Traditional diagnostic strategies for detecting infectious diseases require the use of benchtop instruments that are not suitable for point-of-care testing (POCT). Emerging microfluidics is a highly miniaturized, automated, and integrated technology that is a potential alternative to traditional methods for fast, low-cost, accurate on-site diagnostics. Molecular diagnostic methods are widely used in microfluidic devices as the most effective methods for pathogen detection. This review summarizes recent advances in microfluidic-based molecular diagnostics of infectious diseases from both an academic and industrial perspective. First, we describe a typical on-chip processing of nucleic acids, including sample pretreatment, amplification, and signal reading. The characteristics, advantages and disadvantages of the four types of microfluidic platforms are then compared. Next, we will discuss the use of digital assays for the absolute quantification of nucleic acids. Both classical and recent commercial microfluidic-based molecular diagnostic devices are summarized as evidence of the current state of the market. Finally, we propose future directions for microfluidic diagnosis of infectious diseases.
Infectious diseases are caused by pathogens, including bacteria, viruses, and parasites, that are distributed throughout the world. Unlike other diseases, pathogens quickly become infected and spread between humans and host animals through inoculation, air and water media . Infectious disease prevention is critical as a public health measure. Three main strategies for combating infectious diseases: (1) control the source of infection; (2) interruption of the transmission path; (3) protection of susceptible populations. Among the main strategies, control of the source of infection is considered the most important strategy due to its convenience and low cost. Rapid diagnosis, isolation, and treatment of infected individuals are critical, requiring fast, sensitive, and accurate diagnostic strategies . The current diagnosis of infectious diseases usually combines clinical examination based on signs and symptoms and laboratory studies such as cell culture and molecular diagnostics, which require trained personnel, labor-intensive procedures, and expensive testing equipment [3, 4]. Prevention of infectious disease outbreaks requires rapid, inexpensive, and accurate local diagnosis, especially in resource-limited areas where infectious diseases are common and severe , as well as treatment in the wilderness or on the battlefield, where emergencies are unpredictable. . medical care is limited . In this context, microfluidics is a technology that combines microelectromechanical systems technologies, nanotechnology, or materials science for precise fluid manipulation [7,8,9,10], providing new possibilities for point-of-care detection (POCT). ) infectious agents outside hospitals and laboratories. Compared to traditional time-consuming diagnostics, microfluidic technology offers sample and cost savings for molecular diagnostics during disease outbreaks. The global spread of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), so the importance of microfluidics for the timely prevention and control of the pandemic is again emphasized [11, 12, 13]. Unlike traditional diagnostics, microfluidic POCT uses small portable devices ranging from benchtop analyzers to small sidestream test strips to test near the sampling point . These tests feature simplistic or no sample preparation, fast signal amplification, and sensitive signal readings resulting in short duration and accurate results within minutes. The availability and mass production of microfluidic-based healthcare instruments have expanded their cost-effective and direct diagnostic applications outside the hospital, near the patient, and even at home.
Among the existing strategies for diagnosing infectious diseases, molecular diagnostics is one of the most sensitive [15, 16]. In addition, molecular diagnostics is often used as the gold standard for continuous detection of COVID-19, allowing direct detection of virus-specific regions of RNA or DNA before the onset of an immune response [17, 18]. In the current review, we present the latest advances in microfluidics-based molecular diagnostic processes for infectious diseases, from an academic perspective to future industrial perspectives (Fig. 1). We will start with three key steps in nucleic acid detection: on-chip sample pretreatment, nucleic acid amplification, and signal reading. We then compared different types of microfluidic platforms with their structure and function, showing unique characteristics (strengths and weaknesses). Digital nucleic acid detection is further discussed and given as an example of a third generation technology for the absolute quantification of infectious pathogen molecules. In addition, several typical and latest commercial POCT devices will be presented to demonstrate the current state of the microfluidic POCT market for molecular diagnostics. We will also discuss and explain our vision for future applications.
Modules of microfluidic chips for nucleic acid detection can be divided into three categories (sampling, recognition, and signaling) according to their functions . Among these modules, the sampling module mainly realizes sample lysis and nucleic acid extraction. The sensor module mainly controls the conversion and amplification of nucleic acid signals. The signaling module detects the signal converted and processed by the sensing module. Based on the process of detecting nucleic acids on a chip, we will summarize the various chips that can realize the “input and output” function.
The first step in nucleic acid detection is nucleic acid extraction, i.e. isolating the target nucleic acid from the original sample. Nucleic acid extraction is performed to purify nucleic acids from other molecular contaminants, ensure the integrity of the primary structure of nucleic acid molecules, and optimize results. Nucleic acid extraction requires the necessary sample lysis and nucleic acid capture, the quality and efficiency of which have a huge impact on research and diagnostic results. Any subtle side effects during extraction may limit further detection. For example, polymerase chain reaction (PCR) and loop isothermal amplification (LAMP) methods are inhibited by some residual organic solvents such as ethanol and isopropanol in nucleic acid isolation reagents . Liquid-liquid extraction and solid-phase extraction are the most popular methods for isolating nucleic acids , however, liquid-liquid extraction on a chip is extremely limited, since the reagents used in liquid-liquid extraction cause corrosion of most microfluidic chips. Here, we highlight microarray-based solid phase extraction methods and compare their advantages and disadvantages.
Silicon is a substrate material compatible with nucleic acids due to its biocompatibility, stability, and ease of modification . Importantly, when modified with silica or other materials, this composite exhibits properties to adsorb negatively charged nucleic acids under low pH, high salt conditions while eluting with high pH, low salt solutions. Based on this phenomenon, it is possible to purify the nucleic acid.
