Biochip is the core technology of portable biochemical analyzer. Biochemical treatment of microstructures obtained by micromachining can enable thousands of life-related information to be integrated on a centimeter-square chip. Using biochips can carry out various biochemical reactions involved in life sciences and medicine, so as to achieve the purpose of testing and analyzing genes, antigens and living cells. The ultimate goal of biochip development is to integrate the entire biochemical analysis process from sample preparation, chemical reaction to detection to obtain a so-called micro total analytical system (laboratory on a chip). The emergence of biochip technology will bring a revolution in the fields of life sciences, medicine, chemistry, new drug development, biological weapons warfare, forensic identification, food and environmental sanitation supervision. This paper describes the recent research progress of biochip technology in processing, preparation, function and application.
The goal of the Human Genome Project is to complete the sequencing of 3 billion human genomic DNA bases in 2005. Now the project is expected to be completed in advance by using more advanced capillary array sequencers and commercial operations. Therefore, people have begun to use known genes found in the Human Genome Project to study their functions, that is, functional genomics studies that link the sequence and function of known genes. In addition, research related to disease has shifted from studying the cause of the disease to exploring the pathogenesis, and from disease diagnosis to disease susceptibility research. Since all of these studies are closely related to DNA structure, pathology and physiology, many countries have now begun to consider whether researchers can use effective hardware technology to deal with such huge DNA and protein information in the post-genomic period. Use it. To this end, there have been various solutions, such as DNA mass spectrometry [1], fluorescent single molecule analysis [2], array capillary electrophoresis [3], hybridization analysis [4], etc. But so far, among the various technologies for the analysis of DNA and proteins, the fastest-developing and the most promising technologies are the affinity binding analysis based on biochip technology and capillary electrophoresis analysis [5] And mass spectrometry. In addition, on this basis, by combining with biochip technology and sample preparation methods (chip cell separation technology [6] and biochemical reaction methods (such as chip immunoassay [7] and chip nucleic acid amplification [8]), many Research institutions and industry have begun to build so-called microchip laboratories. The ultimate goal of establishing microchip laboratories is to integrate many discontinuous analytical processes in life science research, such as sample preparation, chemical reactions, and separation testing. Using micro-scale technology such as semiconductor lithography in the manufacturing process of integrated circuits, transplant it into the chip and make it continuous and miniaturized. These years reduced the number of separate computers in the size of several houses to the size of a book. Notebook computers have the same effect. The biochemical analyzers with different uses made by these biochips have the following main advantages, namely, the entire analysis process is automated, the production cost is low, pollution prevention (the chip is a one-time use), analysis The speed can be increased thousands of times, and at the same time, the required samples and chemicals It can obtain hundreds of times reduction, extremely high multi-sample processing capacity, small size, light weight, and easy to carry. The appearance of such instruments will give life sciences, medicine, chemistry, new drug development, biological weapons warfare, Forensic identification, food and environmental hygiene supervision and other fields have brought about a revolution. Therefore, it has attracted the attention of academic and industrial circles in various countries [9].
1 Micromachining of biochips
The processing of biochips borrows some of the more mature microfabrication processes in the microelectronics industry and other processing industries (such as: optical mask lithography, reactive ion etching, micro-injection molding, and polymer film casting) , Micron-sized microstructures such as filters, reaction chambers, micropumps, microvalves, etc. are processed on glass, plastic, silicon wafers and other substrate materials for the separation and reaction of biological samples. Then apply the necessary surface chemical treatment on the microstructure, and then carry out the required biochemical reaction and analysis on the microstructure.
Among the biochips, the fastest growing ones are affinity binding chips (including DNA and protein microarray chips). In addition to the use of some micromachining processes, its processing also requires the use of robotics. There are now four more typical methods of affinity bonding chip processing. One is the light-guided in-situ synthesis method combining optical lithography and photochemical synthesis developed by Affymetrix [10]. The second method is the chemical spraying method used by Incyte pharmaceutical company. Its principle is to spray the oligonucleotide probe synthesized in advance to the designated position on the chip to make a DNA chip. The third is the contact dot coating method used by Stanford University. The realization of this method is to use the pipetting head carried by the high-speed precision manipulator to contact the surface of the glass chip to drop the probe positioning point onto the chip [11]. The fourth method is synthesized by using four piezoelectric nozzles equipped with A, T, G, and C nucleosides as in-situ DNA probes on the chip [12].
