CN1422960A - Electronic gene chip preparation method - Google Patents
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- CN1422960A CN1422960A CN 01129100 CN01129100A CN1422960A CN 1422960 A CN1422960 A CN 1422960A CN 01129100 CN01129100 CN 01129100 CN 01129100 A CN01129100 A CN 01129100A CN 1422960 A CN1422960 A CN 1422960A
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Abstract
The invention is a producing method for electron gene chip, its characters are: a. the DNA probe is pointed at the base point of carrier which has metal film, and accomplishes the fixing of DNA; b. the liquid containing electrochemistry activity base are pointed at the fixed DNA surface, the fixed DNA sample end is attached a electrochemistry activity base; The structure of DNA probe is: -NH2-(CH2)n-******+++++++++++******-(CH2)n-SH-, then the metal surface is immersed in SH-(CH2)6-OH through the whole night.
Description
The invention belongs to the field of gene chip (Genechip) preparation, and particularly relates to a preparation method of an electronic gene chip, which can be used for quantitatively detecting the change of an electric signal generated in a DNA hybridization process by adopting an electronic detector. Namely, the electronic gene chip obtained by the invention realizes the detection of the signal of the gene chip by adopting an electronic detector.
Gene chips (Genechip) are also called DNA chips (DNAChip). It is developed on the basis of gene probe, so-called gene probe is only a section of artificially synthesized base sequence, on the probe several detectable substances are connected, and according to the base complementary principle it utilizes gene probe to identify specific gene in gene mixture. It is performed by immobilizing a large number of probe molecules on a support, hybridizing with a labeled sample, and analyzing by detecting the intensity and distribution of hybridization signals. However, the application of the existing gene chip technology at the present stage has a great obstacle. The existing problems are mainly caused by the detection method, and the detection method is determined by the preparation method, which is particularly shown in the following aspects:
1. the cost of the special equipment of the gene chip and the cost of single detection are high, and the special equipment can only be born by large pharmaceutical companies and scientific research institutions with sufficient expenditure. A commercial gene chip signal detection and analysis system comprises a fluorescence scanner, a computer and software thereof, and is worth about 20 ten thousand dollars. For application type gene chips such as diagnostic chips, a very common infectious disease detection chip requires about two hundred yuan per cent for a single detection cost. Because the cost is the primary problem of realizing wide application, the popularization and application difficulty of the existing gene chip is very high.
2. The fluorescence scanning signal obtained by the existing gene chip technology can only be regarded as a qualitative or semi-quantitative signal result, and cannot be accurately and digitally analyzed. This aspect brings great inconvenience to the further analysis of the large amount of biological data obtained from the gene chip, and also reduces the sensitivity and reliability of signal detection.
3. The existing gene chip technology requires complex pretreatment of samples, mainly representing that the samples need to be subjected to fluorescence labeling. For monochromatic fluorescent labeling, although the pretreatment process is relatively simple, a good signal cannot be obtained; while the four-color fluorescence labeling signal is good, the pretreatment process is relatively complex. This pretreatment process can be accomplished by specialized training of professionals.
In summary, the present gene chip is still only limited to the laboratory stage, limited to the scientific research field, and has a considerable distance from the formal application.
The present invention aims at overcoming the demerits of available technology, and provides the preparation process of electronic gene chip capable of adopting electronic detector to detect the change of electric signal produced in DNA hybridizing process quantitatively to solve the problems of fluorescence detection. The electronic gene chip as one product has electronic detector to detect the signal of the gene chip, and this can reduce the detection cost greatly. The invention makes the gene chip technology become a more cheap, more accurate, more sensitive and perfect technology, thereby realizing the civilization, entering the wide application field and being the artificial septum of the whole mankind.
The technical scheme of the invention is realized by the following technical scheme.
The preparation method of the electronic gene chip is characterized by comprising the following steps:
a. spotting a DNA probe on a base point of the carrier sprayed with the metal thin layer to complete the fixation of the DNA;
b. spotting the solution containing the electrochemically active groups on the surface of the immobilized DNA so that the 5' end of the immobilized DNA sample is provided with the electrochemically active groups;
c. the structure of the DNA probe is as follows:
| 5＇-NH2-(CH2)n-********+++++++++++++++++********-(CH2)n-SH- |
wherein,********two DNA sequences which can be complementarily paired (A is paired with T, and G is paired with C) are shown; specific DNA fragments (e.g., 10-30 bases) to be detected during hybridization; the methyl group (CH) at both ends2) The number n is preferably 0 to 10.
