CN115144451A - Probe for detecting mi-RNA, gaN sensor and detection method - Google Patents
Probe for detecting mi-RNA, gaN sensor and detection method Download PDFInfo
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Abstract
The invention discloses a mi-RNA detection probe, a GaN sensor and a detection method, wherein the mi-RNA probe is arranged on a grid electrode of the GaN sensor; the detection method of the mi-RNA comprises the following steps of preparing a GaN sensor; setting source electrode and drain electrode voltage according to the electrical parameters of the GaN sensor; buffer solutions with different target mi-RNA concentrations are prepared, the GaN sensor is inserted into the buffer solution for testing, and a standard curve is determined; inserting the GaN sensor into the solution to be detected, comparing the output current of the solution to be detected with the output current of the standard curve, judging whether the target mi-RNA exists in the solution to be detected according to the current of the GaN sensor and the current of the solution to be detected, and obtaining the concentration of the target mi-RNA according to the specific current value. The method uses the mi-RNA probe and utilizes a two-dimensional electron gas structure of the GaN semiconductor device to capture the grid potential change generated by the hybridization of the mi-RNA probe and the target mi-RNA; the threshold voltage of the sensor is changed by using the p-type layer structure, the energy consumption of the sensor is reduced, the sensor does not need to be matched with a reference electrode and a counter electrode, and the influence of grid electrification on the measurement accuracy is avoided.
Description
Technical Field
The invention relates to the technical field of mi-RNA detection, in particular to a probe, a GaN sensor and a detection method for detecting mi-RNA.
Background
In recent years, mi-RNA has attracted much attention as an important tumor marker. The detection of the protein can provide effective help for diagnosis and treatment of diseases such as cancer.
Currently, methods for detecting mi-RNA include fluorescence detection, electrochemical detection, polymerase Chain Reaction (PCR), carbon nanotube, and the like. These methods are difficult, expensive, highly dependent on equipment, tedious in testing process, and low in sensitivity.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or problems occurring in the existing mi-RNA detecting methods.
Therefore, the problem to be solved by the present invention is how to provide a probe for detecting mi-RNA for identifying a target mi-RNA.
In order to solve the technical problems, the invention provides the following technical scheme: a probe for detecting mi-RNA has a specific recognition sequence, and can realize base complementary pairing with the target mi-RNA.
It is another object of the present invention to provide a GaN sensor for detecting mi-RNA capable of recognizing a target mi-RNA and causing a change in a channel current of a transistor for subsequent detection.
In order to solve the technical problems, the invention provides the following technical scheme: a GaN sensor for detecting mi-RNA, comprising a mi-RNA probe attached to a gate electrode; the mi-RNA probe can hybridize with the target mi-RNA to generate a change in the gate potential, thereby causing a change in the channel current of the transistor.
As a preferable embodiment of the GaN sensor for detecting mi-RNA according to the present invention, wherein: the solar cell further comprises a substrate, a buffer layer, an intrinsic GaN layer and an intrinsic AlGaN layer which are sequentially arranged from bottom to top, wherein a p-type layer, a source electrode and a drain electrode are arranged on the intrinsic AlGaN layer in parallel, and a grid electrode is arranged on the p-type layer.
As a preferable embodiment of the GaN sensor for detecting mi-RNA according to the present invention, wherein: the p-type layer is a p-GaN or p-NiO layer.
As a preferable embodiment of the GaN sensor for detecting mi-RNA according to the present invention, wherein: the substrate is made of one of Si, siC and sapphire; the source electrode, the drain electrode and the grid electrode are all one or a combination of more of titanium, aluminum, nickel, gold, platinum, molybdenum, iridium, tantalum, niobium, cobalt and tungsten.
As a preferable embodiment of the GaN sensor for detecting mi-RNA according to the present invention, wherein: the thickness of the buffer layer is 1-10000 nm; the thickness of the intrinsic GaN layer is 1-10000 nm; the thickness of the intrinsic AlGaN layer is 3-50 nm; the thickness of the p-type layer is 1-1000 nm.
As a preferable embodiment of the GaN sensor for detecting mi-RNA according to the present invention, wherein: the grid electrode is provided with a Jin Shan layer with the thickness of 1-1000 nm, and the mi-RNA probe is arranged on the gold single layer.
