MicrofluidicsTechnology inMedical tests (poctfield of application
Microfluidic technology is a comprehensive technology involving multiple disciplines. Also known as a lab-on-a-chip (LOC), it can concentrate traditional laboratory biological and chemical experiments on a highly integrated chip of a few square centimeters. Point-of-care testing (POCT), as a rapid, portable, and immediate medical testing method, is of great significance in the fields of infectious disease detection, disease screening, disease prevention, and postoperative testing. Therefore, microfluidic devices have become the main implementation platform for POCT technology. POCT devices based on microfluidic technology combine the advantages of POCT and microfluidics, and are expected to shine in the field of medical testing. This article describes the application of microfluidic technology in combination with other techniques.
At the beginning of 2020, the outbreak of the novel coronavirus (COVID-19) caused immeasurable losses to people's lives and health and damaged economies around the world. This sudden catastrophe exposed the shortcomings of traditional diagnostic methods. Most traditional disease detection methods, while accurate, require expensive and complex instrumentation, operated by specialized technicians in the laboratory, and are complex and time-consuming. In the context of this global pandemic, traditional testing methods do not seem to be suitable for rapid diagnosis, so there is an urgent need to develop timely diagnostics that can be used for rapid screening of the virus, especially in underdeveloped and remote countries and regions, where it is difficult to meet the above-mentioned rigorous experimental conditions of traditional testing methods. Therefore, the development and promotion of rapid and easy detection methods for infectious diseases is of great significance for maintaining global public health.
Point-of-care testing (POCT) is a quick and easy way to test medicine. Poct has the advantages of immediacy, speed, simplicity, portability, and automation, which can reduce complex sample processing time in the laboratory and is not limited by the environment. Therefore, it has become a research hotspot in the field of in vitro molecular diagnostics in recent years. Commonly used diagnostic techniques in POCT detection mainly include immunochromatography, colloidal gold, dry chemistry, and loop-mediated isothermal amplification (LAMP). In recent years, with the progress of science and technology, the development of sensing technology, advanced manufacturing technology and the Internet of Things, micro, convenient and integrated microfluidic chip technology and biosensor technology are increasingly applied to POCT detection, which has obvious advantages in this field.
This article focuses on the key technologies of microfluidics, biosensing technology, and LAMP technology in the field of POCT medical testing (Figure 1).
FigMicrofluidicstechnology and its integrated application in POCT.
MicrofluidicsTechnology
Microfluidics is a science and technology for the precise control and manipulation of micro-nano fluids in micro-nanoscale space. Microfluidic chips, also known as lab-on-a-chip (LOC), are one of the technology platforms and devices used to implement microfluidic technology. Microfluidic chips can integrate the basic functional units involved in biological and chemical experiments on a chip of a few square centimeters, such as sample preparation, reactions, separations, and detection. The fluid flows to each reaction unit through microchannels. The basic feature and advantage of microfluidic chips is the flexible combination and large-scale integration of multiple technical units on a micro-platform, which is an interdisciplinary discipline involving physics, chemistry, materials science, medicine, mechanics, optics, mechanical engineering, micromachining, and bioengineering.
MicrofluidicsApplication of technology in POCT
3.1. Microfluidic chip detection technology
The signal acquisition and detection device of the microfluidic chip is an important part of the microfluidic functional components. In order to complete the detection process on a microfluidic chip of a few square centimeters, there is no doubt that the requirements for the detection method and equipment are more stringent, at least to meet the characteristics of small size, high sensitivity and fast response time. The detection technologies of microfluidic chips include laser-induced fluorescence detection, ultraviolet absorption spectrophotometry detection, chemiluminescence detection, electrochemical detection, mass spectrometry detection, plasma emission spectroscopy detection, thermal lensing spectroscopy detection and biosensor detection. Optical detection and electrochemical detection are the most widely used detection methods.
