Coronavirus (SARS-CoV2) diagnostic with microfluidics

Morphology of Coronavirus SARS-Cov2. Source image: Centers for Disease Control and Prevention.

Coronavirus (COVID-19) information in short:

• The COVID-19 form of coronavirus is an infectious disease that can cause serious illness, especially in the elderly, and in those with underlying medical problems
• We know the virus’ genetic sequence, as well as its morphology and transmission mechanism
• Coronavirus diagnostic tests are being currently developed
• Microfluidics are for infectious disease modelling and treatment, and can be applied to COVID-19 as well

1. Introduction: diagnose coronavirus

Coronavirus disease (COVID-19) is an infectious disease that will cause mild to moderate respiratory illness in most people. It can cause serious illness in older people, and those with underlying medical problems like cardiovascular disease, diabetes, chronic respiratory disease, and cancer [1]. The best way to prevent the spread of this strain of coronavirus is by understanding its way of transmission and taking the right preventative measures.

Since coronavirus spreads primarily through droplets of saliva, or discharge from the nose when an infected person coughs or sneezes, it is important to follow a strict respiratory etiquette (for example, by coughing into a flexed elbow) and protecting yourself and others from infection by washing your hands or using an alcohol-based rub frequently and not touching your face. Currently, no specific vaccine or treatment against coronavirus infections exists, but several clinical trials are ongoing. Coronavirus infections are diagnosed based on the genomic sequence of the virus, its morphology and the specific symptoms of the disease. Through incorporation of microfluidic technology, classic biochemistry-based tests can be made faster and requiring less sample volume. In the future, infections can be modeled in-vitro using human cells and tissues and even replicating connected organ systems through microfluidics.

2. Coronavirus diagnostic tools

After the first occurrence of pneumonia case clusters were linked to a seafood and animal food market in Wuhan, China, several research groups set out to identify the cause.

Ultimately, draft genome sequences of the virus originating from different patients were deposited in the GenBank sequence repository and in the database Global Initiative on Sharing All Influenza Data (GISAID). The coronavirus genome sequence was shown to share over 85% sequence identity with a known SARS-like coronavirus found in bats. It was shown that the virus uses the angiotensin-converting enzyme II (ACE2) to enter host cells, as did SARS-CoV, which infected 8,096 people and caused 774 deaths in a previous outbreak [2]. Since the coronavirus genome was known to the scientific community, the first diagnostic tests focused on the use of sequence-specific primers for polymerase chain reaction (PCR). Those tests were developed rapidly, based on previous technologies used for the detection of former SARS-CoV outbreaks. After initial development at research centers in the absence of commercial tests, companies began with the commercialization for a rapid supply of coronavirus tests.

First, the lab of Christian Drosten, Charité University Hospital, Berlin, developed a real-time PCR (RT-PCR) diagnostic test which formed the basis of 250,000 kits. The World Health Organization (WHO) dispatched them to 159 laboratories across the globe in the past few weeks [3]. At the same time, Chu et al., at the Hong Kong University developed two one-step quantitative RT reverse transcription PCR tests targeting both the open reading frame 1b (ORF1b) and the N regions of the viral genome based on the first sequence deposited at GenBank [4].

In addition to lab tests, several companies have started developing coronavirus detection tests on a commercial scale (for example BGI Group, China; Altona Diagnostics, Germany).

In addition to traditional PCR-based tests, some companies, such as IDbyDNA in the USA, prefer to use metagenomics for coronavirus detection – real-time monitoring through sequencing. Although more expensive than PCR testing, the approach promises real-time monitoring of what is in circulation and thus a rapid update should the viral genome evolve. Monitoring of genetic variations also can give information on the phylogenetic relationships and can help tracking the outbreak of the epidemic [5].

3. Microfluidics to detect coronavirus

Polymerase Chain Reaction (PCR)

As mentioned above, most current coronavirus tests are based on PCR and RT-PCR (Real-Time Polymerase Chain Reaction). Primer and probe sets are designed to react with RNA from the novel COVID-19 coronavirus and its closely related viruses, such as SARS coronavirus [4]. The PCR product has a specific size that can be detected through the emission of fluorescence signals (RT-PCR) or through classical electrophoresis. Coupled to microfluidics, PCR can be accelerated and the diagnostic test results can be obtained faster (from approximately 1 hour to less than 10 minutes) and with a higher accuracy [6].