Various forms of silica-based materials have been used for nucleic acid extraction in microfluidics, such as silica beads, powders, microfiber filters, and silica membranes [23, 24, 25, 26]. Depending on the properties of the material, silicon-based materials can be used in microcircuits in different ways. For example, silica granules, powders, and commercial nanofilters can simply be placed into the pores or microchannels of microfluidic chips and help extract nucleic acids from samples [27, 28, 29]. Surface-modified silica membranes can also be used to rapidly purify DNA from pathogens at low cost. For example, Wang et al.  By combining denaturing amplification reactions with vesicle-mediated chain exchange with silica membranes coated with chitosan oligosaccharides, a versatile portable system was introduced that successfully detected 102–108 colony forming units. (CFU)/ml Vibrio parahaemolyticus. , and the presence of the virus was easily visible. Powell et al.  Silicon-based microarrays were then used to detect hepatitis C virus (HCV), human immunodeficiency virus (HIV), Zika virus, and human papillomavirus and automatic propagation, in which a 1.3 μl tortuous microreactor was developed to capture RNA viruses. and perform in situ amplification. In addition to these methods, surface-modified silica microcolumns also play a key role in nucleic acid extraction, as the geometry and properties of the modifying material greatly increase extraction efficiency. Chen et al.  proposed a microfluidic platform for isolation of low-concentration RNA based on amino-coated silicon microcolumns. This microfluidic device integrates an array of 0.25 cm2 micropillars on a silicon substrate to achieve higher extraction efficiency through a high surface area to volume ratio design. The advantage of this design is that the microfluidic device can achieve up to 95% nucleic acid extraction efficiency. These silicon-based strategies demonstrate the value of rapidly isolating nucleic acids at low cost. In combination with microfluidic chips, silicon-based extraction strategies can not only increase the efficiency of nucleic acid detection, but also facilitate the miniaturization and integration of analytical devices .
Magnetic separation methods use magnetic particles to isolate nucleic acids in the presence of an external magnetic field. Commonly used magnetic particles include Fe3O4 or γ-Fe2O3 magnetic particles coated with silica, amino and carboxyl [33,34,35,36]. The distinguishing feature of magnetic particles compared to silicon-based SPE methods is the ease of manipulation and control with external magnets.
Using the electrostatic interaction between nucleic acids and silica, under conditions of high salt and low pH, nucleic acids are adsorbed on the surface of silica-coated magnetic particles, while under conditions of low salt and high pH, the molecules can be washed again. . Silica-coated magnetic beads make it possible to extract DNA from large volume samples (400 μL) using magnetically controlled motion . As a demonstration, Rodriguez-Mateos et al.  used tunable magnets to control the transfer of magnetic beads to different chambers. Based on silica-coated magnetic particles, 470 copies/mL of SARS-CoV-2 genomic RNA can be extracted from wastewater samples for LAMP reverse transcription detection (RT-LAMP) and the response can be read within 1 hour. naked eye (Fig. 2a).
Devices based on magnetic and porous materials. Conceptual diagram of the IFAST RT-LAMP microfluidic device for SARS-CoV-2 RNA detection (adapted from ). b Centrifugal micro device for dSPE of buccal swab nucleic acid (adapted from ). c Built-in self-powered sample concentrator using an FTA® card (adapted from ). d Fusion 5 filter paper modified with chitosan (adapted from ). SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, RT-LAMP reverse transcription loop mediated isothermal amplification, FTA finders technology partners, NA nucleic acid
Positively charged magnetic particles are ideal for attaching the phosphate backbone of a nucleic acid. At a certain salt concentration, the negatively charged phosphate groups of nucleic acids can be positively charged on the surface of the magnetic composite particles. Therefore, magnetic nanoparticles with a rough surface and a high density of amino groups were developed for the extraction of nucleic acids. After magnetic separation and blocking, magnetic nanoparticles and DNA complexes can be directly used in PCR, which eliminates the need for complex and time-consuming purification and elution operations . Magnetic nanoparticles coated with negative carboxyl groups have also been used to separate nucleic acids adsorbed on surfaces in high concentration polyethylene glycol and sodium chloride solutions . With these surface-modified magnetic beads, DNA extraction is compatible with subsequent amplification. Dignan et al.  described an automated and portable centrifugal microfluidic platform for nucleic acid pretreatment, allowing non-technical personnel to use it on site. In addition, the compatibility of the isolated DNA with LAMP, a method well suited for point-of-care nucleic acid analysis, further demonstrates minimal equipment requirements and suitability for colorimetric assays (Fig. 2b).
Magnetic bead methods offer the possibility of automated extraction, some of which exist in commercial automated nucleic acid extractors [KingFisher; ThermoFisher (Waltham, MA, USA), QIAcube® HT; CapitalBio (Beijing, China) and Biomek®; Beckman (Miami, USA). ), Florida, USA)]. The advantages of combining magnetic beads with microfluidics can be used for efficient automated extraction of nucleic acids, which could potentially advance the development of molecular diagnostics; however, the combination of magnetic beads with microfluidics still relies heavily on complex control systems for precise manipulation of magnetic beads, which explains the popularity of commercial products being bulky and expensive, which limits the further application of magnetic beads in POCT.
Several porous materials such as modified nitrocellulose filters, Finders Technology Associates (FTA) cards, polyethersulfone-based filter papers, and glycan-coated materials have also been used for nucleic acid detection [40, 41, 42, 43, 44]. Porous fibrous materials such as fibrous paper were first used to isolate DNA by physically entangling long-stranded DNA molecules with fibers. Small pores lead to a strong physical restriction of DNA molecules, which positively affects DNA extraction. Due to the different pore sizes of fibrous paper, the extraction efficiency cannot meet the needs of DNA amplification [45, 46]. The FTA card is a commercial filter paper used in the field of forensic medicine and widely used in other areas of molecular diagnostics. Through the use of cellulose filter paper impregnated with various chemicals to lyse the cell membranes in the sample, the released DNA is protected from degradation for up to 2 years. More recently, impregnated cellulose paper has been developed for molecular detection of various pathogens, including SARS-CoV-2, leishmaniasis, and malaria [47,48,49]. HIV in the isolated plasma is lysed directly, and the viral nucleic acid is enriched in the FTA® flow membrane built into the concentrator, which allows efficient production of the nucleic acid  (Fig. 2c). The main problem with nucleic acid detection using FTA cards is that chemicals such as guanidine and isopropanol inhibit subsequent amplification reactions. To solve this problem, we developed Fusion 5 chitosan-modified filter paper, which combines the advantages of both the physical interlacing of DNA molecules and fibrous filter paper, and the electrostatic adsorption of DNA on chitosan-modified compounds to achieve highly efficient nucleic acid extraction. . filter fibers  (Fig. 2d). Similarly, Zhu et al.  demonstrated a chitosan-modified PCR method based on an in situ capillary microfluidic system for rapid isolation and detection of Zika virus RNA. Nucleic acids can be adsorbed/desorbed in a mixed lysate/PCR medium, respectively, based on the on/off switch property of chitosan. on and off”, responsive to pH.
