The microfluidic manipulation of fluids in microfluidic chips is on the order of micrometers, between the macroscale and the nanoscale, where fluid motion exhibits duality. On the one hand, the micrometer scale is still much larger than the mean free path of the molecule in the usual sense. Therefore, for the fluids in it, the continuum theorem is established and the continuity equation is available. The electroosmotic and electrophoretic mobility is independent of the size. On the other hand, relative to the macroscopic scale, the impact of the inertial force on the micrometer scale is small, the influence of the cohesive force increases, the Reynolds number becomes smaller (usually between 106-101), the laminar flow characteristics are obvious, and the mass transfer process follows. Convection dominated by diffusion, and the ratio of surface to surface increased, and surface effects such as cohesive force, surface tension and heat transfer increased, and edge effects increased. The three-dimensional effect was not negligible. At the same time, there are many important differences between microscale and nanoscale. On the nanometer scale, the size of the object is similar to the mean free path of the molecule, so the electrophoretic mobility becomes related to the cross-sectional size, the electrical charge of the even electrical layer overlaps, and the electroosmosis decreases, which in turn affects the momentum imparted to the fluid. In addition, the compression of the space will change the shape of the macromolecules, and the mobility of the macromolecules will also be affected by the non-planar flow velocity vector field, eventually resulting in relatively difficult control of the fluid back.
2. Separation technologySeparation is an important step in microfluidic chip sample analysis. Separation of the capillary slot in the chip loads most of the applied voltage, and its field strength is mostly between 200 and 500V/cm. Therefore, try to reduce the load voltage as much as possible during the design [4. In order to improve the efficiency of separation, many methods are used in the microfluidic chip. For example, according to the method of gradient elution in HPLC, Kutter designs two buffer pools containing different polarity buffers and mixing buffers at different volume ratios. Liquid, and then use this mixture as a supporting electrolyte for the sample, the experiment shows that the effect is better, the separation time is less than 1min [5].
3, micro-droplet technologyThe micro-droplet manipulation includes micro-droplet generation and micro-droplet drive. According to the generation mode, the micro-droplet manipulation method can be divided into two categories. One type is the passive method, that is, through the special design of the micro-channel structure, the velocity of the liquid flow is locally generated to control the micro-droplet, which is mainly a multi-phase flow method. The main feature of this method is that it can quickly generate batches of micro-droplets; the other type is the active method, which uses an external force such as electric field force and thermal energy to generate an energy gradient locally to control the micro-droplets, mainly including electrowetting. Method of mouth and dielectric electrophoresis [same, pneumatic method [? ) and thermocapillary method 0]. The main feature of this method is the manipulation of individual droplets. Compared with traditional continuous-flow systems, the discretized micro-droplet system has a series of potential advantages such as less sample and reagent consumption, faster mixing speed, less cross-contamination, and ease of handling.
4, detection technologyThe high sensitivity detection of the separation is of great significance for the microfluidic chip. At present, the microfluidic chip detection methods can be roughly divided into three categories: optical detection, electrochemical detection, and mass spectrometry detection.
Ultraviolet absorption detection method is a kind of conventional optical detection method, and the corresponding detector has matured. However, because the channel of the chip is small and the sensitivity is not high, the method cannot meet the requirements for the analysis of low concentration and very small amounts of samples. Laser-induced fluorescence detection is the most sensitive method for all fluorescence detections. In most cases, the lower limit of detection is 10*10-10~12 molL, so this method has been widely used.
