WO2001036667A1 - Combinatorial and automatic detection method of target gene, and a detector using the method - Google Patents

Abstract

The present invention is directed to a combinatorial and automatic detection method of target gene, and a detector based on the method. Quartz Crystal Microbalance (QCM) technology in conjunction with gene chip technology is mainly used in the present invention to detect DNA; in a specific method, microfabricating technology is used to etch super-thin quartz resonator array directly on a piezoelectric quartz crystal, then, a number of probes are immobilized, so, a number of DNA or RNA molecule can be detected and analyzed simultaneously. Compared with traditional nucleic acid blot hybridization technique which shortcoming is complex, low automatic degree, less target molecule to be detected; the automatic detection method of target gene and the detector according to present invention can detect in situ without label and obtain detecting information at any time, the detector itself is so small to be carried and can be used conveniently.

Description

 Method for automatically detecting combined target genes and detector using same

 The present invention relates to a method for automatically detecting a combined target gene, and a detector manufactured based on the method. Background technique

 With the implementation of the Human Genome Project and the rapid development of molecular biology-related disciplines, gene sequence databases are growing rapidly at an unprecedented rate.

 However, how to study the biological information of many genes and the functions they play in life has become a problem that researchers in this field need to solve. This has put forward a large number of DNA and RNA sequencing and analysis. Accurate and fast requirements.

 The emergence of gene chips, also known as DNA chips or biochips, has provided possible solutions to such problems. '' Purpose of the invention

 The main purpose of the present invention is to provide an automatic detection method of target genes for genetic diagnosis, genotyping, and forensic and environmental analysis of human genetic diseases, tumors and infectious diseases using in vitro gene chip technology.

 Another object of the present invention is to provide a tester which can be used for scientific research, clinical use, and manufactured based on the above method. A brief description of the detection method of the present invention is to use a microfabrication technique to directly etch an ultra-thin quartz resonator array or a single quartz resonator on a quartz crystal, and then develop a highly sensitive, in-situ hybridization monitor based on this. The miniature quartz resonance gene sensor chip establishes a non-labeled gene sensor detection technology with detection sensitivity equivalent to the current labeled DNA probe technology. At the same time, because the gene sensor chip has a large amount of DNA and probe array fixed, it can solve the existing gene Detection technology can only detect problems with a small amount of genetic information.

 The basic working principle of using the piezoelectric gene sensor for target gene detection according to the present invention is as follows: a large number of probe molecules are fixed on a quartz body support coated with a gold or silver film layer (two sides of the body pass through silver electrodes Apply a certain voltage), and then hybridize with the sample in the reaction cell. Because hybridization will cause the resonance frequency of the quartz crystal to change, the presence or absence of target molecules and the number of samples can be determined by detecting the change in the resonance frequency of the quartz body. BRIEF DESCRIPTION OF THE DRAWINGS

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Replacement page (Article 26) The non-marker gene sensor detection technology equivalent to the needle technology solves the problem that the existing gene detection technology can only detect a small amount of genetic information. BRIEF DESCRIPTION OF THE DRAWINGS

 The following is a description of the drawings of the present invention. Through the following description and the following detailed description, the principle and structure of the present invention can be more clearly understood. A schematic diagram of a DNA chip used; FIG. 2 is a cross-sectional view taken along the line A-A of the chip described in FIG. 1;

 FIG. 3 is a schematic diagram of electrode surface probe fixation in the combined target gene automatic detection method according to the present invention;

 FIG. 4 is a schematic diagram of the detection results of the combined target gene detector according to the present invention on HPV and LT, wherein the Y-axis is a frequency reduction value and the X-axis is time;

 FIG. 5 is a schematic flowchart of an automatic detection method of a combined target gene according to the present invention;

 FIG. 6 is a schematic diagram of a circuit of a combined target gene automatic detector according to the present invention and connection modes of various components.

 The following are the numbers of the components of the above drawings of the present invention, where 1 probe layer, 2 is an upper gold (or silver) film layer, 3 is a quartz crystal, 4 is a lower gold (or silver) film layer, 5 a base , 6 is a silver electrode, 7 is a probe (or known gene fragment), and 8 is a support.

 The following is a detailed description of the detection method and detector according to the present invention. Detailed description

 The method of the present invention is characterized in that a combination of gene chip technology and piezoelectric sensor technology is used to form a unique detection method.

 The invention utilizes microfabrication technology to directly etch an ultra-thin quartz resonator array on a quartz crystal. The resonance frequency of the quartz crystal is very sensitive to fine changes in the surface quality of the crystal, so its quality detection limit can reach pg level. And because the microfabrication technology makes the quartz resonator array easy to prepare in batches, the cost can be greatly reduced.

 The basic technical principles adopted by the method or detector of the present invention are as follows:

 1. Piezoelectric phenomenon and principle

 The effect of external mechanical pressure on a crystal to generate a charge on its surface is called the piezoelectric effect. As early as 1880, researchers discovered the piezoelectric phenomenon of some crystals, such as quartz, and pointed out that when certain dielectric substances are deformed by an external force in a certain direction, polarization will occur inside and a charge will be generated on the surface. When the external force is removed, it returns to the uncharged state again. Moreover, the charge formed on the crystal surface is directly proportional to the applied pressure. The phenomenon of converting this mechanical energy into electrical energy in the prior art is called the "paramagnetic effect" .