Another object of the present invention is to provide a method for detecting mi-RNA, which can better identify the target mi-RNA and determine the content thereof, and solve the problems of low repeatability and insufficient linear range in the prior art.
In order to solve the technical problems, the invention provides the following technical scheme: a method for detecting mi-RNA, comprising the steps of preparing the GaN sensor according to any one of claims 2 to 7; setting source electrode and drain electrode voltage according to the electrical parameters of the GaN sensor; buffer solutions with different target mi-RNA concentrations are prepared, the GaN sensor is inserted into the buffer solution for testing, and a standard curve is determined; inserting the GaN sensor into the solution to be detected, comparing the output current of the solution to be detected with the output current of the standard curve, judging whether the target mi-RNA exists in the solution to be detected according to the current of the GaN sensor and the current of the standard curve, and obtaining the concentration of the target mi-RNA according to the specific current value.
As a preferable embodiment of the method for detecting mi-RNA of the present invention, there is provided a method in which: the preparation method of the GaN sensor comprises the following steps of sequentially growing a buffer layer, a GaN layer, an intrinsic AlGaN layer and a p-type layer on a substrate to form an initial device; dripping photoresist on the upper surface of the initial device, removing part of the P-type layer area by using a photoetching plate for development, and etching by using ICP equipment to form an intermediate device; dripping photoresist on the upper surface of the intermediate device, developing an ohmic electrode area by using a photoetching plate, forming a source electrode and a drain electrode in the ohmic electrode area by using an evaporation coating method, removing the photoresist, and performing rapid thermal annealing to form ohmic contact to form a final device; dripping photoresist on the surface of the final device, selecting a photoetching plate to develop a grid electrode area, growing the grid electrodes in sequence from bottom to top by using an evaporation coating method, and then removing the photoresist to form a primary sensor; packaging the preliminary sensor, coating an isolation layer outside the preliminary sensor, and isolating the source electrode, the drain electrode and the external environment; before the probe is fixed, titrating a probe mi-RNA solution containing sulfhydryl modification in a sensing area by using a prepared buffer solution, incubating in a constant temperature box, forming a stable Au-S bond between gold on the surface of a grid sensing area and sulfhydryl on the probe due to Au-S covalent bonding, forming a self-assembly layer of the mi-RNA probe on the surface, and finally washing off redundant probes by using deionized water to obtain the GaN sensor with the grid attached with the mi-RNA probe.
As a preferable embodiment of the method for detecting mi-RNA of the present invention, there is provided a method in which: the buffer solution for mi-RNA is phosphate buffer solution or hydroxyethyl piperazine ethanethiosulfonic acid buffer solution.
The invention has the beneficial effects that: capturing a grid potential change generated by the hybridization of the mi-RNA probe and the target mi-RNA by using the mi-RNA probe and utilizing a two-dimensional electron gas structure of the GaN semiconductor device; the threshold voltage of the sensor is changed by using the p-type layer structure, the energy consumption of the sensor is reduced, the sensor does not need to be matched with a reference electrode and a counter electrode, and the influence of grid electrification on the measurement accuracy is avoided.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
fig. 1 is a schematic structural view for a GaN sensor.
FIG. 2 is a graph showing the results of testing the GaN sensors of example 5, example 6, and example 7 in mi-RNA solutions of different concentrations.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein, and it will be appreciated by those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the present invention and that the present invention is not limited by the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
For the first embodiment of the present invention, which provides a probe for detecting mi-RNA, the mi-RNA probe 101 has a specific recognition sequence that enables base complementary pairing with the target mi-RNA.
In this example, the mi-RNA of interest was selected as mi-RNA-155 and the mi-RNA probe 101 was selected as mi-RNA-155 single-stranded probe. The sequence is 5'-SH-ACC-CCU-AUC-ACG-AUU-AGC-AUU-AA-FAM-3', and can realize base complementary pairing with the target mi-RNA-155.
Example 2
This embodiment is based on the first embodiment, and differs from the first embodiment in that the target mi-RNA is selected to be mi-RNA-21.
In this example, the mi-RNA probe 101 is selected to be a mi-RNA-21 single-stranded probe having a sequence of 5'-SH-AUC-GAA-UAG-UCU-GAC-UAC-AAC-U-FAM-3' capable of base complementary pairing with the target mi-RNA-21.