The scheme isIn the method, the metal surface of the electronic gene chip with the electrochemical active group can be further immersed in SH- (CH) with a certain concentration2)6And (4) in an OH solution overnight, so that the part of the metal surface, which is not bonded with the DNA, is protected by alcohol to prevent nonspecific adsorption of the matrix. And simultaneously, the DNA arrangement on the surface can be more orderly. The concentration is 1-50 mM.
In the above scheme, the preparation method of the carrier may adopt a general method, such as taking a glass sheet, a ceramic sheet, a plastic sheet or a silicon wafer as the carrier. Covering a mask according to a certain circuit design, vacuum-plating a layer of titanium (adhesive layer) on a carrier or called substrate, then plating a layer of very thin metal, preferably gold, to form base points (up to hundreds to thousands) for placing DNA probes and corresponding circuits, and completing the manufacture of the substrate of the chip and the chip circuit.
In the above scheme, the chip may be further pretreated to obtain a reproducible and smooth gold surface, and the pretreatment may be performed by a conventional method, such as the following methods: a. after being treated by plasma for 30 minutes, the mixture is quickly placed in an ethanol solution; b. treating with ultraviolet-ozone treatment box for 30min, and treating with saturated alkali solution (such as KOH); c. treating with Piranha solution (70% concentrated sulfuric acid/30% hydrogen peroxide) at high temperature (higher than 90 deg.C) for more than 20 min, and performing electrochemical (cyclic voltammetry scanning) cleaning in alkaline solution (such as KOH, NaOH, etc.) at certain potential (-0.4V to-1.2V). All three methods are feasible. The cleaned chip is stored in ethanol or other organic solvents for later use.
In the above scheme, the method for spotting the DNA probe on the base point of the vector to accomplish the immobilization of the DNA can adopt various conventional methods in the prior art, such as: the prepared DNA probe is spotted on a base point, and the reaction is carried out for more than 5 hours at room temperature after the surface is covered. The chemical reaction of 3' -SH and Au forms S-Au bond to realize the self-assembly of surface molecule, thereby completing the fixation of DNA.
The specific DNA probe can be prepared by adopting a conventional solid-phase synthesis method andpurified by reverse phase HPLC, or purchased directly from other companies. In the following:
in the structure of the device, the device is provided with a plurality of grooves,********two DNA sequences which can be complementarily paired (A is paired with T, and G is paired with C) are shown, such as GCATGCGC and GCGCGCATGC; ATATCGCA and TGCGATAT, and the like. In the unhybridized state, the two pieces of DNA can fold upon themselves to form a Stem-loop structure (Stem-loop). The research shows that the stem-loop structure has high affinity and selectivity for the identification of target gene, can well distinguish Single point mutation, and can be used for SNPs (Single Nucleotide Polymorphisms) detection, wherein ++++++++++++++++++++++++++++ show specific DNA fragment to be detected in hybridization process, the base number can be set according to application, for example, 10-30 base, the DNA determines the application of the chip, if the fragment is HIV virus DNA characteristic fragment, the chip can be used for detecting AIDS, and similarly, if the fragment is hepatitis virus DNA characteristic fragment, the chip can be used for detecting hepatitis, methyl (CH) at two ends2) The number can vary from none to tens.
| 5＇-NH2-(CH2)3-********+++++++++++++++++********-(CH2)6-SH-3＇ |
The manufacturing process flow of the electronic gene chip of the invention can be seen in the attached figure 2.
