CN111579541A - Self-calibration dual-signal biosensor and application thereof in miRNA detection - Google Patents

Self-calibration dual-signal biosensor and application thereof in miRNA detection Download PDF

Info

Publication number
CN111579541A
CN111579541A CN202010430048.1A CN202010430048A CN111579541A CN 111579541 A CN111579541 A CN 111579541A CN 202010430048 A CN202010430048 A CN 202010430048A CN 111579541 A CN111579541 A CN 111579541A
Authority
CN
China
Prior art keywords
mirna
self
solution
signal
biosensor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010430048.1A
Other languages
Chinese (zh)
Other versions
CN111579541B (en
Inventor
张丛丛
刘宏
孙铭远
王建
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Jinan
Original Assignee
University of Jinan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Jinan filed Critical University of Jinan
Priority to CN202010430048.1A priority Critical patent/CN111579541B/en
Publication of CN111579541A publication Critical patent/CN111579541A/en
Application granted granted Critical
Publication of CN111579541B publication Critical patent/CN111579541B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/4163—Systems checking the operation of, or calibrating, the measuring apparatus

Landscapes

  • Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Electrochemistry (AREA)
  • Mathematical Physics (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Theoretical Computer Science (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a self-calibration dual-signal biosensor and application thereof in miRNA detection, and belongs to the technical field of biosensors. In the invention, DNA modified by a fluorescent group is fixed on a full covalent bond graphene field effect transistor and is used as a sensitive probe for detecting miRNA. The stability of the device in the solution phase for a plurality of subsequent processing is realized by utilizing the characteristic of covalent bond connection between the layer transition structures; by means of the full-covalent-bond graphene field effect transistor and a fluorescence technology, simultaneous detection of double signals of miRNA is achieved, and a self-calibration function is achieved. Compared with the existing miRNA detection technology, the self-calibration biosensor provided by the invention can meet the detection requirements of stability, reliability, high sensitivity, high selectivity, rapidness, convenience and the like when used for detecting miRNA, and provides a new idea for miRNA detection.