As mentioned above, these strategies combine the advantages of various solid phase materials and increase the efficiency of nucleic acid extraction in microfluidics. In practical applications, the use of these materials in large quantities is uneconomical, and proper surface treatment or surface modification of common materials with these materials can also preserve their function. Therefore, it is believed that the implementation of these strategies after a pilot study can reduce costs.
Nucleic acid testing on microfluidic platforms often uses small sample volumes (< 100 µl), therefore requires amplification of the target nucleic acids with specific probes for conversion to a signal that is convenient for downstream detection (optical, electrical, and magnetic) [53, 54]. Nucleic acid testing on microfluidic platforms often uses small sample volumes (< 100 µl), therefore requires amplification of the target nucleic acids with specific probes for conversion to a signal that is convenient for downstream detection (optical, electrical, and magnetic) [53, 54]. При тестировании нуклеиновых кислот на микрожидкостных платформах часто используются небольшие объемы образцов (< 100 мкл), поэтому требуется амплификация целевых нуклеиновых кислот с помощью специальных зондов для преобразования в сигнал, удобный для последующего обнаружения (оптического, электрического и магнитного) [53, 54]. When testing nucleic acids on microfluidic platforms, small sample volumes (<100 µL) are often used, so the amplification of target nucleic acids with special probes is required to convert it into a signal convenient for subsequent detection (optical, electrical, and magnetic) [53, 54].微流控平台上的核酸检测通常使用小样本量（< 100 µl），因此需要使用特定探针扩增目标核酸，以转换为便于下游检测（光学、电学和磁学）的信号[53, 54]。微流控 平台 上 的 核酸 检测 使用 小样本量 （（<100 µl） ， 因此 需要 特定 探针 扩增 目标 ， 以 转换 为 下游 下游 （光学 、 电学 磁学） 的 信号 [53, 54, 54, 54 ]。 Обнаружение нуклеиновых кислот на микрожидкостных платформах обычно использует небольшие объемы образцов (<100 мкл), что требует амплификации целевых нуклеиновых кислот с помощью специальных зондов для преобразования в сигналы для последующего обнаружения (оптического, электрического и магнитного) [53, 54]]. Detection of nucleic acids on microfluidic platforms usually uses small sample volumes (<100 μl), which requires amplification of target nucleic acids with special probes to convert them into signals for subsequent detection (optical, electrical, and magnetic) [53, 54]]. Nucleic acid amplification in microfluidics can also speed up reactions, optimize detection limits, reduce sample requirements, and improve detection accuracy [55, 56]. In recent years, with the realization of fast and accurate detection, various nucleic acid amplification methods have been applied in microfluidics, including PCR and some isothermal amplification reactions. This section will summarize methods for nucleic acid detection based on microfluidic systems.
PCR is a simulation of the DNA replication process of an organism, the theory of which is described in detail elsewhere and will not be discussed here. PCR can amplify a very small amount of target DNA/RNA at an exponential rate, making PCR a powerful tool for the rapid detection of nucleic acids. In recent decades, many portable microfluidic devices equipped with PCR thermal cycling systems have been developed to meet the needs of point-of-care diagnostics [57, 58]. On-chip PCR can be divided into four types (conventional, continuous flow, spatially switched, and convective PCR) according to different temperature control methods . For example, Gee et al.  developed a direct reverse transcription quantitative PCR (RT-qPCR) method on their own microfluidic platform for the multiplex detection of SARS-CoV-2, influenza A and B viruses in throat swab samples (Fig. 3a) . Park et al.  built a simple pathogen analysis chip by integrating thin film PCR, electrodes, and a finger-operated polydimethylsiloxane-based microfluidic module. However, both works embody the common shortcomings of conventional PCR. PCR requires thermal cycling, which limits further device miniaturization and reduced testing time.
The development of continuous flow based microfluidic and space-switched PCR is critical to address this issue. Using a long serpentine channel or a short straight channel, continuous flow PCR can provide fast amplification by actively circulating reagents in three preheat zones with an off-chip pump. This operation successfully avoids the transition phase between different reaction temperatures and thus significantly reduces the testing time  (Fig. 3b). In another study by Jung et al.  proposed a new rotary PCR genetic analyzer that combines the characteristics of fixed and flow PCR for ultrafast and multiplex reverse transcription PCR (Fig. 3c). For nucleic acid amplification, the PCR microchip will be rotated through three heating blocks at different temperatures: 1. Denaturation block 94°C, 2. Annealing block at 58°C, 3. Expansion block at 72°C.
Application of PCR in microfluidics. Schematic representation of dirRT-qPCR on a microfluidic platform (adapted from ). b Schematic representation of a continuous flow PCR microarray based on a serpentine channel (adapted from ). c Schematic representation of a rotary PCR genetic analyzer, consisting of a microchip, three heating blocks and a stepper motor (adapted from ). d Diagram of thermoconvection PCR with centrifugation and setup (adapted from ). DirRT-qPCR, direct quantitative reverse transcription polymerase chain reaction
Using capillaries and loops or even thin plates, convection PCR can rapidly amplify nucleic acids by natural free thermal convection without the need for an external pump. For example, a cyclic olefin polymer microfluidic platform was developed on a fabricated rotating heating stage that uses thermal cycling with centrifugation in a PCR loop microchannel  (Fig. 3d). The reaction solution is driven by thermal convection, which continuously exchanges high and low temperature in a microchannel with an annular structure. The entire amplification process can be completed in 10 minutes with a detection limit of 70.5 pg/channel.
