Microfluidic-Based Smart Diagnostic Platform for COVID-19 Diagnosis: Comparison
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Please note this is a comparison between Version 2 by Jason Zhu and Version 1 by Ashutosh Kumar.

The pandemic of COVID-19 and its widespread transmission have made uspeople realize the importance of early, quick diagnostic tests for facilitating effective cure and management. The primary obstacles encountered were accurately distinguishing COVID-19 from other illnesses including the flu, common cold, etc. While the polymerase chain reaction technique is a robust technique for the determination of SARS-CoV-2 in patients of COVID-19, there arises a high demand for affordable, quick, user-friendly, and precise point-of-care (POC) diagnostic in therapeutic settings. The necessity for available tests with rapid outcomes spurred the advancement of POC tests that are characterized by speed, automation, and high precision and accuracy. Paper-based POC devices have gained increasing interest in recent years because of rapid, low-cost detection without requiring external instruments. 

  • COVID-19
  • diagnostics
  • POC testing devices

1. Introduction

Microfluidics-based POC diagnostics are valuable tools for detecting SARS-CoV-2, offering integrated platforms through the combination of various techniques on microfluidic chips. These platforms, initially commercialized for analyzing and tracking oncological disease biomarkers, have now become a reality. The current pandemic has further uncovered the boundaries of conventional PCR-based techniques, which rely on skilled personnel and centralized laboratory setups. To overcome these challenges, microfluidics-based diagnostics have shown great potential [57,58][1][2]. Significant technological progress has occurred by incorporating microfluidic-based approaches for genetic material determination in recent years [59,60][3][4]. Successful demonstrations have been conducted, showcasing the detection of viruses, genetic material testing, and outcomes visualization using simple microfluidic-based methods [61,62][5][6]. These devices have been tested for genetic material detection in COVID-19 patients, exhibiting higher overall efficiency compared to assays such as LFA and reverse transcriptase LAMP [27,63][7][8].
It is likely that SARS-CoV-2 will not be the final pandemic people have faced, highlighting the importance of technological preparedness and adaptability for future outbreaks. The need for high-throughput and rapid diagnostic strategies for SARS-CoV-2, in the form of portable POCT systems, remains urgent [64][9]. In recent years, miniaturized biosensors have emerged as promising analytical platforms because of their unique properties, rapid analysis, reliable specificity, high sensitivity, and consistent results [65,66,67][10][11][12]. As an example, Sawank and coworkers have developed a microfluidic nano immunoassay (NIA) called NIA for simultaneous anti-SARS-CoV-2 IgG detection on a single device in 1024 samples. The design of the microfluidic NIA device is shown and demonstrates 100% specificity and 98% sensitivity [68][13]. Furthermore, the creation of an extensive serology platform that is affordable and easily accessible requires the exploration of alternative methods for venipuncture. To address this challenge, Swank et al. devised a sample collection and processing system that allows for non-invasive analysis using dried whole blood samples obtained through a convenient finger prick. The researchers conducted experiments utilizing three distinct approaches for collecting, shipping, processing, extracting, and analyzing dried whole blood samples. They evaluated the performance of two commercially accessible devices designed for the collection of precise 10 μL quantities of whole blood: the Neoteryx Mitra® and the DBS System SA HemaXis™DB10. In forthcoming times, the NIA generates the possibility for individuals to acquire a straightforward blood-sampling kit that comprises a lancet, a blood-sampling tool, and a pre-addressed return envelope. These kits could be obtained conveniently from local pharmacies or supermarkets. The kit’s usage is uncomplicated and user-friendly within the confines of one’s personal space, where a minor finger prick is performed to collect the blood using the provided device. Subsequently, the device containing the blood sample can be dispatched via regular mail to a central laboratory without necessitating specialized biosafety precautions. This central laboratory would then conduct an analysis of the blood sample for one or more biomarkers, interpret the resulting data, and convey the test outcomes back to the individual through means such as smartphones, email, or regular mail.
In a different analysis, a potential microfluidic technique has been proposed for antibody detection towards SARS-CoV-2. The integration capability of the microfluidic substrate with fluorescence, absorbance, and other diagnostic methods provides advantages over conventional diagnostic strategies [69][14]. Significantly, the sensor device enables quantitative measures within a linear detection range of 585.4 copies/μL to 5.854 × 107 copies/μL, having 231 copies/μL sensitivity. Yakoh et al. developed an electrochemical device for testing COVID-19 antibodies, delivering results within 30 min [70][15]. The utilization of a paper-based sensor presents an opportunity to transform the point-of-care testing (POCT) platform, providing the desired sensitivity and characteristics. This approach is particularly advantageous due to its cost-effectiveness, portability, and easy replacement options. Alafeef and his team employed a paper-based electrochemical technique to create detecting probes for genetic material testing [71][16]. Ganguli et al. engineered a portable RT-LAMP device designed to identify SARS-CoV-2 in clinical samples such as nasal swabs.
The cost-effective and validated “SARS-CoV-2 RapidPlex” demonstrates a high level of sensitivity, enabling the simultaneous detection of multiple key biomarkers associated with the SARS-CoV-2 virus. These biomarkers include immune response, viral contamination, and disease severity, making it suitable for home-based diagnostics. In a study, Mateos et al. [72][17] developed an integrated on-chip platform that combines RNA extraction using immiscible filtration assisted by surface tension (IFAST) with colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for RNA amplification and detection. The platform utilizes two sets of primers that target the open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2.

