CN112162027B

CN112162027B - Electrochemical sensor based on triblock probe and application of electrochemical sensor in detection of transgenic double-stranded RNA

Info

Publication number: CN112162027B
Application number: CN202010996358.XA
Authority: CN
Inventors: 刘刚; 宋世平; 闻艳丽; 郑宇�; 王乐乐; 李兰英; 杨雪; 许丽; 杨镇州; 梁文; 李妍; 徐勤; 罗超
Original assignee: Shanghai Institute of Measurement and Testing Technology
Current assignee: Shanghai Institute of Measurement and Testing Technology
Priority date: 2020-09-21
Filing date: 2020-09-21
Publication date: 2023-08-18
Anticipated expiration: 2040-09-21
Also published as: CN112162027A

Abstract

The invention provides an electrochemical sensor based on a triblock probe and application thereof in detecting transgenic double-stranded RNA, wherein the electrochemical sensor comprises a gold electrode and the triblock probe; the triblock probe comprises PolyA and a first capture probe and a second capture probe connected with the PolyA; the first capture probe and the second capture probe comprise a nucleic acid sequence complementary to a transgenic double-stranded RNA; the triblock probe is modified on the surface of the gold electrode through the adsorption of the PolyA and the gold electrode. The electrochemical sensor surface of the invention is modified with the triblock probe, thereby realizing specific, sensitive and stable transgene double-stranded RNA detection, and having the advantages of high sensitivity, short time consumption, low cost, portability and the like.

Description

Electrochemical sensor based on triblock probe and application of electrochemical sensor in detection of transgenic double-stranded RNA

Technical Field

The invention belongs to the technical field of biological detection, relates to an electrochemical sensor based on a triblock probe and application thereof, and particularly relates to an electrochemical sensor based on a triblock probe and application thereof in detecting transgenic double-stranded RNA.

Background

Currently, commercial transgenic crops contain protein-encoding properties, and the expression level of the protein is quantitatively analyzed by immunological methods or LC-MS/MS. The transgenic crops have insect pest resistance besides protein coding property, and the transgenic insect pest resistance character developed based on RNAi technology has become a powerful means for controlling insect pests. Since RNA is an active molecule of RNAi, accurate quantification of RNA is critical to quality monitoring of downstream transgenic foods and feeds during trait development in transgenic crops.

Transgenic crops constructed based on RNAi technology will produce RNA molecules in the form of "hairpin" with stem-loop structures linking double stranded RNA on both sides. It is reported in literature that when the double-stranded RNA region exceeds 60bp, the double-stranded RNA has an insect-resistant effect, and the RNA of the type is easy to form a strong secondary structure, thus causing a great obstacle for accurately and quantitatively detecting RNA molecules.

The RT-PCR has the characteristics of good accuracy and high precision, and is a main method for quantitatively detecting RNA molecules. However, the specificity of RT-PCR is weaker due to the inverted repeat structure of the transgenic double-stranded RNA; in addition, RT-PCR requires expensive laboratory equipment, and can only be used in a laboratory environment, with certain limitations.

Therefore, there is a need to develop a more accurate and reliable quantitative analysis tool for RNAi properties.

Disclosure of Invention

Aiming at the defects and actual demands of the prior art, the invention provides an electrochemical sensor based on a triblock probe and application thereof in detecting transgenic double-stranded RNA, wherein the electrochemical sensor has the advantages of high sensitivity, short time consumption, low cost, portability and the like, and is used for detecting 1000nt double-stranded RNA molecules with complex secondary structures.

To achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an electrochemical sensor based on a triblock probe, the electrochemical sensor comprising a gold electrode and a triblock probe;

the triblock probe comprises PolyA and a first capture probe and a second capture probe connected with the PolyA;

the first capture probe and the second capture probe comprise a nucleic acid sequence complementary to a transgenic double-stranded RNA;

the triblock probe is modified on the surface of the gold electrode through the adsorption of the PolyA and the gold electrode.

In the invention, the triblock probe is adopted as the capture probe of the electrochemical sensor, the target transgenic double-stranded RNA is captured by adsorbing the triblock probe on the surface of the gold electrode through the PolyA, and the first capture probe and the second capture probe are combined with the stacking function of bases, so that the capture capability of the electrochemical sensor on the transgenic double-stranded RNA is remarkably improved, and the stable, sensitive and specific transgenic double-stranded RNA detection is realized.

Preferably, a spacer arm is also included between the polyA and the first and/or second capture probes.

In the invention, the spacer arm is arranged between the polyA and the first capture probe and/or the second capture probe, so that the tri-block probe adsorbed on the gold electrode is not affected by steric hindrance of the gold electrode when being combined with the transgenic double-stranded RNA, thereby being beneficial to improving the detection sensitivity.

Preferably, the triblock probe comprises a first capture probe, a first spacer arm, polyA, a second spacer arm and a second capture probe.

Preferably, the first and second capture probes bind to the same and/or different transgenic double stranded RNAs.

