CN112255297A - Universal nucleic acid detection device and preparation method and application thereof - Google Patents
Universal nucleic acid detection device and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a universal nucleic acid detection device and a preparation method and application thereof, wherein the universal nucleic acid detection device comprises: the three-layer structure comprises the following components in sequence from top to bottom: CNT layer of carbon nanotube and SiO2A layer and a substrate layer, wherein the carbon nanotube CNT is modified with a CW polypeptide, the sequence of the CW polypeptide is: Fmoc-RRMEHRMEWC. The invention has obvious delta Ion electrical signal response to different types of nucleic acid molecules, such as nucleic acid molecules with different lengths and different GC contents, can be used as an electrical response detector of the nucleic acid molecules, and is used for detecting and quantifying the universal nucleic acid molecules.
Description
Technical Field
The invention relates to a universal nucleic acid detection device and a preparation method and application thereof, belonging to the technical field of biological detection.
Background
Nucleic acid detection is of great significance in clinical diagnosis, forensic identification and scientific research. In the postoperative monitoring of many diseases, the content of nucleic acid molecules often reflects the therapeutic effect of the treatment and whether the disease recurs, so in many diseases, nucleic acid molecules are an important marker and help doctors to judge the disease condition of patients.
Common methods for detecting nucleic acid include a fluorescence method and an electric signal method, wherein the fluorescence method is a method for calculating the content of nucleic acid molecules by detecting and analyzing the change of fluorescence spectrum in a system after the change of fluorescence in the system is caused by the nucleic acid molecules. Commonly used fluorescent nucleic acid detection methods are: a gold nanoparticle fluorescence method, a luminol chemiluminescence method and the like.
In addition to fluorescent signals, electrical detection methods for nucleic acid molecules are also an important class of nucleic acid detection methods, because electrical signals are often more sensitive and rapid signal collection and amplification can be achieved by means of electrical devices. In the electrical signal detection of nucleic acids, CNT electrical detection platforms represented by carbon nanotube transistors have received much attention. Due to the excellent electrochemical properties and the characteristics that can be modified of carbon nanotube transistors, biomolecule detection based on carbon nanotube transistors has become an important part of the application of carbon nanotubes. At present, there are many reports on the detection method of specific protein and DNA by using a carbon nanotube biological detection platform.
However, most of these reported methods for detecting nucleic acids are limited to the detection of specific nucleic acid molecules, and the principle is that complementary pairing effect of nucleic acid molecules must be utilized to capture target DNA molecules through probe DNA molecules, so as to further generate electric signals or fluorescent signals. For the general-purpose DNA detection method, the currently reported detection method is very limited. The detection range of the common universal nucleic acid detection method Nanodrop in scientific research is generally only ng/mul level, and for the detection of some precious biological samples, the universal nucleic acid detection method which is more sensitive and has lower detection limit needs to be developed.
Disclosure of Invention
The invention aims to provide a universal nucleic acid detection device, a preparation method and application thereof, and provides a rapid universal nucleic acid detection method.
The present invention provides a universal nucleic acid detecting device, comprising:
the three-layer structure comprises the following components in sequence from top to bottom: CNT layer of carbon nanotube and SiO2A layer and a substrate layer, wherein the carbon nanotube CNT is modified with a CW polypeptide, the sequence of the CW polypeptide is: Fmoc-RRMEHRMEWC.
Further, the universal nucleic acid detecting device of the present invention is also characterized in that: ti is used as metal electrodes on two sides of the CNT layer of the carbon nano tube, and carbon nano tube thin film transistors are uniformly distributed in the middle channel.