Description

Self-calibration dual-signal biosensor and application thereof in miRNA detection
Technical Field
The invention relates to the technical field of biosensors, in particular to a self-calibration dual-signal biosensor and application thereof in miRNA detection.
Background
Micrornas (mirnas) are a class of small non-coding RNAs of about 19 to 23 nucleotides in length that inhibit translation of a corresponding protein by binding to the messenger RNA (mrna) of a target gene. Recent research results show that miRNA can exist in serum and plasma of mammals stably, so the miRNA can be used as a novel disease marker to be applied to aspects of early diagnosis of diseases, indication of individualized treatment and the like. At present, common miRNA detection methods include northern blotting analysis, real-time quantitative polymerase chain reaction and microarray technology, but the methods have some technical limitations, such as semi-quantitative data, complex operation, poor repeatability, poor sensitivity and specificity and the like.
Therefore, the development of a reliable miRNA biosensor with simple operation, high sensitivity and high selectivity has important significance for early diagnosis of diseases, and is an important proposition and challenge to be solved urgently.
Disclosure of Invention
In view of the above prior art, the present invention aims to provide a self-calibration dual-signal biosensor and its application in miRNA detection. The biosensor is characterized in that DNA modified by fluorescent groups is fixed on a full covalent bond graphene field effect transistor and is used as a sensitive probe for detecting miRNA. The stability of the device in the solution phase for a plurality of subsequent processing is realized by utilizing the characteristic of covalent bond connection between the layer transition structures; by means of the full-covalent-bond graphene field effect transistor and a fluorescence technology, simultaneous detection of double signals of miRNA is achieved, and a self-calibration function is achieved.
The invention provides a preparation method of a self-calibration dual-signal biosensor, which comprises the following steps:
(1) carrying out hydroxylation on graphene oxide on Si/SiO through a silane coupling agent2Carrying out layer-by-layer self-assembly on the substrate, and annealing and reducing for 8-10 h at 220-250 ℃ to obtain an RGO film; patterning the RGO film by depositing an aluminum film on the RGO film using a copper mesh template, removing the RGO film not protected by the aluminum film with oxygen plasma, and obtaining an RGO array as source/drain electrodes of the FET; continuing to deposit graphene oxide on and around the source/drain electrode of the FET2Self-assembling layer by layer on the substrate, and annealing and reducing for 6-8 h at 150-180 ℃ to be used as a semiconductor layer of the FET, therebyConstructing a full covalent bond graphene field effect transistor;
(2) soaking the full-covalent-bond graphene field effect transistor constructed in the step (1) in a fluorescein-modified DNA solution, and incubating for 30-60 min; and (3) washing after incubation, soaking in a closed solution for reaction for 1-2h, washing after reaction, and drying to obtain the self-calibration dual-signal biosensor.
Preferably, in step (1), Si/SiO2The hydroxylation treatment of the substrate is specifically as follows:
sequentially cleaning Si/SiO with deionized water and isopropanol2Substrate, Si/SiO cleaned2The substrate was immersed in piranha solution at 100 c for 8 minutes for hydroxylation.
More preferably, the piranha solution is prepared from concentrated sulfuric acid and H2O2Prepared according to the volume ratio of 7: 3.
Preferably, in step (1), the length of the copper mesh template channel is: 205 μm, channel width: 26 microns.
Preferably, in the step (1), the parameter conditions for removing the RGO film which is not protected by the aluminum film by the oxygen plasma are as follows: the time is 10min, the radio frequency power is 100W, and the flow rate is 20 sccm.
Preferably, in step (1), the RGO film has a thickness of 6-8 nm; the thickness of the semiconductor layer is 6-8 nm.
Preferably, in step (2), the fluorescein is 6-carboxyfluorescein (FAM).
Preferably, in step (2), the nucleotide sequence of the fluorescein-modified DNA is 5'-FAM-CCA TCT TTA CCAGAC AGT GTT A-3'.
Preferably, in the step (2), the concentration of the fluorescein-modified DNA solution is 1 × 10-6～1×10-5mol/L。
Preferably, in step (2), the blocking solution is 100-500mM ethanolamine blocking solution.
In a second aspect of the present invention, there is provided a self-calibrating dual-signal biosensor prepared by the above method.
