Research on real-time nucleic acid detection device based on microfluidic technology

Nonisothermal amplification technology involves nucleic acid amplification steps at different temperatures during the reaction process. The most extensively applied nonisothermal amplification technique is the polymerase chain reaction (PCR) [38]. PCR technology mimics the natural process of DNA self-replication and amplification. The PCR process involves repeated cycles of DNA/RNA through three stages: denaturation (93–94°C), annealing (40–60°C), and extension (70–75°C). Each cycle of denaturation, annealing, and extension exponentially amplifies the target sequence. Thus, PCR is an efficient technology that amplifies nucleic acids exponentially. In genetic disease and infectious disease testing, PCR is widely considered the gold standard for detecting target DNA and RNA [39,40]. Performing PCR amplification on a microfluidic chip can enhance various aspects of PCR performance, including amplification efficiency, reducing reaction time, simplifying the procedure, and lowering costs [41]. Temperature control is critical for PCR amplification, as precise thermal cycling is required for the denaturation, annealing, and extension of target sequences. POCT devices, due to their compact nature, often face challenges in maintaining stable and uniform temperatures, leading to potential thermal fluctuations [42]. Inconsistent heating or cooling can result in incomplete denaturation, inefficient primer binding, or suboptimal extension, all of which could decrease amplification efficiency and compromise specificity, leading to false negatives or false positives. Additionally, several POCT devices have been developed and designed [43].

PCR modules incorporated into POCT devices for nucleic acid detection can be broadly classified into fixed microchamber and flow-through types [44]. The PCR module with fixed microchamber usually seals the samples and reagents in the specific chamber of the microfluidic chip and uses the temperature control module with rapid temperature rise and fall function to provide different temperatures required in the PCR reaction process. The PCR module with a fixed microchamber can be heated using various methods, including contact heating devices such as film resistive heaters [45], box heaters [46], hot plates [47], and printed circuit heaters [48], as well as noncontact methods such as infrared heating [49], microwavable [50], wireless induction heating [51]. The flow-through PCR module achieves the desired temperature conditions by managing the reagents’ passage through various constant temperature zones within the microchannel of the reaction chamber [52]. In addition, there is a thermal convection PCR technology [53] that creates a stable temperature gradient within the capillary by applying heat to one end. This temperature gradient will induce the reaction reagent to circulate spontaneously between the cold and hot regions in the capillary and accomplish nucleic acid amplification.

When the target sequence is short (<300 bp), the annealing and extension processes in PCR amplification can be conducted concurrently. For target sequences shorter than 100 bp, the extension time can be reduced to 0 s [54]. The PCR amplification process has been simplified, requiring only two temperatures for target sequence amplification, resulting in a significant reduction in PCR amplification time. Scientists have conducted research on and optimized the temperature differential between the temperature control module and the reaction reagents, the thermal resistance of materials, and the heat capacity of both the microfluidic chip and the reaction reagents. Consequently, some devices capable of performing ultrafast PCR have been developed. Nouwairi et al. [55] designed a nucleic acid amplification device that facilitates ultrafast thermal cycling. The traditional 50-min reverse transcription-polymerase chain reaction (RT-PCR) amplification process was compressed to under 10 min using two thermo-electric Peltier devices. An et al. [56] introduced a temperature control strategy that significantly enhances the temperature ramping rate of the solution by widening the temperature gap between the heating plate and the solution. Within a specific range, the temperature rise rate of the solution is linearly related to the temperature difference, the greater the temperature gap, the more rapidly the rise rate. The maximum heating and cooling ramp rates for a 25 µL solution reached 24.12°Cs⁻¹ and 25.28°Cs⁻¹, respectively, which is 6–8 times faster than that of conventional commercial PCR devices. The PCR amplifier designed using this method can complete 45 PCR cycles in just 9 min. Trauba et al. [57] employed a two-step PCR to amplify the target sequence, configuring the temperature control module with two distinct temperature zones at 90°C and 65°C. In total, 35 temperature cycles were executed within 52 s. Yeom et al. [58] developed an ultrafast PCR amplification device using inexpensive thin glass chips and thermal resistors. This device can heat the reaction reagents at a maximum rate of 28.8°Cs⁻¹, achieving ultrafast PCR amplification while reducing the manufacturing cost of the device. These devices with fast thermal cycling show promise by significantly accelerating the temperature ramp, but the problem of temperature uniformity in the heating region is still a huge challenge, especially in small sample sizes [59]. Moreover, these ultrafast thermal cycling techniques may affect the accuracy of amplification, especially when dealing with highly complex or low abundance targets. Rapid cycling may also increase the possibility of nonspecific amplification or primer dimer formation, thereby reducing specificity. Salman et al. [60] introduced a comprehensive system comprising a PCR chip, thermal cycler, and fluorescence detector. Following nucleic acid amplification, the products can be fluorescently detected to yield results. The system has a heating rate of 1.8°Cs⁻¹ and a cooling rate of 2°Cs⁻¹. This represents a significant reduction in heating and cooling rates compared to the real-time PCR system developed by Petralia et al. [61], which boasts a heating rate of 15°Cs⁻¹ and a cooling rate of 8°Cs⁻¹. Analysis of the two devices suggests that the primary cause of this difference may be the varying sizes of the heating elements and microfluidic chips. The thinner the microfluidic chip, the smaller the reaction reagent volume, resulting in faster temperature ramps. However, too little reagent volume may also lead to unsatisfactory reagent concentration or insufficient reagent mixing, thus reducing the sensitivity. In addition, reducing the volume of reagent may damage the efficiency of downstream detection steps [62]. Excessive miniaturization may also increase the risk of evaporation, especially in closed systems, which may affect the consistency and reproducibility of the reaction, resulting in changes in amplification yield. The thermal cycling required for PCR constrains the further miniaturization of microfluidic devices in POCT. Huang et al. [44] developed an ultrafast and highly sensitive real-time PCR system that integrates the best features of microchamber and flow-through amplification systems. This system includes a microfluidic chip optimized for rapid heat transfer and an amplification device with five temperature zones, complemented by a fluorescence detection module for quantitative nucleic acid assays. It addresses the previous dilemma of achieving both rapid temperature rates and flexibility in amplification modules [63]. The system minimizes mechanical complexity, enabling rapid temperature fluctuations while significantly controlling manufacturing costs. Additionally, the team compared the performance of their real-time PCR system with commercial PCR devices, finding that the results of biological sample tests were in agreement with those from commercial devices, despite reducing the total testing time from 1 h to 15 min. Utilizing the real-time PCR system for nucleic acid testing not only ensures sensitivity but also significantly reduces testing time, offering a promising solution for ultrafast real-time PCR. The preceding text introduced various schemes and structures, and Figure 3 illustrates several representative schemes among them.