Example 3
Referring to fig. 1, a third embodiment of the present invention provides a GaN sensor for detecting mi-RNA, which includes a mi-RNA probe 101 attached to a gate electrode 102; the mi-RNA probe 101 can hybridize with the target mi-RNA to produce a change in the potential of the gate 102, thereby causing a change in the transistor channel current.
Further, the GaN sensor further comprises a substrate 103, a buffer layer 104, an intrinsic GaN layer 105 and an intrinsic AlGaN layer 106 which are sequentially arranged from bottom to top, wherein a p-type layer 107, a source 108 and a drain 109 are arranged on the intrinsic AlGaN layer 106 in parallel, and a gate 102 is arranged on the p-type layer 107.
The GaN sensor in this embodiment is of a GaN/AlGaN structure, and a high-concentration two-dimensional electron gas layer is generated between a GaN layer (channel layer) and an AlGaN layer (barrier layer) based on polarization characteristics of a GaN material. The electron concentration of the two-dimensional electron gas layer is sensitive to grid potential abnormity, and the structure can be used for capturing weak charge changes before and after hybridization of a mi-RNA probe and mi-RNA in a grid region, so that high sensitivity and low detection limit are obtained. According to the invention, the p-type layer is directly grown on the AlGaN layer, so that the threshold voltage of the sensor is changed, the energy consumption of the sensor is reduced, the sensor does not need to be matched with a reference electrode and a counter electrode, a test system is simplified, the power consumption is reduced, the problem that mi-RNA generates electrochemical reaction under the condition of large gate voltage due to the fact that voltage is increased in a solution during testing is avoided, and the test accuracy is improved.
the-SH-RNA probe molecules are fixed on an Au layer on the surface of the AlGaN/GaN heterojunction field effect transistor by utilizing a strong covalent bond between gold and sulfur bonds, potential signals generated by hybridization and combination of the probe molecules and RNA to be detected are converted into simple current output signals, and a two-dimensional electron gas structure of a GaN semiconductor device is utilized for capturing, so that the concentration of the mi-RNA molecules can be detected by only the change of current, and high sensitivity and low detection limit are obtained.
Preferably, the p-type layer is a p-GaN or p-NiO layer, the substrate 103 is made of one of Si, siC and sapphire, the source electrode 108, the drain electrode 109 and the gate electrode 102 are made of one or more combinations of titanium, aluminum, nickel, gold, platinum, molybdenum, iridium, tantalum, niobium, cobalt and tungsten, and the buffer layer 104 has a thickness of 1 to 10000nm; the thickness of the intrinsic GaN layer 105 is 1-10000 nm; the thickness of the intrinsic AlGaN layer 106 is 3-50 nm; the thickness of the p-type layer 107 is 1 to 1000nm.
In a preferred embodiment, the gate 102 is provided with a Jin Shan layer with a thickness of 1-1000 nm, and the mi-RNA probe 101 is provided on a gold single layer.
In this example, the mi-RNA probe 101 is mi-RNA-155, and the mi-RNA-155 single-stranded probe hybridizes with the complementary pair of mi-RNA-155 to form a new working electrode.
Example 4
This embodiment is based on the previous embodiment, and differs from the previous embodiment in that the mi-RNA probe 101 in this embodiment is mi-RNA-21.
The mi-RNA-21 single-stranded probe and the complementary pair of mi-RNA-21 hybridize to form a new working electrode.