Conversely, when an electric field is applied in the direction of dielectric polarization, it will cause mechanical deformation. When the external electric field is removed, the deformation of the dielectric disappears. This phenomenon of converting electrical energy into mechanical energy is called the "reverse piezoelectric effect". In the prior art, dielectric materials having a piezoelectric effect are collectively referred to as piezoelectric materials. The more common piezoelectric materials are quartz, ceramics, etc. Among them, quartz has become a main component of piezoelectric sensing, especially piezoelectric electrochemical and piezoelectric biosensors due to its good mechanical, electrochemical, and temperature properties.

 Quartz is an anisotropic crystal. When the crystal is cut in different directions, its physical properties (such as elasticity, piezoelectric effect, and temperature characteristics) vary widely.

 The researchers of the present invention found that when an alternating excitation voltage is applied to the electrodes on both sides of the piezoelectric crystal, the crystal will generate mechanical deformation and oscillation. When the frequency of the alternating voltage reaches the natural frequency of the crystal, the amplitude will increase and a piezoelectric resonance will be formed. This specific frequency is called the resonance frequency.

 According to the sensitive mechanism of piezoelectric sensing, using a piezoelectric crystal as a resonant structure, it can be found that its output signal (resonant frequency) is closely related to the physical size and properties of the crystal.

 It has also been observed that when a crystal is coated with a thin layer of material, its oscillation frequency changes accordingly.

Sauerbrey first derived the relationship between the mass of the material carried on the surface of the crystal and the resonance frequency shift (2-1) by vibrating in the gas phase through AT-cut quartz crystals, and based on this, proposed the use of piezoelectric crystals as sensitive microbalances. Therefore, this formula is often called the Sauerbrey equation. P « ^A

 AF: Frequency variation (Hz) caused by the coating.

F _q : basic response frequency. = 丄 = 丄, υ: speed of sound wave in air.

t _q : thickness of the quartz (cm).

^: Shear modulus of AT-cut quartz (2.947x10. Cm— '. S— ² ).

P _q : density of quartz (2.648 g. Cm " ³ ).

 Am-. Coating quality of the surface of the quartz wafer (g).

A: The surface area of the quartz wafer (cm ² ).

(Note: A is the total coating mass on both sides of the quartz wafer, and ^ is the surface area of one side of the quartz wafer.) For AT-cut quartz crystals, the shear modulus (/ ^ SS x lO ¹ ^; ^ / ^ ² ) And the density (p _q = 2.648 g. Cm " ³ ) are both fixed values. Substituting the values of ^ and ^ into (2-1) gives:

 The premise of the above two formulas is that it is assumed that a uniformly deposited coating film on a quartz wafer is equivalent to adding a layer of quartz of the same quality. That is, the film and quartz are required to be rigidly bonded, that is, the mass on the electrode surface does not undergo any shear deformation when the crystal oscillates, so that the difference between the film layer and the density and elasticity of the quartz crystal can be ignored.

It can be seen from the Sauerbrey equation that when a quartz wafer oscillates in the gas phase, Af and Δπ show a simple linear relationship. Therefore, the quartz wafer can be used as a very sensitive quality detector, and its detection limit can reach ng level or even pg level. . However, it has been confirmed that the Sauerbrey equation is applied to "rigid" coatings When studying layers, Am / m should be 2% or less (m is the mass of quartz per unit area before deposition).

2. Crystal liquid phase oscillation theory

 Since piezoelectric sensors obtain the required information by measuring changes in the oscillation frequency, the first problems encountered and necessary to understand for crystals that oscillate in the liquid phase are: ① the effect of crystal oscillation activity (viability) What are the factors, or what is the oscillation interval of the crystal in different properties of the solution; ② How does the nature of the solution affect the crystal oscillation frequency. Only by solving these two problems can the crystal oscillation (application) system be broadened and the quality sensing information can be accurately obtained according to the crystal oscillation frequency change.

 In recent years, with the success of the liquid crystal oscillation of quartz crystals, people have become more and more aware of the liquid crystal oscillation characteristics of quartz crystals. The scope of application of quartz crystals has also been broadened, especially in biology. Researchers have discovered that in the liquid phase, quartz crystal microbalances are not only sensitive to mass, but are also excited by external temperature, air pressure, magnetic field fluctuations, shock oscillations, and liquid density, viscosity, dielectric constant, conductance, and flow through the crystal. Influence of factors such as current fluctuations.

 Many people have researched piezoelectric quartz crystal responders as detectors in liquid environments. These studies have confirmed that mass loading and viscous coupling are the two main mechanisms that cause piezoelectric quartz crystals to change frequency.