The technical scheme of the invention ensures that the production of the gene chip adopts a technology completely different from the existing chip production, and breaks through the monopoly of the United states in the field. The technology applies mature microelectronic technology to gene chips, and is in the leading position all over the world at present. The present invention provides a preparation method of electronic gene chip capable of adopting electronic detector to make quantitative detection of change produced by electric signal in the DNA hybridization process. The electronic gene chip, the product obtained by the invention, realizes the detection of the signal of the gene chip by adopting the electronic detector, thereby bringing about the fundamental change of the detection technology of the gene chip, which is specifically represented as follows:
1. the cost of the detection equipment is greatly reduced, and the detection equipment is used as an electrochemical workstation for signal detection, and the cost of the detection equipment is far lower than that of a fluorescence scanner used in the prior gene chip technology. 2. The detection of the signal by this technique is performed by detecting the formation and destruction of a stem-loop structure (step-loop). The stem-loop structure (step-loop) has special selectivity when identifying target genes, and can well distinguish single-point mutation. This is not possible with the prior art and can significantly improve the accuracy of the DNA hybridization signal. 3. The electric scanning signal obtained by the technology is a quantitative signal result and can be accurately and digitally analyzed. This makes it possible to further analyze the great amount of biological data obtained by gene chip and raise the sensitivity and reliability of signal detection greatly. 4. The patent technology makes the sample to be detected not need any mark, so that the pretreatment process only needs simple training and anyone can complete the pretreatment process.
The product of the invention has the advantages that the principle is as follows: before hybridization, since both ends of the probe DNA contain a DNA sequence capable of completely pairing, a stem loop structure (stem loop) can be formed in a free state. At this time, the 5 ' end and the 3 ' end of the probe are close, and the electrochemically active group carried by the 3 ' end can generate an electric signal which can be detected by an electrochemical workstation when the electrochemically active group is close to the Au surface. After hybridization, the specific DNA fragment in the middle section of the probe and the sample are subjected to DNA complementary pairing to form a normal DNA double helix structure, so that the radial-loop structure is damaged. At this point, the 5 'end and 3' end of the probe are no longer in close proximity, resulting in a loss of the original electrochemical signal. By using the principle, whether the DNA completes hybridization pairing can be detected, namely, the hybridization signal of the DNA on the designed gene chip can be detected.
Fig. 1 is a technical schematic diagram.
In the figure: 1. a stem-loop structure; 2. au surface; 3. generating an electrical signal; 4. the electric signal disappears; 5. hybridization pairing with foreign DNA.
The invention is further described below by means of examples, to which the invention is not limited.
Example one
The preparation process comprises the following steps:
1. firstly, a chip substrate is selected from a glass sheet, a silicon wafer and a ceramic sheet for testing. The three substrates were covered with a mask (having a small hole and a circuit extending from the hole), vacuum-plated with about 10nm of titanium, and then with about 300nm of gold to form the DNA probe-immobilized surface and the circuit. And (6) detecting the smoothness of the surface. After detection, the surface of the silicon wafer is the most smooth after gold plating. And selecting a silicon wafer substrate to continue the test.
2. The gold plated silicon wafer was treated by the following three methods: a. treating with plasma, and rapidly placing in ethanol solution; b. treating with ultraviolet-ozone treating box, and treating with saturated KOH; c. treating with piranha solution (70% concentrated sulfuric acid/30% hydrogen peroxide) for 30min, and electrochemically cleaning in 0.5M KOH solution at a certain potential. The results show that the three methods are feasible and have no obvious difference. The cleaned chip was stored in ethanol for further use.
3. Prepared by a conventional solid-phase synthesis method and purified by reversed-phase HPLC to obtain or directly purchase DNA probes with the following structures from other companies: 5' -NH2-(CH2)3-GCG AG- -CT CGC-(CH2)6-SH-3', wherein,the specific DNA sequence selected for this experiment was a fragment of the pUC18 vector.
4. Prepared DNA probes (1uL, 1mM) were manually spotted onto the base dots, covering the surface overnight at room temperature, a procedure called spotting. The chemical reaction of 3' -SH and Au forms S-Au bond to realize the self-assembly of surface molecule, thereby completing the fixation of DNA.
5. A solution containing 5uL, 3mM of electrochemical group (carboxyferrocene) was spotted on the immobilized DNA surface, followed by addition of 10mM EDC/10mM NHS for 1h of reaction.
6. The silicon wafer was immersed in 10mM SH- (CH)2)6-OH in ethanol, waiting for hybridization. And finishing the manufacturing of the gene chip for detecting the electric signals.