Example 5
Referring to FIG. 2, in a 5 th embodiment of the present invention, there is provided a mi-RNA detection method, including the steps of:
s1, preparing a required GaN sensor, which specifically comprises the following steps,
(1) Growing a buffer layer, a GaN layer, an intrinsic AlGaN layer and a p-type NiO layer on a SiC substrate in sequence by using an MOCVD method to form an initial device;
(2) Dripping photoresist on the upper surface of the initial device, selecting a photoetching plate for developing to remove part of the P-type NiO layer area, and etching by using an ICP (inductively coupled plasma) device to form an intermediate device;
(3) Dripping photoresist on the upper surface of the intermediate device, developing an ohmic electrode area by using a photoetching plate, sequentially growing a Ti layer, an Al layer, a Ti layer and an Au layer on the ohmic electrode area from bottom to top by using an evaporation coating method to form a source electrode and a drain electrode, removing the photoresist, and performing rapid thermal annealing to form ohmic contact between Ti metal and AlGaN layer to form a final device;
(4) Dripping photoresist on the surface of the final device, selecting a photoetching plate to develop a grid electrode area, growing a Ni/Au grid electrode from bottom to top in sequence by using an evaporation coating method, wherein the thickness of the Ni layer is 50nm, the thickness of the Au layer is 100nm, and the area of the grid electrode is 800 microns multiplied by 800 microns, and then removing the photoresist to form a primary sensor;
(5) Packaging the preliminary sensor, coating an isolation layer outside the preliminary sensor, isolating a source electrode, a drain electrode and an external environment, and exposing a grid electrode area to 600 micrometers multiplied by 600 micrometers;
(6) Before the probe is fixed, diluting a probe mi-RNA-155 solution containing sulfhydryl modification to 2 mu M by using a prepared PBS solution, titrating 10 mu L of the probe solution into a sensing area each time, incubating for 2 hours in a 37 ℃ thermostat, forming a stable Au-S bond between gold on the surface of a grid sensing area and sulfhydryl on the probe due to Au-S covalent bonding, forming a self-assembly layer of the mi-RNA-155 probe on the surface, and finally washing away redundant probes by using deionized water to obtain the GaN sensor with the mi-RNA-155 probe attached to the grid.
And S2, setting source electrode voltage and drain electrode voltage according to electrical parameters of the GaN sensor, wherein the source electrode is grounded, and the drain electrode voltage is set to be 5V.
S3, the solution to be detected is a mixed solution of mi-RNA-155 solution and Phosphate Buffer Solution (PBS); configuring mi-RNA-155 buffer solutions with different concentrations and the same pH value as the solution to be tested, inserting the GaN sensor prepared in the step S1 into the buffer solution for testing, and determining a standard curve after the output current is stable, as shown in FIG. 2, it needs to be noted that the buffer solution is phosphate buffer solution.
S4, inserting the GaN sensor into the solution to be detected, comparing the output current of the solution to be detected with the output current of the standard curve, judging whether the target mi-RNA exists in the solution to be detected according to the current of the GaN sensor and the current of the standard curve, and obtaining the concentration of the target mi-RNA according to the specific current value.
Example 6
Referring to fig. 2, this embodiment is based on the previous embodiment, and is different from the previous embodiment in that the mi-RNA probe 101 on the GaN sensor in this embodiment is mi-RNA-21, and the solution to be measured is a mixed solution of mi-RNA-21 solution and Phosphate Buffered Saline (PBS).
Example 7
Referring to FIG. 2, and in order to verify the effectiveness of the present invention, this example is a control test, in which a GaN sensor with mi-RNA-155 probe is used to detect the target mi-RNA-21, and the detection results are shown in FIG. 2. As can be seen from the output curves of example 5 and example 6 in FIG. 2, different current values correspond to different target mi-RNA concentrations; furthermore, the output current tends to decrease with the increase of the target mi-RNA concentration, which indicates that the mi-RNA probe 101 on the GaN sensor surface gradually changes from strong polarity to weak polarity as the reaction proceeds. As can be seen from the output curve of example 7, when the mi-RNA probe 101 has a recognition sequence that does not correspond to the target mi-RNA, no current change occurs.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A probe for detecting mi-RNA, characterized in that: the mi-RNA probe (101) has a specific recognition sequence and can realize base complementary pairing with the target mi-RNA.
2. A GaN sensor for detecting mi-RNA, characterized in that: comprises a mi-RNA probe (101) attached to a grid (102);
the mi-RNA probe (101) can hybridize with the target mi-RNA to generate the change of the potential of the grid electrode (102), thereby causing the change of the channel current of the transistor.
3. The GaN sensor for detecting mi-RNA according to claim 2, wherein: the GaN-based light-emitting diode comprises a substrate (103), a buffer layer (104), an intrinsic GaN layer (105) and an intrinsic AlGaN layer (106) which are sequentially arranged from bottom to top, wherein a p-type layer (107), a source electrode (108) and a drain electrode (109) are arranged on the intrinsic AlGaN layer (106) in parallel, and a grid electrode (102) is arranged on the p-type layer (107).