For more insight, you can discover Elvesys’ spin-off company BeforCure. BeforCure develops an ultra-fast PCR on chip system (based on Fastgen technology) to detect pathogens such as SARS-CoV2 in less than 30 minutes.

PCR-Principle. Image source [13].

DNA-sequencing on chip. Image source [14].

DNA sequencing

DNA sequencing on chip takes advantage of the fact that DNA binds to its complementary sequence.

The approach uses immobilized oligonucleotide probes to which the target DNA binds. Several thousand short pieces of DNA of known sequence (oligonucleotides) are bound to a glass or silicon surface at pre-specified locations. Incubation with fluorescently labeled target DNA of  an unknown sequence will result in binding this DNA to a subset of oligonucleotide probes, which can be identified by their change in color (fluorescence). The sequence of the target DNA is deduced by passing this information to a computer and the target sequence is reconstituted from the patterns of oligonucleotide bound to the target [7]. Microfluidic nested PCR followed by MiSeq sequencing enables efficient tracking of the fate of multiple RNA viruses (including coronavirus) in various environments. It has a high sensitivity (detection limit ranged from 10^0 to 10^3 copies/μL in the cDNA sample) and is essential for a better understanding of the circulation of human pathogenic RNA viruses [8]. Microfluidic analyses of DNA can therefore be used in the development of coronavirus tests and gives information on the infection rate and the origin of the infection.

ELISA

Enzyme-linked immunosorbent assay (ELISA) uses an enzyme immunoassay to detect the presence of a ligand in a liquid sample using antibodies against the protein to be measured. The assay is widely used in medical diagnostics and fundamental biological research due to its high specificity and reproducibility. The traditional ELISA suffers from several notable drawbacks, such as long assay time (4–6 hours), burdensome procedures and large sample/reagent volumes (∼100 μl) which significantly limit its application in rapid clinical diagnosis [9].

ELISA assay principle. Image source [10].

Microfluidic-assisted ELISA can overcome those drawbacks. When the immunoassay is performed on the surface of a reaction chamber (the microfluidic channel), the capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Thus, sample volume can be reduced 20-fold, resulting in an overall 5-10-fold reagent saving.

In addition, the assay time can be reduced by more than 50% and thus decrease the overall cost by saving labor cost [10]. Testing against coronavirus through ELISA becomes possible, as more and more information on the virus becomes available. Several companies have already started developing diagnostic kits based on proteins expressed by the coronavirus (e.g. Biocell Biotechnology Co.).

4. Coronavirus disease modelling with microfluidics

In order to understand the infection pathway of the coronavirus and to develop treatments, scientists currently have to use classical biochemical assays, 2D cell culture, animal models and go through laborious clinical studies. This process usually takes a long time until completion, during which animal lives are lost and the process often fails to predict human responses because animal models do not always mimic human pathophysiology correctly. Novel, alternative ways to model the coronavirus infection in vitro are needed in order to allow a fast development of new drugs.

Organ-on-chip device developed by the Wyss Institute at Harvard.

Microfluidic technology allows the reconstitution of complex cellular interactions on the microscale. 3-D architecture, multicellular complexity and physiologically relevant biochemical forces can be reproduced through tissue engineering and the development of organ-on-chip technology. A major challenge in tissue engineering for coronavirus infectious disease research is recreating the cellular microenvironment including biological, chemical and physical cues [11]. Recently, microfluidic devices have been successfully employed for the development of cell-based virus assays [12]. Developing microfluidic tissue-engineering could help understand the coronavirus’ entry strategy, how the infection persists in human cells, and this could accelerate the discovery of novel anti-viral drugs.

5. Summary of coronavirus diagnostics and microfluidics

Given that the coronavirus has a high infection rate, the development of fast diagnostic tools is vital for a rapid identification and treatment of infected patients. Microfluidics can be used to improve existing diagnostic tools, to render them more accurate, efficient and less costly. In addition, microfluidic tissue-engineering can be used to decipher the viral entry strategy and to help develop active drugs.

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References

This review was written by Oore-ofe Akeredolu and Christa Ivanova, in the scope of the MSCA-IF project CAR-OAC, grant agreement number 843279.