As expected, rapid PCR is a powerful tool for fully integrated sample-response molecular diagnostic and multiplex analysis systems. Rapid PCR significantly reduces the time required to detect SARS-CoV-2, which contributes to the effective control of the COVID-19 pandemic.
PCR requires a complex thermal cycler that is not suitable for POCT. More recently, isothermal amplification techniques have been applied to microfluidics, including but not limited to LAMP, recombinase polymerase amplification (RPA), and amplification based on nucleic acid sequences [65,66,67,68]. With these techniques, nucleic acids are amplified at a constant temperature, facilitating the creation of low cost, highly sensitive portable POCT devices for molecular diagnostics.
High-throughput microfluidics-based LAMP assays allow multiple detection of infectious diseases [42, 69, 70, 71]. In combination with a centrifugal microfluidic system, LAMP can further facilitate the automation of nucleic acid detection [69, 72, 73, 74, 75]. The spin-and-react SlipChip was developed for the visual detection of multiple parallel bacteria using LAMP  (Fig. 4a). When using optimized LAMP in the assay, the fluorescence signal-to-noise ratio was approximately 5-fold, and the detection limit reached 7.2 copies/μl of genomic DNA. Moreover, the existence of five common digestive bacterial pathogens, including Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvialis and Vibrio parahaemolyticus, were visualized based on the method in < 60 min. Moreover, the existence of five common digestive bacterial pathogens, including Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvialis and Vibrio parahaemolyticus, were visualized based on the method in < 60 min. Moreover, the presence of five common bacterial pathogens of the digestive tract, including Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvialis and Vibrio parahaemolyticus, was visualized using this method in less than 60 minutes.此外，基于该方法在<60分钟内可视化了五种常见消化道细菌病原体的存在，包括蜡状芽孢杆菌、大肠杆菌、肠沙门氏菌、河流弧菌和副溶血性弧菌。此外 ， 基于 该 方法 在 <60 分钟 内 视化 了 五 种 常见 消化道 细菌病 的 存在 ， 包括 芽孢杆菌 、 大 肠杆菌 、 肠 氏 菌 、 弧菌 和 副溶血性。。。 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 弧菌 HIP In addition, the presence of five common bacterial gastrointestinal pathogens, including Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvius, and Vibrio parahaemolyticus, was visualized using this method in less than 60 minutes.
The advantages of LAMP in microfluidics include, among others, fast response and miniaturized detection. However, due to the reaction temperature (around 70°C), aerosols are inevitably generated during LAMP, resulting in a high false positive rate. Assay specificity, primer design, and temperature control also need to be optimized for LAMP. In addition, chip designs that implement multiple target detection on a single chip are of great value and should be developed. In addition, LAMP is suitable for multi-purpose detection integrated in one chip, which is of great importance, but there is still a lot of room for development.
The high false positive rate of LAMP can be partly reduced with RPA, as the relatively low reaction temperature (~37 °C) results in relatively few evaporation problems . In the RPA system, two opposite primers initiate DNA synthesis by binding to a recombinase and amplification can be completed within 10 minutes [78,79,80,81]. Therefore, the whole RPA process is much faster than PCR or LAMP. In recent years, microfluidic technology has been shown to further improve the speed and accuracy of RPA [82,83,84]. For example, Liu et al.  developed a microfluidic integrated lateral flow polymerase recombinase amplification assay for rapid and sensitive detection of SARS-CoV-2 by integrating reverse transcription RPA (RT-RPA) and a universal lateral flow test strip detection system. into a single microfluidic system. Figure 4b). The limit of detection is 1 copy/µl or 30 copies/sample, and detection can be completed in about 30 minutes. Kong et al. have developed a wearable microfluidic device.  used body temperature and a mobile phone-based fluorescence detection system to rapidly and directly detect HIV-1 DNA using RPA (Figure 4c). The wearable RPA assay detects 100 copies/mL of the target sequence within 24 minutes, demonstrating great potential for rapid diagnosis of HIV-1-infected infants in resource-limited settings.
Isothermal amplification in point-of-care testing (POCT). Development and production of spin and reaction SlipChip. After plasma welding, the top and bottom chips were assembled with a set of nuts to form the final chip (adapted from ). b Schematic of the MI-IF-RPA system for COVID-19 detection (adapted from ). c Schematic of a wearable RPA test for rapid detection of HIV-1 DNA (adapted from ). SE Salmonella enterica, VF Vibrio fluvius, VP Vibrio parahaemolyticus, BC Bacillus cereus, EC Escherichia coli, FAM carboxyfluorescein, human immunodeficiency virus HIV, RPA recombinase polymerase amplification, LED light emitting diode, MI-IF-RPA Microfluidics Integrated Lateral Flow Recombinase- Polymerase Amplification
Microfluidic-based RPA is developing rapidly, however, the cost of chip fabrication and reaction consumption is too high and must be reduced to increase the availability of this technology. In addition, the high sensitivity of RPA can affect the amplification of non-specific products, especially in the presence of contamination. These limitations may affect the application of RPA in microfluidic systems and merit further optimization. Well-designed primers and probes for various targets are also needed to improve the feasibility of RPA-based microfluidic strategies in POCT.
Cas13 and Cas12a have the ability to randomly cleave nucleic acids and thus can be developed as detection and diagnostic tools. Cas13 and Cas12a are activated upon binding to target DNA or RNA, respectively. Once activated, the protein begins to cleave other nearby nucleic acids, after which guide RNAs targeting pathogen-specific nucleic acids can cleave quenched fluorescent probes and release fluorescence. Based on this theory, Kellner et al.  developed a Cas13-based method [Specific High-sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK)], and Broughton et al.  developed another approach based on Cas12a [CRISPR Trans Reporter targeting DNA endonuclease (DTECR)].