2 Examples of biochips
A biochip is a scaled-down biochemical analyzer. By combining the microstructure obtained by micromachining on the chip with biochemical processing, thousands of life-related information can be integrated on a centimeter-square chip. The chip can be used to carry out various biochemical reactions involved in life science and medicine to achieve the purpose of testing and analyzing genes, antigens and living cells. Through analysis, a large amount of biologically and medically meaningful information can be obtained. The biochemical reaction and analysis process usually includes three steps: 1, sample preparation; 2, biochemical reaction; 3, detection and data analysis processing. One of the steps or several steps can be miniaturized and integrated into a chip to obtain biochips with special functions, such as cell filter chips and dielectric electrophoresis chips for sample preparation, gene mutation detection and gene expression. DNA microarray chips and high-throughput micron reaction cell chips for drug screening, etc. Now, scientists from all over the world are working to integrate the entire process of biochemical analysis through the use of different chips to achieve the integration of all functions to achieve the so-called micro-full analysis system or microchip laboratory. Using the microchip laboratory, one can complete a complete set of operations from the original sample to obtain the desired analysis results in a closed system in a short time.
2.1 Sample preparation chip
For DNA analysis, the preparation process usually goes through many aspects such as cell separation, cell disruption, deproteinization, etc., and finally obtains the DNA to be tested with sufficiently high purity. At present, the most prominent methods of cell separation include filtration separation (separation based on the difference in size of biological particles) and dielectric electrophoresis separation (using a high-frequency non-uniform electric field applied on the chip to induce even electrodes in different cells, It causes the cells to be separated from the sample by different dielectric forces), etc .; the cell breaking methods in the chip include chip heating cell breaking, variable voltage pulse cell breaking, and chemical cell breaking. In terms of capturing DNA, Cepheid uses wet etching, reactive ion etching / plasma etching and other processes to process 5000 pieces of DNA extraction chips with a fine column structure with a height of 200 microns and a diameter of 20 microns, specifically for DNA Extraction [13].
2.2 Biochemical reaction chip
Because the sensitivity of the detection instruments currently used is not high enough, the DNA extracted from the samples still needs to replicate hundreds of thousands or even millions of identical DNA fragments using amplification and replication technology such as PCR before labeling and application.
At present, the success of the nucleic acid amplification reaction in the chip is the University of Pennsylvania research group [8, 14], the Lawrence-Livermore National Laboratory [15] and Perkin-Elmer company [16]. The amplification reaction done by the University of Pennsylvania research team is carried out in silicon-glass chips. The external heating and cooling of the chips are computer-controlled Peltier electric heaters. After inert treatment of the chip surface, that is, after growing a layer of 2000 Angstrom silicon oxide on the surface of the silicon wafer, they successfully completed a series of different nucleic acid amplification reactions in the silicon-glass chip, such as RT-PCR, LCR, multiplex PCR and DOP-PCR. The heating method adopted by the silicon chip processed by Lawrence-Livermore National Laboratory is the thin-film polysilicon heating jacket built in the chip, and the temperature rise and fall speed is very fast. Perkin-Elmer's PCR reaction is completed on a plastic chip. Researchers at Imperial College London have developed a PCR chip that allows samples to flow continuously in a constant temperature range at different temperatures [17]. All the above-mentioned work is done with the DNA or RNA purified in advance as the substrate for the amplification reaction. In order to integrate sample preparation and amplification reactions, the University of Pennsylvania research team recently succeeded in directly amplifying the DNA released after the isolated human leukocytes were lysed by heating in a dam-type microfiltration chip. In the first case, the results of research that integrated sample preparation and amplification reaction as a whole [14].