In a third aspect of the invention, the self-calibration dual-signal biosensor is provided for use in the preparation of a device for detecting miRNA.
The fourth aspect of the present invention provides a method for detecting miRNA by using the self-calibration dual-signal biosensor, comprising the following steps:
testing the transfer curve of the self-calibration dual-signal biosensor and obtaining the grid voltage value D of the Dirac point1(ii) a Taking miRNA solutions with a series of concentration gradients, and respectively testing the fluorescence intensity I of the miRNA solutions1(ii) a Then, respectively soaking the self-calibration dual-signal biosensor in miRNA solutions with series concentration gradients, and incubating for 15-60 min;
testing the transfer curve of the self-calibration dual-signal biosensor after soaking and obtaining the grid voltage value D of the Dirac point2(ii) a Calculating Δ VCNP＝D2-D1The differential pressure delta V is used as an electrical signal to sense and establish the Dirac point grid differential pressure delta VCNPLinear equation I with miRNA concentration;
testing the fluorescence intensity I of the miRNA solution after soaking2Calculating Δ I ═ I2-I1Taking the miRNA as a fluorescence signal for sensing, and establishing a linear equation II between the difference value delta I of the fluorescence intensity and the miRNA concentration;
soaking the self-calibration dual-signal biosensor in a solution to be tested, incubating for 15-60min, and testing the Dirac point grid voltage difference delta V of the self-calibration dual-signal biosensor before and after soakingCNPAnd the difference value delta I of the fluorescence intensity of the solution to be detected before and after soaking is substituted into the linear equation I and the linear equation II respectively, so that the electrical signal sensing detection result and the fluorescence signal sensing detection result of the miRNA in the solution to be detected are obtained respectively.
Preferably, the concentration of the miRNA solution of the series of concentration gradients is 1 × 10-6mol/L、1×10- 7mol/L、1×10-8mol/L、1×10-9mol/L、1×10-10mol/L、1×10-11mol/L and 1 × 10-12mol/L；
The solvent of the miRNA solution was DEPC water and rnase inhibitor was added at the time of formulation.
Preferably, the linear equation I is Δ VCNP＝0.67318+0.05207 logC; the linear equation II is Δ I-15761.6678 +1282.9992 logC.
The invention has the beneficial effects that:
compared with the existing miRNA detection technology, the self-calibration dual-signal biosensor provided by the invention can be used for detecting miRNA, can meet the detection requirements of low price, reliability, rapidness, convenience and the like, and provides a new idea for miRNA detection. The self-calibration dual-signal biosensor can achieve simultaneous detection of electrical signals and fluorescence signals, achieves a self-calibration function, reduces false positives, and effectively improves the reliability of detection and the sensitivity and selectivity of sensing.
Drawings
FIG. 1: the invention discloses a schematic sensing mechanism of a self-calibration dual-signal biosensor.
FIG. 2 is a standard curve (a) for electrical signal and a standard curve (b) for fluorescence signal of the self-calibrating dual-signal biosensor according to the present invention.
FIG. 3: the self-calibration dual-signal biosensor provided by the invention has an electrical response (a) and a fluorescence response (b) to different miRNA species.
FIG. 4: the self-calibration dual-signal biosensor provided by the invention is used for detecting a transfer characteristic curve (a) of miRNA in fetal calf serum and a fluorescence spectrum (b) of a miRNA solution.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background art, the existing miRNA detection method has the problems of semi-quantitative data, complex operation, poor repeatability, poor sensitivity and specificity and the like. A nano Field Effect Transistor (FET) biosensor attracts a wide attention in the field of life sciences due to its high degree of miniaturization and integration. Because the nano material has unique physicochemical properties such as surface effect, micro-size effect, quantum effect, macroscopic quantum tunneling effect and the like, the nano FET biosensor has the characteristics of high sensitivity and selectivity, high analysis speed, no mark, simplicity in operation and the like, is used for detecting various biomolecules, and is more and more widely applied to biomedical detection. However, the nano FET sensors developed at present have some problems as follows:
(1) the small size of the nanometer material leads to the difficulty in controllable preparation of the sensing device, and particularly the poor stability of subsequent processing in a solution phase.