In recent years, various methods for the detection of nucleic acids based on CRISPR have appeared [89, 90]. Conventional CRISPR based methods are often time consuming and labor intensive due to multiple procedures including nucleic acid extraction, amplification and CRISPR detection. Exposure of liquids to air may increase the chance of false positive results. Given the above, CRISPR-based systems are in dire need of optimization.
A pneumatically controlled microfluidic platform that can perform 24 analyzes in parallel has been developed for CRISPR-Cas12a and CRISPR-Cas13a detection applications . The system is equipped with a fluorescence detection device that bypasses nucleic acid amplification and automatically detects femtomolar DNA and RNA samples. Chen et al.  integrated recombinase amplification with the CRISPR-Cas12a system in centrifugal microfluidics (Fig. 5a). This work overcomes the difficulty of integrating these two processes because Cas12a can digest messenger DNA and inhibit the amplification process. In addition, Chen et al.  additionally pre-stored the reaction reagents in a centrifugal microfluidic control to automatically complete the entire process. In another work, Silva et al.  developed a diagnostic method without CRISPR/Cas12a amplification and a smartphone to detect SARS-CoV-2 (Fig. 5b). This assay, known as a cell phone-based amplification-free system, includes a CRISPR/Cas-dependent enzyme that is based on smartphone visualization of catalase-generated bubble signals in microfluidic channels. Sensitive detection of less than 50 copies/µl of nucleic acid without pre-amplification, the whole process from sample injection to signal reading takes only 71 minutes.
Nucleic acid detection methods based on CRISPR. Centrifugal POCT for integrated molecular diagnostics based on CRISPR (adapted from ). b Development of the CASCADE test for smartphone-based analysis of SARS-CoV-2 (adapted from ). RAA recombinase amplification, PAM adjacent protospacer motif, CRISPR clustered short palindromic repeats at regular intervals, CASCADE system without cell phone amplification with CRISPR/CAS-dependent enzymes, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride EDC
As the last step in nucleic acid detection, signal detection directly reflects diagnostic results and is a critical factor in the development of an efficient, sensitive, and accurate POCT. Signals can be read using various methods such as fluorescent, electrochemical, colorimetric and magnetic strategies. In this section, we describe the rationale for each approach and compare the molecular diagnostics of infectious diseases in microfluidics.
Fluorescence-based strategies are widely used for POCT diagnostics of infectious diseases due to their remarkable advantages of excellent sensitivity, low cost, ease of operation, and point-of-care analysis [94, 95]. These strategies use labeled fluorophores such as fluorescent dyes and nanomaterials to create a detectable signal (fluorescence enhancement or quenching). This finding suggests that fluorescence-based strategies can be divided into direct fluorescent labeling, signal-on, and signal-off fluorescent detection . Direct fluorescent label detection uses special fluorescent labels to label specific ligands that generate a specific amount of fluorescence when selectively bound to a target. For signal-based fluorescence detection, the quality of the fluorescent signal is positively related to the magnitude of interest. Fluorescence intensity is negligible in the absence of a target and is detectable when a sufficient amount of target is present. Conversely, the intensity of fluorescence detected by “signal-off” fluorescence is inversely proportional to the amount of target, initially reaching a maximum value and gradually decreasing as the target is enlarged. For example, using the CRISPR-Cas13a target-dependent trans-cleavage mechanism, Tian et al.  developed a novel recognition strategy to detect RNAs that bypass reverse transcription directly (Fig. 6a). Upon binding to complementary target RNAs, the CRISPR-Cas13-RNA complex can be activated, triggering transcollateral cleavage by non-specific reporter RNAs. The fluorescently labeled reporter [fluorophore (F)] is quenched by the quencher (Q) intact and fluoresces when cleaved by the activated complex.
The advantage of electrochemical detection is high detection speed, easy production, low cost, easy to carry and automatic control. It is a powerful analytical method for POCT applications. Based on graphene field-effect transistors Gao et al.  developed a nanobiosensor for the multiplex detection of Lyme disease antigens from Borrelia burgdorferi bacteria with a detection limit of 2 pg/mL (Fig. 6b).
Colorimetric assays have been used in POCT applications, benefiting from the advantages of portability, low cost, ease of preparation, and visual reading. Colorimetric detection can use the oxidation of peroxidase or peroxidase-like nanomaterials, the aggregation of nanomaterials, and the addition of indicator dyes to convert information about the presence of target nucleic acids into visible color changes [99, 100, 101]. Notably, gold nanoparticles are widely used in the development of colorimetric strategies, and due to their ability to induce rapid and significant color changes, there is increasing interest in the development of POCT colorimetric platforms for in situ diagnosis of infectious diseases . With an integrated centrifugal microfluidic device , foodborne pathogens in contaminated milk samples can be automatically detected at the level of 10 bacterial cells, and the results can be read visually within 65 minutes (Fig. 6c).
Magnetic sensing techniques can accurately detect analytes using magnetic materials, and there has been significant interest in POCT applications in recent decades. Magnetic sensing techniques have some unique advantages such as low cost magnetic materials rather than expensive optical components. However, the use of a magnetic field improves detection efficiency and reduces sample preparation time . In addition, the results of magnetic probing demonstrate high specificity, sensitivity, and high signal-to-noise ratio due to the insignificant magnetic background signal of biological samples . Sharma et al. integrated a magnetic tunnel junction based biosensor into a portable microchip platform.  for multiplex detection of pathogens (Fig. 6d). Biosensors sensitively detect subnanomolar nucleic acids isolated from pathogens.
Typical signal detection method. The concept of hyperlocalized detection of Cas13a (adapted from ). b Graphene nanobiosensor FET in combination with Lyme GroES scFv (adapted from ). c Colorimetric indications for multiplex detection of foodborne pathogens in a centrifugal microfluidic chip: No. 1 and No. 3 samples with target pathogens, and No. 2, No. 4 and No. 5 samples without target pathogens (adapted from ). d Biosensor based on a magnetic tunnel junction, including a platform, a built-in blocking amplifier, a control unit, and a power supply for signal generation/acquisition (adapted from ). GFET Graphene FET, Escherichia coli, Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus, Vibrio parahaemolyticus, Listeria monocytogenes, PC PC, PDMS Dimethicone, PMMA polymethyl methacrylate
Despite the excellent characteristics of the above detection methods, they still have disadvantages. These methods are compared (table 1), including some applications with details (pros and cons).