There are three basic modes of electrochemical detection: amperometry, conductance and potentiometry. Amperometry is the most commonly used method. The basic principle is that when the measuring compound is subjected to an oxidation or reduction reaction on the surface of the electrode, it loses or obtains electrons and generates an electrode current proportional to the concentration of the analyte. By measuring the current in the microchannel, the change in the concentration of the solution can be obtained. The sensitivity of electrochemical detection can be comparable to fluorescence detection, and at the same time, microelectrodes can be processed into chips. Therefore, it is more suitable for the detection of microchips. The principle of mass spectrometric detection 14 is to achieve the purpose of detection based on the difference in molecular mass to charge ratio. Its greatest advantage is that it can provide molecular space structure information, so it has unique features in the study of the structure of biological macromolecules (such as proteins). However, since the mass spectrometry detection system itself is larger than the chip, it is also difficult to realize the miniaturization of the entire system. A single detection method will be difficult to complete all detection tasks, so the joint use of multiple detection methods and new detection methods should be studied.
The research of microfluidic chip technology in the analysis of water environment pollution is still in its infancy, so it focuses on the relevant reports of priority pollutants, mainly including heavy metals, nutrients, organic pollutants, and microorganisms.
1. Microfluidic chip system for detecting heavy metals in water
With the development of industry and agriculture, more and more heavy metals such as mercury, chromium, lead, copper, nickel, vanadium, etc. are discharged into water bodies, which will not only produce toxic effects on aquatic animals and plants, but also enter the biological chain through enrichment. It poses a serious threat to the entire ecological environment. For the detection of the above-mentioned heavy metals, high-accuracy methods such as atomic absorption spectroscopy and atomic fluorescence spectrometry can be used. But responding to a sudden pollutant leak or connecting a region
In the case of continued monitoring, there is still a need for rapid and efficient detection tools. Using photolithography and wet etching technology, a microfluidic chip was successfully developed. This chip successfully measured the cobalt nitrate using Luminol luminescence properties. At the same time, after a simple transformation, the micro total analysis system can also become a device for detecting hydrogen peroxide or nitrogen dioxide, and can be combined with a signal transmission device to become a device with its own wireless signal transmission function.
Paper-based microfluidic devices have also developed rapidly in recent years. Compared with microfluidic devices with similar functions, they have the advantages of simple operation, no need for external aid equipment, multiple detection, and the like. Paper chips that detect multiple heavy metals show good sensitivity.
2, for the determination of nutrients in water body chip system
Microfluidic chip systems for nutrient measurement are mostly based on the principle of spectrophotometric detection. Modern microfabrication techniques are used to integrate various optoelectronic components. For example, a microfluidic chip system for the detection of phosphate in water. The system is equipped with a data launching device, which can be used to arrange all-round real-time monitoring of the phosphate pollution in the area at different locations in the target area. The minimum detection limit is 0.3 mg/L.
Jia Hongxin et al. proposed a three-layer hybrid microfluidic chip, which processes micro-reaction channels on glass slides, processes gas-permeable membranes with PDMS and PDMS negatives with accepting channels, and realizes the reaction of ammonium ions in solution to produce ammonia. Diffusion, separation, absorption, bromothymol blue coloration and photometric detection on microfluidic chips.
3, for the analysis of organic pollutants in water
In addition to inorganic contaminants in water bodies, a greater number of them are organic contaminants. They affect the ecosystem in ways that are toxic and reduce dissolved oxygen in water, and endanger human health. Therefore, the quantity of organic contaminants is the evaluation of water pollution. An extremely important indicator of the situation. Due to the low content of this type of pollutants, it is usually necessary to carry out pre-treatment. The advantages of the microfluidic chip is that it can integrate the pre-treatment and the later detection, and has a high extraction/enrichment efficiency. Wait.
4, for microbiological detection chip system in water
The micro-organisms in the water body belong to the granular organic carbon range according to their particle size, and their species groups can reflect the ecological characteristics of the water body and some important pollution conditions. It is a routine monitoring indicator in the ecological investigation of water bodies. During its measurement, flow cytometry is the most accurate and rapid method. However, its equipment is expensive, bulky and requires special operations. It is not suitable for on-site and continuous monitoring requirements. The appearance of microfluidic chip based on sheath-flow control overcomes these limitations to a certain extent, and may realize the integration of instruments. Miniaturization, automation and portability.
