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

**Figure 3.**

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

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.

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 healthrelated 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.