2.3 Detection chip
2.3.1 Capillary electrophoresis chip
Chip capillary electrophoresis was developed by DuPont's Pace in 1983 [18]. Subsequently, Ciba-Geigy of Switzerland and Alberta University of Canada cooperated to complete the separation of oligonucleotides using glass chip capillary electrophoresis [19]. The first time that chip hair array electrophoresis was used to detect DNA mutations and sequence DNA was completed by a research team led by Mathies at the University of California, Bergley [20,21]. By adding high-voltage direct current to the chip, they completed the rapid separation of many DNA fragments from 118bp to 1353bp in nearly two minutes. In addition, the Mathies team, in collaboration with the research team of Lawrence-Livermore National Laboratory Nothrup, reported the first multiplex PCR detection work that integrated the nucleic acid amplification reaction with chip capillary electrophoresis [22]. The Wilding team of the University of Pennsylvania and the Ramsey team used chip capillary electrophoresis to separate multiple DNA fragments amplified by the chip for the diagnosis of Duxin-Baker muscular atrophy [14]. Other foreign companies and academic institutions that use chip capillary electrophoresis to detect mutations include Perkin-Elmer, Caliper technologies, Aclara biosciences, and MIT.
2.3.2 DNA mutation detection chip
The reason why dNA can hybridize is because nucleosides A and T, G and C can be hydrogen-bonded and complementary at the same time. Many classic molecular biology methods such as Sanger DNA sequencing and PCR are based on this. Several recent techniques, such as photolithographic masking technology for light-guided in situ DNA synthesis [23], electronic hybridization technology [24], high-sensitivity laser scanning fluorescence detection technology [25], etc., have enabled hybridization-based The application has been greatly improved. Some recent authoritative journals in English have reported on the use of chip hybridization to detect mutations. Hacia et al. Used a hybrid chip composed of 96,000 oligonucleotide probes to complete 24 heterozygous mutations (single nucleoside mutation polymorphism) on the exons of hereditary breast cancer and ovarian tumor genes BRCA1. Of detection. They introduced the color difference analysis between the reference signal and the detected signal to make the hybridization specificity and detection sensitivity improved [26]. In addition, Kozal et al. Used high-density HIV oligonucleotide probe chips to analyze the polymorphism of HIV strains [27]. Cronin et al. Used a chip with 428 probes to detect the mutant genes that cause cystic fibrosis in the lungs [28]. American companies that use biochips for hybridization mutation detection include Beckman Instruments, Abbot laboratory, Affymetrix, Nanogen, Sarnoff, Genometrix, Vysis, Hyseq, Molecular dynamics, etc .; British and American academic institutions include the University of Pennsylvania, Baylor Medical College, Oxford University, Whitehead institute for Biomedical Research, Naval Research Laboratory, Argonne National Laboratory, etc.
Another application technique for analyzing DNA by hybridization is repeated sequencing. So, how does repeated sequencing work? First, people synthesize and fix probes with a length of 8-20 nucleosides to a silicon or glass chip the size of a fingernail. When the DNA containing the complementary sequence of the probe is placed on the chip associated with the probe, the solidified probe will bind by hybridizing with the DNA fragment complementary to its sequence [10]. By using a fluorescence detection system with a computer to analyze and combine the fluorescence intensity on each grid of the "checkerboard" and the sequence of known probes on each grid, the base sequence contained in the sample DNA can be known. Recently, Science magazine in the United States reported on sequencing using chip hybridization technology. Chee et al. Repeated sequence determination of the entire human mitochondrial DNA with a length of 16.6 kbp on a silicon chip with 135,000 oligonucleotide probes (each probe was 25 nucleosides in length). The space between each probe is 35 microns. The sequencing accuracy is 99%. In addition, Hacia also reported that a minisequencing-based assays provides a powerful means for detecting all possible changes in base sequence. In this method, four kinds of ddNTPs labeled with fluorescent dyes of different colors need to be added to the enzymatic reaction of the primers. The oligonucleotides cured on the microarray are used as primers for the enzymatic reaction. Base changes in sequence. Affymetrix and Hyseq [29] are American companies that use biochips for hybrid sequencing.