4. The GaN sensor for detecting mi-RNA according to claim 3, wherein: the p-type layer is a p-GaN or p-NiO layer.
5. The GaN sensor for detecting mi-RNA according to claim 3, wherein: the material of the substrate (103) is one of Si, siC and sapphire;
the source electrode (108), the drain electrode (109) and the grid electrode (102) are all one or a combination of more of titanium, aluminum, nickel, gold, platinum, molybdenum, iridium, tantalum, niobium, cobalt and tungsten.
6. The GaN sensor for detecting mi-RNA according to claim 3, wherein: the thickness of the buffer layer (104) is 1-10000 nm; the thickness of the intrinsic GaN layer (105) is 1-10000 nm; the thickness of the intrinsic AlGaN layer (106) is 3-50 nm; the thickness of the p-type layer (107) is 1-1000 nm.
7. The GaN sensor for detecting mi-RNA according to claim 3, wherein: the grid electrode (102) is provided with a Jin Shan layer, the thickness of the layer is 1-1000 nm, and the mi-RNA probe (101) is arranged on the gold single layer.
8. A method for detecting mi-RNA, comprising: comprises the following steps of (a) carrying out,
preparing a GaN sensor according to any of claims 2 to 7;
setting source electrode and drain electrode voltage according to the electrical parameters of the GaN sensor;
buffer solutions with different target mi-RNA concentrations are prepared, the GaN sensor is inserted into the buffer solution for testing, and a standard curve is determined;
inserting the GaN sensor into the solution to be detected, comparing the output current of the solution to be detected with the output current of the standard curve, judging whether the target mi-RNA exists in the solution to be detected according to the current of the GaN sensor and the current of the standard curve, and obtaining the concentration of the target mi-RNA according to the specific current value.
9. The method for detecting mi-RNA according to claim 8, wherein: the fabrication of the GaN sensor includes the steps of,
sequentially growing a buffer layer, a GaN layer, an intrinsic AlGaN layer and a p-type layer on a substrate to form an initial device;
dripping photoresist on the upper surface of the initial device, selecting a photoetching plate for developing and removing part of the P-type layer area, and etching by using an ICP (inductively coupled plasma) device to form an intermediate device;
dripping photoresist on the upper surface of the intermediate device, developing an ohmic electrode area by using a photoetching plate, forming a source electrode and a drain electrode in the ohmic electrode area by using an evaporation coating method, removing the photoresist, and performing rapid thermal annealing to form ohmic contact to form a final device;
dripping photoresist on the surface of the final device, selecting a photoetching plate to develop a grid electrode area, growing the grid electrodes from bottom to top in sequence by using an evaporation coating method, and then removing the photoresist to form a primary sensor;
packaging the preliminary sensor, coating an isolation layer outside the preliminary sensor, and isolating the source electrode, the drain electrode and the external environment;
before the probe is fixed, titrating a probe mi-RNA solution containing sulfhydryl modification in a sensing area by using a prepared buffer solution, incubating in a constant temperature box, forming a stable Au-S bond between gold on the surface of a grid sensing area and sulfhydryl on the probe due to Au-S covalent bonding, forming a self-assembly layer of the mi-RNA probe on the surface, and finally washing off redundant probes by using deionized water to obtain the GaN sensor with the grid attached with the mi-RNA probe.
10. The method for detecting mi-RNA according to claim 8 or 9, wherein: the buffer solution for the mi-RNA is phosphate buffer solution or hydroxyethyl piperazine ethanethiosulfonic acid buffer solution.
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| CN107064260A (en) * | 2017-04-28 | 2017-08-18 | 中国科学院半导体研究所 | Nucleic acid aptamer sensor for tumor-marker analyte detection and preparation method thereof |
| CN112881485A (en) * | 2021-01-14 | 2021-06-01 | 西安电子科技大学 | GaN sensor for detecting hypochlorite and detection method |
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| CN107064260A (en) * | 2017-04-28 | 2017-08-18 | 中国科学院半导体研究所 | Nucleic acid aptamer sensor for tumor-marker analyte detection and preparation method thereof |
| CN112881485A (en) * | 2021-01-14 | 2021-06-01 | 西安电子科技大学 | GaN sensor for detecting hypochlorite and detection method |
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