The invention also provides a preparation method of the universal nucleic acid detection device, which is characterized by comprising the following steps:
the method comprises the following steps: preparing a CNT-TFT electrical device, wherein carbon nano tubes are uniformly distributed in a channel;
step two: incubating a first predetermined amount of N-1-pyrenemaleimide solution with the prepared CNT-TFT electrical device at room temperature for a first predetermined time using DMF and ddH2After the O alternate wash, a second predetermined amount of a CW-polypeptide solution is used, the sequence of the CW-polypeptide being: Fmoc-RRMEHRMEWC, incubating at room temperature for a second predetermined time,
step three: by ddH2O cleaning and then drying.
The invention also provides application of the PEPTIDIZED CNT-TFT electric device in general nucleic acid detection.
Further, the application of the electrical device of the PEPTIDIZED CNT-TFT in general nucleic acid detection also has the characteristics that the electrical device comprises the following steps:
the method comprises the following steps: taking a series of nucleic acid standard solutions with different concentrations, respectively incubating the nucleic acid standard solutions with the polypeptide CNT-TFT device to obtain corresponding delta Ion signal responses, and then fitting the relation between the DNA concentration and the delta Ion responses by using a Hill-Langmuir model to obtain a fitting formula;
step two: preparation of nucleic acid sample: obtaining a nucleic acid sample according to a preparation scheme adapted to the target nucleic acid;
step three: diluting the obtained nucleic acid sample, incubating the diluted nucleic acid sample with the polypeptide CNT-TFT device, analyzing the delta Ion response by using a semiconductor device analyzer, substituting the delta Ion response into a fitting formula in the step one, and calculating to obtain the nucleic acid content.
Further, the use of the present invention is also characterized in that: wherein the nucleic acid is DNA or RNA.
Further, the use of the present invention is also characterized in that: wherein the nucleic acid is cDNA.
Further, the use of the present invention is also characterized in that: when used to detect the amount of cDNA in a cell, the procedure is as follows:
the method comprises the following steps: taking a series of DNA standard solutions with different concentrations, respectively incubating the DNA standard solutions with the polypeptide CNT-TFT device to obtain corresponding delta Ion signal response, and fitting the relation between the DNA concentration and the delta Ion response by using a Hill-Langmuir model to obtain a fitting formula;
step two: preparation of cellular cDNA samples: adding a predetermined amount of TRIzol reagent into cells in a good growth state, uniformly mixing at normal temperature, standing for 5min, adding 100 mu l of chloroform, standing for 5min, centrifuging at 4 ℃, transferring supernatant into an EP tube without nuclease, adding equal amount of isopropanol, incubating on ice for 10min, centrifuging at 4 ℃, collecting precipitate, washing with 75% ethanol once, drying on ice for 10min, adding DEPC (diethyl phthalate) for treatment, taking 1-2 mu l of dissolved solution, adding Oligo-dT, incubating at 65 ℃ for 5min, immediately placing on ice, sequentially adding dNTP (dNTP), reverse transcriptase and reverse transcriptase buffer solution into the system, and carrying out PCR (polymerase chain reaction) reaction by using a PCR (polymerase chain reaction) instrument to obtain a cDNA (complementary deoxyribonucleic acid) sample of the cells;
step three: diluting the obtained cDNA sample of the cell, incubating the diluted cDNA sample with a polypeptide CNT-TFT device, analyzing delta Ion response by using a semiconductor device analyzer, substituting the delta Ion response into a previously fitted formula, and calculating to obtain the cDNA content of the cell.
The polypeptide modification method of the carbon nano tube comprises the following specific steps: firstly, preparing a CNT-TFT electric device with good properties, wherein a large number of carbon nano tubes are uniformly distributed in a channel; then incubating the prepared CNT-TFT electrical device with 6mM N-1-pyrenemaleimide solution for 4-5h at normal temperature, alternately cleaning DMF and ddH2O, incubating with 150 μ M CW polypeptide solution (sequence: Fmoc-RRMEHRMEWC) for 14-18h at normal temperature, cleaning with ddH2O, and drying N2 for later use.
