀).

As seen in Figure 1, R₀ represents a well-established epidemiological concept that measures the potential for an infectious disease to spread by determining the average number of secondary infections that one primary case will generate [5]. Assuming nobody is either immune or vaccinated, an R₀ value less than zero would represent the infections eventually stopping on their own, whereas an R₀ greater than zero would estimate exponential growth in the number of cases in the given population. With R₀ values averaging over 2.0 in most parts of the world, COVID-19 was expected to reach pandemic levels by the start of the second year [6]. Prior to December 2020 (when vaccines became mass-produced and widely distributed), SARS-CoV-2 was a global virus that would endure for many years to come. Without an accurate means of diagnosing individuals for SARS-CoV-2, there were no reliable means for quickly managing potential outbreaks and secondary infections. The high risk of lethality and seemingly permanent post-infection symptoms thus prompted urgent and necessary advancements in vaccine technology, but more importantly in diagnostic technology [1, 2, 3, 4].

Figure 1.

A schematic for visualizing the practical significance of R₀ values. Adapted from Ref. [5].

2. The COVID-19 “Experience”

SARS-CoV-2 has become the most persistent and lethal virus seen in the last century. After more than 2 years, to date, the COVID-19 pandemic has drastically changed the lives of people across the globe. Although the pandemic is an ongoing issue, there are two major time periods which each contain several important milestones in the evolution and management of life during the COVID-19 pandemic. The first year preceded the mass administration and distribution of vaccines and diagnostic testing [2]. While the second year saw more focus on the amelioration of the post-mass-vaccination testing capabilities and societal norms of countries worldwide [6]. By highlighting some of the milestones within the 2 years, the progression of the COVID-19 “experience” can be traced.

2.1 Year one

In the pre-vaccination stage of the COVID-19 pandemic, between December 2019 and 2020, most people’s “experience” with COVID-19 included a constant fear of unknowingly being infected with SARS-CoV-2 [1, 2, 3, 4]. At the time, a significant amount of immune-compromised and seemingly healthy individuals infected with SARS-CoV-2 were readily being admitted to the hospital with a range of symptoms as mild as incessant coughs to more severe symptoms such as shortness of breath and even death [4]. As vaccines had not yet been developed, most people had not yet developed innate or induced immunological defences to this novel virus and feared the uncertainty in symptoms severity [4, 6, 7]. This concern was further magnified after reports of hospitalized COVID-19 patients presenting little to no symptoms, symptoms often indistinguishable from the symptoms of the more common seasonal flu, during the early stages of infection. The ambiguity of symptom development coupled with the high transmissibility of SARS-CoV-2 resulted in an increased likelihood of unreported transmission, and an even greater difficulty in tracking the propagation and transmission of the virus throughout the population [7]. Therefore, without an effective and reliable means to diagnose the early stages of the COVID-19 infection (< 1 week), the ability of hospitals and healthcare professionals to control massive outbreaks and effectively treat the outcomes of infected patients, was severely limited [7, 8]. Thus, the need for improved diagnostic capabilities became the most essential goal in combating the continued spread of SARS-CoV-2.

Polymerase chain reaction (PCR) and immunohistochemistry assays were two of the most commonly used assays being employed to combat the spread and exponential transmission of SARS-CoV-2 [9]. PCR and immunohistochemistry assays required the collection of bodily fluids (> 1 mL) such as saliva, blood, and sinus fluid. These tests needed to be administered by trained technicians at clinics, hospitals, and pop-up testing centres to later be processed in specialized facilities [6]. In most cases, due to the limited number of testing centres and test-availability, wait-times and lines were always very long [7]. Despite the robustness and utility of conventional diagnostic platforms, such as polymerase chain reaction (PCR) and immunohistochemistry assays, the first year of COVID-19 became difficult for diagnostic technologies to match the increasing global demand [9, 10]. The need for improved diagnostic capabilities became increasingly apparent, forcing the requirements for conventional diagnostic platforms to evolve as well. PCR tests sported a limit of detection (LOD) and specificity that was yet unmatched by alternative testing technology like immunohistochemistry assays [2, 9]. In the early to middle stages of the first year, this practice of diagnosing patients experiencing flu-like symptoms, who are potentially infected with SARS-CoV-2, with PCR tests became the gold standard. For a time, it allowed healthcare professionals to better manage and track primary infections, from mild to severe symptomatic infections, by providing a superficial means to control potential outbreaks through contact tracing [2, 9, 10]. In addition, it allowed for better-directed resources and healthcare efforts for those positively infected with SARS-CoV-2. This, however, would prove to eventually become less and less viable as a diagnostic method, due to the test speed at which transmissions between primary and secondary infections were occurring.

Although PCR tests are normally able to process tests within 1 week of submission, the delay in onset of symptoms and high transmissibility of SARS-CoV-2, severely hindered its effectiveness to facilitate tracing and resource management in the healthcare system [11]. To further hinder the efforts of PCR testing, testing backlogs at processing facilities resulted in a delay of over two weeks to receive testing results. This coupled with the understanding that SARS-CoV-2 was often transmitted before the onset of symptoms (< 1 week) meant that people were now not able to confirm their state of infectiousness until their infectious period had already passed. The sheer speed and virality of SARS-CoV-2 left PCR tests incapable of forewarning primary infections of their risk of transmission. PCR tests, shown in Figure 2, remained the gold standard for the diagnosis of SARS-CoV-2. However, exploring other detection methods may address some tests’ inherent limitations.

Figure 2.

A visualization of the testing procedures involved in PCR and immunohistochemistry assays. Adapted from Ref. [12].