Laser-induced fluorescence detection is the earliest, most widely used, and most sensitive optical detection method. The principle is that atoms absorb light of a certain wavelength under an excitation light source of a specific frequency. Atoms change from a low energy level to a high energy level and release low-frequency fluorescence. Laser-induced fluorescence is a method of inducing electron transitions to produce fluorescence by using a laser as an excitation light source. In 2006, the Whitesides team developed a PDMS system-on-a-chip that uses fluorescence detection.
Chemiluminescence detection is also a highly sensitive detection method. It determines the content of a substance by detecting the luminous intensity by using the luminescence phenomenon, in which the ground state molecule absorbs energy to transform into an excited state in the form of light radiation during a chemical reaction and returns to the ground state. Compared with optical detection, chemiluminescence detection does not require a light source, the equipment is simple, easy to miniaturize and integrate, and is more suitable for microfluidic chip detection. In 2006, Lin's group designed a rotational scanning chemiluminescence detection device for multi-channel detection.
Electrochemical detection is a detection method that converts the chemical signal of a substance into an electrical signal for analysis and testing. It has the advantages of high sensitivity, simple equipment and low cost. Mathies et al. designed it to detect analytes using sheath flow techniques and capillary electrophoresis.
3.2. LAMP-based microfluidic chips
The combination of microfluidic technology and nucleic acid diagnosis is one of the most promising application directions of microfluidic technology. This combination greatly simplifies the complex steps of nucleic acid amplification and detection, eliminates heavy equipment, and is therefore ideal for POCT molecular diagnostics. The nucleic acid amplification process of isothermal amplification technology only needs to be completed at a constant temperature. It does not require a complex heating and cooling temperature system, which greatly reduces the reaction time and is more suitable for miniaturized instruments. Therefore, the combination of microfluidic technology and isothermal amplification technology can take advantage of the advantages of both while improving the detection sensitivity and detection throughput, which makes it have great potential for development in the field of molecular POCT.
The principle of the LAMP technique, proposed by Notomi et al. in 2000, is to amplify nucleic acids at approximately 65 using four specific primers and one strand instead of active DNA polymerase. Microfluidic technology can provide LAMP with flexible structural platforms, such as stand-alone reaction microcavities and serpentine flow channels for sample transfer. This not only speeds up the reaction and saves reaction reagents, but also avoids cross-contamination and aerosol leakage. In recent years, researchers have paid more and more attention to the combination of lamps and microfluidic chips.
China's classic LAMP microfluidic chip is a centrifuge disk constant temperature microfluidic chip designed by CapitalBio Technology. The chip has 24 detection holes, each with a volume of 14 l, results in 30 minutes. It can realize the parallel detection of multiple indicators, which greatly reduces the detection time and cost. In 2012, Kong et al. designed a portable PoCT microfluidic chip based on LAMP technology for bacterial identification. The chip has a simple structure and builds microchannels on PDMS. Reagent dispensing is carried out by combining air plasma with glass plates, using the capillary siphon effect, and fluid control by torque valves. During amplification, use a 65 water bath for 45 min to visualize the results. It is also possible to identify bacteria with "sample input, result output" and POCT. In 2010, Jiang's team proposed a microfluidic chip for the quantitative detection of pathogens based on LAMP technology, called microlamp (LAMP). The structure is a PDMS glass composite microfluidic chip with eight conical inlet and outlet channels, which can