CN1422961A - Electronic gene chip detecting method - Google Patents
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- CN1422961A CN1422961A CN 01129104 CN01129104A CN1422961A CN 1422961 A CN1422961 A CN 1422961A CN 01129104 CN01129104 CN 01129104 CN 01129104 A CN01129104 A CN 01129104A CN 1422961 A CN1422961 A CN 1422961A
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Abstract
The invention is a testing method of electron gene chip, the gene chip which has electrochemistry activity base is hybridized with sample DNA, then the electron testing device replaces the fluorescence scanner to test the change of electrical signal before and after hybridization, it judges if the tested sample contains special DNA segment need to be tested, the sample DNA needn't to be fluorescence processed and can change the nature directly.
Description
The invention belongs to the technical field of DNA hybridization detection, and particularly relates to a detection method of an electronic gene chip.
In the prior art, a conventional detection method of a gene chip, i.e., a fluorescence scanning method, requires pretreatment of sample DNA, i.e., fluorescence labeling of a sample, hybridization of the gene chip and the sample DNA with a fluorescence scanning signal, i.e., fluorescence labeling, and detection of the hybridized fluorescence signal by a fluorescence scanner, to determine whether the detected sample contains a DNA fragment to be detected.
However, the conventional detection method of the above-mentioned conventional gene chip, i.e., the fluorescence scanning method, is still limited to the laboratory stage for the following reasons, and is still quite apart from the official application: 1. the detection cost is high, and the detection cost can be only borne by large pharmaceutical companies and scientific research institutions with sufficient expenditure. A commercial set of GeneChip signal detection and analysis systems, including fluorescence scanners, computers and software, cost about 20 million dollars, for example, the international market price of a set of chip systems (including 5 oligonucleotides and 1 cDNA chip) of Affymetrix in the United states exceeds 15 million 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. The cost is the first problem to achieve wide application, so that the popularization and application difficulty are very high. 2. The existing detection method needs to carry out complicated pretreatment on sample DNA, and mainly shows that the sample needs to be subjected to fluorescent 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 process requires specialized training by professionals. 3. The fluorescent signal obtained by hybridizing the gene chip and the sample DNA with the fluorescent label can only be regarded as a qualitative or semi-quantitative signal result, and cannot be directly and accurately analyzed in a digital mode. 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.
The invention aims to make up for the defects of the prior art and provides a detection method of an electronic gene chip, which is convenient to operate, low in cost, high in detection sensitivity and reliability and applicable to large-scale popularization.
The purpose of the invention is realized by the following technical scheme.
The invention relates to a detection method of an electronic gene chip, which comprises the steps of hybridizing the gene chip with an electrochemical active group with sample DNA, detecting the change of an electric signal before and after hybridization of the gene chip by using an electronic detector instead of a fluorescence scanner, and judging whether a detected sample contains a specific DNA fragment to be detected.
In the above scheme, the hybridization process is:
a. sample DNA was directly denatured without fluorescent pretreatment: heating the sample in water bath until the sample is boiled, and keeping the sample for more than 1 minute; taken out of the boiling water and immediately put on ice until completely cooled. At this point, the sample DNA has been completely denatured into single strands.
b. Sample DNA hybridization: mixing the denatured DNA sample (liquid) with the probe on the base point of the chip, and incubating for 1-24 hours at 20-45 deg.C (varying with different probe DNA sequences) for hybridization.
c. Elution of the chip: after hybridization, the chip is rinsed several times with hybridization solution without DNA sample, the temperature is controlled at 20-55 deg.C (varying with different probe DNA sequences), the time lasts more than half an hour, until the sample DNA which can not be hybridized and the non-specific adsorption of the probe are washed away.
In the above embodiment, the gene chip of the electrochemically active group is preferably a DNA probe having the following structure:
| 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 (10-30 bases) to be detected during hybridization are shown as ++++++++++++++; the methyl group (CH) at both ends2) The number n is 0 to 10.
In the above embodiment, the electrochemically active group may be a group of any structure capable of generating an electrochemical signal.
In the above scheme, the electrochemically active group may be carboxyferrocene or other methyl violet carboxyl derivative electrochemically active groups.