(2) When the biomolecule is detected, a single sensing signal is mostly adopted, a calibration process is lacked, and a false positive detection result is easy to appear.
Based on this, the present invention develops a self-calibrating dual-signal biosensor. The biosensor fixes fluorescent group modified DNA on a full covalent bond graphene field effect transistor through pi-pi stacking between aromatic rings of GO and heterocyclic rings of single-chain DNA, and the DNA is used as a sensitive probe for detecting miRNA. The stability of the device in the solution phase for a plurality of subsequent processing is realized by utilizing the characteristic of covalent bond connection between the layer transition structures; through the full-covalent-bond graphene field effect transistor and the fluorescence technology, the simultaneous detection of the double signals of the miRNA is realized, so that the self-calibration function is realized, and the occurrence of false positive detection results is avoided.
In one embodiment of the present invention, a process for preparing the self-calibrating dual-signal biosensor is provided, which comprises the following steps:
(1) sequentially cleaning SiO with deionized water and isopropanol2A silicon wafer with an oxide layer. Next, they are placed in piranha solution (H)2SO4：H2O27: 3) the mixture was immersed at 100 ℃ for 8 minutes for hydroxylation.
(2) The hydroxylated silicon wafer was immersed in a 3-Aminopropyltrimethylsilane (APTMS) solution (ethanol: water 95: 5, APTMS 2 vol%) for 30 minutes, rinsed and sonicated in Mill-Q water for three 10 minutes, followed by N2To obtain an aminated substrate. The aminated substrate was immersed in an aqueous Graphene Oxide (GO) solution (0.1mg/ml) for 45 minutes, then the sample was rinsed and sonicated in Mill-Q water for three 15 minutesTreating and using N2Drying to obtain a bilayer (APTMS/GO) membrane. By repeating the above steps 5 times, covalently assembled (APTMS/GO) is obtained5The membrane, where 5 denotes the number of layers of a bilayer (APTMS/GO) membrane, each layer having a thickness of 1.3 nm.
Assembled by the layer-by-layer self-assembly (LBL) method described above (APTMS/GO)5The membrane was vacuum annealed at 240 ℃ for 8 hours to obtain an RGO membrane. The RGO film was patterned by depositing an aluminum film (about 40nm thick) on the assembled RGO film using a copper mesh template (channel length: 205 microns, channel width: 26 microns). The RGO film not protected by the aluminum film was removed by oxygen plasma (time 10min, rf power 100W, flow rate 20 sccm). The sample was immersed in a dilute nitric acid solution (10 vol%) at 50 ℃ for 30 minutes to etch the aluminum film. The RGO array obtained by the above procedure was used as the source/drain electrodes of the FETs. To obtain the FET, growth on the source/drain electrode and the substrate around the source/drain electrode according to the method described above was continued (APTMS/GO)5The film was thermally annealed at 160 deg.C for 6 hours to serve as a semiconductor layer of the FET device. Thus, the full covalent graphene field effect transistor is constructed, and the type of the full covalent graphene field effect transistor is an FET device with a bottom gate and a bottom electrode.
(3) Soaking a full covalent graphene field effect transistor in 600 mu L of a solution with the concentration of 1 × 10-6～1×10-5And (2) incubating the FET in a 6-carboxyfluorescein (FAM) modified DNA (FAM-DNA) solution at room temperature for 30min, washing the surface of the FET with DEPC water to remove redundant DNA sequences, soaking the surface of the FET in 600 mu L of ethanolamine blocking solution with the concentration of 100mM, reacting for 1h, washing with DEPC water, and drying at room temperature of 20-25 ℃ for at least 2s to obtain the self-calibration dual-signal sensor.
In the preparation method of the self-calibration dual-signal biosensor, a substrate (with SiO) is firstly coated2Silicon wafer of oxide layer) is subjected to hydroxylation treatment, so that interface contact is improved, and the bonding performance of the substrate and a graphene oxide film driven to be self-assembled on the substrate through covalent bonds is improved; meanwhile, the stability of the device in the solution phase in multiple subsequent processing is realized by combining and utilizing the characteristic of covalent bond connection between layer-transfer structures of the device.
In order to realize self calibration through double signals, the invention innovatively fixes fluorescent group modified DNA on a semiconductor layer of the constructed full-covalent graphene field effect transistor through pi-pi accumulation between an aromatic ring of GO and a heterocyclic ring of single-chain DNA, the DNA is used as a sensitive probe for detecting miRNA, and a DNA sequence is designed according to the sequence of the detected target miRNA and is complementary with the target miRNA, thereby realizing specific combination.