The invention has the beneficial effects that: experiments of an Atomic Force Microscope (AFM), a scanning tunnel microscope (SEM), an Agilent B1500 type semiconductor device analyzer and the like prove that the CNT-TFT electric device can be successfully modified by polypeptide, and has obvious delta Ion electric signal response for different types of nucleic acid molecules, such as nucleic acid molecules with different lengths and different GC contents. Can be used as an electrical response detector of nucleic acid molecules and is used for detecting and quantifying general nucleic acid molecules.
The universal nucleic acid detection device is simple to operate, quick in response and capable of being used as a quick and effective nucleic acid molecule detection method within 5min of the detection process.
Compared with the prior art, the invention has remarkable technical progress. First, the current general purpose type of nucleic acid molecule electrical signal detector is not uncommon, and most of them are response detectors for specific nucleic acid molecules. Secondly, compared with the known widely used universal nucleic acid molecule detector Nanodrop, the universal nucleic acid electric signal detection device (CNT-TFT) has a significantly lower detection limit (0.88 mu g/L) and response detection interval (1.6x10-4 mu mol/L to 5 mu mol/L). Provides a method for universal nucleic acid detection of some precious biological samples.
Drawings
FIG. 1 is an AFM image of a CNT-TFT device prior to polypeptide modification; it can be seen that a large number of carbon nanotubes are uniformly distributed in the channel.
FIG. 2 is an AFM image of a CNT-TFT device after polypeptide modification; it can be seen that a large amount of carbon nanotubes are still distributed in the channel, and the morphology of the carbon nanotubes is intact.
FIG. 3 is an electrical representation of Id-Vg of a CNT-TFT device after polypeptide modification; it can be seen that the polypeptide molecule and DNA are modified to induce obvious response change of delta Ion after incubation.
FIG. 4 is a graph of the Δ Ion response of a generic nucleic acid detector (CNT-TFT) incubated in DNA solution for various periods of time: it can be seen that within 5min, the response of Δ Ion reaches saturation.
FIG. 5 is a graph showing the detection response of a universal nucleic acid detector (CNT-TFT) to different lengths of DNA: it can be seen that the universal nucleic acid detector (CNT-TFT) can be used for analytical detection of DNAs of different lengths.
FIG. 6 is a graph showing the detection response of a universal nucleic acid detector (CNT-TFT) to different types of DNA: it can be seen that the universal nucleic acid detector (CNT-TFT) can be used for analytical detection of different types of DNA.
FIG. 7 is the detection response of a universal nucleic acid detector (CNT-TFT) to DNA of different GC contents: it can be seen that the universal nucleic acid detector (CNT-TFT) can be used for analytical detection of DNA with different GC contents.
FIG. 8 is a Δ Ion signal response of a generic nucleic acid detector (CNT-TFT) to different concentrations of DNA: the response rule of the method is consistent with a Hill-Langmuir model, and the correlation coefficient is 0.98.
FIG. 9 is a Δ Ion signal response of a generic nucleic acid detector (CNT-TFT) to different concentrations of RNA: the response rule of the method accords with a Hill-Langmuir model, and the correlation coefficient is 0.99.
FIG. 10 shows the results of quantitative detection of cDNA from T47D cells using a general-purpose nucleic acid detector (CNT-TFT): it can be seen that the quantitative analysis result is similar to the nanodrop control.
FIG. 11 shows the preparation of the universal nucleic acid detector of the present invention and the procedure for performing nucleic acid detection.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
Example 1: polypeptization modification of CNT-TFT device:
firstly, a carbon nanotube thin film transistor device (CNT-TFT) with good electrical property is prepared, and after AFM and SEM characterization, a large number of carbon nanotube thin film transistors are confirmed to be uniformly distributed in a channel. The device was then placed in 6mM N-1-pyrenemaleimide solution and incubated at room temperature for 4h, during which time the liquid was allowed to contact the CNT-TFT device uniformly with gentle shaking. Then taking out the device, using DMF and ddH2After O alternate washing, the solution is further washed by CW polypeptide solution 150 mu mol/L, and the polypeptide sequence is as follows: Fmoc-RRMEHRMEWC, incubating the CNT-TFT device for 14h, and then adding ddH2O washing, N2And (5) drying. By AgilentAnd analyzing the Id-Vg curve change condition of the CNT-TFT device by using a B1500 type semiconductor device analyzer before and after modification, and verifying that the polypeptide is successfully modified on the CNT-TFT. The preparation of the universal nucleic acid detector and the brief process for carrying out the nucleic acid detection are shown in FIG. 11.
