key: cord-0804568-2p7z2xs5
authors: Berkenbrock, José Alvim; Grecco-Machado, Rafaela; Achenbach, Sven
title: Arsenal of microfluidic testing devices may combat COVID-19 pandemic
date: 2020-09-27
journal: MRS Bull
DOI: 10.1557/mrs.2020.181
sha: b9e9a11aae7831fe52d16dbff8c8f48bf6f1f6eb
doc_id: 804568
cord_uid: 2p7z2xs5

nan

tion. The high number of cases around the world led the World Health Organization (WHO) to declare a pandemic situation on March 11, 2020 . Although viruses have a variety of arrangements and compositions, they are generally composed of genetic material (DNA or RNA), a protein shell, and a lipid bilayer. Detection approaches focus either on the proteins that form the shell or on the genetic material and its sequence of nucleotides, the building blocks of DNA and RNA.

The first publications on SARS-CoV-2 identified it as an RNA-based virus of the order Nidovirales, the family Coronaviridae, and the genus Betacoronavirus. Although many coronavirus infections in humans cause only mild symptoms, SARS-CoV-2 was determined to be similar to other aggressive strains, such as the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and

the Middle East Respiratory Syndrome coronavirus (MERS-CoV). The similarities among SARS-CoV-2, SARS-CoV, and MERS-CoV provided scientists with a strong indication of where to start working. Even before samples from patients infected with SARS-CoV-2 became available, Christian Drosten, director at the Institute of Virology at Berlin's Charité University Hospital, together with an international research group, published a WHO-endorsed diagnostic protocol for SARS-CoV-2 based on its similarities to other viruses. 1 Currently, new information about SARS-CoV-2 continues to be published daily. To tain the spread of SARS-CoV-2 have been social distancing, testing widely, isolating and closely monitoring infected patients, and tracing the people who had contact with them (See Figures 1 and 2 ).

Testing is key to identifying infected individuals so they can be isolated and treated and so that deaths related to the limited capacity of health systems can be avoided. A news feature published in Nature observed how the United Arab Emirates, Singapore, and South Korea required their test kits and required extensive testing of the population as well as the tracing of each patient's circle of contacts. 2 A variety of experiments are being developed to test for COVID-19, but the technique known as real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) is the most widely used. Recommended by both the WHO and the US Centers for Disease Control and Prevention (CDC), this test uses a biological sample extracted This sample is tested for the presence of viral genetic material. Some of the key elements necessary for qRT-PCR are thermocyclers to amplify the complementary DNA, primers to bind to the target seto function correctly. These elements genetic material in the patient's samples to obtain a measurable result.

Despite the ability of the qRT-PCR test to detect viral material in patient samples, it has substantial drawbacks. First, the test is a lengthy process. Although in the best cases reported, the time to collect samples, perform the reaction, and analyze the results takes from 60 minutes to 4 hours, this process typically takes a few days to complete. Other time-limiting factors include extracting the sample from the patient, storing and delivering the sample to the testing center, and reporting results to the physicians who requested the test (not always an online process).

Second, shortages of biotechnology primers can, as has occurred during the current pandemic, result in a bottleneck for widespread testing of populations. Third, only a few companies have the capacity to produce the qRT-PCR test kits. The rapid spread of the virus has obliged governments to import large amounts of the test kits. While larger economies can import tests, many countries are facing shortages and have not been able to perform enough tests in their populations. These limitations on the production of screen tests have reduced the chances of punctual isolation, jeopardized positive outcomes for thousands of patients, and have become a potential threat to the safety of thousands of people worldwide.

Alternatives to the problematic benchtop assays such as real-time RT-PCR can be fabrication. Advancements achieved by the electronics industry have allowed for with at least one dimension in the mideals with devices with dimensions in the nanometer range.

In the last few decades, this field has rapidly evolved to create new solutions for health-related applications. The sciences has become evident in lab-ona-chip, organs-on-a-chip, and point-ofcare testing (PoCT) devices. Sometimes referred to as "bedside testing," PoCT devices facilitate the extraction of information from patient samples without extensive laboratory work. Although these devices do not always achieve the same levels of sensitivity and reliability as the well-established laboratory assays, they have several advantages over traditional methods: the ability to extract information from reduced sample volumes; decreasing the need for reagents, waste, and sometimes energy consumption; shorter reaction times since smaller volumes are processed; and parallel operations for multiple concurrent detections.

Several studies have demonstrated the versatility of this class of devices. Currently, at elements to indicate detection and signal to the user presence of the target element cent elements, called labels, such a group of devices have the basic triad of binder, 

contained in the sample. The wells without the target virus remained shiny while those with samples containing the target became dark. The detection principle is based on reverse transcription loop-mediquiring more primers to bind the target. Additionally, while PCR needs multiple temperature cycles, LAMP requires only one temperature. Thus, LAMP can use a simpler technology apparatus that allows portable equipment to perform PoCT.