Second, the application of microfluidic chip in cell biologyWith the continuous development of microfluidic chips, microfluidic analysis chip technology is constantly infiltrating the research field of cellomics. Microfluidic chip applications in cell biology include cell culture, cell isolation and manipulation, cell component analysis, and cell-wide analysis system.
For example, Carlson et al. reported the use of hydrostatic pressure-driven methods to separate cells from blood samples. Because the volume of red blood cells is much smaller than that of white blood cells and the viscosity is small, the red blood cells pass through the micro-flow network at a faster rate. The whole cell analysis system refers to a microfluidic system that integrates cell three-dimensional culture, cell stimulation, cell isolation, lysis, and separation and analysis of cell components. This system can not only quickly analyze cells but also
The microfluidic chip analysis system brings great advantages in analytical performance by miniaturizing microchannels and structures.
1) Shorten the reaction time and improve the analysis efficiency. Many analysis processes can be completed in a few minutes;
2) The saving of reagents and samples, the consumption of samples and reagents for microfluidic analysis has been reduced to the level of a few microliters, and with the improvement of the technical level, it may be further reduced;
3) Easy to integrate, portable, easy to operate, and easier to automate.
1, enzyme analysis
A simple cross channel or reflection chamber can be constructed on a silicon wafer, a glass chip, a quartz chip, or a polymer polymer chip, and a simple enzyme assay can be performed by using an electrochemical detector, an optical detector, or other detection system. For example, Hadd-AG produced an enzyme detection system with 5 solution inlet and outlet channels on the chip. First the fluorophore substrate RBG was mixed with the Tris buffer, with the B-galactosidase solution and the competitive inhibitor PETG solution. After mixing, the substrate enzymatic hydrolysate produces a fluorescent substance that is detected by a laser-induced fluorescence detector. The system uses only 120pg and 7.5ng of enzyme and substrate, which is 4 orders of magnitude lower than that of conventional analysis methods. This shows that the microfluidic analysis chip enzymatic analysis has a good application prospect in the field of clinical diagnostics for drug development and other fields.
2, immune analysis
Immunoassay is one of the most important analytical methods. Routine immunoassay requires a long analysis time, the liquid handling process is also troublesome, and more expensive antibody reagents are needed. The microfluidic analysis chip can effectively overcome this shortcoming. Integrating the analysis system on the chip can enhance the reaction efficiency, simplify the analysis process, reduce the analysis time, and reduce the reagent consumption. For example, Sato et al. pre-coated polystyrene beads with anti-CEA antibodies and introduced them into channels. A cofferdam was constructed in the channel to block the beads, and then it was labeled with serum samples containing CEA, primary antibodies, and colloidal gold. Anti-response, sandwich immunoassay with three antibodies can detect ultra-trace CEA in serum minutes, reducing the overall analysis time to about 35 minutes.
3. Proteomics research
Protein column analysis is considered to be the most promising area following genomic analysis. With the development of technology, proteome research has evolved from gel-based electrophoretic separation toward the use of mass spectrometry. Because proteome studies require large-scale, high-throughput protein analysis and identification methods, low-volume, high-throughput microfluidic chip analysis technology combined with highly sensitive mass spectrometry detection technology has great advantages. For example, Gao integrated an integrated device for proteolysis, peptide separation, and mass spectrometry on the chip. This device enables the work done in the original number J to be completed within 5 minutes, and the amount of reagents is less than or equal to nanograms or nanograms.
IV. Application of Microfluidic Chip Gene Analysis1. Polymer-based PCR microfluidic chip
PCR as an in vitro method for amplifying nucleic acids has long been an indispensable tool for studying molecular biology. Although the conventional PCR operation is simple, it has a slow heating cycle and is inefficient because of its large heating volume. In order to solve this problem, the reaction volume of the PCR was reduced to 5 ouls or even 1 pl, but the volume reduction also correspondingly limited the yield. PCR microfluidic chips are developed in this situation.