Figure 3:

Microfluidic chips and temperature control devices for ultrafast PCR. (A) Ultrafast PCR design and hardware device [56]. (B) Ultrafast PCR chip with 35 cycles in 1 min and fast rising and cooling device [57]. (C) Microfluidic chip to improve RTDs performance and the temperature control device [62]. PCR, polymerase chain reaction; RTDs, resistance temperature detectors.

To sum up, now there are many POCT devices that can achieve rapid PCR amplification, which greatly improves the efficiency of nucleic acid amplification. However, there are still challenges in detecting low copy target sequences. As shown in Table 1, compared with other PCR techniques, digital PCR (dPCR) technology has extremely high sensitivity and absolute quantification advantages. In the future, as dPCR technology becomes more economical and compact, integrating it into POCT system can completely change the sensitivity by allowing absolute quantification of target sequences, even at very low concentrations [64]. Developing rapid heating and cooling devices with good temperature uniformity using new materials can help reduce thermal cycling errors [65]. Developing integrated temperature sensors and more powerful thermal control modules will improve sensitivity and specificity. Integrating and optimizing existing on-chip sample preparation technologies, such as automatic nucleic acid purification or improved inhibitor removal methods, will help to eliminate sample interference and reduce false negative effects caused by PCR inhibition. POCT devices shall be designed with built-in pollution prevention function, such as closed disposable reaction chamber, which will reduce cross contamination and improve the specificity of results.

Compared to PCR amplification technology, the advent of isothermal amplification technology provides a bright strategy to the challenge of further reducing the size of POCT devices, which was previously impossible due to the necessity of temperature control modules in nonisothermal amplification devices [66]. Common isothermal amplification techniques include Loop mediated adiabatic amplification (LAMP), recombinant polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA). The subsequent review will solely concentrate on POCT devices for nucleic acid amplification employing LAMP technology.