Another example of portability potential is evident in work by Mengqi Kong of China and Singapore. They developed a wearable device to identify HIV (AIDS) that used the body's temperature to sup- 4 The copies of viruses in a sample were increased with the heat produced by the human wrist (33-34°C). Since the patient samples may contain only a few copies of the virus, number of copies and facilitates detection and signaling. This polymer-based device detected a concentration of 100 copies of HIV-1 per mL of solution in a 24-minute-long assay. The research team developed a smartphone-based scanner Another 2019 study, by Carlton F.O. Hoy of The University of Tokyo and colleagues from Japan, focused on delinked immunosorbent assay. 5 The research team used a polymer cassette to perform a MERS immunoassay and used immobilized His-MERS-NP (protein) to detect anti-MERS-NP (antibodies) in samples. Although experiments with patient samples are still to be done, the initial laboratory results showed a fair limit

Label-free detectors are a class of devices that avoid extra reagents for assays by usthe Universidade de São Paulo in Brazil presented an electrochemical DNA bio- 6 Their sensor is based on gold electrodes functionalized with thiol chemical groups. Thiol functional groups form a self-assembling monolayer on gold surfaces that facilitate the attachment of antibodies (or DNA probes) to the device (see Figure 4) . The research group used impedance analysis to observe the variations caused by adding chemical layers (thiol), binding elements (DNA detection by the electrodes. Similarly, P.R. Bueno and colleagues at São Paulo State University presented a device to detect DENV based on the electrochemical capacitive method. 7 They proposed a new label-free and reagentless methodology based on a ferrocene-tagged peptide attached to antibodies. The proposed detection principle was based on altering the surface capacitance of the electrode upon antigen-antibody binding.

The two research groups were able to identify a low concentration of target elements without using labels or amplielectronics were necessary to process the electrical measurements. Both teams used electrochemical impedance spectroscopy to analyze the samples on their electrode DNA (i.e., NS5), while Buenos's team aptein (i.e., NS1) in the sample. For SARSalready used for qRT-PCR may be adapted free detectors can reduce the number of reagents and be extended to platforms for the detection of multiple targets.

that rely on well-established techniques such as electrochemical measurements and immunoassays, emerging technologies and innovative micro-and 9 The research team developed a porous membrane funcsingle-stranded RNA called oligoprobes to detect and discriminate four subtypes of DENV (DENV-1 to 4). The team was able to detect down to 100 copies of RNA per mL in human plasma in about 90 minutes. In the proposed platform, a single PCR was performed to interrogate the sample against the four subtypes of the virus. Then the revealed the presence of the target subtype. An ion-selective membrane was attached to each of the reservoirs to selectively detect the virus subtypes by a system monitoring the ion current through the membranes.

The fabrication of membranes with require sophisticated technological equipment such as focused ion beams and cleanroom facilities. Vincent Tabard-Cossa and colleagues from the University of Ottawa proposed an ingenious new technique for the fabrication of nanopores in silicon nitride membranes (SiN x ), which was described in the journal Nature Protocols. 10 tion around a SiN x membrane, the research team opened pores with diameters of 1-20 nm. Their work has shown promising results in detecting generic double-and single-stranded DNA. The team suggested that this fabrication method could democratize access to nanopore-based membranes for researchers globally. Researchers from Imperial College London presented another way of making the fabrication of microdetectors more accessible in a non-peer-reviewed paper recently made available at bioRxiv. 11 Corresponding authors Estefania Nunez-Bajo and Firat Güder, with colleagues also from the University of Turku (Finland) and Moredun Research Institute (Scotland), proposed a silicon-based microdevice to chemically amplify and detect pathogenperiments were performed with synthetic SARS-CoV-2 since patient samples were not available. Also, the manuscript detailed the fabrication of the chip and electronics setup to enable easy replication of the device and use for screening. These last two works demonstrate simple fabrication processes that can facilitate the widespread production of PoCT devices.

Although it may take some time for microto become widely available, the experience As Drosten and colleagues were able to establish a detection protocol even before they had viral samples, it is expected that engineers will be able to use the knowledge as summarized to create SARS-CoV-2-

The similarities among viruses and the need to be harnessed to generate rapid tests that are more widely available, easier to use and transport, cheaper to produce, and quicker to generate results than current tests. Critical situations, such as the current pandemic, tend to propel the development of technologies for innovative approaches to detect viruses and explorations of the capabilities of micro-and nanotechnology. New small devices designed to rapidly collect information from a drop of blood or saliva are being developed to detect a variety of diseases and biomarkers. These innovations will accelerate diagnosis of disease, so crucial in a pandemic.

bioRxiv (forthcoming)