2. LAMP Tests

Isothermal amplification techniques, like loop-mediated isothermal amplification (LAMP), play a crucial role in isothermal amplification testing (IAT) and offer the advantage of detecting various targets simultaneously within a single reaction. Several studies have demonstrated that LAMP exhibits higher sensitivity and accuracy compared to RT-PCR for SARS-CoV-2 detection. The LAMP process is depicted [74][18], involving the amplification of viral nucleic acids and LAMP-specific primers extracted at a constant 60–65 °C temperature. The outcomes are typically generated through fluorescence or colorimetry, which could be seen by a compact device or the naked eye. The detection and interpretation of LAMP results do not rely on bulky instruments, making it suitable for community and household self-screening.
Natsuhara and colleagues introduced a microfluidic chip with dispensing and mixing sections capable of continuous fluid dispensing [75][19]. This chip enables the analysis of communicable diseases like influenza and COVID-19 through LAMP-based colorimetry within individual chambers. Lyu and co-authors devised a droplet array slip-chip for high-throughput COVID-19 determination [76][20]. The facile movement of fluid was achieved by chip sliding which can avoid the precise bonding challenges associated with conventional chips featuring high-precision microchannels. In a study by de Oliveira and co-workers, a manually controlled centrifuge microfluidic device was employed for LAMP-based SARS-CoV-2 detection, utilizing a fidget spinner [77][21]. Although this device is portable and independent of a specific instrument, it exhibits inaccurate speed control, which may lead to errors if not operated properly. While the aforementioned works primarily focus on designing microfluidic chip structures for fluid control, there arises a requirement for a portable signal readout device. Smartphones have emerged as reliable signal readout devices for LAMP, offering improved computing power and imaging capabilities. Colbert and co-workers proposed a technique for SARS-CoV-2 identification by merging LAMP with particle diffusometry [78][22]. The smartphone is utilized for capturing images of samples comprising fluorescent beads after RT-LAMP, facilitating the detection process.

3. RT-PCR Tests

The RT-PCR technique integrates complementary DNA (cDNA) PCR and RNA reverse transcription technologies. By leveraging mass and heat transfer methods based on fundamental hydrodynamic principles, microfluidics has the potential to enhance detection accuracy and reduce the time required for RT-PCR, particularly in terms of the necessary temperature variations. Incorporating microfluidic devices into RT-PCR testing can lead to more compact and rapid processes. Turiello and co-authors introduced an automated, rotationally driven microfluidic platform designed for the purification and enrichment of SARS-CoV-2 RNA [79][23]. The isolation of the virus for sample enrichment in the device is achieved using nanotrap magnetic particles, which effectively remove complex matrices and prevent the inhibition of RNA amplification and detection. The utilization of portable and microfluidic biochips determination devices that employ the RT-PCR technique greatly enhances the speed and accuracy of SARS-CoV-2 detection. Centrifugal microfluidic chips have found extensive application in disease diagnosis [80,81][24][25]. Furthermore, these chips enable highly automated and integrated multiple determinations, thereby enhancing practicality and functionality [82][26]. 