Example 2: polypeptization modification of CNT-TFT device:
first, a carbon nanotube thin film transistor device (CNT-TFT) with good electrical properties is prepared, and after AFM and SEM characterization, it is confirmed that a large number of carbon nanotube thin film transistors are uniformly distributed in the channel as shown in fig. 1 and 2. The device was then placed in a 6mM N-1-pyrenemaleimide solution and incubated at room temperature for 4.5h, during which time the liquid was allowed to uniformly contact the CNT-TFT device with gentle shaking. Then taking out the device, using DMF and ddH2After O alternate washing, the solution is further washed by CW polypeptide solution, 150 mu mol/L, and the polypeptide sequence is: Fmoc-RRMEHRMEWC, incubating the CNT-TFT device for 16h, and then adding ddH2O washing, N2And (5) drying. And (3) analyzing the Id-Vg curve change condition of the CNT-TFT device before and after modification by using an Agilent B1500 type semiconductor device analyzer, and verifying that the polypeptide is successfully modified on the CNT-TFT, as shown in figure 3.
Example 3: polypeptization modification of CNT-TFT device:
firstly, a carbon nanotube thin film transistor device (CNT-TFT) with good electrical property is prepared, and after AFM and SEM characterization, a large number of carbon nanotube thin film transistors are confirmed to be uniformly distributed in a channel. The device was then placed in 6mM N-1-pyrenemaleimide solution and incubated at room temperature for 5h, during which time the liquid was allowed to contact the CNT-TFT device uniformly with gentle shaking. Then taking out the device, using DMF and ddH2After O alternate washing, the solution is further washed by CW polypeptide solution 150 mu mol/L, and the polypeptide sequence is as follows: Fmoc-RRMEHRMEWC) was incubated for 18h with CNT-TFT devices, followed by ddH2O washing, N2And (5) drying. And (3) analyzing the Id-Vg curve change condition of the CNT-TFT device before and after modification by using an Agilent B1500 type semiconductor device analyzer, and verifying that the polypeptide is successfully modified on the CNT-TFT.
Example 4: delta Ion response induced by incubation of a Universal nucleic acid Detector (CNT-TFT) in DNA solution for various periods of time.
A1. mu. mol/L DNA solution was prepared and incubated with a universal nucleic acid detector to detect the Δ Ion response at different times to determine the saturation time. The results are shown in FIG. 4, and it can be seen that within 5min, the response of Δ Ion reaches saturation.
Example 5: the detection response of the universal nucleic acid detector (CNT-TFT) to different lengths of DNA.
Firstly, synthesizing DNA molecules with different lengths, wherein the sequences are shown in a table 1,
table 1: DNA molecules of different lengths
The universal nucleic acid detector prepared in the previous example was then used to perform the responsive detection of DNA of varying lengths in Table 1.
As a result, as shown in FIG. 5, it can be seen that the nucleic acid detector of the general type (CNT-TFT) can be used for analytical detection of DNAs of different lengths.
Example 6: the detection response of the universal nucleic acid detector (CNT-TFT) to different types of DNA.
Single-stranded and double-stranded DNA molecules were synthesized separately, the sequences being shown in table 2:
table 2: different types of DNA molecules
The universal nucleic acid detector prepared in the previous example was then used to perform responsive detection of the different types of DNA in Table 2.