The sensing mechanism of the self-calibration dual-signal biosensor is shown in figure 1, and as can be seen from the figure, the biosensor can simultaneously realize dual-signal sensing of an electrical signal and a fluorescent signal, and can obtain detection results of two different signal mechanisms through one-time detection, so that self-calibration is performed by using the two signal sensors, false positives are reduced, and the detection reliability is effectively improved.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier. Wherein:
example 1: preparation of self-calibration dual-signal biosensor
(1) Sequentially cleaning SiO with deionized water and isopropanol2A silicon wafer with an oxide layer. Next, they are placed in piranha solution (H)2SO4：H2O27: 3) the mixture was immersed at 100 ℃ for 8 minutes for hydroxylation.
(2) The hydroxylated silicon wafer was immersed in a 3-Aminopropyltrimethylsilane (APTMS) solution (ethanol: water 95: 5, APTMS 2 vol%) for 30 minutes, rinsed and sonicated in Mill-Q water for three 10 minutes, followed by N2To obtain an aminated substrate. The aminated substrate was immersed in an aqueous Graphene Oxide (GO) solution (0.1mg/ml) for 45 minutes, then the sample was rinsed and sonicated in Mill-Q water for three 15 minutes andwith N2Drying to obtain a bilayer (APTMS/GO) membrane. By repeating the above steps 5 times, covalently assembled (APTMS/GO) is obtained5The membrane, where 5 denotes the number of layers of a bilayer (APTMS/GO) membrane, each layer having a thickness of 1.3 nm.
Assembled by the layer-by-layer self-assembly (LBL) method described above (APTMS/GO)5The membrane was vacuum annealed at 240 ℃ for 8 hours to obtain an RGO membrane. The RGO film was patterned by depositing an aluminum film (about 40nm thick) on the assembled RGO film using a copper mesh template (channel length: 205 microns, channel width: 26 microns). The RGO film not protected by the aluminum film was removed by oxygen plasma (time 10min, rf power 100W, flow rate 20 sccm). The sample was immersed in a dilute nitric acid solution (10 vol%) at 50 ℃ for 30 minutes to etch the aluminum film. The RGO array obtained by the above procedure was used as the source/drain electrodes of the FETs. To obtain the FET, growth on the source/drain electrode and the substrate around the source/drain electrode according to the method described above was continued (APTMS/GO)5The film was thermally annealed at 160 deg.C for 6 hours to serve as a semiconductor layer of the FET device. Thus, the full covalent graphene field effect transistor is constructed, and the type of the full covalent graphene field effect transistor is an FET device with a bottom gate and a bottom electrode.
(3) Soaking a full covalent graphene field effect transistor in 600 mu L of a solution with the concentration of 1 × 10-6～1×10-5In mol/L6-carboxyfluorescein (FAM) modified DNA (FAM-DNA) solution, the sequence of the DNA is 5'-FAM-CCA TCT TTACCA GAC AGT GTT A-3'. And (3) incubating for 30min at room temperature, washing the surface of the FET with DEPC water to remove redundant DNA sequences, soaking the surface of the FET in 600 mu L of ethanolamine blocking solution with the concentration of 100mM, reacting for 1h, washing with DEPC water, and drying at room temperature of 20-25 ℃ for at least 2s to prepare the self-calibration dual-signal sensor.
Example 2: performance investigation of self-calibration dual-signal biosensor for detecting miRNA
1. Linear curve and sensitivity:
(1) the self-calibration dual-signal biosensor was prepared as in example 1, the transfer curve of the self-calibration dual-signal biosensor was measured, and the value D of the Dirac point grid voltage was obtained1；
(2) The self-calibration dual-channelSoaking the biosensor in 600 μ L of complementary miRNA (5'-UAA CAC UGU CUG GUA AAG AUG G-3') solutions with different concentrations, incubating at room temperature for 20min, washing with DEPC water to remove excessive miRNA sequences, drying at room temperature of 20 deg.C for at least 2s, testing the transfer curve of the self-calibration dual-signal biosensor, and obtaining the grid voltage value D of Dirac point2；
The solvent of the miRNA solution was DEPC water, and rnase inhibitor was added at the time of formulation so that the final concentration of rnase inhibitor was 10 μ M.
(3) Testing the fluorescence spectrum of the miRNA solution before soaking the self-calibration dual-signal biosensor in the miRNA solution to obtain fluorescence intensity I1And testing the fluorescence spectrum of the miRNA solution after soaking the self-calibration dual-signal biosensor to obtain the fluorescence intensity I2。
