Microfluidic technology is a technology to accurately control and manipulate microscale fluid (micro-rise to skin-lift level). In the past two or three decades, thanks to the maturity of nano-manufacturing technology and the demand of biochemical technology for the manipulation of trace liquids, microfluidic technology has made rapid development. Compared with the traditional detection methods, the detection technology based on microfluidic platform has the advantages of saving samples and reagents, faster reaction speed, high throughput, easy portability, high automation potential and so on.
In 1998, Burns put forward the concept of “lab-on-a-chip (LOC)”, which integrates a variety of biological and chemical analysis functions on a single microchip, which shows the bright prospect of the application of microfluidic technology in clinical detection and precision medicine. In recent years, the development of “chip laboratory”, also known as “miniature total analysis system”, has developed into a new interdisciplinary research field of physics, microelectronics, materials, chemistry, biology, medicine and so on. This paper mainly introduces the classification and principle of microfluidic technology and its application in clinical nucleic acid detection, immune protein detection and drug screening, in order to show the application prospect and challenge of microfluidic technology in the field of clinical detection.
Application in nucleic acid detection.
Before analyzing DNA or RNA in clinical samples, nucleic acid molecules need to be extracted and purified from the original samples. The most commonly used method of nucleic acid purification in microfluidic chip is magnetic beads method. Sista et al. successfully purified human genomic DNA from human whole blood samples by magnetic beads on a digital microfluidic chip and used it in subsequent polymerase chain reactions (polymerase chain reaction, PCR). In this system, blood samples are successively combined with lysate, DNA capture beads, cleaning solution and eluent on the chip. By activating the electrode moving droplets and cooperating with the magnetic device located at the bottom of the chip, the combination and separation of different droplets and magnetic beads can be realized, thus the purification process of DNA can be completed. Many microfluidic chips accelerate cell lysis by heating when extracting nucleic acid. In view of the fact that temperature control devices are usually needed for subsequent analysis of nucleic acids, this design can improve the efficiency of nucleic acid extraction without increasing the complexity of the equipment. In addition to magnetic beads method, heating method and other methods, some microfluidic chips use the unique techniques in the field of microfluidic control, such as dielectric electrophoresis effect (dielectrophoresis trapping) and isokinetic electrophoresis separation, to complete the purification of nucleic acid molecules. In addition, in order to meet the needs of the second generation sequencing technology for higher purity nucleic acid samples, Choi et al used inertial focusing technology (inertial focusing) to separate cytomegalovirus particles from whole blood and plasma samples after passing through a spiral microflow channel, reducing the proportion of human genes in the samples and reducing the background noise of subsequent sequencing.
In view of the small volume of reaction solution in the field of microfluidic, highly sensitive detection methods are needed to detect nucleic acid molecules in samples. At present, the most commonly used real-time PCR method developed on microfluidic chip is fluorescence detection, which detects the target gene by non-specific embedded dyes such as SYBR or specific fluorescence probes such as Taqman probe and molecular beacon probe. Small light-emitting diodes can be integrated into microfluidic systems to replace larger mercury lamps and mercury lamps as excitation light sources. In addition to real-time PCR, researchers have integrated nucleic acid analysis methods such as nucleic acid hybridization, capillary electrophoresis, pyrophosphate sequencing and DNA optical atlas on various microfluidic platforms as follow-up detection and analysis methods for nucleic acid amplification products.
In view of the small volume of reaction solution in the field of microfluidic, highly sensitive detection methods are needed to detect nucleic acid molecules in samples. At present, the most commonly used real-time PCR method developed on microfluidic chip is fluorescence detection, which detects the target gene by non-specific embedded dyes such as SYBR or specific fluorescence probes such as Taqman probe and molecular beacon probe. Small light-emitting diodes can be integrated into microfluidic systems to replace larger mercury lamps and mercury lamps as excitation light sources. In addition to real-time PCR, researchers have integrated nucleic acid analysis methods such as nucleic acid hybridization, capillary electrophoresis, pyrophosphate sequencing and DNA optical atlas on various microfluidic platforms as follow-up detection and analysis methods for nucleic acid amplification products.