With the development of microfluidics, microelectromechanical systems, nanotechnology and materials science, the use of microfluidic chips for the detection of infectious diseases is constantly advancing [55,96,107,108]. Precise manipulation of miniature equipment and fluids contributes to diagnostic accuracy and cost-effectiveness. Therefore, for further development, efforts have been made to optimize and upgrade the chips, resulting in various microfluidic chips with different structures and functions. Here we briefly introduce several common types of microfluidic platforms and compare their characteristics (pros and cons). In addition, most of the examples listed below focus primarily on combating SARS-CoV-2.
LOCCs are the most common miniaturized complex analytical systems and their operations are highly miniaturized, integrated, automated and parallelized from sample injection and preparation, flow control and liquid detection [109, 110]. Liquids are manipulated through carefully designed geometry and the interaction of many physical effects such as pressure gradients, capillary action, electrodynamics, magnetic fields and acoustic waves . LOCC shows excellent advantages in high-throughput screening and multiple detection, with fast analysis speed, small sample size, low power consumption, and high management and operation efficiency; however, LOCC devices are very delicate, and manufacturing, packaging, and interfacing. However, multiplexing and reuse face enormous difficulties . Compared to other platforms, LOCC has unique advantages in terms of maximum application diversity and best technology compatibility, but its disadvantages are also obvious, namely high complexity and poor repeatability. Dependence on external pumps, which are often bulky and expensive, further limits their use in POCT.
During the COVID-19 outbreak, LOCC received a lot of attention. At the same time, there are several new chips that combine several technologies. For example, smartphones are now widely used as portable analytics devices and have great potential for LOCC integration. Sun et al.  fabricated a microfluidic chip that allows multiplexing specific nucleic acid sequences of five pathogens, including SARS-CoV-2, using LAMP and analyzed them using a smartphone within 1 hour after the end of the reaction. As another example, Sundah et al.  created a molecular switch [catalytic amplification by molecular transition state switch (CATCH)] for direct and sensitive detection of SARS-CoV-2 RNA targets using smartphones. CATCH is compatible with portable LOCC and achieves superior performance (approximately 8 RNA copies/μl; < 1 h at room temperature) . CATCH is compatible with portable LOCC and achieves superior performance (approximately 8 RNA copies/μl; < 1 h at room temperature) . CATCH совместим с портативным LOCC и обеспечивает превосходную производительность (примерно 8 копий РНК/мкл; < 1 ч при комнатной температуре) . CATCH is compatible with portable LOCC and provides excellent throughput (approximately 8 RNA copies/µl; < 1 h at room temperature) . CATCH 与便携式LOCC 兼容并具有卓越的性能（大约8 RNA 拷贝/μl；室温下< 1 小时）。 CATCH 与便携式LOCC 兼容并具有卓越的性能（大约8 RNA 拷贝/μl；室温下< 1 小时）。 CATCH совместим с портативными LOCC и обладает превосходной производительностью (примерно 8 копий РНК/мкл; < 1 часа при комнатной температуре) . CATCH is compatible with portable LOCCs and has excellent performance (approximately 8 RNA copies/µl; < 1 hour at room temperature) . In addition, LOCC devices for molecular diagnostics also use some driving forces such as vacuum, stretch, and electric fields. Kang et al.  demonstrated a real-time, ultra-fast nanoplasma-on-a-chip PCR for rapid and quantitative diagnosis of COVID-19 in the field using a vacuum plasmonic liquid PCR chip. Li et al.  subsequently developed a stretch-driven microfluidic chip that enabled the diagnosis of COVID-19. The platform uses the RT-LAMP amplification system to determine if a sample is qualitatively positive or negative. Subsequently, Ramachandran et al.  achieved appropriate electric field gradients using isotachophoresis (ITP), a selective ion focusing technique implemented in microfluidics. With ITP, target RNA from raw nasopharyngeal swab samples can be automatically purified. Then Ramachandran et al.  Combining this ITP purification with ITP-enhanced LAMP and CRISPR assays detected SARS-CoV-2 in human nasopharyngeal swab and clinical specimens in about 35 minutes. In addition, new ideas are constantly emerging. Jadhav et al.  proposed a diagnostic scheme based on surface-enhanced Raman spectroscopy in combination with a microfluidic device containing either vertically oriented gold/silver-coated carbon nanotubes or disposable electrospun micro/nanotubes. Membrane-functionalized built-in filter microchannels are disposable. The device adsorbs viruses from various body fluids/exudations such as saliva, nasopharynx and tears. Thus, the virus titer is abundant and the virus can be accurately identified by the Raman signature.
LOAD is a centrifugal microfluidic platform in which all processes are controlled by a frequency protocol that rotates a microstructured substrate . The LOAD device is characterized by using centrifugal force as an important driving force. Liquids are also subject to capillary, Euler and Coriolis forces. Using a centrifuge device, analyzes are performed in continuous liquid operation from a radial inward to outward position, eliminating the need for additional external tubing, pumps, actuators, and active valves. In short, a single control method simplifies operation. The forces acting on the liquid in the same microfluidic channel at the same distance from the load center are equal, which makes it possible to repeat the channel structure. Thus, LOAD equipment is simpler and more economical to design and manufacture than conventional LOCC equipment, while the reactions are largely independent and parallelized; however, due to the high mechanical strength of centrifugal equipment, available chip material is limited and small volumes are difficult. to the car. At the same time, most LOAD devices are designed for single use only, which is expensive for large-scale detection [96, 117, 118, 119].