The newly observed limitations of PCR testing, highlighted by the exponential growth of SARS-CoV-2 cases across the globe, demonstrated that future prospective diagnostic tests required less turnaround time and greater accessibility to the public [12]. Near the end of the first year, in response to the growing need for even faster SARS-CoV-2 diagnostic technology, funding and research into “rapid” immunohistochemistry tests began to grow exponentially [2, 13]. At the time, rapid tests based on immunohistochemistry were difficult to mass-produce with enough rigor to reliably diagnose patients for SARS-CoV-2, while also being logistically difficult to meet its demand [9, 13, 14]. At minimum, future rapid tests would now need to be more accessible such that people might be able to test in any environment without specialized equipment while producing results within the first week of suspected infection to help prevent primary infections from spreading. Moreover, the production and distribution of these potential tests would require great logistical improvements, which would not be possible without a great deal of continued funding. These obstacles meant PCR tests, for a little while longer, would remain the gold-standard for SARS-CoV-2 diagnostics over other detection methods, including immunohistochemical assays. This growing list of requirements jumpstarted the need for the technological advancements that would lead to the mass-production of rapid, accurate, low-cost, on-site tests seen in the second year of the COVID-19 pandemic [2, 8]. The improvements that earlier quantitative and qualitative rapid tests required to meet the needs of society could have been addressed more readily, in the short-term, with immunohistochemical detection methods, but were not the most viable long-term solution [12, 13].

2.2 Year two

By early 2021, the fear of unknowingly acquiring and transmitting SARS-CoV-2 prompted governments, healthcare professionals and businesses to enter a state of stasis; with the hopes, it would mitigate the transmission and persistence of SARS-CoV-2 [2, 14]. Under the advisement of healthcare professionals, people were now forced to restrict their social interactions and to experience heightened levels of caution between one another for necessary tasks. This time of stasis created a loop of financial and emotional hardships between consumers and businesses in ways the world had never experienced before. The speed at which SARS-CoV-2 was transmitted between individuals was not the only concern researchers were trying to address by improving our qualitative and quantitative diagnostic capabilities. Variants of the SARS-CoV-2 virus were slowly emerging and becoming an increasing cause for concern during the spring of 2021 [15]. The emergence of the SARS-CoV-2 variants had now provoked a resurging fear across the globe.

With the introduction of vaccines in early 2021, the potential prevention and reduction of SARS-CoV-2 infections quickly turned much of the first year’s fear, uncertainty, and general unease into hope [2, 11, 15]. The hopeful end to COVID-19, at this time, was further supported by a large increase in the production and accessibility of rapid (< 30 min) diagnostic tests that helped to control the spread of SARS-CoV-2 variants. Despite the emergence of the virus variants, people were hoping to return to some sense of normalcy. To facilitate this transition, some governments and healthcare professionals began relaxing health policies as vaccinations were being regularly administered to a large portion of the population. This hope would, however, prove to be short-lived as novel discoveries demonstrated the virus’s ability to replicate and mutate into several increasingly more infectious variants [15]. Almost exclusively across the second year of the pandemic, five COVID-19 variants of note were recorded by the World Health Organization (WHO) [15]. These variants of note included Alpha and Beta (discovered in December 2020), Gamma (January 2021), Delta (April 2021), and Omicron (November 2021) [15]. Each variant of note is structurally similar to that of the original strain, often referred to as the “index virus,” in that they all contain a lipid shell that houses viral genetic material and spike proteins on the surface of the lipid shell, which facilitates the anchoring and fusion of viral cells to healthy cells [2]. However, each subsequently discovered variant of SARS-CoV-2 demonstrates slight differences in the structure of the spike proteins that occupy the outer lipid shell [2]. Normally, our bodies are built to naturally defend against repeated infections from viruses by producing antibodies that specifically target the surface proteins of a previously introduced virus, as well as antibodies with slight modifications to try and prevent infections from similarly structured viruses [7, 9]. Unfortunately, the slight variations in spike proteins of subsequent SARS-CoV-2 variants proved to be significant enough that the body could not recognize or defend against them, resulting in increased infectiousness that bypasses both the induced and innate immune response against infections. This meant that despite the induced protection from the SARS-CoV-2 vaccines, these new variants could once again infect a person without presenting symptoms in the early stages of infection. Mass-production of diagnostic rapid tests was now, more than ever, in direct need of improvements to aid in combating the spread of SARS-CoV-2 and its variants. The previously established quantitative and qualitative tests, such as PCR and **ELISA, offered their own strengths and limitations, but neither could meet the needs of the healthcare system to help control the spread of SARS-CoV-2 independently. Therefore, alternative diagnostic technologies were being explored. Microfluidic systems continued to show great promise in addressing the shortcomings of the diagnostic testing platforms currently available, as well as the logistical limitations in mass-producing these tests [16].

3. The evolution of diagnostic technology throughout the COVID-19 pandemic

By December of 2019, SARS-CoV-2 became a newly established virus, which meant that researchers and scientists did not yet have a thorough understanding of how SARS-CoV-2 transmission occurred or exactly how infectious and deadly it would become [12, 16]. This meant that many of the established conventional diagnostic technologies were not yet optimized for the accuracy, sample processing speed, and capacity for mass production that was required to help control mass outbreaks like the COVID-19 pandemic. As a result, many of the deaths and infections unknowingly caused by COVID-19 in the first year could not have been averted. The obstacles to outbreak management and tracing caused by a strain on the healthcare systems around the world almost certainly led to the underestimation of both the transmission rate, severity, and the capacity of COVID-19 as a pandemic-level threat. In effect, the world was not prepared for the rampant shortages of hospital beds, cleaning supplies, and masks which were essential for mitigating people’s exposure risk to the virus [16]. Eventually, SARS-CoV-2 diagnostic tests would be able to accurately diagnose patients in a reasonable amount of time. However, it took roughly 1 year and a half to achieve a sufficient level of reliable testing capabilities utilizing improvements on previously established diagnostic systems [16].