quickly and sensitively provide specific and quantitative identification of target nucleic acids, meeting the requirements of multi-channel parallel detection. The amplification reaction conditions were 1 h in a 63 water bath, and the results were visually visible and confirmed by gel electrophoresis. Real-time quantitative analysis of products can be achieved through turbidity absorbance measurement, multi-integrated optical fibers, digital fiber sensors, and optical transistors. Bau's research team reported a LAMP reactor-integrated separator membrane for POCT detection of infectious diseases. This disposable cassette includes a single lamp chamber in which nucleic acids can be isolated, concentrated, purified, and amplified. The separation membrane captures and eliminates nucleic acids from the elution step. LAMP reactors can be used to detect nucleic acids associated with other pathogens carried in saliva, urine, and other body fluids, as well as water and food. Luo et al. designed a real-time microfluidic multiplex electrochemical lamp chip for distinguishing bacteria. This microfluidic system is known as a multiplex electrochemical (me-lamp) system. The ME-LAMP system combines a LAMP and an indium tin oxide (ITO) electrode-based microfluidic chip to qualitatively and quantitatively analyze multiple genes by measuring electrochemical signals. This has the potential to be applied to clinical diagnosis. Lee et al. proposed a hydrophilic thin-film-based lamp for digital quantification of DNA microfluidic chip coating. This microfluidic chip does not require an external pump and uses capillary force to drive the flow of liquid. This design has a promising future in POCT diagnosis. LAMP technology can also be used to detect foodborne and waterborne pathogens to ensure the safety of food and drinking water. Microfluidic chips with disc or butterfly structures are used to increase detection throughput and efficiency in multiplex detection of sexually transmitted diseases, and these chips have been commercially applied. There are still many improvements in LAMP-based microfluidic chips, which will definitely occupy a place in the field of POCT diagnostics in the future.
4 Paper-based and hybrid microfluidic chips
Paper discs have the advantages of low cost, simple processing, flexible structure, environmental friendliness, and good biocompatibility. The combination of microfluidic paper-based chip and microfluidic technology combines the advantages of both and is a development direction with good application prospects. Paper-based microfluidic chips typically use filter paper as the substrate and filter paper as the reaction or circulation zone for hydrophilic or hydrophobic treatment. The paper material's unique three-dimensional pore structure provides capillary force to drive liquid flow, and the hydrophilic-treated surface of the material allows reagents to adsorb and react there. In addition, the hydrophobic surface facilitates the flow of liquids. Papers commonly used for substrates include Whatman series filter papers, nitrocellulose membrane filter papers, ink papers, chromatography papers, kleenex papers, and various composite papers. Paper-based microfluidic chip processing methods include lithography, wax printing, flexographic printing, inkjet printing, 3D printing, and laser cutting. There are one-step and two-step methods for hydrophilic and hydrophobic treatments. The one-step method applies a single hydrophilic and hydrophobic treatment to all material surfaces, and the two-step method refers to the local treatment of the material using physical deposition, plasma treatment, and other methods. Unlike polymer-based microfluidic chips, some paper-based microfluidic chips do not need to be turned off, and these are called open channels because the liquid in the paper-based microfluidic chip moves inside the paper fibers, which simplifies the processing of the microfluidic chip. In addition, hybrid microfluidic chips combined with polymers, glass, hydrogels, and other materials integrate the advantages of various materials to make the chip more comprehensive, which is one of the most promising development directions for microfluidic chips in the future.