As a result, as shown in FIG. 6, it can be seen that the nucleic acid detector of the general type (CNT-TFT) can be used for analytical detection of different types of DNA.
Example 7: the detection response of the universal nucleic acid detector (CNT-TFT) to DNA with different GC contents.
DNA molecules of different GC contents were prepared and the sequences are shown in Table 3.
Table 3: DNA molecules of different GC contents
The universal nucleic acid detector prepared in the previous example was then used to perform response detection on DNA of varying GC content in Table 3.
FIG. 7 is the detection response of a universal nucleic acid detector (CNT-TFT) to DNA of different GC contents: it can be seen that the universal nucleic acid detector (CNT-TFT) can be used for analytical detection of DNA with different GC contents.
Example 8: the nucleic acid detector of general purpose type (CNT-TFT) responds to the delta Ion signal of DNA of different concentrations.
Different concentrations of DNA molecules were prepared and detected using the universal nucleic acid detector prepared in the previous example, resulting in a Δ Ion signal response. And fitting is performed. As shown in FIG. 8, it can be seen that the response rule conforms to the Hill-Langmuir model, and the correlation coefficient is 0.98.
According to the standard curve determined by the graph in FIG. 8, the response relation is obtained by the Hill-Langmuir model fitting, wherein the corresponding response interval is 1.6x10-4 mu mol/L to 5 mu mol/L, the detection limit is 0.88 mu g/L by combining the molecular weight of the selected DNA.
Example 9: and detecting the RNA molecules with different concentrations by using the universal nucleic acid detector.
Different concentrations of RNA molecules were prepared and detected using the universal nucleic acid detector prepared in the previous example, resulting in a Δ Ion signal response. And fitting is performed. The results are shown in FIG. 9, and it can be seen that the response rule conforms to the Hill-Langmuir model, and the correlation coefficient is 0.99.
Example 10: polypeptide CNT-TFT device for detecting cDNA of T47D cell
The method comprises the following steps: a series of DNA standard solutions with different concentrations are respectively incubated with the polypeptide CNT-TFT device to obtain corresponding delta Ion signal responses, and then a Hill-Langmuir model is used for fitting the relation between the DNA concentration and the delta Ion responses, which is shown in FIG. 8.
Step two: the preparation method of the T47D cell cDNA sample comprises the following specific steps: T47D cells in good growth state were taken, and a certain amount of TRIzol (Invitrogen) reagent was added thereto, mixed well at room temperature, and left to stand for 5 min. Then 100. mu.l of chloroform was added, and after standing for another 5min, centrifugation was carried out at 4 ℃ under the following conditions: 12000rpm,15 min. The supernatant was then transferred to a nuclease-free EP tube, added with equal amounts of isopropanol, incubated on ice for 10min, and then centrifuged at 4 ℃ under the following conditions: 12000rpm,15min, collect the precipitate, and use 75% ethanol washing, put on ice to dry for 10min, add DEPC treated water to dissolve. Then 1-2 mul of the dissolved solution is taken, Oligo-dT (Invitrogen) is added, the solution is incubated for 5min at 65 ℃, then the solution is immediately put on ice, dNTP, reverse transcriptase and reverse transcription buffer are sequentially added into the system, and PCR reaction is carried out by a PCR instrument, thus obtaining cDNA of T47D cells.
And finally diluting the obtained cDNA of the T47D cell, incubating the cDNA with a polypeptide CNT-TFT device, analyzing the delta Ion response by an Agilent B1500 type semiconductor device analyzer, substituting the delta Ion response into a previously fitted formula, and calculating to obtain the cDNA content of the T47D cell. FIG. 10 shows the results of quantitative detection of cDNA from T47D cells using a general-purpose nucleic acid detector (CNT-TFT): it can be seen that the quantitative analysis result is similar to the nanodrop control.