4. CRISPR-Associated Proteins System (Cas) Tests

CRISPR, recognized as a powerful gene-editing tool, has been referred to as “molecular scissors” [88][27]. Zhang et al. devised a system that combines CRISPR with fluorophore-quencher DNA probes for signal amplification and detection purposes [89][28]. Ramachandran et al. implemented isotachophoresis (ITP) on a microfluidic chip in combination with CRISPR and LAMP, enabling the diagnosis of COVID-19 within a 35-min timeframe [90][29]. In this system, Cas12, along with the guide DNA, selectively binds to the target DNA, leading to fluorophore–quencher DNA probe cleavage. However, the efficiency of CRISPR is hindered by time-consuming amplification and nucleic acid processes as well as the dependence on huge instruments for fluorescent signal readout. Silva and co-authors developed a catalase-mediated assay for CRISPR-based detection of SARS-CoV-2 [91][30]. Certain scientists have devised testing techniques that operate autonomously without the need for external apparatus. Li and his group introduced a lateral flow microfluidic device that utilizes a hand-warmer pouch serving as a heat source [92][31]. In this approach, the reagent’s freeze-dried powder is preloaded into a reaction chamber. Then, the handler can manually manipulate the liquid as well as observe the outcomes with the bare eye. While numerous RNA detection methods that are both highly sensitive and suitable for POC application exist, they are marred by a susceptibility to non-specific amplification when conducted under isothermal conditions. This susceptibility consequently results in inaccurate positive outcomes. To counteract this concern, the incorporation of isothermal amplification techniques with CRISPR technology has proven to be instrumental in mitigating the likelihood of non-specific detection [93,94][32][33]. Leveraging these attributes, a multitude of approaches that combine CRISPR technology with isothermal amplification have emerged. These innovative techniques effectively enhance the amplification of target genes and consequently elevate the overall efficacy of COVID-19 detection procedures.

5. Electrochemical Biosensors

Electrochemical biosensors have received enormous attention owing to their simplicity, affordability, and potential for miniaturization. These tiny devices utilize tailored electrodes that serve as receptors or transducers, depending on the specific requirements, to enable real-time, specific, and accurate target monitoring [95,96,97][34][35][36]. By extracting potentiometric or amperometric signals from the sensing electrode, information linked to the analyte presence can be obtained [98,99,100][37][38][39].
In their study, Sanati-Nezhad and his group fabricated an electrochemical immunosensor approach to determine the nucleocapsid protein antigens of SARS-CoV-2. They utilized a bbZnO/rGO nanocomposite coating on carbon screen-printed electrodes (SPEs) to enhance antibody adsorption [101][40]. The resulting device system was connected to a readout system, which generated electrochemical signals in COVID-19 presence. Similarly, Ali et al. employed improved 3D printing technology to construct a 3D reduced-graphene-oxide (rGO) electrode and integrated it with a microfluidic device, serving as an electrochemical sensor [102][41]. This setup allowed for the antibodies specific determination towards SARS-CoV-2, achieving a 2.8 × 10−15 M limit of detection (LOD). Additionally, Fabiani and the group developed a miniaturized electrochemical sensor utilizing magnetic beads and a carbon black-based electrode for SARS-CoV-2 determination [103][42]. The utilization of magnetic beads offers advantages in terms of preconcentration, reduced washing steps, and enhanced sensitivity and reliability. Additionally, the application of an external magnetic field enables the removal of interference from seasonal H1N1 influenza virus during detection. Zhao et al. demonstrated the feasibility of using a smartphone-based electrochemical sensor for the determination of SARS-CoV-2 RNA, eliminating the necessity for extensive laboratory processes and equipment [104][43]. This “plug-and-play” setup opens up possibilities for portable testing. Another recent study integrated the electrochemical platform with a wireless module, facilitating the development of graphene electrodes for rapid COVID-19 identification [105][44]. The “SARSCoV-2 RapidPlex” test, which is cost-effective and highly sensitive, can determine SARS-CoV disease and offer information on COVID-19 key aspects (disease severity, immune response, and viral infection). This test has the efficiency to be performed at home, offering convenience and accessibility to individuals.

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