Compared with traditional PCR, the main advantages of the PCR chip are its large specific surface area, fast heat transfer rate, and greatly improved reaction rate; furthermore, the internal temperature is uniform and the reaction process is easy to control. At the same time, the amount of sample and reagent required for the PCR chip reaction is small, which greatly reduces the cost. For example, KOPP used the PCR microfluidic chip to complete the PCR amplification of the 176 bp fragment of the Neisseria gyrase gene in 20 cycles over 1.5-18.7 min.
2. Separation and analysis of nucleic acid restriction fragments
Microfluidic chips can be used to rapidly isolate DNA restriction fragment PCR products much faster than conventional capillary electrophoretic separations. Oligonucleotide mixtures have been isolated since the successful application of microfluidic chip capillary electrophoresis techniques such as Manz et al. The length of the DNA fragments isolated by the microfluidic chip is gradually expanding, and a multi-channel array chip capable of parallel analysis has emerged.
3, DNA sequencing
Using the microfluidic chip four-color labeling method, 150 bases can be isolated at 540S with an accuracy of over 97%. Conventional DNA sequencing requires the preparation of micro-upgraded samples. The consumption of reagents is large. It has been reported that the nanoscale sample preparation system is micro-scaled onto a chip for sequencing, and excess primers, salts, and nucleotides can be removed before separation. It is 1/300 of the Sanger dideoxy termination method, the cost of sequencing is significantly reduced, and solid-phase sequencing can be performed.
V. Application of Microfluidic Chips in Bionic ResearchAlong the research direction and ideas of bionic simulation, the ability of microfluidic chip technology to control the spatio-temporal control of cells and microenvironment has been fully demonstrated in animal cell biological correlation research. HO et al [30] designed and prepared a cell trapping chip, which can induce the radial beaded arrangement of hepatocytes in the microcavity through the electric field of the bottom concentric electrode array of the chip, and then perfuse the human umbilical vein endothelial cells in the human gap. To simulate liver tissue. This study confirmed the possibility of reconstructing the hepatic lobules in vitro. Liu et al. used integrated microfluidic chip technology to build a chip system for the dynamic study of the interaction between cells and the microenvironment. The chip is manufactured using multilayer soft lithography technology. Through pneumatic microvalve control of liquid flow, cells and cell microenvironment, a variety of micro-environmental cell stimuli responses can be studied. This study achieved continuous experimental operations such as on-chip surface treatment, cell positioning loading, and heterotypic cell co-cultivation. A series of dynamic and operational studies on the interaction between tumor cells (HepG2 hepatoma cells) and stromal cells (3T3 fibroblasts) were performed.
Weiwei Li et al. 2 focused on the influence of in vitro environment on fertilization and embryonic development. The current artificial assisted reproductive technology has the problem of low fertilization success rate and high fetal risk. A microfluidic chip was developed and the chip contains 3 layers. The structure consists of PDMS for the top and bottom layers and a porous PC film for the middle layer. The top layer of the chip contains a 500-μm wide and 110-μm-high channel. A series of curved screens for trapping egg cells are interleaved in the channel. The screen diameter is 150 μm. Six micro-pillars with a diameter of 35pm surround. The bottom layer of the chip contains four parallel rectangular channels (6mmX3mmX110pm) with both ends connected to a common inlet and outlet. The bottom of the channel has a 4X3 microcolumn array for supporting the PC membrane. Embryogenesis is promoted by careful simulation of the uterine environment using endometrial cell-embryonic co-culture and continuous perfusion. Using the above-mentioned chip uterus, a series of experimental procedures such as ovulation, fertilization, implantation and embryogenesis were completed. The chip uterus is not only easy to operate, but also can obtain a higher embryo formation rate than the traditional method.
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