In recent decades, LOAD, considered one of the most promising microfluidic devices, has received considerable attention from researchers and manufacturers. Thus, LOAD has gained wide acceptance and has been used for molecular diagnostics of infectious pathogens [120, 121, 122, 123, 124], especially during the COVID-19 outbreak. For example, at the end of 2020, Ji et al.  demonstrated a direct RT-qPCR assay for rapid and automated parallel detection of SARS-CoV-2 and influenza A and B infections in throat swab specimens. Then Xiong et al.  presented a LAMP-integrated discoid microfluidic platform for rapid, accurate, and simultaneous detection of seven human respiratory coronaviruses, including SARS-CoV-2, within 40 minutes. In early 2021, de Oliveira et al.  demonstrated a polystyrene toner centrifugal microfluidic chip, manually operated with a fingertip rotator, for RT-LAMP molecular diagnosis of COVID-19. Subsequently, Dignan et al.  presented an automated portable centrifuge microdevice for purification of SARS-CoV-2 RNA directly from buccal swab sections. Medved et al.  proposed an inline SARS-CoV-2 aerosol sampling system with a small volume rotating microfluidic fluorescent chip with a detection limit of 10 copies/μL and a minimum cycle threshold of 15 minutes. Suarez et al.  recently reported the development of an integrated modular centrifugal microfluidic platform for the direct detection of SARS-CoV-2 RNA in heat-inactivated nasopharyngeal swab samples using LAMP. These examples demonstrate the great benefits and promise of LOAD in the molecular diagnostics of COVID-19.
In 1945 Muller and Clegg  first presented microfluidic channels on paper using filter paper and paraffin. In 2007, the Whitesides group  created the first functional paper platform for protein and glucose testing. Paper has become an ideal substrate for microfluidics. The paper has inherent properties such as hydrophilicity and porous structure, excellent biocompatibility, light weight, flexibility, foldability, low cost, ease of use and convenience. Classical µPADs consist of hydrophilic/hydrophobic structures built on paper substrates. Depending on the three-dimensional structure, μPADs can be divided into two-dimensional (2D) and three-dimensional (3D) μPADs. 2D µPADs are produced by forming hydrophobic boundaries to form microfluidic channels, while 3D µPADs are usually made from stacks of layers of 2D microfluidic paper, sometimes by paper folding, slip techniques, open channels, and 3D printing . Aqueous or biological fluids on the μPAD are primarily controlled by capillary force without an external power source, facilitating pre-storage of reagents, sample handling, and multiplex detection. However, accurate flow control and multiplex detection are hampered by insufficient detection speed, sensitivity, and reusability [96, 127, 128, 129, 130].
As an unusual microfluidic platform, μPAD has been widely promoted and developed for the molecular diagnosis of infectious diseases such as HCV, HIV, and SARS-CoV-2 [131, 132]. For selective and sensitive detection of HCV, Tengam et al.  developed a novel biosensor based on fluorescent paper using a highly specific nucleic acid probe based on pyrrolidinyl peptide. Nucleic acids are covalently immobilized on partially oxidized cellulose paper by reductive alkylation between amino groups and aldehyde groups, and detection is based on fluorescence. These signals can be read by a specially made gadget with a portable fluorescent camera in combination with a cell phone camera. Subsequently, Lu et al.  designed a paper-based flexible electrode based on nickel/gold nanoparticles/carbon nanotubes/polyvinyl alcohol organometallic framework composites for HIV target detection by DNA hybridization using methylene blue as a DNA redox indicator. More recently, Chowdury et al.  presented a hypothetical platform design for point-of-care µPAD testing using raw patient saliva in combination with LAMP and portable imaging technology for COVID-19 analyte detection.
Lateral flow tests guide fluids by capillary forces and control fluid movement by the wettability and characteristics of porous or microstructured substrates. The lateral flow devices consist of sample, conjugate, incubator and detection, and absorbent pads. The nucleic acid molecules in the LFA recognize specific binders that are pre-stored at the binding site and bind as complexes. As the liquid passes through the incubation and detection plates, the complexes are captured by the capture molecules located on the test and control lines, showing results that can be read directly to the naked eye. Typically, LFA can be completed in 2-15 minutes, which is faster than traditional discovery. Due to the special mechanism, LFA requires few operations and does not require additional equipment, which makes it very user-friendly. It is easy to manufacture and miniaturize, and the cost of paper-based substrates is lower. However, it is only used for qualitative analysis, and quantitative detection is very difficult, and the multiplexing ability and throughput are very limited, and only one sufficient nucleic acid can be detected at a time [96,110,127].
Although most applications of LFA are focused on immunoassays, the use of LFA for molecular diagnostics in microfluidic chips is also effective and popular . In the case of hepatitis B virus, HIV and SARS-CoV-2 LFA Gong et al.  proposed an up-conversion nanoparticle LFA platform and demonstrated the versatility of this miniaturized and portable platform through sensitive and quantitative detection of multiple targets such as HBV nucleic acid. In addition, Fu et al.  demonstrated a novel LFA based on surface-enhanced Raman spectroscopy for the quantitative analysis of HIV-1 DNA at low concentrations. For rapid and sensitive detection of SARS-CoV-2, Liu et al.  developed a microfluidic-integrated RPA lateral flow analysis by combining RT-RPA and a universal lateral flow detection system into a single microfluidic system.
The application of various microfluidic platforms varies depending on specific studies, taking full advantage of the capabilities and advantages of the platforms. With affordable valves, pumps and ducts, LOCC is the most comprehensive platform for application diversity and interoperability with the greatest room for development. Therefore, we hope and recommend that the newest studies be carried out at LOCC as a first attempt and that the conditions be optimized. In addition, more efficient and accurate methods are expected to be discovered and used in the system. LOAD excels in precise control of fluids from existing LOCC devices and demonstrates unique advantages in single drives by centrifugal force without the need for external drives, while parallel responses can be separate and synchronized. Thus, in the future, LOAD will become the main microfluidic platform with less manual operations and more mature and automated technologies. The µPAD platform combines the benefits of LOCC and paper based materials for low cost, single use diagnostics. Therefore, future development should focus on convenient and well-established technologies. In addition, the LFA is well suited for naked eye detection, promising to reduce sample consumption and speed up detection. A detailed platform comparison is shown in Table 2.