(4) Calculating Δ VCNP＝D2-D1And recording the obtained difference Dirac point grid voltage difference as an electrical sensing output signal.
(5) Calculating Δ I ═ I2-I1And recording the obtained difference fluorescence intensity difference delta I as a fluorescence sensing output signal.
(6) Plotting Δ V separatelyCNPAnd a linear curve between Δ I and miRNA concentration (fig. 2), the detection limits of the calculated electrical and fluorescent signals were 2.21 × 10, respectively-13M and 7.10 × 10–12M (signal-to-noise ratio ═ 3).
2. And (3) selectivity:
(1) the self-calibration dual-signal biosensor prepared in example 1 was immersed in 600. mu.L of 1 × 10-6M different kinds of miRNA solution and incubating for 20min, washing with DEPC water to remove excessive miRNA sequence, drying at room temperature of 20 deg.C for at least 2s, testing transfer curve of the FET, and obtaining gate voltage value D of Dirac point2；
The different kinds of miRNA solutions include: miRNA complementary with DNA of the self-calibration dual-signal biosensor, miRNA mismatched with single base, non-complementary miRNA and blank; wherein:
the sequence of the complementary miRNA is: 5'-UAA CAC UGU CUG GUA AAG AUG G-3' are provided.
The sequence of the single base mismatched miRNA is: 5'-UAA CAC UGU CUG GUG AAG AUG G-3' are provided.
The sequence of the non-complementary miRNA is: 5'-CGG ACU GAG AGU UCG GGA CGU U-3' are provided.
Blank is DEPC water.
The solvent of the miRNA solution was DEPC water and rnase inhibitor was added at the time of formulation so that the final concentration of rnase inhibitor was 10 μ M.
Testing the fluorescence spectrum of the miRNA solution before soaking the self-calibration dual-signal biosensor in the miRNA solution to obtain fluorescence intensity I1And testing the fluorescence spectrum of the miRNA solution after soaking the self-calibration dual-signal biosensor to obtain the fluorescence intensity I2。
(2) Calculating Δ VCNP＝D2-D1And recording the obtained difference Dirac point grid voltage difference as an electrical sensing output signal.
(3) Calculating Δ I ═ I2-I1And recording the obtained difference fluorescence intensity difference delta I as a fluorescence sensing output signal.
The result is shown in figure 3, the self-calibration dual-signal biosensor can well distinguish complementary miRNA, miRNA with single base mismatch and non-complementary miRNA, and has good selectivity.
Example 3: serum sample analysis
(1) The self-calibration dual-signal biosensor was prepared as in example 1, the transfer curve of the self-calibration dual-signal biosensor was measured, and the value D of the Dirac point grid voltage was obtained1；
(2) Fetal bovine serum was diluted 20-fold with DEPC water and 6 × 10 was added thereto–10M (5'-UAACAC UGU CUG GUA AAG AUG G-3') to prepare miRNA test solutions.
The rnase inhibitor was added when preparing the miRNA test solution so that the final concentration of the rnase inhibitor was 10 μ M.
(3) Soaking the self-calibration dual-signal biosensor in 600 mu L of miRNA test solution and incubating for 20min, washing with DEPC water to remove redundant miRNA sequences, and drying at room temperature of 20 ℃ for at least 2sThen testing the transfer curve of the FET and obtaining the grid voltage value D of the Dirac point2；
Soaking the self-calibration dual-signal sensor in miRNA test solution before testing the fluorescence spectrum of the miRNA test solution to obtain fluorescence intensity I1And testing the fluorescence spectrum of the miRNA test solution after soaking the self-calibration dual-signal sensor to obtain the fluorescence intensity I2。
(4) Calculating Δ VCNP＝D2-D1And recording the obtained difference Dirac point grid voltage difference as an electrical sensing output signal.
(5) Calculating Δ I ═ I2-I1And recording the obtained difference fluorescence intensity difference delta I as a fluorescence sensing output signal.
(6) Calculating the miRNA concentration in the serum to be 5.74 × 10 through electrical signals–10M, 95.7% recovery, 5.70 × 10% concentration in serum calculated from fluorescence signal–10M, the recovery rate is 95%.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> university of Jinan
<120> self-calibration dual-signal biosensor and application thereof in miRNA detection
<130>2020
<160>4
<170>PatentIn version 3.5
<210>1
<211>22
<212>DNA
<213> Artificial sequence
<400>1
ccatctttac cagacagtgt ta 22
<210>2
<211>22
<212>RNA
<213> Artificial sequence
<400>2
uaacacuguc ugguaaagau gg 22
<210>3
<211>22
<212>RNA
<213> Artificial sequence
<400>3
uaacacuguc uggugaagau gg 22
<210>4
<211>22
<212>RNA
<213> Artificial sequence
<400>4
cggacugaga guucgggacg uu 22