In experiments, the steps 2 and 6 can be omitted, but the quality of the product is better after the method is adopted. If the chip in the step 2 is adopted for pretreatment, a reproducible and smooth gold surface can be obtained, which is beneficial to the following sample application operation; the part of the metal surface not bound with DNA can be protected by alcohol by step 6 to prevent nonspecific adsorption of the matrix. Meanwhile, the DNA arrangement on the surface can be more ordered, so that the detection accuracy is more accurate when the product is applied to quantitative detection on the change generated by an electric signal in the DNA hybridization process.
Example two
This example is a detection application example of the first embodiment.
1. The chip of example one was removed and the electrical signal at the base point was measured using an electrochemical workstation at 9uA (200 mv for Ag/AgCl).
2. As for the DNA used in the hybridization, pUC18 vector was used as the DNA, and pUC18 plasmid DNA (10uL, about 1ug) was extracted, placed on ice quickly in a boiling water bath for 5 minutes to denature the DNA.
3. The denatured pUC18 plasmid was spotted on the base points and incubated at 37 ℃ for 2 hours.
4. The chip was washed with double distilled water and rinsed several times for about 30 minutes in double distilled water at 45 ℃.
5. The chip was removed and the electrical signal from the base point was measured again using an electrochemical workstation at 2uA (for Ag/AgCl, 200mv, this signal is the detection background signal). It is proved that the electric signal group is far away from the Au surface on the chip due to the damage of the stem-loop structure, so that the disappearance of the electrochemical signal is caused. It was confirmed that the sample contained a DNA sequence that matched with the probe DNA sequence, i.e., contained the pUC18 plasmid.
EXAMPLE III
This example is the preparation and detection example of electronic gene chip, wherein the preparation and treatment of the chip are the same as those of the first example. Briefly, the process is as follows:
DNA sequences of the structure shown below were synthesized or purchased: 5' -NH2-(CH2)3-ATGCGT- -ACGCAT-(CH2)6-SH-3＇The specific DNA sequence selected for this experiment was a fragment of pUC18 vector as in example 1, and the DNA used for hybridization was pUC18 vector. Prepared DNA probes (1uL, 1mM) were manually spotted onto the base dots, and the surfaces were covered overnight at room temperature. The chemical reaction of 3' -SH and Au forms S-Au bond to realize the self-assembly of surface molecule, thereby completing the fixation of DNA.
A solution containing 5uL, 3mM of electrochemical group (carboxyferrocene) was spotted on the immobilized DNA surface, followed by addition of 10mM EDC/10mM NHS for reaction for about 1 h.
The silicon wafer was immersed in 10mM SH- (CH)2)6-OH in ethanol, waiting for hybridization. And finishing the manufacturing of the gene chip for detecting the electric signals.
The chip was removed and the electrical signal at the base point was measured using an electrochemical workstation at 8.7uA (vs. Ag/AgCl, 200 mv).
Plasmid DNA (10uL, about 1ug) of pUC18 was extracted, placed on ice quickly in a boiling water bath for about 5 minutes, and the DNA was denatured.
The denatured pUC18 plasmid was spotted on the base point, and the resulting spot was incubated at about 37 ℃ for about 2 hours.
By ddH2O cleaning the chip and placing the chip in ddH at 45 DEG C2Rinse in O several times for about 30 minutes.
The chip was removed and the electrical signal from the base point was measured again using an electrochemical workstation at 2uA (for Ag/AgCl, 200mv, this signal is the detection background signal). It is proved that the electric signal group is far away from the Au surface on the chip due to the damage of the stem-loop structure, so that the disappearance of the electrochemical signal is caused. It was confirmed that the sample contained a DNA sequence that matched with the probe DNA sequence, i.e., contained the pUC18 plasmid.
In this example, compared with the first example, only the sequence forming the stem-loop structure is changed, and the result shows that the DNA sequence of the stem-loop structure region in the patented method can be changed.
Example four
This example is still the preparation and detection example of electronic gene chip, wherein the preparation and treatment of the chip are the same as those of the first example. Briefly, the process is as follows:
DNA sequences of the structure shown below were synthesized or purchased:
5 ＇-NH2-(CH2)3-GCG AG- 
-CT CGC-(CH2)6-SH-3＇the specific DNA sequence selected for this experiment is a section of Bacillus pumilus alkaline protease gene, and the DNA used for hybridization is a plasmid vector containing the section of gene.
