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WO2001036667A1 - Procede de detection automatique d'un gene cible et applications d'un detecteur utilise dans ce procede - Google Patents

Procede de detection automatique d'un gene cible et applications d'un detecteur utilise dans ce procede Download PDF

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Publication number
WO2001036667A1
WO2001036667A1 PCT/CN1999/000192 CN9900192W WO0136667A1 WO 2001036667 A1 WO2001036667 A1 WO 2001036667A1 CN 9900192 W CN9900192 W CN 9900192W WO 0136667 A1 WO0136667 A1 WO 0136667A1
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Prior art keywords
array
gene
quartz
dna
detection method
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PCT/CN1999/000192
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English (en)
Chinese (zh)
Inventor
Weiling Fu
Jianghua Wang
Minghua Liu
Yingying Wang
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Medical Laboratory Center Of South Western Hospital Third Military Medical University
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Priority to PCT/CN1999/000192 priority Critical patent/WO2001036667A1/fr
Priority to AU12576/00A priority patent/AU1257600A/en
Publication of WO2001036667A1 publication Critical patent/WO2001036667A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/022—Fluid sensors based on microsensors, e.g. quartz crystal-microbalance [QCM], surface acoustic wave [SAW] devices, tuning forks, cantilevers, flexural plate wave [FPW] devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/036—Analysing fluids by measuring frequency or resonance of acoustic waves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00608—DNA chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00704—Processes involving means for analysing and characterising the products integrated with the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0255—(Bio)chemical reactions, e.g. on biosensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/04—Wave modes and trajectories
    • G01N2291/042—Wave modes
    • G01N2291/0426—Bulk waves, e.g. quartz crystal microbalance, torsional waves

Definitions

  • the present invention relates to a method for automatically detecting a combined target gene, and a detector manufactured based on the method. Background technique
  • 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.
  • 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
  • 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 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.
  • the microfabrication technology makes the quartz resonator array easy to prepare in batches, the cost can be greatly reduced.
  • the effect of external mechanical pressure on a crystal to generate a charge on its surface is called the piezoelectric effect.
  • 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" .
  • piezoelectric 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.
  • 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.
  • A The surface area of the quartz wafer (cm 2 ).
  • 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.
  • 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.
  • the researchers of the present invention fix the probe on the piezoelectric crystal. This will cause the overall frequency to change.
  • 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.
  • 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 ....
  • 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.
  • 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.
  • hybridization 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.
  • the hybridization process can be used to detect whether the unknown DNA sample contains DNA complementary to the known sequence.
  • 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 is as follows: a section of a gene is fixed on a solid support.
  • 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.
  • the fixation of the known gene fragments on the support is shown in Figure 3.
  • gene sensors also known as DNA and nucleic acid biosensors
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • QCM Quadrat Crystal icrobalance
  • 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.
  • 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.
  • 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).
  • SPR Surface plasmon resonance
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • piezoelectric gene sensors were mostly used to detect changes in the quality of the dry surface of the sensor before and after hybridization.
  • 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.
  • 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.
  • adsorption method adsorption method
  • covalent bonding method adsorption method
  • combination method adsorption method
  • the latter two types of methods in the prior art are adopted.
  • a solid probe modification layer can be obtained, the surface hybridizes.
  • Anti There should be few active sites and the method is complicated.
  • terminally modified probes to form stable, highly ordered monolayers (SAMs) on the surface by using the self-assembly of molecules with certain structures.
  • SAMs highly ordered monolayers
  • PCR Polymerase chain reaction
  • 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.
  • the detection steps of the automatic detection method of the target genome according to the present invention are as follows:
  • 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. .
  • 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.
  • different types of probes that is, a plurality of specific gene fragments-nucleotide sequences
  • 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
  • hepatitis B virus a certain pathogen
  • 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 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 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:
  • 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 .;
  • the detector according to the present invention preferably uses a one-time chip
  • 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.
  • Table 1 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 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. .
  • FIG. 6 is a schematic circuit diagram of the detector according to the present invention.
  • Example 1 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
  • 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.
  • the negative specimens were 1, 8, 9, 10, 11, 14, 15, 18, 19, 20, 21, 22, 23, 26, 27, and the rest were positive;
  • the negative specimens were 1, 6, 7, 9, 11, 18, 19, 21, 22, 27, 28, and the rest were positive.
  • 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.
  • MGI one + ten + + + ten + one-sample number 11 12 13 14 15 16 17 18 19 20 cultivation- ⁇ ten-one one ⁇ -

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Abstract

L'invention concerne un procédé de détection automatique d'un gène cible combiné et le détecteur utilisé dans ce procédé. L'invention utilise essentiellement la technique de microbalance à cristal de quartz et la technique associée à la puce génique pour déceler l'ADN. Le procédé consiste à mettre en oeuvre la technique du microusinage pour graver un réseau de résonateurs de quartz très fin directement sur un cristal de quartz piézo-électrique, puis un certain nombre de sondes sont immobilisées de manière à détecter et analyser en une seule étape des molécules d'ADN ou d'ARN. Cette invention a pour objectif de simplifier les techniques classiques d'hybridation d'acide nucléique, d'abaisser le niveau d'automatisation, de réduire les molécules cibles à détecter. Le procédé de détection automatique d'un gène cible et le détecteur portable peuvent déceler in situ et sans marqueur et obtenir des informations à tout moment. Ce détecteur présente l'avantage d'être petit et facile à manipuler.
PCT/CN1999/000192 1999-11-16 1999-11-16 Procede de detection automatique d'un gene cible et applications d'un detecteur utilise dans ce procede WO2001036667A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN1999/000192 WO2001036667A1 (fr) 1999-11-16 1999-11-16 Procede de detection automatique d'un gene cible et applications d'un detecteur utilise dans ce procede
AU12576/00A AU1257600A (en) 1999-11-16 1999-11-16 Combinatorial and automatic detection method of target gene, and a detector using the method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1999/000192 WO2001036667A1 (fr) 1999-11-16 1999-11-16 Procede de detection automatique d'un gene cible et applications d'un detecteur utilise dans ce procede

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WO2001036667A1 true WO2001036667A1 (fr) 2001-05-25

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5501986A (en) * 1988-04-06 1996-03-26 E. I. Du Pont De Nemours And Company Piezoelectric specific binding assay with mass amplified reagents
US5661028A (en) * 1995-09-29 1997-08-26 Lockheed Martin Energy Systems, Inc. Large scale DNA microsequencing device
WO1998010122A1 (fr) * 1996-09-03 1998-03-12 Northeastern University Reseau capillaire hybride a microstructure et ensemble de detection a canaux multiples

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5501986A (en) * 1988-04-06 1996-03-26 E. I. Du Pont De Nemours And Company Piezoelectric specific binding assay with mass amplified reagents
US5661028A (en) * 1995-09-29 1997-08-26 Lockheed Martin Energy Systems, Inc. Large scale DNA microsequencing device
WO1998010122A1 (fr) * 1996-09-03 1998-03-12 Northeastern University Reseau capillaire hybride a microstructure et ensemble de detection a canaux multiples

Also Published As

Publication number Publication date
AU1257600A (en) 2001-05-30

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