 The prior art shows that after a voltage is applied to the two ends of the piezoelectric quartz crystal, the frequency of the piezoelectric quartz crystal under pressure is fixed. On the basis of this, the researchers of the present invention fix the probe on the piezoelectric crystal. This will cause the overall frequency to change. However, the frequency after the change is still a fixed value; however, because external factors will cause the frequency to drift, the present invention adopts the following measures, that is, setting a reference detection. Only one probe is cured, then a crystal with the same probe cured is used as a reference test. If an ultra-thin quartz resonator array is obtained by etching the same crystal, the same probe is selected to be cured in the array. One is used as a reference test so that a stable reference frequency and true and reliable experimental results can be obtained.

 The change in the frequency of the quartz crystal brought by the different solidified probes can be easily detected by the prior art.

 3.Introduction to the principle of hybridization

 Watson and Crick et al. (1953) proposed the concept of a double helix model of DNA. They point out:

 (1) A DNA molecule is composed of two parallel polynucleotide strands in opposite directions, and the two strands have chemically opposite directions. That is, the structure of P_5'-ribose- 3'_P ... is opposite to the structure of P-3'-ribose- 5'_P ....

 (2) There are certain rules for base pairing: Chargaff et al. Used chromatography to analyze the base composition of DNA in various organisms and found that the number of adenine (A) and thymine (T) in the DNA are equal, and cytosine The number of (C) is equal to the number of guanine (G). Therefore, there are four possible base pairs in DNA: A-T, T_A, G-C> C ~ G.

(3) The two chains are mainly connected by hydrogen bonds between the bases: the plane of the base pair passes through the spiral axis and is approximately perpendicular to the spiral axis. Two hydrogen bonds can be formed between ATs and three hydrogen bonds can be formed between GCs. Meanwhile, for DNA double For the stability of the spiral structure, the force of a hydrophobic bond is also required.

 (4) Since four base pairs are suitable for this model, each chain can have any base sequence, but due to the regularity of base pairing, if the base sequence of one chain is determined, the other chain must be There is a corresponding base sequence.

 Since the double helix structure of DNA is mainly maintained by hydrogen bonds and hydrophobic bonds, all factors that can destroy the hydrogen and hydrophobic bonds, such as heating, acid-base, and organic solvents, can cause denaturation, making the double helix structure of DNA into random clusters. . The "renaturation" between different denatured DNA fragments by complementary base pairing is called hybridization. Hybridization can occur not only between DNA and DNA strands, but also between homologous sequences of DNA and RNA strands. During the hybridization process, two complementary single-stranded DNAs form a double bond hybrid in a non-covalent manner.

When the sequence of one of the strands is known, the hybridization process can be used to detect whether the unknown DNA sample contains DNA complementary to the known sequence.

 Although long-stranded DNA molecules are not rigid, and each nucleotide residue has 6 single-stranded strands that can rotate freely, the DNA double helix may exist in different conformations; however, short-stranded DNA molecules in dry conditions are still It has a certain rigidity, and the space distance of the oligonucleotides fixed on the electrodes is very small. Because the fixed DNA is formed on the wafer, the mass of the fixed oligonucleotide probe is relative to the mass of the crystal itself. It is very small, so it can meet the requirements of the present invention.

 The basic working principle of using a piezoelectric gene sensor for target gene detection according to the present invention is as follows: a section of a gene is fixed on a solid support. Specifically, the fixed support may be a piezoelectric quartz crystal. A voltage is applied to the two ends of the piezoelectric quartz crystal through a silver electrode to obtain a fixed frequency, and then it is used to hybridize with a complementary oligonucleotide in solution. The change in mass load and viscous coupling in the hybridization process results in The frequency of the quartz piezoelectric crystal changes. By analyzing the value of the frequency change, whether or not to hybridize and the number of hybridizations can be obtained, thereby realizing the detection of specific DNA in the liquid phase. The fixation of the known gene fragments on the support is shown in Figure 3.

 4. Overview of the development of existing technologies

 In recent years, research on gene sensors (also known as DNA and nucleic acid biosensors) at home and abroad is becoming a research hotspot in biosensor technology. Gene sensors are unique in molecular biology and medicine due to their unique advantages of simplicity, fastness, and low cost. The fields of inspection and environmental monitoring have a wide range of application prospects. In addition to gene sequence analysis, gene mutation, gene detection and diagnosis, it also involves research on the interactions between DNA, drugs, and proteins.

 Reversible hybridization of complementary DNA is the basis of biological processes such as replication, transcription, and translation. Nucleic acid hybridization is essential for understanding these important biological processes at the molecular level.

At present, the method of gene analysis mainly detects the specific DNA sequence in a heterogeneous system. The more commonly used method is nucleic acid hybridization. Nucleic acid hybridization is a process in which two complementary single-stranded DNAs form a double bond hybrid by non-covalent bonding. When the sequence of one of the strands is known, this is a very useful analysis technique. By detecting the hybridization process, you can find out whether the unknown DNA sample contains DNA complementary to the known sequence. The most commonly used method is A solid support is immobilized with a known sequence of genes, and then used in the solution Hybridization is performed with complementary oligonucleotides in the liquid, so that specific DNA in the liquid can be detected.