3.1 The cost of technological advancement

Given the rampant fear brought on by the uncertainty of the first year of the pandemic, it was given that a collective effort across worldwide industry, healthcare professionals, and governments would be necessary to tackle the logistical and technological shortcomings of developing rapid diagnostic tests. In response, many resource-abundant countries promised to commit billions of dollars worth of funding to promote the development and manufacturing of rapid tests [17]. Due to the large influx of funding, the attraction for developing increasingly robust rapid tests prompted many small and large-scale companies to try and compete to mass-produce effective tests faster than each other. By the summer of 2021, there was increased access to rapid, 30-minute tests, which allowed people to slowly gain clarity on how to go more carefully about their daily lives while reducing the spread of COVID-19 [17]. These rapid tests empowered people to monitor their own social behaviours and individual health to a greater degree than what was previously possible, providing them with the knowledge to minimize their risk for exposure and transmission. This short-term competition between companies had lent itself to exponential advancements in the development and production of rapid tests which resulted in some countries eventually being able to give out limited numbers of rapid tests for free. Within countries that were fortunate enough to have ready access to a combination of vaccines and rapid tests, the number of COVID-19 cases saw a gradual reduction, over an extended period of time [2, 10]. Conversely, the risk for continued viral transmission was not effectively addressed in resource-limited countries. Other less-equipped countries were unable to produce, receive or distribute as many rapid tests as their more fortunate counterparts, to their own citizens. The disparity between countries for access to rapid tests was observed to have, in part, a direct correlation with population size and density, as well as the economic state of a country’s wellbeing [7, 10]. Thus, the short-term reliance on rapid diagnostic tests that were too costly to produce or distribute in resource-limited countries highlighted the possibility of conventional diagnostic techniques not being ideal in globally addressing the importance of fair access to accurate and rapid testing.

3.2 Progression of PCR and immunohistochemical diagnostics

Accurately tracking the state of active infectiousness in a person with SARS-CoV-2 is an essential tool in managing and combating potential outbreaks by providing people with primary infections some insight into a timeline for when and how long they should be self-isolating to reduce the risk of propagating secondary infections [17]. The progression of an individual’s infection, otherwise known as active infectiousness, can ideally be monitored by quantifying the viral load of a person, the number of viral copies in a unit volume of bodily fluid. Measuring the viral load can in turn provide healthcare professionals and researchers with useful data pertaining to the time required for the onset of symptoms and the state of a person’s active infectiousness.

Quantifying the concentration of individual units of double-stranded template genetic material (DNA) in a unit volume of sample is possible through PCR. By cycling through the three major steps of PCR (denaturation, annealing, and elongation), one individual copy of DNA can be multiplied exponentially [8]. However, assessing the “active infectiousness” of a person involves quantifying individual units of viral genetic material, which are instead, units of single-stranded mRNA [8, 17]. To use single-stranded mRNA as the substrate for the replication of genetic material, we first require a molecular complex known as a reverse-transcriptase (RT) to create a complementary strand of DNA (cDNA) to the template single-stranded mRNA [8, 9]. By introducing the RT to the template mRNA, a double-stranded DNA template that can be further amplified in a downstream PCR reaction, is created. This technique combines these two processes to create RT-PCR (rtPCR) which will inevitably allow for the amplification of genetic material from mRNA. To later quantify mRNA samples previously amplified through rtPCR, the use of a fluorescent reporter molecule must be used [8]. This reporter molecule could be in the form of a dye or a molecular complex, such as a probe. These reporter molecules will then bind to double-stranded DNA, if it is a dye or a specific target sequence if it is a genetic probe [8]. By measuring the increase in fluorescence of the sample, a quantitative PCR (qPCR) analysis can be achieved. The fewer number of cycles it takes for the sample to fluoresce beyond a certain threshold, the greater the concentration of genetic material, and vice versa. This process allows for the detection of targeted sequences in very low concentrations as well as quantification of viral loads through the addition of fluorescence-based reporter probes. PCR assays have shown their use in reliably diagnosing infectious diseases, with a reasonable amount of accuracy, but require higher costs and turnaround times than other methods such as immunohistochemistry assays.

Immunohistochemical assays, such as in Figure 3, offer qualitative detection methods that do not rely on a genetic component but instead on the intrinsic binding properties of antibodies and aptamers to biomarkers of interest [17, 18]. Qualitative, immunohistochemical assay, rapid 30-minute tests were designed to target some of the most common antibodies present in people infected with SARS-CoV-2, namely IgG, IgM, and IgA, but lacked the ability to track the progression of an individual’s infection [2, 18]. Due to the potential specificity of antibodies and aptamers to biomarkers of interest, false positives in immunohistochemical assays are not common, however, false negatives are not so uncommon in rapid diagnostic systems [19]. Due to the strictly qualitative nature of the test, the concentration of target molecules must reach a certain threshold before resulting in an observable positive result and can result in false negatives [19]. As a trade-off, these assays can instead gain a greater amount of modularity, selectivity, and specificity compared to other assays by researching and testing combinations of detector and target molecules to optimize the detection technique. One important example of an immunohistochemical assay with proven applications in medical diagnostics is the enzyme-linked immunosorbent assay (ELISA). ELISA assays bind antigens onto an absorbent surface that would facilitate the even distribution of a test buffer and sample mixture across the entire surface through capillary action [17, 19]. When a target antibody moves across some of the bound antigens on the surface, they will bind and be fixed onto the surface in a particular orientation [17]. Afterward, the secondary detection antibody, designed to be complimentary to the antibody at the other end of the target antibody, then subsequently becomes fixed onto the surface allowing for conformational changes in the detection antibody. This conformational change then allows for a reporter gene to be activated and create a colour change that can be qualitatively assessed by the naked eye [17]. However, unlike PCR tests, this technology is not capable of achieving quantitative diagnostic capabilities.