As a nucleic acid amplification method with high specificity, high sensitivity and rapid reaction, the application of PCR in the field of microfluidic has also been fully developed. Accurate temperature control system is the basic condition to complete PCR. Based on the implementation method of temperature control, the PCR methods on microfluidic chips can be simply divided into two categories: static PCR and dynamic PCR. In the static PCR method, the position of the reaction solution remains unchanged in the PCR process, and the thermal cycle is realized by raising and cooling the fixed reaction point. For example, Chang et al used platinum as heating element and temperature sensor to control the temperature of fixed PCR reaction site, and successfully amplified dengue virus type 2 nucleic acid on digital microfluidic chip. In the dynamic PCR method, the reaction liquid moves back and forth controllably between several constant temperature regions with specific temperatures, so as to realize the thermal cycle of the reaction solution. The microfluidic chip for PCR developed by Sista et al has a constant temperature range of 60 ℃ and a constant temperature range of 95 ℃. The reaction liquid moves between the two constant temperature regions, and 40 cycles of PCR reaction can be completed in 18 min. The combination of PCR and microfluidic technology also breaks through the limit of reaction speed of traditional PCR method. For droplet microfluidic, the high surface area to volume ratio of droplets makes the heat conduction faster and more uniform, which can greatly accelerate the PCR reaction process. Wheeler et al. explored the limit of ultra-fast PCR on microfluidic chip from the point of view of reaction thermokinetics and DNA polymerase. Using SpeedSTAR HS DNA polymerase or KAPA2G DNA polymerase suitable for rapid PCR and reducing the residence time in denaturation, annealing and extension steps, 35 cycles of PCR amplification were completed within 3 min. The digital microfluidic chip designed by Chen Tianlan et al uses the cymbal electrode as the temperature control system, which can make the droplet ultra-fast rise and fall in temperature, and complete the dissolution curve analysis of SYBR and specific molecular beacon probe within 7 seconds. Compared with traditional PCR devices, PCR on most microfluidic chips can reduce reaction time and sample consumption by at least 50% and 70% without losing sensitivity.
In addition to combining with PCR technology, various isothermal nucleic acid amplification techniques based on microfluidic chips have also been developed. Although it is different from PCR in sensitivity and specificity, because the isothermal amplification technology requires less precise temperature control, developers are more likely to miniaturize and portable the corresponding devices. The portable microfluidic device developed by Wan Ren et al can realize ring-mediated isothermal amplification (loop mediated isothermal amplification, LAMP) and detection of Brucella genes on a digital microfluidic platform less than a shoebox size. After the amplification reaction at 67 ℃ for 40 min, the fusion curve of the molecular beacon probe was analyzed, and the detection of the target gene could be completed in less than 5 min. The microfluidic nucleic acid detection chip developed by Laili et al combines rolling ring amplification (rolling circle replication, RCA) and microfluidic electrophoresis. After RCA amplification at 37 ℃ and 60 min, the sample can be separated by electrophoresis in the microfluidic channel to detect whether the sample contains the target gene of Vibrio cholerae.
Application in immunoassay.
Immunoassay based on specific binding between antigens and antibodies is one of the most commonly used detection methods in the field of clinical diagnosis. Traditional immunoassay methods, such as enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA), have the advantages of low cost and easy operation, but their time-consuming, labor-consuming and additional equipment such as enzyme labeling instrument also hinder their application in real-time detection. The immunoassay method based on microfluidic platform can promote the adsorption between antigens and antibodies, reduce the reaction time, realize automatic control, integrate small optical probes to miniaturize the equipment, and achieve the goal of real-time detection.
The coating of bait antigen (or antibody), the transfer of liquid phase and the capture of reaction signal are the three most basic aspects of enzyme immunoassay.
The coating of bait antigen (or antibody) has a great influence on the sensitivity and specificity of enzyme immunoassay. Planar media (such as PDMS, glass, etc.), magnetic beads and non-magnetic microspheres are the three most commonly used coating media in the field of microfluidic. Kevin et al integrated protein coating into the process of PDMS polymerization. Because of its simplicity and high coating efficiency, this method has great potential for application in PDMS microfluidic chips.
In channel microfluidic chips, liquid phase transfer is often accomplished by manipulating pumps and valves, or by means of centrifugal force. Wang et al. completed the enzyme immune detection of hepatitis C virus (HCV) antibody on PDMS chip by micro pneumatic pump and miniature pneumatic valve. The enzyme immunity analysis chip developed by Lai et al based on micro-centrifugal force can integrate the enzyme immunity test into a microfluidic device the size of an ordinary optical disc. In digital microfluidic chips, the replacement of liquid phase is usually completed by electrode-driven liquid movement combined with magnetic devices. In the digital microfluidic chip designed by Wheeler et al, the magnetic device at the bottom of the chip is used to fix or move the magnetic beads coated with rubella virus surface antigen, drive the electrode to control the fusion and separation of different droplets such as samples, enzyme markers, chromogenic solution and magnetic beads, and complete the automatic detection of rubella virus IgM and IgG at the same time.