 In recent years, the research on the detection of specific DNA sequences in the liquid phase by hybridization methods has become more and more intensive. The detection of DNA sequences by hybridization methods has important application values. The main applications are: clinical genetic diagnosis, forensics, food, biochemistry And environmental protection, and gene detection methods have become more convenient and safe due to the application of non-radioactive markers such as biotin, digoxin, and fluorescent dyes, especially the application of polymerase chain reaction PCR technology, which makes gene detection More sensitive.

 Traditional DNA hybridization reactions require the use of labeling methods to detect hybridization signals. These methods allow in situ detection and can be highly sensitive. For example: The detection limit of PCR technology can reach nmol / 1; DNA computer technology also provides a method for detecting a specific DNA sequence from a large number of mixed systems; Due to the application of short-wave fluorescence and confocal microscope technology, fluorescent labeling has become a detection Trace legs are very sensitive and commonly used methods. However, these methods also have certain shortcomings: ① Probe labeling and modification are expensive and cumbersome; ② Pre- and post-processing of labeled probes are complicated; ③ It is difficult to accurately obtain quantitative information such as the absolute number and time required for hybridization ④ Long hybridization time, which often takes several hours or even days; ⑤ Special test equipment and conditions are often required; ⑥ The technical requirements of the operator are very high and it is not easy to master. Many researchers should use the SPR method to achieve quantitative and in situ monitoring of liquid-labeled and unlabeled DNA hybridization, but this method has the disadvantages of expensive equipment and inaccurate quantification.

 Recently, more and more researchers have begun to use non-marker methods to detect gene sequences. More researches are DNA biosensor systems, which detect and identify DNA sequences through hybridization methods, and can carry out quantitative DNA research. This detection method is simple, time-consuming, and does not require signal molecules to perform quantitative analysis directly. It can be used not only for the determination of DNA sequences and gene point mutations, but more importantly, it can monitor the progress of hybridization reactions dynamically and quantitatively, without the need to clean the electrodes or dry, and directly obtain hybridization information in the liquid state. A DNA biosensor immobilizes a modified DNA probe on a conversion unit that converts a physical or chemical signal into an electrical signal. DNA biosensors can be divided into electrochemical, optical, and piezoelectric crystal sensors according to the selected medium and transducer.

 In the present invention, the researchers mainly applied the quartz crystal microbalance QCM (Quartz Crystal icrobalance) technology, which is a piezoelectric sensor technology combined with gene chip technology to detect DNA, and compared the response time and hybridization efficiency of the sensors with different fixing methods. Impact.

 In order to more clearly illustrate the inventiveness of the present invention, the applicant hereby makes the following description of the research progress of the prior art on gene sensors:

 A. Optical Gene Sensor

 At present, there are mainly three types of fluorescent optical fiber gene sensors, surface enhanced Raman gene probes and surface plasmon resonance gene sensors.

Henke et al. Used an ethidium bromide hybridization indicator and total internal reflection fluorescence method to determine the hybridization reaction on the fiber surface, studied the preparation of fluorescent fiber DNA sensors, and compared the single-stranded DNA on the fiber surface by means of scattering and UV-UIS spectroscopy. The results of the two immobilization methods indicate that it is difficult to directly fix the oligonucleotide to the amino terminal of the hydrophobic linker on the surface, but the amide coupling reaction can successfully immobilize. Uddion et al. Used a DNA synthesizer to synthesize oligonucleotides directly on the surface of the quartz fiber after the linker treatment. dsDNA embedded in ethidium bromide has developed a fiber-optic DNA sensor for fluorescence detection, which is used to detect the formation of triple helix DNA. Abel et al. [ ^1] developed an automatic optical DNA sensor system. The principle is to fix a biotin-labeled probe on the surface of an optical fiber with avidin or streptavidin, and use the fluorescence excitation and detection of the loss field of quartz fiber to realize the probe. On-site monitoring of hybridization with fluorescein-labeled complementary strands, with a sensitivity of 132 pmoL. Compared with fluorescence detection, surface-enhanced Raman detection has higher sensitivity. Therefore, the surface-enhanced Raman (SERS) reagent modified gene probe can be directly used for gene detection without amplification. Graham et al. Reported a SERS gene probe with a sensitivity of 0.8 pmol. Isola et al. Combined SERS technology with spectral selectivity and high sensitivity with PCR technology to detect HIV-Gag genes. Although the SERS technology requires expensive and complicated equipment, based on its extremely small spectral bandwidth (half-peak width <11 ^ 1), it is expected that multiple SERS gene probes can be used to simultaneously detect multiple target genes on one chip. This is difficult to achieve by general optical detection technology (such as the half-peak width of 50-100nm for fluorescence detection).

 Surface plasmon resonance (SPR) gene sensors are based on the sensitivity of SPR to changes in the sensor's surface, without the need for hybridization indicators or DNA markers, and directly monitor the hybridization process on site. Peterlinz et al. Reported the use of two-color SPR spectroscopy to achieve unlabeled on-site quantitative detection of DNA hybridization, and tracked and studied the kinetics of hybridization and thermotropic processes. Corn and Smith reports that scanning SPR measurement and micro-SPR technology are used to characterize the hybridization and adsorption of DNA on the surface of gold film and its combination with streptavidin, which is formed by the action of biotin-streptavidin Streptavidin / DNA multi-layer to amplify the SPR signal of probe hybridization. At the same time, the laboratory constructed an array of 2x2 oligonucleotide probes (probe spot diameter about 2.0mm) on the surface of the gold membrane. Detecting multiple DNA hybrids simultaneously using on-site microscopy SPR technology. This study has initially demonstrated the feasibility of using SPR technology for DNA chip detection.