In 2006, Wang et al. designed a single-use microfluidic device that can be used for DNA amplification and detection. This is an early nucleic acid detection system that combines microfluidic technology with lateral chromatography technology to greatly improve the detection efficiency. The device introduces a temperature-sensitive hydrogel valve as a control switch that not only closes the PCR chamber but also inhibits the formation of air bubbles. The detection method of the microfluidic box is to excite the fluorescent particles with an infrared laser to produce fluorescence. The entire process of cell lysis, DNA isolation, purification, amplification, and detection can be completed on a single chip, enabling one-time, independent and rapid detection of pathogens and body fluids. In 2007, a team at Harvard University Whiteside first proposed a paper-based microfluidic chip and used it to analyze metabolites such as blood glucose, uric acid, and lactate. Dou et al. (2016) developed a PDMS paper-glass hybrid microfluidic chip for the detection of multiple pathogens. The chip has three layers. The upper layer of PDMS is used to transfer reagents, and it includes four microchannels and an injection port. The middle layer is the reaction zone, which consists of eight pores connected by four microchannels. The assay chamber, negative control chamber and chromatography paper are also placed in the middle layer. The 3D porous structure of cellulose paper is beneficial in protecting DNA primers from adverse environmental influences and can effectively prevent the formation of aerosols, which can lead to the loss of primers in the air. As a result, polymer paper-based composite chips have a longer service life. The ground floor is supported by glass shards. The processing method for hybrid chips is laser cutting, which is fast and easy to handle. The bonding method is oxyionic bonding to bond glass and PDMS. Inlet and outlet are encapsulated with epoxy resin. The by-product pyrophosphate binds to manganese ions to form complexes, and calcein combines with manganese ions to produce fluorescence, which can be directly observed with the naked eye within 1 hour. Hybrid microfluidic chips can be used to detect a wide range of pathogens with high sensitivity and specificity, low cost, speed, and no instrumentation required. The YIN group introduced the Kirigami technique into the 3D structure, extended the idea of 2D decoupage to 3D space decoupage, and designed a universal 3D decoupage module with multiple deformation modes. Li and Liu used origami technology to design a three-dimensional multifunctional integrated paper-based microfluidic analysis system, which can realize the parallel detection of four tumor markers. The researchers believe that modular design and assembly will be independent of size and materials, and their research is expected to find potential applications in the fields of reconfigurable metamaterials, robotics, and architectural design. The introduction of origami technology provides a new idea for the development of paper-based microfluidic chips. Combining LAMP technology, microfluidic technology, and biosensing technology, the LI team designed a paper-polymer hybrid microfluidic nanosensor based on an optical disc (CD) structure for the detection of meningitis and other infectious diseases. At just a few tens of cents and the size of a coin, the chip can effectively store primers for 73 days, facilitating subsequent amplification and improving detection sensitivity and stability. The chip is small in size, portable, low in cost, fast in speed, and can quantitatively detect a variety of pathogenic bacteria. It is very suitable for remote, underdeveloped and impoverished areas and is of great significance in promoting medical diagnosis in these areas. In 2019, Dr. Ning's research group designed an autonomous capillary microfluidic chip (ACMC) that enabled POCT point-of-care infarction detection by using capillary drive, self-focusing lens optical detection, and a customized mobile app. The test results are transmitted to both the hospital and the user. In addition, in the field of analytical chemistry, there are paper-based POCTs combined with electrochemical biosensors for the detection of secretions and biomarkers. Paper-based microfluidic chips have great application potential in the field of POCT.
MicrofluidicsBiosensors
In recent decades, people have become more health-conscious, and there is a growing need for the prevention and diagnosis of certain diseases, especially infectious diseases. Biosensors integrated with microfluidic chips for POCT diagnostics meet the requirements for rapid detection and have become a research hotspot. The combination of microfluidic technology and advanced biosensing technology has led to the invention of many excellent small analytical platforms that can enable precise control of micro-nano liquids and the integration of various types of biological arrays on small platforms. This microfluidic integrated biosensor device offers many advantages, such as low reagent consumption, short reaction times, automated sample preparation, high-throughput analysis, minimal hazardous substance handling, parallel detection, high detection accuracy, and flexible design and miniaturization, portability, low cost, and single-use.