2.3.3 DNA chip used for gene expression analysis
With the sequence of the Human Genome Project, more and more human gene sequences that can be expressed and various mutations that cause diseases and predict diseases are gradually being recognized. In order to be able to search for multiple possible genetic mutations at the same time and speed up the process of functional genomics research, people have now paid more and more attention to the so-called parallel molecular genetics that can provide information about multiple genes and their sequences at the same time. Scientific analysis (parallel molecular genetic analysis) method. Functional genomics studies the expression of genes in specific tissues, at different stages of development, or at different stages of disease. Therefore, its requirement is to be able to obtain the results of multiple molecular genetic analysis at the same time. For example, many disease-causing genes may have hundreds of specific mutations related to characterization. Therefore, effective methods for screening these mutations simultaneously are required. In addition, thousands of genes are expressed in any cell. The differences in gene expression between cells often reflect whether these cells are developing normally or are developing toward malignant tumor cells. The advantage of using chip technology to analyze gene expression by hybridization is that it can provide information about differential expression of multiple genes with very little cellular material, thus providing unprecedented amount of information for disease diagnosis and drug screening [30]. Lockhart et al. Used a probe chip with a length of 20 nucleosides immobilized with 65,000 different sequences to quantitatively analyze 21 different messenger RNAs in the entire RNA population of a mouse T cell line. These specially designed probes can hybridize with 114 known mouse genes. The analysis found that after inducing the generation of cell division, the expression of 20 other messenger RNAs also changed. The detection results show that the detection rate of the system for RNA is 1: 300,000, and the quantitative benchmark for messenger RNA is 1: 300 [32]. When Wang et al. Studied the apoptosis of cells induced by epipodophyllotoxin glucopyranoside (etoposide), using DNA chip technology, an oligonucleotide microarray capable of detecting 6591 human cell messenger RNA was prepared and detected. The amount of 62 messenger RNAs in the cells changed after induction. By selecting 12 genes related to induction for further study, they discovered two new p53 target genes [33]. DeRisi et al. "Brushed" 870 different cDNA probes obtained from a malignant tumor cell line onto a glass slide by a manipulator to observe the expression of oncogenes. After comparing the hybridization results of two cell messenger RNA groups marked with different fluorescent markers, they analyzed the gene expression results in cells whose tumor genes were suppressed after introduction of normal human chromosomes [34]. In addition, Shoemaker et al. Reported a so-called molecular bar coding technique that uses biochips to determine the biological functions of many newly discovered zymogen genes. The advantage of this technique is that it allows us to quantitatively analyze very complex nucleic acid mixtures in parallel [35]. Lueking et al. Recently used protein microarray technology to immobilize the protein used as a probe on a polyvinylidene difluoride at a high density, and detected a 10 pg trace protein test sample. The products expressed by 92 human cDNA clone fragments were tested, and the parallel analysis using monoclonal technology confirmed the low detection rate of false positives. Since protein microarray technology is not limited to the antigen-antibody system, it can provide a new and effective way for efficiently screening gene expression products and studying receptor-ligand interactions [36].
2.4 Microchip Lab
The ultimate goal of the development of biochips is to integrate the entire analysis process from sample preparation, chemical reaction to detection to obtain a so-called micro-full analysis system or microchip laboratory. In June 1998, Dr. Cheng Jing of Nanogen Company and his colleagues reported for the first time the entire analysis process from sample preparation to reaction result display realized by the chip laboratory. They used this device to successfully isolate bacteria from blood mixed with E. coli. After high-pressure pulse disruption, the protein was incubated with proteinase K to prepare purified DNA. Finally, it was confirmed by another electronically enhanced DNA hybridization chip. The extract is the DNA of E. coli. This is a successful breakthrough towards the microfilm laboratory [37]. At present, chips containing heaters, micropumps, microvalves, microflow controllers, electronic chemistry and electron luminescence detectors have been developed, and there are also chips that combine sample preparation, chemical reaction, and analytical detection , Sample preparation and PCR [38]; competitive immunoassay and capillary electrophoresis separation [39]). It is believed that in the near future, microchip laboratories containing all steps will continue to emerge.
3 End
After nearly ten years of unremitting efforts, the development of biochip technology has begun to bring impacts and even revolutions in many fields of life science research. Western developed countries, led by the United States, have already made dazzling achievements in this field. Up to now, the three steps from sample preparation, chemical reaction to detection have been partially integrated, and the integration of all three parts has begun to emerge. China has not yet started in this respect. If all parties pay attention and invest a certain amount of human and material resources, I believe we will have a place in this field in the near future.
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