Prepared DNA probes (1uL, 1mM) were manually spotted onto the base dots, and the surfaces were covered overnight at room temperature.
A solution containing 5uL, 3mM of electrochemical group (carboxyferrocene) was spotted on the immobilized DNA surface, followed by addition of 10mM EDC/10mM NHS for 1h of reaction.
The silicon wafer was immersed in 10mM SH- (CH)2)6-OH in ethanol, waiting for hybridization. And finishing the manufacturing of the gene chip for detecting the electric signals.
The chip was removed and the electrical signal at the base point was measured using an electrochemical workstation at 10uA (vs. Ag/AgCl, 200 mv).
Plasmid DNA (10uL, about 1ug) containing the Bacillus pumilus alkaline protease gene was extracted, incubated in boiling water for 5 minutes, and quickly placed on ice to denature the DNA.
The denatured plasmid was spotted on the base point and incubated at 37 ℃ for 2 hours.
By ddH2O cleaning the chip and placing the chip in ddH at 45 DEG C2Rinse in O several times for about 30 minutes.
The chip was removed and the electrical signal from the base point was measured again using an electrochemical workstation at 2uA (for Ag/AgCl, 200mv, this signal is the detection background signal). It is proved that the electric signal group is far away from the Au surface on the chip due to the damage of the stem-loop structure, so that the disappearance of the electrochemical signal is caused. It was confirmed that the sample contained a DNA sequence that matched with the DNA sequence of the probe, i.e., contained the Bacillus pumilus alkaline protease gene.
In this example, compared with the first example, only the specific DNA sequence to be detected is changed, and the result shows that the sequence of the specific DNA region in the patented method can be changed, i.e., the patented method can be used for detecting the existence of various specific DNAs.
EXAMPLE five
This example is still the preparation and detection example of electronic gene chip, wherein the preparation and treatment of the chip are the same as those of the first example. Briefly, the process is as follows:
DNA sequences of the structure shown below were synthesized or purchased: 5' -NH2-(CH2)3-GCG AG- -CTCGC-(CH2)6-SH-3＇The specific DNA sequence selected for this experiment is a section of Bacillus pumilus alkaline protease gene, and the DNA used for hybridization is the PCR product containing this section of gene.
Prepared DNA probes (1uL, 1mM) were manually spotted onto the base dots, and the surfaces were covered overnight at room temperature. The chemical reaction of 3' -SH and Au forms S-Au bond to realize the self-assembly of surface molecule, thereby completing the fixation of DNA.
A solution containing 5uL, 3mM of electrochemical group (carboxyferrocene) was spotted on the immobilized DNA surface, followed by addition of 10mM EDC/10mM NHS for 1h of reaction.
The silicon wafer was immersed in 10mM SH- (CH)2)6-OH in ethanol, waiting for hybridization. And finishing the manufacturing of the gene chip for detecting the electric signals.
The chip was removed and the electrical signal at the base point was measured using an electrochemical workstation at 10uA (vs. Ag/AgCl, 200 mv).
The PCR product (5uL, ca. 0.5ug) of the Bacillus pumilus alkaline protease gene was boiled in a water bath for 5 minutes and quickly placed on ice to denature the DNA.
The denatured pUC18 plasmid was spotted on the base points and incubated at 37 ℃ for 2 hours.
By ddH2O cleaning the chip and placing the chip in ddH at 45 DEG C2Rinse in O several times for about 30 minutes.
The chip was removed and the electrical signal from the base point was measured again using an electrochemical workstation at 2uA (for Ag/AgCl, 200mv, this signal is the detection background signal). It is proved that the electric signal group is far away from the Au surface on the chip due to the damage of the stem-loop structure, so that the disappearance of the electrochemical signal is caused. It was confirmed that the sample contained a DNA sequence that matched with the DNA sequence of the probe, i.e., contained the Bacillus pumilus alkaline protease gene.
In this example, only the source of the sample is changed compared with the fourth example, in which the sample is extracted plasmid DNA and the sample is PCR product. The results show that the samples required for the patented method can come from a number of different methods.