In the above scheme, the electronic detector may be an electrochemical workstation or other electronic detection equipment. And detecting the change of the electric signals before and after hybridization by using a sensitive alternating current voltammetry so as to judge whether the detected sample contains the specific DNA fragment to be detected.
Normally, an electrical signal is present at the base point prior to hybridization. If the detection sample contains a probe sequence (specific DNA fragment to be detected), the electric signal on the base point disappears after hybridization; otherwise, the electrical signal still exists.
The detection process flow of the electronic gene chip of the invention can be seen in FIG. 2.
The principle of the technical scheme of the invention is as follows: before hybridization, since both ends of the probe DNA contain a DNA sequence capable of completely pairing, a radial loop structure (stemloop) 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. For a further understanding of the above principles, reference may be made to FIG. 1.
The detection method of the electronic gene chip can carry out quantitative detection on the change generated by the electric signal in the DNA hybridization process. The technology applies mature microelectronic technology to gene chips, and is in the leading position all over the world at present.
The technology uses an electronic detector to detect the signal of the gene chip, directly applies the mature technology and the achievement of the microelectronic industry, and compared with the prior gene chip, the technology has the following advantages: 1. the cost of the detection equipment is greatly reduced, and the cost of the detection equipment used as an electronic detector for signal detection, such as an electrochemical workstation, is far lower than that of a fluorescence scanner used in the prior art, thereby being beneficial to realizing civilization and entering a wide application field; 2. in the hybridization process, a sample to be detected does not need any label, so that a complex fluorescent label pretreatment process is omitted, and the pretreatment process can be finished by anyone only through simple training. 3. The electric scanning signal obtained by the method 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. Table 1 shows the comparison between the present invention and the conventional detection method.
TABLE 1 comparison with existing detection methods
| Existing detection methods | The detection method of the invention | |
| Sample pretreatment | Extracting DNA and carrying out fluorescence labeling | Extraction of DNA |
| Hybridization process | Conventional solid-liquid hybridization procedure | Conventional solid-liquid hybridization procedure |
| Detecting the signal | Fluorescent signal | Digitized electrical signal |
| Detection device | Confocal fluorescence scanning microscope | Electrochemical workstations or other electrical signal detection apparatus |
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 following are examples of the present invention, and the present invention is not limited to the examples.
Example one
First, the DNA probe is structured to be 5' -NH2-(CH2)3-GCG AG--CT CGC-(CH2)6-SH-3' gene chip for detecting electric signal of carboxyl ferrocene as electrochemical group
The specific DNA sequence selected for this experiment was a fragment of pUC18 vector, corresponding to which the DNA used for hybridization was pUC18 vector, and the electric signal from the base point was measured by an electrochemical workstation and found to be 9uA (200 mv for Ag/AgCl). For hybridization of the sample DNA described below in this example. The preparation method of the gene chip for detecting the electric signal can be seen in other patents.
The hybridization process of the sample DNA of this example was:
a. denaturation of sample DNA: plasmid DNA pUC18 (10uL, ca. 1ug) was extracted, placed in a boiling water bath for 5 minutes, quickly placed on ice to denature the DNA, at which point the sample DNA had been completely denatured into single strands.
b. Sample DNA hybridization: the denatured DNA sample (liquid) was mixed with the probe on the base of the gene chip for electrical signal detection prepared above, i.e., denatured pUC18 plasmid was spotted on the base, and the mixture was incubated at 20 ℃ to 45 ℃ for 1 to 24 hours (varying depending on the DNA sequence of the probe), followed by hybridization, in this case, at 37 ℃ for 2 hours.
c. Elution of the chip: and (3) rinsing the chip for several times by using the hybridization solution without the DNA sample, controlling the temperature to be 20-55 ℃ (changing with different probe DNA sequences), and lasting for more than half an hour until the sample DNA which is not hybridized is washed away and the non-specific adsorption of the probe is carried out. After completion of hybridization in this example, the chip was washed with double distilled water and rinsed several times for about 30 minutes in double distilled water at 45 ℃.
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 two
In this example, a gene chip for detecting an electric signal with an electrochemical group of carboxyferrocene was purchased or synthesized from a DNA probe having the following structure: 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 one, and the DNA used for hybridization was pUC18 vector.
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) was extracted from pUC18, placed in a boiling 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 coreSlicing, and subjecting the chip to ddH at 45 deg.C2Rinse in O several times for about 30 minutes. ddH2And O is double distilled water.
