Microfluidic Platform for COVID-19: History
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Contributor:
Qi Li
,
Xingchen Zhou
,
Qian Wang
,
Wenfang Liu
,
Chuanpin Chen

Spread of coronavirus disease 2019 (COVID-19) has significantly impacted the public health and economic sectors. It is urgently necessary to develop rapid, convenient, and cost-effective point-of-care testing (POCT) technologies for the early diagnosis and control of the plague’s transmission. Developing POCT methods and related devices is critical for achieving point-of-care diagnosis. The POCT devices based on microfluidic technology on the market, including paper-based microfluidic, centrifugal microfluidic, optical fluid, and digital microfluidic platforms.

  • microfluidic
  • COVID-19
  • molecular assays

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the global spread of coronavirus disease 2019 (COVID-19) [1][2], and the World Health Organization reported that, by August 2022, more than 620 million infections and more than 6.5 million deaths had been confirmed worldwide [3]. Characteristics such as a rapid and widespread transmission, an uncertain incubation period, and nonspecific symptoms are why this outbreak is challenging to control [4][5][6]. Furthermore, as the epidemic progressed, the genetic material of the virus was altered, which affected the rate of transmission, the degree of symptoms after infection, and the effectiveness of drugs and vaccines, ultimately making the epidemic more difficult to control [7]. So far, the epidemic has lasted for nearly three years, during which the blow to both global public health and the economy has been tremendous. On the one hand, the rampant spread of the virus has posed a severe threat to human life and health. On the other hand, the shutdown and repeated closures to control the epidemic have seriously slowed down the global economy; thus, it is worth thinking about how to prevent and control COVID-19 and promote economic recovery effectively.
There are three main ways to control COVID-19 as an infectious disease: controlling the source of the infection, cutting off the channels of transmission, and protecting susceptible populations [8][9][10][11][12]. Among these approaches, the first one is considered the most important. The main sources of infection of COVID-19 are confirmed cases and asymptomatic patients, and the early detection, isolation, and treatment of these patients is necessary to effectively control the infectious source. However, the uncertain incubation period and the presence of asymptomatic patients make this initiative very difficult; therefore, it is of great importance to develop rapid, sensitive, and specific field detection methods and kits [13]. COVID-19 detection is divided into two main categories: molecular detection methods for detecting nucleic acids of SARS-CoV-2 and immunoassays for detecting antibodies or antigens [14]. Molecular detection methods usually use respiratory samples, such as nasal or pharyngeal swabs, under laboratory conditions, and the most commonly used method is reverse transcription polymerase chain reaction (RT-PCR). Isothermal nucleic acid amplifications have also been widely studied due to the fact of their advantages of no thermal cycler, simplicity, and rapidity, which make them more suitable for field detection. In immunoassays, antigen detection is often performed using nasal swabs or pharyngeal swabs as a complementary method to nucleic acid detection, while antibody detection is performed using serum or whole blood as a sample, mainly as a classification and screening tool for IgM/IgG potency [15]. Both molecular detection and immunoassays require specialized personnel in the laboratory, which is time consuming, and they require complex instruments and professional operation. In this context, the market demand and outstanding advantages of point-of-care testing (POCT) products have led to the rapid development of this industry. The ideal POCT system should be user friendly, easy to use, and low cost, with high clinical sensitivity, specificity, and accuracy, and, more importantly, provide immediate results [16], which would make them suitable for large-scale immediate diagnosis, enable rapid detection, and provide timely results in primary hospitals and communities for early diagnosis, isolation, and treatment.
Microfluidics is a powerful technology for the realization of POCT diagnosis, which can integrate and miniaturize the multiple steps involved in sample detection on a small chip. Furthermore, due to the small reaction chamber of the chip, the consumption of detection reagents and sample volume is also small, which can effectively reduce costs. In addition, microfluidic chips have the advantages of high throughput and less contamination. The combination of microfluidic technology with traditional laboratory assays is expected to lead to automated POCT, monitoring of COVID-19 disease transmission in a potential home diagnostic model, and provide new solutions for both developed and developing countries [17].

2. Microfluidic Platform

2.1. Passive Microfluidic Platform—μPADs

Passive microfluidic technology can be divided into capillary driving and gravity driving. Passive microfluidic technology has many unique advantages. Firstly, passive microfluidic platforms are usually easier to make and easier to expand production. Secondly, a passive microfluidic platform is usually easier to operate and can conduct self-tests at home without the operation of professional technicians. Thirdly, compared with an active microfluidic platform, the passive microfluidic platform does not need an external power supply, so the cost is lower, and it is undoubtedly a promising platform for areas with limited resources.