Biosensor technology refers to technology that is able to sense or respond to chemical and biological information and convert chemical or biological signals into electrical or optical signals that can be identified according to certain rules. POCT is an advanced technology that integrates biology, chemistry, materials, optics, microelectronics, and other disciplines. It is fast, sensitive, low-cost, and the inspection equipment is easy to automate and miniaturize. POCT has broad application prospects in medical diagnosis. A biosensor is a sensor that uses a biological entity as an identification element to convert biological signals into easily measurable signals such as light, electricity, heat, pressure, and mass through specific targets to detect various biological entities. There are three main parts to biosensors. One consists of biological entities used for detection, including DNA, animal and plant tissues, bacteria, microorganisms, cells, enzymes, antibodies, proteins, nucleic acids, glucose, amino acids, and lactate. The second is a detection converter, which is used to detect biological signals and convert them into other measurable signals. The third is the display equipment and signal processing equipment used to display the analysis results. Biosensors can be divided into two categories: microarray biosensors and microchannel biosensors, the latter being biosensors with integrated microfluidic chips. In addition, according to different biomolecules, microchannel biosensors can be divided into nucleic acid biosensors, enzyme biosensors, immune biosensors, bacterial biosensors, microbial sensors, cell sensors, and biomimetic sensors. According to the different principles of signal detection, microchannel biosensors can be divided into electrochemical biosensors, optical biosensors, thermistor sensors, field-effect transistor sensors, piezoelectric quartz crystal sensors, mass sensors and surface plasmon resonance sensors. Biosensors can be used for both qualitative and quantitative analysis of biomolecules. Pregnancy tests and blood glucose meters are currently commercially successful biosensors, but most of the biosensors currently in use rely on laboratory instruments for sensing and are not suitable for POCT rapid test systems. In recent years, scientists have worked on the development of portable, miniaturized, low-cost, and easy-to-operate biosensors. Therefore, microfluidic biosensors that combine microfluidic technology and biosensors have emerged. POCT devices with integrated microfluidic biosensors have great application potential in the fields of clinical detection, biochemical analysis, disease diagnosis, food safety, environmental monitoring, national defense, anti-terrorism measures, and biological warfare agents. They are more effective, more specific, more sensitive, more portable, easier to automate, and easier to read results. A schematic diagram and application of a biosensor is shown in Figure 2.
Figure 2: Schematic diagram of biosensor application.
Sia et al. designed a microfluidic biochip based on optical detection for the diagnosis of HIV, bringing hope for low-cost disease prevention and diagnosis in remote areas. Combining a paper-based microfluidic chip with a biosensor, Crook's team used printing and origami techniques to produce a three-dimensional paper-based microfluidic chip biosensor for adenosine detection. Rushling et al. used a printed carbon electrode as a substrate on CD to fabricate PDMS microchannels and inserted silver and platinum electrodes to form electrochemical sensing for the detection of serum markers. There is also some research on the detection of bioluminescent bacteria. These microfluidic biosensors typically consist of microfluidic chips containing microchannels, active cells for sensing targets, and transducers. Depending on the state of the bacteria, these studies can be divided into bacterial suspension, freeze-dried, and fixed types. Bacterial suspension refers to bacteria in suspension in a liquid, bacterial lyophilization refers to bacteria that make lyophilized powder, and bacterial fixation refers to the immobilization of bacteria on disposable sample cells, optical fibers, or chips. For example, the fiber-optic monitoring system developed by Eltzov's group to detect toxic contaminants in water is to fix bacteria on optical fibers. Jouanneau et al. designed a microfluidic biosensor for the detection of heavy metals. Today, with the popularity of smartphones, microfluidic biosensors integrated with mobile phones can provide real-time, synchronized detection result feedback to users and the cloud using the phone's features such as photography, sensing and Bluetooth. Microfluidic biosensors integrated with smartphones are a new generation of smart biosensors with huge market prospects. Ozcan's team has developed a POCT rapid detection diagnostic device installed on a smartphone, which acquires images from the camera and connects them to a customized app for synchronous processing. It then automatically outputs the diagnostic results and sends them to users and medical institutions. Researchers can successfully use this device to detect HIV, AIDS, tuberculosis, malaria, and various infectious diseases. It greatly facilitates the prevention, surveillance and diagnosis of epidemics, and also provides conditions for the promotion of rapid diagnosis of infectious diseases in remote areas.