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 III
In this example, a gene chip for detecting an electric signal with an electrochemical group of carboxyferrocene was purchased or synthesized from a DNA probe having the following structure: 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 a plasmid vector containing the section of gene.
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 with an electrochemical workstation as 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 four
This example is an example of the whole process of preparing and detecting an electronic gene chip.
Firstly, a glass sheet, a silicon wafer and a ceramic sheet are selected as a substrate of a core substrate 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.
The gold plated silicon wafer was treated by the following three methods:
a. treating with plasma, and rapidly placing in ethanol solution;
b, treating by using an ultraviolet-ozone treatment box, and then treating by using 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.
Synthetic junctionThe DNA sequence shown below: 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.
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, the source of the sample is changed compared with the sample in the third example, the sample is extracted plasmid DNA, and the sample in this example is PCR product. The results show that the samples required for the patented method can come from a number of different methods.
Claims (7)
1. A detection method of electronic gene chip is characterized by that the gene chip with electrochemical active group and sample DNA are hybridized, then the electronic detector is substituted for fluorescent scanner to detect the change of electric signal before and after hybridization of gene chip, and judge that the tested sample contains the specific DNA fragment to be detected.
2. The method for detecting electronic gene chip according to claim 1, wherein the gene chip having the electrochemically active group is preferably one having the electrochemically active group thereonA DNA probe of the following structure:
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 (10-30 bases) to be detected during hybridization are shown as ++++++++++++++; the methyl group (CH) at both ends2) The number n is 0 to 10.
3. The method for detecting an electronic gene chip according to claim 1, wherein the hybridization process is:
a. the sample DNA was directly denatured: heating the sample in water bath until the sample is boiled, and keeping the sample for more than 1 minute; taken out of the boiling water and immediately put on ice until completely cooled. At this point, the sample DNA has been completely denatured into single strands.
b. Sample DNA hybridization: mixing the denatured DNA sample (liquid) with the probe on the base point of the chip, and incubating for 1-24 hours at 20-45 deg.C (varying with different probe DNA sequences) for hybridization.
c. Elution of the chip: after hybridization, the chip is rinsed several times with hybridization solution without DNA sample, the temperature is controlled at 20-55 deg.C (varying with different probe DNA sequences), the time lasts more than half an hour, until the sample DNA which can not be hybridized and the non-specific adsorption of the probe are washed away.
4. The method for detecting an electronic gene chip according to claim 1, wherein the electrochemically active group is a group having any structure capable of generating an electrochemical signal.
5. The method for detecting an electronic gene chip according to claim 4, wherein the electrochemically active group is carboxyferrocene or other methyl violet carboxyl derivatives.
6. The method for detecting an electronic gene chip according to claim 1, wherein the electronic detector is an electrochemical workstation or other electronic detection equipment.
7. The method for detecting an electronic gene chip according to claim 1, wherein the detection process comprises: a. pretreating a sample, and extracting DNA; b. sample DNA denaturation; c. hybridizing with the chip; d. cleaning a chip; e. detecting an electric signal; f. and (5) carrying out post-digitized signal analysis.
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| CNB011291044A CN1313621C (en) | 2001-11-23 | 2001-11-23 | Electronic gene chip detecting method |
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| CNB011291044A CN1313621C (en) | 2001-11-23 | 2001-11-23 | Electronic gene chip detecting method |
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| CN1313621C CN1313621C (en) | 2007-05-02 |
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Cited By (2)
| 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1273364A (en) * | 1999-05-06 | 2000-11-15 | 杨梦甦 | Detection method of special DNA chip for diagnosis of pathogenic bacteria and disease-related gene mutation |
| JP2001194298A (en) * | 1999-10-28 | 2001-07-19 | Nippon Telegr & Teleph Corp <Ntt> | Surface plasmon resonance enzyme sensor and method for measuring surface plasmon resonance |
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2001
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Cited By (2)
| 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 |
| US7803542B2 (en) | 2005-11-29 | 2010-09-28 | The Regents Of The University Of California | Signal-on architecture for electronic, oligonucleotide-based detectors |
Also Published As
| Publication number | Publication date |
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| CN1313621C (en) | 2007-05-02 |
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