 B. Electrochemical gene sensor

 Generally, the electrochemical gene sensors produced are difficult to be popularized due to their poor accuracy and relinearity. Therefore, the current research is mainly on the development of electrochemical gene sensors that are expected to be disposable, using microfabrication technology that is easy to mass-produce. Wang's laboratory has developed a series of miniature DNA thick-film carbon electrodes using screen printing technology. Applications include detection of nucleic acid sequences based on hybridization methods, embedding of dsDNA by drugs and agricultural products, or determination of these small molecules by their effects on the oxidation signal of nucleobases. As well as direct adsorption stripping voltammetry for ultra-small amounts of nucleic acids, the laboratory has proposed the use of a highly sensitive constant current stripping chronopotentiometry to detect DNA hybridization recognition. The DNA sequences measured include M. Tuberculosis, HIV-I, E .Coli, Protozoan crypyosporidium parvum, etc. Hashimoto et al. Used photolithographic microfabrication technology to etch out a 0.3mm diameter micro-gold electrode with a fixed DNA probe, which can be used once, and the electroactive substance Hoechst 33258 was used as a hybridization indicator to detect the patient's serum HBV-DNA. concentration. Singhal and Kuhr proposed the use of electrocatalytic oxidation of ribose and amino groups on the copper surface, and proposed a DNA electrochemical sensor different from the current detection of adenine and guanine nucleobase oxidation. Since all nucleotides and DNA molecules contain ribose and amino groups, this type of sensor is suitable for the determination of various types of nucleotides.

In addition to the aforementioned gene detection, another important application of electrochemical gene sensors is the detection of gene mutations and injuries. Domestic Sun Xingyan et al. Used ssDNA to covalently immobilize on the surface of graphite electrodes, and used daunorubicin (DR) as a hybridization indicator to hybridize ssDNA on the electrode surface with complementary ssDNA in solution to form dsDNA. The electrochemically active DRN is embedded in the double-helix structure of the DNA during the hybridization process to form a DNA electrochemical sensor. Based on the difference in the degree of hybridization and the degree of hybridization under the irradiation of ultraviolet light and nitrous acid, the DNA structure variation is detected, and The extent of DNA damage caused by the above two factors and the possible mutation mechanism were discussed. Previously, Wang et al. Directly used dsDNA-immobilized miniature electrochemical sensors to explore the damage of DNA caused by ultraviolet radiation based on changes in the oxidation signal of guanine in DNA, including the conformational changes of DNA and the photochemical reaction of guanine.

 C. Piezoelectric gene sensor

 Piezoelectric gene sensors are based on the sensitivity of bulk acoustic wave devices-piezoelectric quartz resonators to changes in their surface quality (mass sensitivity can reach ng level). No hybridization indicator is needed to directly detect the DNA hybridization reaction on the sensor surface. Such sensors are also called Bulk acoustic and quartz (crystal) micro / nanobalance DNA sensors. In the study of the sensor DNA probe immobilization method, Nicolili et al. Used LB membrane technology to deposit a monolayer of ssDNA mixed with fatty amine on a quartz resonator using LB membrane technology, which has good hybridization activity; Fawcett et al. Respectively on the sensor surface The application of macromolecules in the development of DNA sensors was preliminarily discussed by using hydrophobic polystyrene, polyethylene and acrylic copolymers to cross-link and immobilize probes. Because biological macromolecules are easily adsorbed on hydrophilic surfaces, in order to maximize the activity of the probe and reduce non-specific adsorption, the bioaffinity of the surface of the sensor except the probe site should be reduced as much as possible. The reaction site on the surface may be an ideal surface for hybrid DNA sensors.

 In the past, piezoelectric gene sensors were mostly used to detect changes in the quality of the dry surface of the sensor before and after hybridization. With the maturity of liquid-phase piezoelectric sensor technology, the on-site monitoring of the hybridization process makes piezoelectric gene sensors easier and faster; The study of the kinetics of the surface hybridization process provides a basis for the optimization of gene sensors. The earliest research in this area was Okahato Lab. They determined the kinetic parameters such as the equilibrium constant of the 10 to 30-mer oligonucleotide double-strand complementary binding, the rate constant of binding and dissociation, and the amount of hybridization on the surface; and The method of fixing the probe, the length of the probe and the target gene, the number of mismatched bases, the hybridization temperature, and the ionic strength of the hybridization solution were changed, and the dynamic characteristics of the hybridization process on the sensor surface were studied in detail.