Claims (8)
1. A preparation method of an electronic gene chip is characterized by comprising the following preparation steps:
a. spotting a DNA probe on a base point of the carrier sprayed with the metal thin layer to complete the fixation of the DNA;
b. spotting the solution containing the electrochemically active groups on the surface of the immobilized DNA so that the 5' end of the immobilized DNA sample is provided with the electrochemically active groups;
the structure of the DNA probe is as follows:
5＇-NH2-(CH2)n-********+++++++++++++++++********-(CH2)n-SH-
wherein,********two DNA sequences which can be complementarily paired (A is paired with T, and G is paired with C) are shown; specific DNA fragments (e.g., 10-30 bases) to be detected during hybridization; the methyl group (CH) at both ends2) The number n is preferably 0 to 10.
2. The method for preparing an electronic gene chip according to claim 1, wherein the metal surface of the electronic gene chip having the electrochemically active group is further immersed in SH- (CH)2)6-OH solution overnight.
3. The method for preparing electronic gene chip according to claim 1, wherein the SH- (CH)2)6The concentration of the-OH solution is 1-50mM
4. The method for preparing an electronic gene chip according to claim 1, wherein the carrier is prepared by using a glass plate, a ceramic plate, a plastic plate or a silicon wafer as a carrier, covering a mask (mask) according to a certain circuit design, vacuum-plating a titanium (adhesion layer) on the carrier or a substrate, and then plating a very thin metal layer to form base points (up to hundreds to thousands) for placing DNA probes and corresponding circuits.
5. The method for preparing an electronic gene chip according to claim 4, wherein the metal is gold.
6. The method for preparing an electronic gene chip according to claim 4, wherein the chip is further pretreated by: a. after being treated by plasma for 30 minutes, the mixture is quickly placed in an ethanol solution; or b, treating the mixture for 30 minutes by using an ultraviolet-ozone treatment box and then treating the mixture by using saturated alkali solution (such as KOH); or c, treating the mixture by Piranha solution (70 percent of concentrated sulfuric acid/30 percent of hydrogen peroxide) at high temperature (higher than 90 ℃) for more than 20 minutes, and then carrying out electrochemical (cyclic voltammetry scanning) cleaning in alkali solution (such as KOH, NaOH and the like) at a certain potential (-0.4V to-1.2V). The cleaned chip is stored in ethanol or other organic solvents for later use.
7. The method for preparing an electronic gene chip according to claim 1, wherein the DNA probe spots perform DNA immobilization on the base spots of the carrier by: the prepared DNA probe is spotted on a base point, and the reaction is carried out for more than 5 hours at room temperature after the surface is covered.
8. The method for preparing an electronic gene chip according to claim 1, wherein the preparation process comprises: a. plating gold on a silicon chip or a glass slide to manufacture a base point and a circuit; b. carrying out pretreatment; c. spotting DNA on a base point; d. synthesizing an electrochemical radical block; e. surface protection; f. and finishing the preparation of the chip.
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| CN 01129100 CN1227368C (en) | 2001-11-23 | 2001-11-23 | Electronic gene chip preparation method |
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| CN 01129100 CN1227368C (en) | 2001-11-23 | 2001-11-23 | Electronic gene chip preparation method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7803542B2 (en) | 2005-11-29 | 2010-09-28 | The Regents Of The University Of California | Signal-on architecture for electronic, oligonucleotide-based detectors |
| US8003374B2 (en) | 2003-03-25 | 2011-08-23 | The Regents Of The University Of California | Reagentless, reusable, bioelectronic detectors |
| CN1572887B (en) * | 2003-06-20 | 2014-01-08 | 佳能株式会社 | Setting device with point pattern encrypting function and detecting device corresponding to the encryption |
-
2001
- 2001-11-23 CN CN 01129100 patent/CN1227368C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8003374B2 (en) | 2003-03-25 | 2011-08-23 | The Regents Of The University Of California | Reagentless, reusable, bioelectronic detectors |
| CN1572887B (en) * | 2003-06-20 | 2014-01-08 | 佳能株式会社 | Setting device with point pattern encrypting function and detecting device corresponding to the encryption |
| US7803542B2 (en) | 2005-11-29 | 2010-09-28 | The Regents Of The University Of California | Signal-on architecture for electronic, oligonucleotide-based detectors |
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| Publication number | Publication date |
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| CN1227368C (en) | 2005-11-16 |
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