Sequence listing
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Claims (8)
1. A universal nucleic acid detecting device, comprising:
the three-layer structure comprises the following components in sequence from top to bottom: CNT layer of carbon nanotube and SiO2A layer and a substrate layer, wherein the carbon nanotube CNT is modified with a CW polypeptide, the sequence of the CW polypeptide is: Fmoc-RRMEHRMEWC.
2. The universal nucleic acid detecting device according to claim 1, wherein:
ti is used as metal electrodes on two sides of the CNT layer of the carbon nano tube, and carbon nano tube thin film transistors are uniformly distributed in the middle channel.
3. A method for preparing a universal nucleic acid detection device is characterized in that:
the method comprises the following steps: preparing a CNT-TFT electrical device, wherein carbon nano tubes are uniformly distributed in a channel;
step two: incubating a first predetermined amount of N-1-pyrenemaleimide solution with the prepared CNT-TFT electrical device at room temperature for a first predetermined time using DMF and ddH2After the O alternate wash, a second predetermined amount of a CW-polypeptide solution is used, the sequence of the CW-polypeptide being: Fmoc-RRMEHRMEWC, incubating at room temperature for a second predetermined time,
step three: by ddH2O cleaning and then drying.
4. Use of the polypetized modified CNT-TFT electrical device of claim 1 in universal nucleic acid detection.
5. The use of the polypetized modified CNT-TFT electrical device of claim 4 in universal nucleic acid detection, comprising the steps of:
the method comprises the following steps: taking a series of nucleic acid standard solutions with different concentrations, respectively incubating the nucleic acid standard solutions with the polypeptide CNT-TFT device to obtain corresponding delta Ion signal responses, and then fitting the relation between the DNA concentration and the delta Ion responses by using a Hill-Langmuir model to obtain a fitting formula;
step two: preparation of nucleic acid sample: obtaining a nucleic acid sample according to a preparation scheme adapted to the target nucleic acid;
step three: diluting the obtained nucleic acid sample, incubating the diluted nucleic acid sample with the polypeptide CNT-TFT device, analyzing the delta Ion response by using a semiconductor device analyzer, substituting the delta Ion response into a fitting formula in the step one, and calculating to obtain the nucleic acid content.
6. Use according to claim 5, characterized in that:
wherein the nucleic acid is DNA or RNA.
7. Use according to claim 5, characterized in that:
wherein the nucleic acid is cDNA.
8. Use according to claim 5, characterized in that:
when used to detect the amount of cDNA in a cell, the procedure is as follows:
the method comprises the following steps: taking a series of DNA standard solutions with different concentrations, respectively incubating the DNA standard solutions with the polypeptide CNT-TFT device to obtain corresponding delta Ion signal response, and fitting the relation between the DNA concentration and the delta Ion response by using a Hill-Langmuir model to obtain a fitting formula;
step two: preparation of cellular cDNA samples: adding a predetermined amount of TRIzol reagent into cells in a good growth state, uniformly mixing at normal temperature, standing for 5min, adding 100 mu l of chloroform, standing for 5min, centrifuging at 4 ℃, transferring supernatant into an EP tube without nuclease, adding equal amount of isopropanol, incubating on ice for 10min, centrifuging at 4 ℃, collecting precipitate, washing with 75% ethanol once, drying on ice for 10min, adding DEPC (diethyl phthalate) for treatment, taking 1-2 mu l of dissolved solution, adding Oligo-dT, incubating at 65 ℃ for 5min, immediately placing on ice, sequentially adding dNTP (dNTP), reverse transcriptase and reverse transcriptase buffer solution into the system, and carrying out PCR (polymerase chain reaction) reaction by using a PCR (polymerase chain reaction) instrument to obtain a cDNA (complementary deoxyribonucleic acid) sample of the cells;
step three: diluting the obtained cDNA sample of the cell, incubating the diluted cDNA sample with a polypeptide CNT-TFT device, analyzing delta Ion response by using a semiconductor device analyzer, substituting the delta Ion response into a previously fitted formula, and calculating to obtain the cDNA content of the cell.
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