 Compared with the hybrid detection technology of labeled nucleic acids such as radioactive isotopes, the sensitivity of gene sensors is still far behind. In addition to further improving the detection sensitivity of the sensor itself, Chen Yuhua and others in China took the herpes simplex virus and human endoplasmic reticulum molecular chaperone Grp94 genome as examples, and for the first time tried the development of a primer chain extension reactive piezoelectric gene sensor. The principle is to use the primer chain extension reaction after primer-template hybridization in DNA sequence analysis, so that the short-chain probes can be gradually extended using the target gene as a template after hybridization, which results in a larger surface mass and increased sensitivity. In addition, Wang et al. Used peptide nucleic acid (PNA) probes to achieve on-site discrimination of single-base mismatches, significantly improving the specificity of the sensor, and reducing the hybridization time to 3-5 minutes.

 Gene probes-Immobilization of single-stranded DNA fragments (oligonucleotides) on the sensor surface is the primary and basic condition for gene sensors.

The method used in the present invention includes three probe immobilization methods: adsorption method, covalent bonding method, and combination method. The latter two types of methods in the prior art are adopted. Although a solid probe modification layer can be obtained, the surface hybridizes. Anti There should be few active sites and the method is complicated.

 The researchers of the present invention have noticed that it is undoubtedly ideal to prepare terminally modified probes to form stable, highly ordered monolayers (SAMs) on the surface by using the self-assembly of molecules with certain structures. For reactivity, in the present invention, it is preferable to use a thiol group for terminal modification, that is, it is preferable to use a thiol covalent bond method.

 Among the aforementioned various gene sensors, SPR and piezoelectric sensors that do not require labeling or hybridization indicators are easy to prepare in large quantities, the technology is simple and fast, real-time on-site monitoring is possible, and the required equipment is relatively simple and cheap. These two types of sensors have become the mainstream of gene sensor research.

 Polymerase chain reaction (PCR) technology has been widely used in various fields of medicine, but its current application requires the product to be separated and analyzed after PCR amplification. Therefore, on-site monitoring of polymerase chain reaction, that is, real-time PCR technology has important application value.

 The signal generated by the combination of some specimens and probes is relatively weak. The researchers of the present invention took the detection of human papilloma virus oncogene transcripts as an example, and for the first time tried to combine a field-capable piezoelectric gene sensor with PCR technology Construct a new type of real-time PCR technology; in other words, in order to increase the signal intensity, the present invention adopts the method of first performing PCR amplification on the specimen, or adopting the method of real-time PCR amplification to improve the signal intensity and detection sensitivity.

 An important trend in the development of gene sensors is the construction of miniature gene sensor arrays, that is, gene sensor chips. So far, in addition to the previously mentioned attempts using SPR microscopy, Arlinghaus et al. Have used sputter-excited resonance ionization microprobe technology to detect hybridization of isotope-labeled DNA with gene sensor chips, and the present invention uses a difference from the above The prior art miniature piezoelectric quartz resonator array constructs the target genome automatic detection system of the present invention.

 The detection steps of the automatic detection method of the target genome according to the present invention are as follows:

 First, the specimen collection process is performed, and then the specimen is added to the hybridization reaction system through the sampling system. The hybridization reaction system described here is composed of a base, a chip, and a lead; the hybridization reaction system changes the frequency parameters of the specimen and the probe after contact, and The data of the comparison detection parameters are fed back to a data acquisition and processing system, which includes a multi-channel test acquisition and real-time central signal processing. The multi-channel test acquisition is actually performed on multiple piezoelectric quartz crystals. The probes on the probe or a plurality of probes on a piezoelectric quartz crystal array are sequentially collected for the parameter changes caused by the combination with the specimen; the real-time central signal processing is finally represented by the channelized data display, data comprehensive processing, and image display. .

 In fact, the miniature piezoelectric quartz resonator described in the present invention can be a single quartz crystal solidified probe, or a miniature piezoelectric quartz resonator array can be used, so that a large number of etched quartz crystal A large number of probes are provided on the small block, as shown in FIG. 1, and usually at least 9 kinds of probes are provided.

In the present invention, different types of probes (that is, a plurality of specific gene fragments-nucleotide sequences) can be labeled on one chip. Different types of probes have different genetic information; if only one probe is labeled, multiple samples of different sources can be detected and analyzed for the same genetic information (such as a certain pathogen: hepatitis B virus, etc.); such as By labeling multiple probes, you can diagnose and analyze multiple genetic information of a specimen (for example, when detecting hepatitis, multiple probes can be fixed to detect hepatitis A, B, and C). The miniature quartz resonance array gene sensor chip of the present invention uses a microfabrication technology to directly etch an ultra-thin quartz resonator array on a quartz crystal, and then fixes a large number of probe molecules to the quartz crystal plated with gold or silver film. On the support, a certain voltage is applied to both sides of the crystal through a silver electrode, and then the probe is hybridized with the specimen. The hybridization will cause the resonance frequency of the quartz crystal to change. The sample can be judged by detecting the change value of the resonance frequency of the quartz crystal. The presence or absence of target molecules and the number.