Claims (10)

1. A preparation method of a self-calibration dual-signal biosensor is characterized by comprising the following steps:
(1) carrying out hydroxylation treatment on the graphene oxide on the Si/SiO through a silane coupling agent2Carrying out layer-by-layer self-assembly on the substrate, and annealing and reducing for 8-10 h at 220-250 ℃ to obtain an RGO film; patterning the RGO film by depositing an aluminum film on the RGO film using a copper mesh template, removing the RGO film not protected by the aluminum film with oxygen plasma, and obtaining an RGO array as source/drain electrodes of the FET; continuing to deposit graphene oxide on and around the source/drain electrode of the FET2Carrying out layer-by-layer self-assembly on the substrate, and annealing and reducing for 6-8 h at 150-180 ℃ to serve as a semiconductor layer of the FET, so as to construct a full covalent bond graphene field effect transistor;
(2) soaking the full-covalent-bond graphene field effect transistor constructed in the step (1) in a fluorescein-modified DNA solution, and incubating for 30-60 min; and (3) washing after incubation, soaking in a closed solution for reaction for 1-2h, washing after reaction, and drying to obtain the self-calibration dual-signal biosensor.
2. The method according to claim 1, wherein in the step (1), Si/SiO2The hydroxylation treatment of the substrate is specifically as follows:
sequentially cleaning Si/SiO with deionized water and isopropanol2Substrate, Si/SiO cleaned2The substrate was immersed in piranha solution at 100 c for 8 minutes for hydroxylation.
Preferably, the piranha solution is prepared from concentrated sulfuric acid and H2O2Prepared according to the volume ratio of 7: 3.
3. The preparation method according to claim 1, wherein in step (1), the RGO film has a thickness of 6 to 8 nm; the thickness of the semiconductor layer is 6-8 nm.
4. The method according to claim 1, wherein in the step (2), the nucleotide sequence of the fluorescein-modified DNA is 5'-FAM-CCA TCT TTA CCA GAC AGT GTT A-3';
preferably, the concentration of the fluorescein-modified DNA solution is 1 × 10-6～1×10-5mol/L。
5. The method according to claim 1, wherein the concentration of the blocking solution in the step (2) is 500 mM.
6. A self-calibrating dual signal biosensor prepared by the method of any one of claims 1-5.
7. Use of the self-calibrating dual-signal biosensor of claim 6 in the preparation of a device for detecting miRNA.
8. A method for detecting miRNA using the self-calibrating dual-signal biosensor of claim 6, comprising the steps of:
testing the transfer curve of the self-calibration dual-signal biosensor and obtaining the grid voltage value D of the Dirac point1(ii) a By taking a series of concentration gradientsmiRNA solution, and respectively testing fluorescence intensity I of the miRNA solution1(ii) a Then, respectively soaking the self-calibration dual-signal biosensor in miRNA solutions with series concentration gradients, and incubating for 15-60 min;
testing the transfer curve of the self-calibration dual-signal biosensor after soaking and obtaining the grid voltage value D of the Dirac point2(ii) a Calculating Δ VCNP＝D2-D1The differential pressure delta V is used as an electrical signal to sense and establish the Dirac point grid differential pressure delta VCNPLinear equation I with miRNA concentration;
testing the fluorescence intensity I of the miRNA solution after soaking2Calculating Δ I ═ I2-I1Taking the miRNA as a fluorescence signal for sensing, and establishing a linear equation II between the difference value delta I of the fluorescence intensity and the miRNA concentration;
soaking the self-calibration dual-signal biosensor in a solution to be tested, incubating for 15-60min, and testing the Dirac point grid voltage difference delta V of the self-calibration dual-signal biosensor before and after soakingCNPAnd the difference value delta I of the fluorescence intensity of the solution to be detected before and after soaking is substituted into the linear equation I and the linear equation II respectively, so that the electrical signal sensing detection result and the fluorescence signal sensing detection result of the miRNA in the solution to be detected are obtained respectively.
9. The method of claim 8, wherein the miRNA solutions of the series of concentration gradients are each at a concentration of 1 × 10-6mol/L、1×10-7mol/L、1×10-8mol/L、1×10-9mol/L、1×10-10mol/L、1×10- 11mol/L and 1 × 10-12mol/L；
The solvent of the miRNA solution was DEPC water and rnase inhibitor was added at the time of formulation.
10. The method of claim 8, wherein the linear equation I is avCNP0.67318+0.05207 logC; the linear equation II is Δ I-15761.6678 +1282.9992 logC.
CN202010430048.1A 2020-05-20 2020-05-20 Self-calibration dual-signal biosensor and application thereof in miRNA detection Active CN111579541B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010430048.1A CN111579541B (en) 2020-05-20 2020-05-20 Self-calibration dual-signal biosensor and application thereof in miRNA detection