6 Research progress of microfluidic chips
In addition, microfluidic technology has also been applied to disease diagnosis, bacterial screening, immune and biochemical reactions, enzyme protein recognition, organoid technology and other fields. In 2021, the exodff microfluidic chip, developed by Hou et al. at Nanyang Technological University in Singapore, was used to assess the vascular health of diabetic patients. exodff uses centrifugal force and fluid mechanics to separate vesicular cells from blood for analysis. Compared to traditional blood separators, the cost of exodff chips is only a few yuan, which greatly reduces the cost of detection. The chip helps patients quickly monitor the health of their blood vessels and has great commercial potential. In July 2021, Science reported that a microfluidic glass chip called High-Throughput Microfluidic Enzyme Kinetics (HT-MEK) could simultaneously detect mutations in more than 1,000 enzymes in just a few hours. At just $10, it's about 7 square centimeters in size and contains 1,568 microwells, each of which can test an enzyme. Droplet microfluidics is one of the commonly used microfluidic techniques for high-throughput detection. Meng Wang's team from the Institute of the Chinese Academy of Sciences (CAS) designed a droplet embedding microfluidic technique for high-throughput screening of 10,000 strains of Streptomyces per hour. The work was published in the journal Nature. Researchers at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, have designed a microfluidic chip that can isolate high-purity circulating tumor cells (CTCs) and circulating fusion cells (CFCs) from whole blood samples in a single step and perform high-throughput single-cell transcriptome sequencing. A digital microfluidic chip is a high-throughput microfluidic chip that uses an array of electrodes on a chip to form electrical signals to control droplets. The principle is to use the difference in surface tension to control the droplets, and the core technology is how to efficiently and stably produce the droplets. Recently, researchers at the Suzhou Institute of Biomedical Engineering and Technology of the Chinese Academy of Sciences proposed a digital microfluidic chip that can split a large droplet into three smaller droplets. Smaller droplet sizes can break the minimum volume limit. This technology successfully achieves the effective separation of small droplets. In combination with other functional modules, a fully automated digital microfluidic analysis platform was developed that can detect 5 samples in parallel in 10 minutes. The platform can be used for immunodiagnosis, disease surveillance, guidance and prognostic assessment. The researchers have designed a microfluidic platform that can accurately quantify and analyze the formation process of single-cell bulges and use microfluidic technology to enable rapid screening of mitochondria-specific drugs, which will have a positive impact on the development of novel anti-cancer drugs. The work was published in Analytical Chemistry. The fluid circuit board is combined with a microfluidic device for automated cell culture and precise fluid control. Users have more flexibility with this new modular microfluidic system.
7 Summary and outlook
In recent years, POCT, as an important part of in vitro diagnostics (IVD), has attracted the attention of major universities, research institutions and IVD companies. As a multidisciplinary technology, microfluidic technology has been used as the main implementation platform for POCT technology, both from the advantages of the technology itself and from the national level. Fast, portable, highly sensitive and specific POCT molecular diagnostic products have become the new favorite in the market. Due to its rapid development, microfluidic technology has gradually become the main means of POCT diagnosis, but there are also challenges and opportunities. However, most of the current microfluidic products are limited to the laboratory stage of scientific research, and there are not many mature products on the market. There are many problems with the application of microfluidic technology, such as being limited by the requirements of ultra-high-precision processing, as well as the difficulty of precisely controlling liquids in micro-nano size, the challenges of microchannel surface modification, and how to achieve rapid mass production at low cost. Cost and efficiency have always been issues that need to be addressed. After years of research, more and more emerging technologies have begun to be applied to microfluidic technology, involving a new generation of manufacturing technology, chip material selection, amplification and detection methods, as well as sensing and other fields, such as 3D printing technology, LAMP technology, biosensing technology, paper-based microfluidic chips, nanomaterial-based microfluidic chips, and hybrid-based microfluidic chips. The introduction of a variety of functional materials has made it possible to develop multifunctional microfluidic chips, which has greatly expanded the application field of microfluidic chips. Relevant researchers and practitioners should seize the opportunity to respond to the challenges in a timely manner and make microfluidic technology shine in the biomedical industry.