Digital analyzes divide the sample into many microreactors, each of which contains a discrete number of target molecules [139, 140]. Digital assays offer significant advantages for performing absolute quantitation by performing thousands of parallel biochemical experiments simultaneously and individually in micron scale compartments rather than in a continuous phase. Compared to traditional microfluidics, compartment reactions can reduce sample volume, increase reaction efficiency, and be easily integrated with other analytical methods without the need for channels, pumps, valves, and compact designs [141, 142, 143, 144, 145, 146, 147] . The following two methods are used in digital assays to achieve uniform and accurate separation of solutions, including reagents and samples such as cells, nucleic acids, and other particles or molecules: (1) drop emulsions exploiting liquid interface instability; (2) array division is carried out by the geometric constraints of the device. In the first method, droplets containing reagents and samples in microchannels can be created by passive methods such as co-current, crossflow, flow focusing, staged emulsification, microchannel emulsification, and membranes through viscous shear forces and emulsification with channel change. localization [143, 145, 146, 148, 149] or using active methods [150, 151], which introduce additional energy through electrical, magnetic, thermal and mechanical control. In the latter approach, the best fluid volume uniformity in microfluidic chambers is shared by keeping spatial structures of the same size, such as micropits and surface arrays [152,153,154]. Notably, droplets are major flow sections that can also be generated and manipulated on electrode arrays based on digital microfluidics (DMF). Electrowetting of dielectrics is one of the best studied DMF theories, since electrowetting of dielectrics allows precise manipulation of individual drops, controlling the shape of the liquid and asymmetric electrical signals passing through different sides [141, 144]. The main operations with droplets in DMF include sorting, splitting, and merging [151, 155, 156], which can be applied in various fields of analysis, especially in molecular detection [157, 158, 159].
Digital nucleic acid detection is a third-generation molecular diagnostic technology following conventional PCR and quantitative real-time PCR (qPCR), in parallel with high-throughput sequencing and liquid biopsy. In the last two decades, digital nucleic acids have rapidly developed in the field of molecular diagnostics of infectious pathogens [160, 161, 162]. Absolute quantification of digital nucleic acid detection begins with packing samples and reagents into individual compartments to ensure that each target sequence has the same probability of entering each individual compartment. Theoretically, each section may be assigned multiple target sequences, or there may not be an independent microreaction system. Through the various sensing mechanisms described above, compartments with microbial target sequences that generate signals above a certain threshold can be visualized with the naked eye or by a machine and are labeled as positive, while other compartments that generate signals below the threshold are labeled as positive. negative ones, which make the signal for each section a boolean. Thus, by calculating the number of compartments created and the rate of positive results after the reaction, the original copies of the test samples can be matched using the Poisson distribution formula without the need for a standard curve, which is required for routine quantitative analyzes such as qPCR.  Compared with traditional molecular diagnostic methods, digital nucleic acid detection has a higher degree of automation, higher analysis speed and sensitivity, fewer reagents, less contamination, and simpler design and manufacture. For these reasons, the use of digital assays, especially drop-based methods, for molecular diagnostics, combining amplification and signal readout techniques, has been well studied during the critical outbreak of SARS-CoV-2. For example, Yin et al.  combined droplet digital and fast PCR methods to detect the ORF1ab, N, and RNase P genes in SARS-CoV-2 in a microfluidic chip. Notably, the system was able to identify a positive signal within 115 seconds, which is faster than conventional PCR, indicating its effectiveness in point-of-care detection (Figure 7a). Dong et al. , Sow et al. , Chen et al.  and Alteri et al.  also applied droplet digital PCR (ddPCR) to detect SARS-CoV-2 in a microfluidic system with impressive results. To further improve the detection rate, Shen et al.  achieved ddPCR-based chip imaging in as little as 15 s without the use of image stitching techniques, speeding up the ddPCR technology process from lab to application. Not only thermal amplification methods such as PCR are applied, but also isothermal amplification methods are used to simplify reaction conditions and fast response. Lu et al.  developed SlipChip for droplet analysis, capable of generating droplets of various sizes at high densities in one step and quantifying SARS-CoV-2 nucleic acids using digital LAMP (Figure 7b). As a rapidly evolving technology, CRISPR can also play an important role in digital nucleic acid detection through convenient colorimetric imaging without the need for additional nucleic acid stains. Ackerman et al. developed a combinatorial matrix reaction for multiplex evaluation of nucleic acids.  detected 169 human-associated viruses, including SARS-CoV-2, in droplets containing CRISPR-Cas13-based nucleic acid detection reagents in a microwell assay (Figure 7c). In addition, isothermal amplification and CRISPR technology can be used in the same system to combine the benefits of both. Park et al.  A CRISPR/Cas12a digital assay was developed in a commercial microfluidic chip for the detection of extracted and heat-killed SARS-CoV-2 based on a single-stage RT-RPA with a shorter and higher signal-to-background detection time ratio. , wider dynamic range and better sensitivity (Fig. 7d). Some descriptions of these examples are given in Table 3.
Typical digital platform for nucleic acid detection. a The rapid digital PCR workflow consists of four key steps: sample preparation, distribution of the reaction mixture, amplification process, and target quantification (adapted from ). b Schematic showing analysis of SlipChip droplets for droplet formation at high density (adapted from ). c CARMEN-Cas workflow diagram13 (adapted from ). d Overview of advanced digital virus detection with CRISPR/Cas in one pot (adapted from ). W/O water-in-oil, polydimethylsiloxane PDMS, PCR polymerase chain reaction, DAQ data collection, PID proportional integral derivative, CARMEN combinatorial matrix reaction for multiplex nucleic acid evaluation, SARS-CoV-2, severe acute respiratory syndrome, coronavirus 2 , RT Amplification of reverse transcriptase recombinase polymerase-RPA, S/B signal in the background
Post time: Sep-15-2022