Figure 3.

A visualization of the qualitative nature of a rapid immunohistochemistry diagnostic test. Adapted from Ref. [11].

Despite the numerous technological advances in diagnostic technology, many conventional detection techniques such as immunohistochemistry and PCR have yet to comprehensively address the requirements for a mass-producible, rapid, on-site diagnostic test that is capable of both quantitative and qualitative results [19]. Many of these diagnostic tests are too costly to manufacture in resource-limited parts of the world, increasing their intrinsic cost and restricting their access from the general-public. In addition, the on-site requirements of many of these available tests are too great either structurally or logistically to be reliably used and shipped across the globe. For example, several types of cost-effective tests cannot withstand extreme changes in weather conditions through transit and could be compromised in terms of their accuracy. Many currently available rapid tests (< 30 min) have insufficient detection limits to provide an accurate diagnosis across the early stage (0–7 days) of infection, where primary cases might already be infectious to surrounding people [2, 19]. The limitations of conventional diagnostic techniques are continuously magnified by the seemingly endless need for tests as more and more variants, with greater infectiousness than their predecessor, begin to make our healthcare practices less effective over time. Moreover, the benefits of such technological and health-related advancements were limited to wealthier countries, which celebrated earlier access to both vaccines and rapid tests, long before other less fortunate countries had access to either of these life-saving resources [16]. It would therefore be of great importance to continue to innovate existing diagnostic technologies, or develop new ones, to try and address the socioeconomic disparity between countries in the pursuit of preventing further mass outbreaks.

Throughout the COVID-19 pandemic, the main goals of conventional rapid-test manufacturers and researchers were to increase the reproducibility and reliability of the unit tests, while reducing overall costs in production and logistics [11, 16]. Improving the technology of more readily available diagnostic tests, such as rapid PCR and immunohistochemistry tests, would prove to be an extremely useful tool in reducing the transmission of COVID-19 around the world. However, it would still be necessary to explore alternate diagnostic technologies which were also expected to advance our diagnostic capabilities, such as microfluidic devices. Microfluidic devices offer not only some promising prospects to address many of the limitations of previous conventional devices through their modularity and reproducibility, but also offer the ability to enhance previously established diagnostic techniques.

4. Microfluidic devices as diagnostic platforms for SARS-CoV-2/COVID-19

Microfluidic devices utilize fluid systems on the micro-scale that behave differently than that of fluids at volumes seen in day-to-day life. The main difference between micro and macro-scale fluid dynamics is seen in the readiness of micro-scale fluids to achieve a state of flow known as laminar flow [2]. Laminar flow describes a state of fluid flow where the fluid moves in continuous parallel layers, without any disruption between the layers of fluid [20]. In practice, this means that even at lower velocities, the fluid is not subjected to unwanted lateral mixing and the particles within the fluid itself are moving in straight, parallel, lines with one another. This directly contrasts with the type of flow ordinarily seen in macro-scale volumes, turbulent flow, which would instead experience rapid and chaotic variations in pressure and flow velocity across any given period. In turbulent fluids, particles are unevenly distributed and are subjected to random changes, which are undesirable traits when attempting to measure or work with systems that require a great deal of accuracy [20]. In the context of microfluidics, by taking advantage of the readily achievable laminar flow states, researchers and scientists can perform extremely useful and precise experiments that would normally be impossible with larger volumes [20, 21]. Interestingly, microfluidic technology was originally invented in the 1950s by Siemens-Elema, not as a diagnostic technique but as a subset of printing technology used to efficiently transport ink [21].

It was not until the turn of the millennium that this microfluidic technology really began seeing more practical medical applications [21]. As seen in Figure 4, the number of microfluidic applications has seen a steady increase since the early 2000s, largely in part to the extremely fast development of computing power and computational hardware, which allowed for many combinations of microfluidic technology, integrated with digital processing [19, 20, 21]. Therefore, the number of potential future microfluidic applications appears limitless.

Figure 4.

A timeline of the historical milestones achieved with microfluidics [21].

4.1 Microfluidic classification and scientific relevance

Over the years, microfluidics has grown in a way that several classifications of microfluidic technology exist to aid in organizing the myriad of its applications. The biggest classification of microfluidics distinguishes microfluidic systems between continuous-flow and droplet-based systems, sometimes called segmented flow.

As seen in Figure 5, Continuous-flow microfluidic systems utilize the characteristics of laminar flow to facilitate experimental processes that often require controlled mixing of micro-scale reagent and sample volumes [20]. Moreover, these continuous-flow systems can serve as ideal platforms for other slow-processing experiments such as microfluidic-based PCR quantification and even real-time sample separation techniques. Conversely, droplet microfluidics allows for more instances of chaotic mixing to occur in a highly controlled environment, essentially facilitating numerous independent mixing phenomena to occur [20].

Figure 5.

A visualization of (a) droplet, and (b) continuous-flow microfluidics fluid dynamic mixing [20].

The differences in how these techniques can be achieved or improved upon with microfluidics depend on the micro-channel design of the system. In continuous-flow microfluidics, the four main categories of channel design include serpentine, spiral, oscillating-flow, and straight microchannels [22]. Each design channel design serves its own unique purpose when being used in microfluidics. For instance, serpentine microchannels can be used to extend the time in which a fluid is exposed to external factors, such as UV light for crosslinking or heat to promote a particular reaction, across a more unified and even distribution of the fluid [18, 22]. Oscillatory continuous-flow microfluidics has been used to increase the effectiveness of liquid-liquid separations as well as aid in sample concentration techniques to improve detection outcomes in downstream processes [22]. These continuous-flow adaptations allow microfluidic systems to intrinsically offer more freedom for the experimental design than conventional techniques and can use a smaller working volume to make better use of smaller and costlier reagents. In general, continuous-flow microfluidic systems have seen an increase in popularity over the years due to their modularity, but do not compare in popularity to droplet microfluidic systems.

Droplet microfluidic systems forego the continuous laminar flow physics that is innate in continuous-flow systems, but instead make use of the micro-scale laminar flow physics to facilitate the production of individual droplet microenvironments [23]. These droplet microenvironments, shown in Figure 6, are discrete microlitre volumes that are formed through the controlled flow of a sheath (transporter) fluid across one or more sample fluids at a common junction [23]. Thus, repeatedly partitioning the sample fluids in a controlled manner and encapsulating the mixture of sample fluids into droplets. What makes droplet microfluidics so important and unique, even compared to continuous-flow microfluidics, is that it can allow for both high throughput experimentation and an increase in resolving power [2, 24]. Droplet microfluidic systems have been demonstrated to effectively process, in high throughput, experimental analysis in resolutions as high as single-cell resolutions; something many other platforms cannot achieve on their own [23, 24]. The various forms of droplet microfluidic systems can be classified into three main categories: high and ultrahigh throughput droplet microfluidics (htDM/uhtDM), digital droplet microfluidics (dDM), and controlled droplet microfluidics (cDM) [23]. In htDM and uhtDM, the main purpose of the system is to generate as many stable and uniform droplet microenvironments as possible to facilitate increased resolution and turnaround time. The production of thousands of comparable droplet microenvironments in a controlled manner promotes increased reaction efficiency as well as both qualitative and quantitative analysis through integration with digital technology. This integration between htDM and uhtDM with digital technology does not necessarily overlap with the dDM classification but does experience some similarities in that they can both utilize automation to enhance their analyses [24]. dDM systems aim to achieve complete automation through their integration with droplet microfluidics. However, it does not often utilize high throughput processing [23, 24]. Instead, purely dDM systems often generate multiple types of droplets in one system and have these different droplets interact with one another, in a highly controlled manner [23]. By utilizing components of both htDM/uhtDM systems and dDM systems, researchers can achieve systems that are classified as controlled droplet microfluidic systems. These controlled droplet microfluidic systems gain some of the advantages of the other two systems and offer even greater modularity than either of those systems independently. As a result, the use of digital integration along with controlled droplet microfluidic systems microfluidics permits microfluidic platforms to be one of the most promising cost-reducing diagnostic platforms in both current and future diagnostic research.

Figure 6.

A graphic highlighting the various droplet microfluidic classifications [23].

4.2 Advantages of microfluidic integration in diagnostic tools

The increased efficiency of sample-reagent interactions is a particularly attractive characteristic of microfluidic devices which drives research towards improved diagnostic applications of microfluidic technology [25]. Normally, diagnostic technologies utilizing sample-reagent interactions between molecules of relatively weak binding affinities are regarded as non-optimal, in macro-scale volumes, due to the inefficiencies and potential overconsumption of resources [25]. Resources such as reagents, samples, and equipment can often be extremely resource-intensive and expensive, which can result in fewer tests per unit volume of sample and may discourage researchers from moving forward with a particular combination of cost-effective and practical diagnostic material. Ideal detection molecules for diagnostic purposes should offer an acceptable balance between cost, binding efficiency, and selectivity to the target molecule of interest. Finding this balance of characteristics in a detection molecule often requires lengthy amounts of research and testing, which would further incur operational costs. However, these limitations associated with non-microfluidic technologies can be improved upon with the use of microfluidic technology. Therefore, microfluidic integration can overcome many of the limitations associated with non-microfluidic systems and gain some advantages unique to microfluidics.

4.2.1 Cost-reduction

Conventional diagnostic techniques often require costly materials as well as logistical and operational costs, all of which create barriers limiting the access of these techniques to resource-limited settings [26]. Furthermore, due to the COVID-19 pandemic, unmet demand for conventional diagnostic tests had significantly increased the prices of individual tests, leaving several countries and low-income individuals with fewer options for obtaining these potentially lifesaving items [19, 26]. In 2021, it was found that a single COVID-19 rapid antigen test could have costed consumers upwards of $20 USD, per test [2, 19, 25]. These essential diagnostic tools are often subject to the whim of the companies that produce and distribute the tests, which can only further limit access to lower-income individuals [16, 17]. By integrating microfluidic technology with previously existing platforms, microfluidics has the potential to reduce the overall costs of mass-producing rapid and accurate diagnostic tests for the detection and quantification of SARS-CoV-2.

4.2.1.1 Fabrication

To better optimize the costs for mass-producing diagnostic tests, microfluidic integration may help to reduce costs by permitting more variety in the materials used to fabricate tests. As seen in Table 1, commonly used materials in producing several microfluidic integrated systems including silicon, glass, polymers, and paper; each material, offers its own respective advantages and disadvantages [2, 26].

	Glass	Silicon	Polymer	Paper
Fabrication Techniques	Photolithography Etching	Bulk or surface micromachining Nano-imprint lithography Electron beam irradiation	Soft lithography Injection moulding 3D printing	Wax and inkjet printing Photolithography
Advantages	Transparent Inert and stable Solvent compatible Hydrophilic	Mechanically strong Thermostable Chemical resistance	Transparent Easy fabrication Low cost	Flexible and lightweight Low cost Biocompatible Recyclable
Limitations	Brittle Not flexible High cost	High cost Biocompatibility	Hydrophobic Short shelf life	Humidity and temperature sensitive Difficult to design and integrate into single chip

Table 1.

A summary of several different microfluidic substrate types and their respective characteristics.

Adapted from Ref. [2] with permission.

Silicon and glass were two of the most used materials in the fabrication of microfluidic devices due to their abundance [2]. These two materials offered great modularity in the type of potential applications the microfluidic systems would supplement. However, both suffered from high material costs [2, 26]. As technology advanced and other synthesized materials became more affordable, these two abundant microfluidic materials became less and less viable for the mass-production of diagnostic tests [16, 26]. As a result, materials such as polymers and paper became more attractive microfluidic substrates. Coupled with their simplified fabrication and processing methods, both paper-based and polymer microfluidic devices share stronger potential as on-site SARS-CoV-2 diagnostic tests, as compared to glass or silicon-based systems [16, 18, 19].

In addition to the reduction of microfluidic substrate costs, microfluidics reduces the inherent costs associated with reagent use since only microlitre volumes are used in the system. Each individual microfluidic test would, ideally, only require microlitre volumes of any reagents and samples to be effectively analyzed. Lower fluid volumes required for the successful operation of each test reduce the cost of fabrication per test and might allow for a better redistribution of financial resources.

4.2.1.2 Operations

A microfluid integrated diagnostic device has the potential to greatly reduce operational costs associated with processing samples. By reducing the number of experienced operators required to pilot the device or removing the need for expensive equipment to process samples, a large portion of the cost (per test) incurred by the consumer can be greatly reduced. This operational cost reduction can be achieved by developing a system with self-contained microfluidic tests that are qualitatively and quantitatively analyzed by a small portable test analyzer, with a single operator.

By developing a portable microfluidic analyzer that can independently process multiple diagnostic tests, in sequence or in parallel, only one unit operator would be required to perform on-site diagnoses. Depending on the design and software of the analyzer, the operator might not always require a great deal of experience to collect, process, and record samples. In the case where diagnostic sample processing includes whole-sample mediums, such as whole blood, saliva, and mucosal liquids, a potential biohazardous risk to the collector could exist [24, 25]. These potentially biohazardous samples can be collected by trained personnel swabbing the patient’s sinuses or mouth, as well as through the drawing of blood [25]. These collection techniques are used to require trained operators to wear personal protective equipment (PPE) to minimize potential transmission and required processing off-site. In the early stages of the pandemic, it was common for diagnostic tests being administered by a healthcare professional wearing PPE to take more than a week to produce results. Many of these early tests still required millilitre-volume samples that lead to lowered detection-sensitivity issues [17]. Conversely, in the later stages of the pandemic, more and more diagnostic tests were being self-administered, and taking less time, as diagnostic technology continued to advance. However, these self-administered tests were often restricted to qualitative detection methods that could not yet provide a measure of an individual’s “active infectiousness”. In theory, microfluidic technology is expected to allow for a single portable microfluidic analyzer to both qualitatively and quantitatively diagnose large numbers of samples, with only one operator.

An equally important feature of microfluidic systems is its ability to facilitate on-site and on-device pre-treatment of samples. Some form of molecular separation or enhanced sample concentration was usually required to accurately process samples for diagnostic purposes [2]. In addition, this pre-treatment usually required some equipment that was both non-portable and difficult to operate. However, microfluidic technology has allowed for some of these pre-treatment techniques to be automated and contained within the test itself, for improved on-site capabilities [11]. The separation of unwanted molecules, from the sample, and the enhanced concentration of the target analyte could now be performed on-device and increase accuracy and sensitivity. One way in which microfluidics can facilitate this is by introducing a mixture of channel designs and reagents into the microfluidic device to initiate this pre-treatment of samples, directly inside the microfluidic device. The self-containing design principle intrinsic to most on-site microfluidic systems readily facilitates this feat and can be integrated into a myriad of diagnostic techniques such as qPCR [16]. Furthermore, by integrating these on-device pre-treatment steps into each individual test, the microfluidic analyzer would be able to capture and process data in real-time. Thereby, reducing some operational costs while improving the assay’s sensitivity and limit of detection (LOD) significantly. By enhancing the modularity of diagnostic tests through microfluidic integration and a portable microfluidic analyzer, not only can the number of operators and trained personnel be reduced but can also provide quantitative analysis.

4.2.1.3 Logistics

Diagnostic platforms integrated with microfluidic systems may offer advantages over other non-microfluidic platforms such as portability, size, structural integrity, and reproducibility that can reduce logistical costs associated with the mass-production and distribution of tests. One of the most important measures of on-site diagnostic validity is the ability to efficiently reproduce, store and distribute tests without affecting quality or accuracy of the device [19].

Paper-based and polymer-based microfluidic designs offer a greater amount of reproducibility and modularity compared to non-microfluidic tests [2]. The moulds used to imprint upon the paper and polymer substrates in microfluidic devices can be readily fabricated through methods such as lithography and etching [2]. These fabrication methods allow for microfluidic devices to remain relatively small and highly reproducible. Despite the moulds requiring expensive equipment to fabricate, only a few moulds need to be made to continuously produce a large number of microchannels for the intended devices. Thus, testing devices can be manufactured at high capacity. By separating the testing platform and the analyzer, one can significantly reduce costs associated with quality control and large-scale production.

Storage of the diagnostic tests depends on both the reagent lifespan used in the tests as well as the rigor of the test itself. Reagents used in diagnostic analysis often have expiration dates that are meant to limit quality control issues in the reagents themselves, which may lead to inaccurate testing results [22]. In general, most diagnostic reagents have a recommended shelf-life of 1 year, which would provide ample time for a microfluidic-based device to be stored and distributed with little worry. The additional rigor added to diagnostic devices through microfluidic integration refers to the thermostability and structural integrity of the devices themselves. If a test must be stored and transported in a temperature-controlled environment, the logistical costs for transporting those tests would significantly increase. Similarly, if the tests are not structurally sound, the need for more delicate transportation would also add to the costs of storing and transporting the tests. Thus, microfluidics offers a useful advantage in both scenarios as the self-containing principle behind ideal microfluidic diagnostic devices, can be applied. Therefore, through microfluidic integration, these important logistical metrics may be readily met, promoting continued mass-production of diagnostic tests.

4.2.2 Decreased turnaround time

Prior to the advent of the technological revolution of the 2000s, microfluidic technologies were extremely limited in their automation capacity due to many of the automation-driven adaptations being hindered by the cost of computational processing, the size of the equipment required to process the experiments, and the time required for the experiment to complete [21]. The increased accessibility for computational processing and digital analysis over the last two decades has allowed microfluidics to achieve significant improvements in diagnostic technology, such as increased automation and high throughput analysis. Turnaround times in both microfluidic and non-microfluidic diagnostic tests have significantly improved over the last 2 years, from times greater than 1 week to less than 30 minutes [25]. Microfluidic automation and high throughput processing improved transmission prevention, and potentially improved treatment outcomes might be possible by more accurately diagnosing SARS-CoV-2 at an earlier stage.

4.2.2.1 Automation

Microfluidic integration of digital automation can allow for several simultaneous processes to occur, which can exponentially decrease the time it takes to analyze a certain number of samples. Greater computational processing power and more robust software and hardware have provided microfluidic technologies with the means to explore previously unfathomable feats in diagnostic testing [23, 24]. Inconsistencies such as human error in sample analysis or device fabrication can more easily be avoided, further reducing costs to the producer. This in turn will create a greater incentive for microfluidic research, further driving down prices and effectively creating a feedback loop. Equipment can be coded to operate machinery, treat samples, and even provide qualitative and quantitative insight that would have been difficult for operators to observe. Through automation, the reliance on manual operation, experienced operators, and expensive equipment for diagnostic techniques is severely mitigated and can be adapted to process samples at speeds impossible for humans to do, otherwise known as high throughput processing.

4.2.2.2 High throughput processing

The benefits of high throughput processing achieved through microfluidic integration can be easily observed in microfluidic-based techniques such as droplet microfluidics. In droplet microfluidics, hundreds if not thousands of individual droplet microenvironments can be formed to initiate microscopic bioreactions in series, which can be later processed or analyzed in parallel [23, 27]. The processing speed in which high throughput analyses works, coupled with the micro-scale volumes used in microfluidics, presents a unique advantage that is not easily achieved in other detection methods [24]. Therefore, by integrating high throughput analysis through microfluidics, exponentially shorter turnaround times can be achieved.

4.2.3 Accuracy and LOD

The effectiveness of conventional non-microfluidic diagnostic techniques can often be hindered by their limit of detection (LOD) and their accuracy [19]. Microfluidic devices can, in some cases, overcome these limitations. Accuracy is defined by the ability of the test to correctly discern between a state in which a target condition is met and when that target condition is not met [2, 19]. By establishing benchmarks for accuracy in non-microfluidic diagnostic techniques, we can compare it to the accuracy seen in similar microfluidic techniques. In many situations, since microfluidic integration does not necessarily change the detection method primarily used, the accuracy might not significantly improve. In most immunohistochemical assays, the accuracy of a diagnostic test varies significantly with differences in viral loads [17, 18]. As the concentration of virus particles increases, so does the accuracy of most immunohistochemical tests, not necessarily in a linear manner. Conversely, through microfluidic implementation, a pre-treatment step can be applied to concentrate and separate the target molecules from other unwanted material into standardized volumes [22]. In doing so, the variance in accuracy from samples with low viral loads can be mitigated. The modularity of microfluidic systems may offer a means to improve the LOD, as well as improved accuracy.

Target molecules at low concentrations found in macro-scale volumes of sample fluid are not always readily detected by non-microfluidic devices [23, 24, 25]. This limitation of non-microfluidic devices becomes more important when dealing with molecules that are not easily replicated by conventional means, such as protein biomarkers found on the surface of a SARS-CoV-2 cell. As a result, single-cell-resolution droplet microfluidic systems can be designed to generate thousands of microlitre droplet microenvironments to capture biomarkers, in extremely low concentrations, normally too difficult to detect through conventional means [27].