Since its invention by Hase et al. in 2000, LAMP technology has been a primary focus in the development of POCT, owing to its simplicity, high sensitivity, and speed. LAMP is carried out at 60–65°C and can amplify DNA to 10⁹–10¹⁰ times within 15–60 min. The LAMP reaction generates >50 times the number of amplicons compared to any other PCR-based amplification technique. LAMP also has the potential to amplify target DNA of various sizes ranging from 130 bp to 300 bp, which can be used for multiplex amplification of pathogens [67,68]. Compared to other isothermal amplification techniques, LAMP employs 4–6 primers that offer superior specificity and can amplify both DNA and RNA. But if the primer design is unsatisfactory, it will increase the possibility of non-specific binding. Improper primer selection or unsatisfactory primer concentration will lead to the formation of primer dimer, which competes with the target amplification and reduces the reaction efficiency. In addition, if the primers are combined with unexpected sequences, nonspecific amplification may occur, resulting in false positive. LAMP is a one-step amplification technique that requires no additional sample processing, utilizes a single DNA polymerase, and demonstrates outstanding resistance to polymerase inhibitors [69]. Therefore, LAMP is currently the most prevalent isothermal amplification technology in the field of POCT. Jiang et al. [70] developed a microfluidic chip for rapid nucleic acid detection using LAMP, achieving a detection limit of 102 copies µL⁻¹. Xiao et al. [71] leveraged microfluidic technology to develop a handheld microfluidic lamp colorimetric chip. This chip enables direct DNA extraction from meat samples using microneedles and swiftly transfers the samples to the microfluidic chip for LAMP amplification. El Tholoth et al. [72] used LAMP to realize real-time detection of multichannel pathogens, which can detect multiple different virus samples at the same time. Loo et al. [73] developed a highly integrated nucleic acid detection platform based on LAMP, utilizing a disk platform to provide a sample-answer solution. A silicon membrane is employed for on-chip solid-phase extraction and purification of bacterial DNA; centrifugal force drives the movement of reagents within the chip, and SYTO-9 (New England Biolabs) fluorescent dye is used for real-time fluorescence detection. This microfluidic platform can complete the entire nucleic acid detection process within 2 hr, analyzing multiple samples simultaneously. Liu et al. [74] developed a fully automated centrifuge chip that can simultaneously detect five different bacteria and designed a corresponding automated supply platform. Nucleic acid testing can be completed within 70 min, with a detection limit of 10 copies µL⁻¹ at the limit of detection. LAMP is qualitative in nature, which means that although it has high sensitivity, it cannot provide absolute quantification of target molecules. For applications requiring accurate quantification, such as viral load measurement or therapeutic effect monitoring, the lack of quantitative accuracy may be a major limitation. Since the introduction of the digital LAMP (d-LAMP) method by Gansen et al. [75], it has been extensively applied for precise quantification of nucleic acids. Rane et al. [76] developed a microfluidic device leveraging d-LAMP technology to perform all the steps required for digital nucleic acid detection. The device can produce 1 million droplets every 110 min, with an average droplet size of 10 pL. Following amplification, fluorescence detection was conducted, achieving a detection limit of 600 copies µL⁻¹. Although d-LAMP improves the accuracy of quantification, it still faces challenges related to the consistency of droplet formation and potential cross contamination between droplets. The detection limit of d-LAMP also depends on the sensitivity of the system. Suboptimal droplet size distribution or incomplete droplet sealing may affect the sensitivity and specificity. In addition, the integration of d-LAMP into compact POCT devices will bring additional complexity, which may increase the cost and require more complex fluid control. Xie et al. [77] developed a simple and cost-effective nucleic acid detection platform using colorimetric LAMP technology. Simple on-site disease diagnosis can be directly performed by visually discriminating colors with the naked eye. The detection limit stands at 101 copies µL⁻¹. The preceding text introduced multiple cases, and Figure 4 presents several representative ones among them.

Figure 4:

Microfluidic chips and temperature control devices for LAMP. (A) Portable device for multichannel real-time nucleic acid detection [72]. (B) Schematic diagram of the principle of the microfluidic chip (a quarter of the disk chip) for the whole process detection and schematic diagram of the matching automatic detection platform [73]. (C) Schematic diagram of disk microfluidic chip based on LAMP and its supporting automatic detection platform [74]. LAMP, loop-mediated adiabatic amplification; LED, light-emitting diode.

The characteristic of LAMP constant temperature amplification is beneficial for making nucleic acid detection POCT devices more compact and convenient. As shown in Table 2, applying LAMP technology to nucleic acid detection POCT devices eliminates the need for cumbersome temperature control devices and has higher sensitivity and specificity compared to PCR, which helps to construct smaller POCT devices in resource limited environments. However, due to the high difficulty of primer design for LAMP and the expensive reagents used, the cost of POCT devices using LAMP technology is also higher, and their popularity in rural and remote areas is not as good as POCT devices using PCR technology.