Microfluidic paper-based analytical devices (µPADs) are a representative passive microfluidic platform, which use patterned paper as the carrier and distributes liquid through capillarity. μPADs are suitable for multiplex assays of various samples and are compatible with various assays. In contrast to traditional lateral flow strips, μPADs can autonomously drive multistep sequences, while the timing of the reagent’s movement can be controlled by adjusting the length and fluid volume of each channel [18]. Compared to traditional microfluidic labs, using paper sheets as a carrier also has unique advantages. Firstly, paper sheets have good biocompatibility, easy absorption of reagents, and are suitable for clinical analysis. Secondly, the inherent white background of paper sheets can provide contrast for color-based methods. Thirdly, paper is an ideal material for manufacturing environmentally friendly portable equipment because of its lightweight and low cost. Based on these advantages, μPADs have received attention in POCT applications, with rapid POCT detection of infectious diseases, especially in resource-poor areas, being one of the most popular areas. Herein, the following will discuss the development of μPADs for COVID-19 POCT.

Some researchers have made great efforts in the system integration, portability, and operation procedure simplification of paper-based microfluidic analysis. Li et al. designed a paper-based electrochemical impedance sensor by synthesizing ZnO nanowires directly on the working electrode by the hydrothermal growth method on the μPADs [19]. This paper-based electrochemical impedance spectroscopy solves the problems of the paper-based enzyme-linked immunosorbent assay (ELISA) method that requires labeling and extra steps to amplify the signal but retains the advantages of the simple fabrication and low cost of μPADs, and the whole process can be completed within 30 min. It is simple, environmentally friendly, and fast, making COVID-19 antibody testing easier.
In addition, some researchers have worked hard to improve the automation and sensitivity of the detection method. Yin et al. designed an automated paper-based microfluidic multiplex detection platform that combines clustered regularly interspaced short palindromic repeats (CRISPR) technology and recombinase polymerase amplification (RPA) to detect COVID-19, with a total detection time of less than 1 h and a sensitivity of 100 copies, which is comparable to RT-PCR [20]. The paper-based microfluidic chip’s CRISPR detection chamber is designed and fabricated using wax-printing technology. The printed RPA reaction chamber is connected to the CRISPR detection chamber using a programmable, normally closed paper-based sucrose valve, which automatically opens when a preset time is reached. At this time, the RPA amplicon migrates to the CRISPR detection chamber, enabling the automatic transfer of products, and carry-over contaminations are reduced. Furthermore, the μPADs can be stored at room temperature for 30 days without needing cold chain transport, significantly improving the feasibility of detecting SARS-CoV-2 infection in the field. In conclusion, this experimental platform enables simple, low-cost, and automated multigene detection and is a meaningful research attempt to detect COVID-19 in resource-limited areas.
Compared with traditional microfluidic platforms, μPADs are more convenient to use, less expensive to manufacture, simple, and nonpolluting for post-processing. They can be performed by simple and safe combustion, but using paper as a carrier provides benefits but also has some limitations. For some samples with low surface tension, the hydrophobic zone is not necessarily hydrophobic enough and may be prone to leakage; in addition, samples tend to remain in the paper channel leading to low sample utilization; most importantly, the paper chip is still lacking in the issue of sensitivity.