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010430048.1A CN111579541B (en) 2020-05-20 2020-05-20 Self-calibration dual-signal biosensor and application thereof in miRNA detection

Publications (2)

Publication Number Publication Date
CN111579541A true CN111579541A (en) 2020-08-25
CN111579541B CN111579541B (en) 2022-10-11

Family

ID=72123101

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010430048.1A Active CN111579541B (en) 2020-05-20 2020-05-20 Self-calibration dual-signal biosensor and application thereof in miRNA detection

Country Status (1)

Country Link
CN (1) CN111579541B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105021680A (en) * 2015-06-30 2015-11-04 上海集成电路研发中心有限公司 Graphene sensor based method for detecting MicroRNA
WO2017112941A1 (en) * 2015-12-23 2017-06-29 The Regents Of The University Of California Nano-sensors for nucleic acid detection and discrimination

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105021680A (en) * 2015-06-30 2015-11-04 上海集成电路研发中心有限公司 Graphene sensor based method for detecting MicroRNA
WO2017112941A1 (en) * 2015-12-23 2017-06-29 The Regents Of The University Of California Nano-sensors for nucleic acid detection and discrimination
CN108700535A (en) * 2015-12-23 2018-10-23 加利福尼亚大学董事会 Nano-sensor for detection of nucleic acids and discriminating

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GAO JIANWEI等: ""Ultrasensitive Label-free MiRNA Sensing Based on a Flexible Graphene Field-Effect Transistor without Functionalization"", 《ACS APPLIED ELECTRONIC MATERIALS》 *
尹长青等: "纳米场效应晶体管生物传感器在肿瘤早期检测中的应用", 《武汉大学学报(医学版)》 *
蔡冰洁: ""基于石墨烯场效应晶体管的核酸生物传感器研究"", 《中国优秀硕士学位论文全文数据库 信息科技辑》 *
迟景元等: "DNA-二维纳米片层材料传感平台的构建及其应用", 《化学学报》 *

Also Published As

Publication number Publication date
CN111579541B (en) 2022-10-11

Similar Documents

Publication Publication Date Title
US20230333039A1 (en) An active-electrode integrated biosensor array and methods for use thereof
Kilic et al. microRNA biosensors: Opportunities and challenges among conventional and commercially available techniques
Yuan et al. Ratiometric electrochemical assay for sensitive detecting microRNA based on dual-amplification mechanism of duplex-specific nuclease and hybridization chain reaction
Labib et al. Electrochemical sensing of microRNAs: avenues and paradigms
Taufik et al. Bismuth oxide nanoparticles/chitosan/modified electrode as biosensor for DNA hybridization
Sánchez et al. Multiplex PCB-based electrochemical detection of cancer biomarkers using MLPA-barcode approach
CN109738503B (en) Positive feedback amplification electrochemical sensor based on exonuclease III
CN104807869A (en) Two-dimensional nanomaterial-based biosensor and manufacturing and integration method thereof
CN113008967A (en) Unmarked graphene field effect transistor flexible biosensor and preparation method and application thereof
Zamani et al. Recent advances in gold electrode fabrication for low-resource setting biosensing
Mujica et al. New trends in the development of electrochemical biosensors for the quantification of microRNAs
Cui et al. Catalytic hairpin assembly-based electrochemical biosensor with tandem signal amplification for sensitive microRNA assay
WO2009119972A2 (en) Highly sensitive bio-sensor, biochip including same and method for manufacturing same
CN112986355A (en) Graphene field effect transistor biosensor with double-gate structure and preparation method thereof
Vais et al. A novel and ultrasensitive label-free electrochemical DNA biosensor for Trichomonas vaginalis detection based on a nanostructured film of poly (ortho-aminophenol)
CN114410749B (en) Electrochemiluminescence detection kit and method based on click chemistry and ARGET-ATRP amplification strategy
CN111579541B (en) Self-calibration dual-signal biosensor and application thereof in miRNA detection
Song et al. Detection of microRNA based on three-dimensional graphene field-effect transistor biosensor
Kong et al. Graphene-based liquid gated field-effect transistor for label-free detection of DNA hybridization
CN114199969A (en) Nucleic acid aptamer-based nano-electrode biosensor and application thereof
WO2018056744A1 (en) Nanoplasmonic biosensor and method for detecting disease marker by using same
CN212568599U (en) Flexible biosensor of no mark graphite alkene field effect transistor and wearable equipment
Dickens et al. Label-free detection of synthetic, full genomic length HIV-1 RNA at the few-copy level
CN113130648A (en) Tumor marker sensor based on fin field effect transistor manufacturing process
CN115518701B (en) Preparation and detection method of gold nanoflower microarray chip for miRNA detection

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant