**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 [1–4]. 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.

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

*Perspective Chapter: Microfluidic Technologies for On-Site Detection and Quantification… DOI: http://dx.doi.org/10.5772/intechopen.105950*

#### **Figure 2.**

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

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].