2.2. Active Microfluidic Platforms

2.2.1. Centrifugal Microfluidic Platform

Centrifugal microfluidics is a kind of microfluidic system that uses centrifugal force as the driving force of the liquid flow to realize reagent detection and analysis. Centrifugal microfluidics has many advantages. First, centrifugal forces are present on all surfaces of the disk (Figure 1), so liquid transport is simple and efficient. Secondly, the physical and chemical properties of the sample have little effect on the microfluidic panel, which is good for body fluid samples. Thirdly, centrifugal microfluidics can be highly integrated. The whole test process, such as sample pretreatment, mixing, and valve control, can be realized on a single disk [21][22]. For example, Malic et al. fabricated a disk for the rapid detection of SARS-CoV-2 from whole blood based on a centrifugal microfluidic platform [23]. The priming enzyme and reaction buffer were prefixed in the reaction chamber, and the disk could be rotated in different directions by the spindle motor to move the solution to the waste chamber or collection chamber for purification, amplification, and detection. The device was subsequently shown to successfully detect SARS-CoV-2 in whole blood samples from purified samples to result in a limit of detection (LOD) as low as 0.5 copies/uL. Moreover, the platform could complete the process in less than 60 min. All these device characteristics demonstrate its potential for the diagnosis of SARS-CoV-2 by POCT in resource-poor areas.
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Figure 1. Schematic diagram of the centrifugal chip principle.
Centrifugal microfluidics has great advantages in sample mixing, step simplification, and high integration, but in order to further promote it, efforts need to be made to reduce the volume of equipment and reduce the use of external equipment. Dignan et al. developed the first centrifugal microdevice to prepare high-purity nucleic acids comprehensively from virgin oral swab samples [24]. Unlike previous centrifugal microfluidic devices, this system automatically uses controllable laser-on-board microvalves instead of passive valves sensitive to temperature fluctuations and changes in channel surface energy, providing better reproducibility under various environmental conditions and achieving tight spatiotemporal control of the fluid flow. In this study, centrifugal microfluidic technology was combined with dynamic solid-phase extraction to achieve on-disk extraction of DNA or RNA and with loop-mediated isothermal amplification (LAMP) technology to fabricate an automatable, portable microfluidic platform-based nucleic acid preparation device, which allows for its practical use in the field by nontechnical personnel.

2.2.2. Optical Fluid Microfluidic Platform

As an emerging technology research field over the past decade, optical fluid is very suitable for the biochemical analysis of small volume analytes. Combined with traditional detection methods, optical microfluidics can accurately control, operate, and monitor the analysis process in real time. For example, Sampad et al. developed an optical flow control platform integrated with FPGA, which was used to detect and analyze single-labeled biological particles in the flow while extracting the target concentration and other experimental parameters [25]. The real-time implementation by the customized Verilog program processes the photon counting of the signal in three parallel blocks, thus eliminating the time limit of the serial processing scheme. This method is proved by real-time analysis of fluorescent nanorods and single bacterial plasmid DNA. Compared with the post-processing analysis in MATLAB, the target detection is reliable, and the accuracy is 99%. Moreover, the real-time detection of each target can accurately determine the clinically relevant concentration between 3.4 × 104 and 3.4 × 106 per milliliter in seconds to minutes. Furthermore, the optical flow control platform can use many optical properties, including refractive index and fluorescence and Raman scattering, for signal detection alone or in combination, which is worth considering to improve the sensitivity of the detection signal.

2.2.3. Digital Microfluidics Platform

Digital microfluidics is a disruptive technology for the design, integration, and operation of microfluidic systems based on a single droplet that utilizes precise droplet control in the microliter to nanoliter range to enable complex laboratory analyses. Digital microfluidic devices usually adopt a dielectric wetting method for droplet bioanalysis, which has the advantages of a simple structure, high sealing, avoidance of cross-contamination, and a high degree of automation. For example, Ho et al. proposed an N gene detection platform using SARS-CoV-2 based on digital microfluidics technology [26]. N1 and N2 primers and probes were used, and the chip integrated electrical, thermal, and optical modules to achieve uniform temperature control and ideal fluorescence reading. The reagent volume of this platform is only 1.5 uL, which is more than 13 times smaller than the traditional desktop PCR instrument, but the amplification performance is not affected, which is comparable to that of the desktop PCR instrument.
According to the different driving forces of the fluid, these contents briefly introduce the principles, advantages, and improvement directions of paper-based microfluidic, centrifugal microfluidic, optical, and digital microfluidic platforms. Compared to traditional laboratory instruments, the development of these microfluidic platforms is clearly necessary for resource-constrained areas. However, there are still many difficulties for microfluidics to gain further adoption in the COVID-19 testing market. Firstly, most active microfluidic platforms still require a lot of power, which is not friendly to areas with power shortages. Secondly, although a passive microfluidic platform does not require an external pump, compared with an active microfluidic platform, the control ability and accuracy of a passive microfluidic system inevitably have some deficiencies. Finally, with the microfluidic pursuit of miniaturization and low cost at the same time, the performance of the system may decline, which is an important issue that must be balanced.

This entry is adapted from the peer-reviewed paper 10.3390/bios13020163

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