Although these rapid amplification systems using PCR and lamp technology show great prospects, the current research lacks the actual performance verification in outdoor environment. GeneXper^™ system is manufactured by Cepheid using PCR amplification and ID NOW^™ platform is produced by Abbott Laboratories using isothermal amplification have been tested and validated in remote areas such as Uganda and South Africa and have shown good performance during the pandemic [78,79]. The accuracy and efficiency of POCT devices that have not been tested in practical application scenarios under on-site conditions (such as rural clinics, disaster areas, and emergency response situations) have not been fully determined. It is very important to test these systems in the environment with changeable temperature and humidity to ensure their reliability and adaptability in practical applications. The ability to maintain high sensitivity and specificity outside the laboratory environment remains a major challenge. To improve the performance of POCT device in the actual scene, efforts should be made to develop devices that can handle rapid temperature changes while maintaining temperature uniformity. The integrated temperature sensor and powerful thermal control module will help to reduce the thermal cycle error and improve the sensitivity and specificity of the results.

The microfluidic platforms can effectively extract and amplify nucleic acids, but it is still challenging to ensure that these POCT devices can detect low concentrations of nucleic acids in complex clinical samples (such as blood and sputum). The presence of inhibitors (such as proteins, salts, or metabolites) in the sample will interfere with nucleic acid extraction or PCR amplification, resulting in false negative or reduced detection accuracy [97]. It is essential to develop robust systems that can overcome these challenges while maintaining high robustness.

The POCT device for nucleic acid detection usually needs to integrate multiple steps of the detection process in a single, compact platform, including sample collection, nucleic acid extraction, amplification, and detection. This inevitably involves trade-offs between device size, function, and performance. As the size of the devices shrinks, the physical space required for reagent storage, thermal management (PCR or other amplification technologies), and necessary components such as sensors or detectors may be limited. Although miniaturization enhances portability, it also limits the ability to incorporate other functions or effectively execute certain programs [98]. Designing a system that is compact and capable of performing all necessary tasks without sacrificing performance is a major challenge. In addition, most POCT devices used for nucleic acid detection require external component power supply, data analysis, or connection (such as smart phones or computers). It is difficult to integrate these components into a single device without making the system too large or complex. Powering devices in remote or resource limited settings can be challenging, especially for devices that need to work in an energy-scarce environment.

The microfluidic system, micro electromechanical system (MEMS), and optical detection components need to be integrated into a compact portable system. These processes will significantly increase the production cost of POCT device. Moreover, the demand for high-quality components that can operate reliably in different environments and reagents that perform well in different environments further increases the total cost. General POCT devices are often customized for specific diseases, sample types, or certain specific environmental conditions. Customization of these POCT devices may require energy conversion components or reagents to obtain the best performance. In addition, the overall material sustainability of POCT devices should also be considered [99]. Adding environmentally friendly materials, such as biodegradable plastics or recycled polymers, to the construction of POCT devices will help reduce the impact on the environment. In the future, the waste management system can be integrated into the devices, such as the automatic treatment protocol of used components, which can help guide users to correctly and effectively dispose of materials. To sum up, manufacturers will face great challenges in developing and producing economical, efficient, and universal POCT devices that can be applied to a variety of scenarios.

For POCT device, ensuring user friendliness is a key factor for successful adoption, especially in clinics, remote areas, and even home-based nonlaboratory environments. POCT devices must be easy to use, require minimal training, and provide clear and reliable results. POCT devices for nucleic acid detection usually involve complex multistep processes, including sample processing, nucleic acid extraction, amplification (such as PCR), and detection. For users without technical background, it is a major challenge to have a simple and clear operation interface and as few operation steps as possible. Many devices require precise control of fluid flow, temperature, and reaction conditions, which may be difficult to manage manually. Ensuring that users can handle these tasks without advanced training while maintaining high performance and accuracy requires careful system design and automation. Simplifying the interface and reducing the number of steps that users need to perform is critical to creating a user-friendly system. An intuitive user interface is essential to ensure that healthcare providers and even patients with minimal technical expertise can operate the device effectively. The human-computer interaction interface shall clearly guide the user to complete each step of the test process, from example to result interpretation. Designing a human-computer interaction interface that provides clear instructions, visual feedback, and easy to understand results is very important to improve the user experience [100]. In addition, the devices should include mechanisms such as visual or audible alarms to notify users of problems (e.g., insufficient sample size, incorrect reagent use, or device failure).