 The basic working principle of using a piezoelectric gene sensor chip for target gene detection according to the present invention is as follows: a section of a gene is fixed on a solid support, specifically, the fixed support may be a piezoelectric quartz crystal, A voltage is applied to the two ends of the piezoelectric quartz crystal through a silver electrode to obtain a fixed frequency, and then it is used to hybridize with a complementary oligonucleotide in solution. The change in mass load and viscous coupling in the hybridization process passes As a result, the frequency of the quartz piezoelectric crystal is changed. By analyzing the change value of the frequency, whether to hybridize and the number of hybridizations can be obtained, thereby realizing the detection of specific DNA in the liquid phase. The fixation of the known gene fragments on the support is shown in Figure 3.

 The following is a summary of the miniature piezoelectric quartz resonator array gene sensor chip of the present invention: wherein, the miniature piezoelectric quartz resonator array gene sensor chip of the present invention includes an AT-cut quartz crystal 3 etched into an array, A metal film layer 4 is plated on the lower surface of the quartz crystal, a metal film layer 2 in the same array is plated on the upper surface of the quartz crystal array, and a probe array layer 1 is cured on the upper surface of the upper metal film layer 2 in the array. The electrode 6 is fixed at the corresponding probe 7, and the lower metal film layer is thermally bonded to the base 5. The number of the array is at least one, preferably at least six, and most preferably at least nine. The number of blocks of the array is the same as the number of cured probe layers 1; grooves 9 are provided between the blocks presented by the array; the metal film layer may be a gold film layer or a silver film layer; The electrode is a silver electrode; the base is a glass base; in order to reduce the influence of other factors, one of the same probes cured in the array is used as a reference detection; the curing used in the present invention method Be an adsorption method, covalent bonding method and the combination method, but preferably sulfhydryl terminus modification covalent bonding method.

 The steps of the detection method according to the present invention are as follows:

 1. Specimen processing: Extraction of nucleic acids from specimens, endonuclease treatment (different enzymes for different specimens), separation and purification of target fragments according to different samples, etc .;

 2. If the chip is not for one-time use, perform electrode cleaning before the sample injection test. The detector according to the present invention preferably uses a one-time chip;

 3. Start the computer operation software program, which includes sampling, real-time monitoring, signal processing, image display system and result analysis;

 4. Contact the specimen with the probe through the injection hole to cause a hybridization reaction;

 5. Obtain test results based on computer data and images;

 6. Clean the injection and reaction cells;

 7. Cycle operation.

The multi-channel (well) reaction device according to the present invention has been produced by the applicant and sold publicly. The automatic method of the target genome according to the present invention uses bioengineering, molecular biology, sensors, micro-processing technology, etc., and its main uses include the diagnosis of diseases (infectious diseases, genetic diseases, tumors, cardiovascular diseases, etc.), Detection of genetic mutations, development of new drugs and environmental monitoring.

 The detection performance of the target gene automatic detector constructed by the method of the present invention is shown in Table 1 below: Table 1

 The main technical and economic indicators

• Minimum detection-5 pg, equivalent to isotope labeling

 • Checkout time: 10 min

 • Sensor chip size: 10 mm x 10 mm

 • Sensor and array size: 1.0 mm x 1.0 mm, 7 x 7

 • Number of target genes that can be detected by the chip: 49

 • Sensor chip life: 10 times, preferably one-time use

 • Service life of detection system: 10,000 times or more

 The advantages of using the method of the present invention or the target genome automatic detector constructed by the method are as follows: the chip constructed by the miniature piezoelectric quartz resonator array used by the present invention has a sensitivity of up to pg; the specificity and the currently used label detection The technology is the same.

 The target gene automatic detector constructed by the method of the invention has the advantages of in situ measurement, no labeling, detection information available at any time, small size, easy to carry, convenient to use, and low cost, and is suitable for clinical and field environment detection. .

 Comparison of the performance / price of the detection method and the detection device according to the present invention with conventional methods

 Note: ELISA and culture methods are currently widely used in clinical detection of pathogens

The protection scope of the detection method described in the present invention can be seen in the claims, but it is not limited to this. Changes in the order and combination of the detection methods described in the present invention under the concept of the present invention should be within the protection scope of the present invention. FIG. 6 is a schematic circuit diagram of the detector according to the present invention.

 Except for the chip, each part of the circuit of the present invention can adopt circuits in the prior art. One of the contributions of the present invention is to combine the detection circuits with the chip of the present invention to achieve the present invention. Purpose The following are examples, which are only used to illustrate the present invention, but not to limit the present invention. Example 1

 Using the method of the present invention and the detector constructed according to the method, a 30 bp Hpvl 8 (7021-7050) fragment and a 20 bp heat-resistant enterotoxin (LT) fragment produced by toxinogenic E. coli (ETEC) were fixed on the On the chip, the specimen is tested, as shown in Figure 4, and it can be seen from Figure 4 that when the reaction reaches equilibrium in about 5 minutes, the chip oscillation frequency is reduced by about 40-50HZ, where the 其中 line is the specimen negative control and the A line For the test result of HPV, X line is the test result of LT. Example 2 Tables 3 and 4 are comparisons of the results of detection of Mycobacterium tuberculosis and Neisseria gonorrhoeae (specified specimens) with conventional culture methods, PCR methods, and the methods and detectors described in the present invention.