In the traditional enzyme immunoassay methods carried out in the laboratory, the absorbance of the enzyme labeling instrument is usually used to confirm the reaction results. However, most of the microfluidic devices choose to use fluorescence signals to detect the results of enzyme immunoreaction on the chip. Compared with absorbance, the sensitivity and specificity of fluorescence signal are higher, and multiple detection can be realized by using the existing fluorescence labeling system on microfluidic chip. The PDMS microfluidic chip designed by Tohid et al can perform multiple detection of five antibodies in a single channel. In addition to optical signals, electrochemical signals are also often used in microfluidic chips to detect enzyme immunoreaction results. Quantitative results can be obtained by detecting the potential, current, voltage, capacitance, resistance and other factors changed by immune reaction. Compared with the optical signal, the electrochemical signal does not need additional excitation light source, and it is easier to be integrated into the miniaturized microfluidic system, and the detection sensitivity of the electrochemical signal is not affected by the optical path, liquid turbidity and other factors. it has natural advantages in microfluidic system.
Application in cell analysis.
The development of microfluidic chips capable of cell culture, sorting and analysis is also a research hotspot in the field of microfluidic. The miniaturization and high-throughput characteristics of microfluidic technology make it have the potential to use precious and rare tissue and cell samples for high-throughput analysis, which provides support for precision medicine and personalized medicine. Irena et al verified the feasibility of cell culture and drug cytotoxicity analysis on digital microfluidic chip. In this experiment, Plannick F68 was added to the droplets as an additive to reduce the adsorption of cells and proteins on the chip surface, thus reducing the voltage needed to drive the droplets so as not to cause damage to the cells. The PDMS microfluidic chip developed by Ada et al uses specially designed microfluidic channels to generate droplets containing single cells, which can be used for drug screening of tumor cell lines and primary tumor tissue cells on a chip of 2.4 cm × 2.4 cm.
3D cell culture technology based on microfluidic chip is also a development direction of microfluidic technology applied in biomedical field in recent years. Compared with the traditional 2D cell culture method, cell culture in 3D environment can better simulate the real cell growth environment in vivo and reflect the interaction between cells and extracellular matrix. In the PDMS chip designed by Yu et al, multiple parallel cell culture cavities are cross-linked with microchannels to simulate the interaction between tissue and capillary network in vivo. The experimental results of Raty et al showed that the growth rate of mouse embryonic cells in microfluidic channel was closer to the real growth rate in vivo than that in traditional plate culture. Yeong et al. combined the special structure formed by PDMS with porous cell culture plates, and infused hydrogel to form several parallel 3D cell culture cavities, and constructed a high-throughput 3D cell culture microfluidic platform which is completely compatible with traditional cell analysis and drug screening methods.
In addition to microfluidic chips based on cell culture, micro-cell sorting chips that can replace large-scale flow cell devices are also a research focus. In addition to sorting by fluorescence labeling, size and other factors, microfluidic chips can also sort cells through the effect of dielectrophoresis. In the non-uniform electric field, cells with different polarities migrate to different electric field intensities. Using the effect of dielectric electrophoresis and specially designed microfluidic channels, Takahashi et al completed high-throughput cell sorting. In addition, inertial microfluidic technology is often used in cell sorting. Kwon et al use spiral microfluidic channels to make dead cells and cell fragments precipitate from the cell culture medium after passing through the microfluidic chip, which can be further used in perfusion cell culture. The microfluidic chip cascaded with multiple microfluidic channels designed by Abdulla et al can separate circulating tumor cells (circulating tumor cells, CTC) from diluted whole blood samples without any labeling, and the isolated CTC maintains a high survival rate, which is convenient for subsequent drug screening and other tests.
Prospect.
In recent years, the application of microfluidic technology in the field of clinical diagnosis has greatly promoted the development of on-site, real-time detection and accurate and personalized medicine. Although the comprehensive promotion of microfluidic technology is still limited by a series of problems such as the complex operation in the preparation process and the adsorption of biochemical components by chips, with the continuous emergence of new materials, new technologies and new discoveries, there is no doubt that researchers from various disciplines have made continuous research and improvement on microfluidic technology. There is no doubt that microfluidic technology has great potential in the field of clinical diagnosis and even basic medical research. The industrialization of microfluidic technology will also promote the development of clinical testing methods in the direction of miniaturization, high speed, high throughput, portability and automatization, and the wide application of “lab on a chip” is just around the corner.