5. Standards for ideal on-site diagnostic microfluidic devices

Microfluidic devices that would like to be tested and sold as on-site diagnostic devices, must first adhere to a standardized set of criteria set out by the WHO. The WHO published a list of criteria that states that on-site diagnostic devices must be “affordable, sensitive, user-friendly, rapid and robust, equipment-free and deliverable to end-users” [2]. These criteria are the 6 basic standards that prospective on-site diagnostic devices should strive to achieve, otherwise known as ASSURED. The ASSURED criteria allow researchers and healthcare professionals to assess the effectiveness of prospective on-site diagnostic devices [2]. The six criteria can be further divided into two main categories for evaluation, the technical and the practical criteria. The technical criteria include: “sensitivity”, “rapid and robust”, as well as “equipment-free”. While the practical criteria include: “affordable”, “user-friendly” and “deliverable to end users”. While some criteria are not necessarily related to one another, they are nevertheless all equally important in designing an idealized on-site microfluidic device. However, the ASSURED criteria are not easy to be simultaneously and effectively implemented. Therefore, to improve the overall potential of a microfluidic device as an effective on-site diagnostic tool, several other more measurable criteria must also be addressed; as seen in Figure 7.

Figure 7.

The 7 pillars for assessing effective on-site diagnostic devices. Adapted from Ref. [2] with permission.

Effective diagnostic microfluidic devices that manage to address the ASSURED criteria, as best as possible, may still struggle to measure how they are addressing these criteria [2]. Figure 7 summarizes a list of seven more measurable criteria that simplify and quantify the characteristics that best represent an ideal on-site microfluidic device used for diagnostic purposes. These “seven pillars” of integrated microfluidic device design will aid in providing developers with quantifiable metrics to better evaluate their device’s efficacy and on-site diagnostic potential.

6. Current state of microfluidic systems in SARS-CoV-2 diagnostics

Advancements in diagnostic technology over the past 2 years have allowed for infectious diseases, such as SARS-CoV-2, to be more readily and accurately diagnosed [2, 9, 11]. Improvements made to costs, accuracy, turnaround times, and processing speeds in non-microfluidic diagnostic tests have greatly improved the ability of healthcare workers and governments to better prevent and manage mass outbreaks [12]. For example, rapid antigen tests have demonstrated their effectiveness in reducing the number of potential secondary infections by providing people with a qualitative means of diagnosing for COVID-19 [14]. However, this does not mean that these tests and their detection technology cannot still be improved upon. Microfluidic technology offers a means to continuously advance the world’s diagnostic capabilities and preparation for the next potential pandemic-level virus.

The past decade has demonstrated the effectiveness of microfluidics in the on-site diagnosis of various infectious pathogens, such as Zika, HIV-1 and more recently SARS-CoV-2 [2]. The errant lack of tests, medical devices, and human resources seen throughout the pandemic, prompted a large increase in the demand for technology capable of being remotely operated and readily analyzed [18, 20]. Advancements in information technology and computational processing created a revolution of new approaches by which microfluidic devices can accurately diagnose COVID-19 patients, on-site [21, 24]. Figure 8 highlights several of the most current, digitally integrated, microfluidic platforms capable of diagnosing SARS-CoV-2.

Figure 8.

Various microfluidic diagnostic devices for SARS-CoV-2 [25].

In many of the diagnostic platforms highlighted in Figure 8, smartphones are used as an imaging processor to read fluorescent signals through machine learning and artificial intelligence [25]. With the ever-increasing global access to the internet, smartphone-enabled microfluidic diagnostic devices can produce results that can be uploaded to a data-cloud to be immediately stored. Moreover, these smartphone-enabled devices can reduce the turnaround time for qualitatively diagnosing SARS-CoV-2 to, on average, less than 15 minutes.

Despite the processing speed advantages which smartphone-enabled systems offer, there is still unmet need for faster and more robust microfluidic devices capable of quantitative analysis [26]. As seen in Table 2, several types of existing quantitative microfluidic diagnostic tools are slowly becoming more comparable even the most current rapid diagnostic tests for SARS-CoV-2. It is only a matter of time before quantitative microfluidic-based tests will be able to either perform comparably, or better, than the rapid antigen tests.

	Immunoassay (rapid antigen test)	RT-PCR	Nanoparticle	Microflow Cytometry
Reagent consumption	10 μg (in tube)	20 μL (in tube)	Negligible	50 μL (in tube)
Target of detection	IgG, IgA, IgM	N gene, E gene	Gold-spiked	IgM, IgG
Limit of detection	0.4 ng/L	1–10 copy/μL	0.08 mg/L	0.06–0.10 mg/L
Total assay time	30 minutes	2 hours	2–5 hours	30 minutes
Sample volume	20 μL	120 μL	1 μL	10 μL
Assay control	Automated	Manual	Manual	Automated
Cost per test	~ 6 (USD)	~ 4 (USD)	~ 10 (USD)	~ 5 (USD)
Quantitative	No	Yes	Yes	Yes
Mobile	Yes	Yes	No	No

Table 2.

Comparison of rapid test to microfluidic diagnostic tests.

Adapted from Ref. [2] with permission.

7. Conclusions

This chapter deals with the experience of COVID-19 over the last 2 years and highlights the significance of microfluidics to the history and advancement of SARS-CoV-2 diagnostic technology. In these years, conventional rapid PCR and ELISA COVID-19 technology has advanced greatly. However, limitations in their modularity, sensitivity, turnaround time and cost greatly reduce their future viability as on-site diagnostic tools. The current state of microfluidic, information and smartphone technology allow microfluidic-based diagnostics to address many limitations associated with conventional on-site/rapid tests. The advantages of microfluidic integration into medical diagnostics are discussed throughout this chapter. The expectation is that microfluidics will advance our future diagnostic abilities to help better prepare for, and manage, the next possible pandemic-level threat.

Acknowledgments

The authors would like to acknowledge Alexander Diab-Liu and Kamaya Bosland for their help in proofreading this work.

Conflict of interest

The authors declare no conflict of interest.

Notes/thanks/other declarations

Thank you to Dr. Xu and IntechOpen for the opportunity to work on this chapter.