 Among the 30 N. gonorrhoeae specimens shown in Table 3, the negative specimens were 1, 8, 9, 10, 11, 14, 15, 18, 19, 20, 21, 22, 23, 26, 27, and the rest were positive;

 Among the 30 tuberculosis specimens shown in Table 4, the negative specimens were 1, 6, 7, 9, 11, 18, 19, 21, 22, 27, 28, and the rest were positive.

 It can be seen from the comparison of the test results that the conventional culture method is more accurate and objective, but has limited sensitivity. The PCR method has high sensitivity but is prone to false positives. From the experimental data, regardless of accuracy or sensitivity, the automatic target gene detector according to the present invention The detection effect is better than these two methods.

 The target gene automatic detection method and the detector constructed by the method are characterized by a combination of a DNA chip and a sensor technology, and have the following advantages.

 1. Low price and easy to carry;

 2, easy to use, can be monitored in situ and in real time;

 3. No labeling required, can be stored for a long time, and the third sensitivity is high;

 4. Multiple groups of specimens can be detected simultaneously.

12

Correction page (Article 91) Table 3 Report of clinical test results of Neisseria gonorrhoeae

Sample No. 1 2 3 4 5 6 7 8 9 10 Cultivation-+ + +---

PCR-+ + + + +-one +

MGI one + ten + + + ten + one-sample number 11 12 13 14 15 16 17 18 19 20 cultivation-÷ ten-one one ―-

PCR-+ one + + one ―-

MGI-÷ + one one +--― Sample number 21 22 23 24 25 26 27 28 29 30 Cultivation---÷ ÷---÷-

PCR ―-+ + +-+

MGI ―---― +

Table 4 Report of clinical test results of Mycobacterium tuberculosis

Sample number 1 2 3 4 5 6 7 8 9 10 Cultivate one + + + + ― one-―

PCR-+ + + ― + +-+

MGI-+ + +--+ one + + sample number 11 12 13 14 15 16 17 18 19 20 culture-+ + + one one +

PCR-+ + +-― one ÷

MGI one + + + + +--one sample number 21 22 23 24 25 26 27 28 29 30 culture-one + + +-―-÷

PCR ― + + + + +

MGI ―-+ 4- + + ―-Ten +

Claims

Rights request 1. An automatic detection method for a combined target gene, characterized in that the detection method uses a microfabrication technique to directly etch an ultra-thin quartz resonator array on a quartz crystal, and fixes a large number of probes correspondingly to metal plating A miniature quartz resonance array gene sensor chip is formed on the quartz resonator array of the film layer. A fixed frequency is obtained by applying a voltage to the two ends of the chip through a silver electrode, and then the probe is in solution with a complementary sample (oligonucleotide). ) Perform hybridization and determine the change in mass load and viscous coupling during the hybridization process. By causing the frequency value of the chip, whether or not to hybridize and the number of hybridizations can be obtained.  2. The detection method according to claim 1, wherein the miniature quartz resonant array gene sensor chip comprises an AT-cut quartz crystal 3 etched into an array, and a metal film layer 4 is plated on the lower surface of the quartz crystal. The upper surface of the quartz crystal array is plated with a metal film layer 2 in the same array, the probe array layer 1 is cured on the upper surface of the upper metal film layer 2 in the array, and the electrodes 6 are fixed at the corresponding probes 7, The lower metal film layer is thermally bonded to the base 5.  3. The detection method according to claim 1 or 2, characterized in that the number of blocks formed by the array is at least one, preferably at least six, and most preferably at least nine;  4. The detection method according to claim 1 or 3, characterized in that the number of blocks of the array and the number of solidified probes 1 are the same; grooves are provided between the blocks presented by the array Slot 9;  5. The detection method according to claim 1 or claim 1, wherein the metal film layer is a gold film layer or a silver film layer; the electrode is a silver electrode; the base is a glass base;  7. The detection method according to claim 1 or claim 1, characterized in that one of the same probes solidified in the array in the array is used as a reference detection probe; the probe is a known gene fragment; The chip array can be labeled with the same or different kinds of probes. .  8. The detection method according to claim 1, wherein the method for curing the probe comprises an adsorption method, a covalent bonding method, and a combination method; preferably a thiol group is used for terminal modification, that is, a thiol covalent bonding method.  9. The detection method according to claim 1 or claim 1, characterized in that said specimens are first amplified by PCR, or are amplified by real-time PCR.  10. The target genome automatic detector constructed according to claim 1, characterized in that said detector is provided with a micro quartz resonance array gene sensor chip, and the chip is arranged in a hybridization reaction system, and said hybridization reaction system is based on a base The hybridization reaction system feeds back the data of the frequency change parameters and the contrast detection parameters of the specimen and the probe to the data acquisition and processing system. The data acquisition and processing system includes multi-channel test acquisition. And real-time central signal processing, the multi-channel test acquisition is actually a parameter change caused by a plurality of probes on a piezoelectric quartz crystal or a plurality of probes on a piezoelectric quartz crystal array due to combination with a specimen Sequential acquisition; real-time central signal processing is finally expressed in lane data display, data comprehensive processing, and image display.