At present, POCT devices have made significant progress in the laboratory environment, but there are still many challenges to ensure that these devices can perform well in real application scenarios. In real application scenarios, especially in outdoor or remote areas, there will be many uncertain factors interfering with POCT performance. The collection of on-site samples may have different sources and have a high degree of complexity, so that the samples may contain impurities that affect the subsequent amplification and detection. There are huge temperature fluctuations and humidity changes, which will affect the stability of the devices and lead to inconsistent thermal cycle accuracy, thus affecting the amplification efficiency and sensitivity. In the detection process, interference signals generated by strong light, ultraviolet light, or other environmental light sources in the outdoor environment will affect the precision of the optical detection system of the devices, resulting in the decline of sensitivity [101]. There are still many challenges to improve the sample adaptability, amplification stability, and anti-interference ability of equipment in complex field environments.

In conclusion, although POCT nucleic acid detection device has broad development prospects, it still faces many challenges. This requires continuous research, innovation, and collaboration of multidisciplinary integration, and focus on improving the portability and intelligence of devices in combination with current new technologies.

Future POCT devices should be committed to making greater breakthroughs in ease of operation and automation. Many existing devices still require operators to have certain professional knowledge, and the links of sample processing and data analysis are relatively complex. Future POCT devices must pay more attention to user friendliness, adopt more intuitive human-computer interaction interface, use less manual operation, and realize “fool type” operation. In addition, with the integration of artificial intelligence (AI) and machine learning technology, the devices should actively integrate with these technologies and strive to automate a series of processes such as sample collection, processing, and analysis, so as to further reduce the operation complexity and human error.

Real-time data monitoring and remote diagnosis: with the popularity of 5G and Internet of things (IoT) technology, future POCT devices will be able to transmit detection data in real time and connect with telemedicine platforms. This means that doctors or professionals can remotely monitor the detection process, timely obtain the detection results, and make rapid diagnosis and treatment decisions based on the data. This function is especially suitable for remote areas or emergency situations and can realize rapid disease screening and emergency response. However, this combination also has potential security and privacy issues, especially in clinical and sensitive environments involving patient data. Sensitive patient data may be intercepted or tampered with during transmission, which may result in personal health information being exposed to unauthorized third parties or altered diagnostic data, leading to incorrect diagnostic results [102]. These issues must be carefully addressed, and only through rigorous security testing and regular software updates to patch vulnerabilities can patient information be guaranteed. In addition, the device should be equipped with a secure boot process and authentication mechanism to ensure that the potential advantages of POCTIoT integration are achieved without compromising patient safety, confidentiality, and trust.

These technologies have been applied to the rapid diagnosis of pathogens, gene mutation fragments, and cancer markers in POCT devices. To further improve the performance of nucleic acid detection POCT devices, the future development direction should focus on the complete automation of hardware control by the main control chip [103], the high integration of microfluidic modules in POCT devices [104], and the high intelligence of combining with smartphone software to perform nucleic acid analysis, provide results without the upper computer [105], and even try to combine with IoT technology to upload data to the cloud to form a data network [106]. By analyzing and processing uploaded data, disease monitoring and early warning can be achieved.

POCT devices have enormous potential to revolutionize healthcare services, especially in low- and middle-income countries. For example, during the COVID-19 pandemic, the necessity of POCT device research has been highlighted. These devices provide fast, cost-effective, and user-friendly diagnostic solutions that can be deployed in resource limited environments. However, due to the significant cost investment required for the early development of POCT devices, it is necessary for local governments and international organizations to work together to provide subsidies or financial assistance for the research and development of POCT devices [107]. In the early stages of development, it is possible to simplify the design of the flow channel structure, reduce manufacturing complexity, and use inexpensive but functionally reliable materials to replace traditional expensive materials, thereby reducing manufacturing and research and development costs. The driving force for the development of POCT devices comes from the commercialization of POCT devices developed by relevant research and development devices. Commercial POCT devices should be simpler and more efficient to use and provide accurate and reliable detection results. Enable clinical doctors to easily access diagnostic information at a glance. In addition, before commercialization, the devices must comply with international regulatory standards for diagnostic tools, which is extremely important for ensuring the quality and performance of the devices and ensuring the confidentiality of patient privacy. This requires developers to comply with the ISO 13485 standard when researching and designing, ensuring that the device can meet user needs and regulatory requirements throughout its entire lifecycle. Develop clear devices specifications and implement risk management processes in accordance with ISO 14971 to identify, assess, and mitigate potential risks, particularly those related to patient safety, misdiagnosis, or devices failure. Devices related to IoT technology also need to ensure encryption of information transmission to ensure compliance with global data protection regulations [108]. Before commercialization, it is essential to conduct validation in actual application scenarios to ensure that the device can function properly in different scenarios. When quantifying production, relevant standards should be strictly followed to ensure consistent devices quality. Even after actual circulation in the market, it is necessary to actively collect adverse event reports and user feedback, regularly reevaluate the performance of the devices to ensure that it can continue to comply.