In a second aspect, the invention provides a kit for detecting transgenic double stranded RNA, the kit comprising an electrochemical sensor according to the first aspect.

Preferably, the kit further comprises a detection probe, wherein the detection probe comprises a nucleic acid sequence complementary to the transgenic double-stranded RNA, and biotin is marked at the 3' end.

Preferably, the kit further comprises a spacer probe, wherein the spacer probe comprises a nucleic acid sequence complementary to the transgenic double-stranded RNA, and is used for reducing interference among different detection probes and improving detection sensitivity.

Preferably, the kit further comprises dimethyl sulfoxide, avidin labeled HRP, TMB or H ₂ O ₂ Any one or a combination of at least two of these.

In the invention, dimethyl sulfoxide is used for opening the strong secondary structure of the transgenic double-stranded RNA, preventing the secondary structure of the double-stranded RNA from being formed again, and obviously improving the detection sensitivity.

Preferably, the triblock probe on the electrochemical sensor comprises the nucleic acid sequences shown in SEQ ID NOs 1-3.

Preferably, the detection probe comprises the nucleic acid sequence shown in SEQ ID NOS.4-20.

Preferably, the spacer probe comprises the nucleic acid sequences shown in SEQ ID NOS.21 to 22.

In a third aspect, the present invention provides a method for detecting a transgenic double-stranded RNA using the electrochemical sensor of the first aspect and/or the kit of the second aspect.

Preferably, the detection method comprises the steps of:

(1) Incubating the target transgenic double-stranded RNA, the detection probe and the interval probe together, and then placing the incubated double-stranded RNA, the detection probe and the interval probe on ice;

(2) Dripping the modified product of the transgenic double-stranded RNA combined with the detection probe and the interval probe on the surface of the electrochemical sensor to perform hybridization reaction, and forming a sandwich structure on the gold electrode;

(3) Adding an avidin-labeled HRP, and incubating;

(4) Addition of TMB and H ₂ O ₂ Electrochemical detection is performed.

Preferably, the temperature of the co-incubation in step (1) is 85 to 95℃and may be, for example, 85℃86℃87℃88℃89℃90℃91℃92℃93℃94℃or 95℃and preferably 90 ℃.

Preferably, the incubation time in step (1) is 5 to 20min, for example, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min, preferably 10min.

Preferably, the time of placing on ice in the step (1) is 5-20 min, for example, may be 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min, preferably 20min.

Preferably, dimethyl sulfoxide is added to the co-incubation system in step (1) to help open the secondary structure of the double-stranded RNA and prevent the secondary structure of the transgenic double-stranded RNA from being formed again.

Preferably, the hybridization reaction in step (2) is carried out at a temperature of 40 to 60℃and may be carried out at 40℃41℃42℃43℃44℃45℃46℃47℃48℃49℃50℃51℃52℃53℃54℃55℃56℃57℃58℃59℃59℃60℃60℃or preferably 50 ℃.

Preferably, the hybridization reaction time in the step (2) is 40-60 min, for example, 40min, 41min, 42min, 43min, 44min, 45min, 46min, 47min, 48min, 49min, 50min, 51min, 52min, 53min, 54min, 55min, 56min, 57min, 58min, 59min or 60min, preferably 60min.

Preferably, the incubation time in the step (3) is 10 to 30min, for example, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min or 30min, preferably 20min.

Preferably, the electrochemical detection in step (4) employs cyclic voltammetry.

Preferably, the cyclic voltammetry has a sweep rate of 80 to 100mV/s, which may be, for example, 80mV/s, 81mV/s, 82mV/s, 83mV/s, 84mV/s, 85mV/s, 86mV/s, 87mV/s, 88mV/s, 89mV/s, 90mV/s, 91mV/s, 92mV/s, 93mV/s, 94mV/s, 95mV/s, 96mV/s, 97mV/s, 98mV/s, 99mV/s or 100mV/s, preferably 100mV/s.

Preferably, the sweep voltage of the cyclic voltammetry is 0 to 0.7V.

In a fourth aspect, the invention provides the use of an electrochemical sensor according to the first aspect and/or a kit according to the second aspect for the preparation of an RNAi-based transgenic crop toxicity detection reagent.

Compared with the prior art, the invention has the following beneficial effects:

(1) The surface of the electrochemical sensor is modified with the triblock probe, and two sections of capture probes of the triblock probe capture the transgenic double-stranded RNA simultaneously, and the specific, sensitive and stable transgenic double-stranded RNA detection is realized by combining the stacking effect of bases;

(2) According to the invention, a plurality of detection probes and interval probes are added into a detection system, so that detection signals are amplified, and the signal-to-noise ratio is improved;

(3) The electrochemical sensor has low detection cost and diversified functions, and has wide application prospect in the field of RNAi transgenic double-stranded RNA detection.

Drawings

FIG. 1 is a schematic diagram of the electrochemical detection principle;

FIG. 2A is a data graph of cyclic voltammetry for 10nM transgenic double stranded RNA and blank, and FIG. 2B is a data graph of chronoamperometry for 10nM transgenic double stranded RNA and blank;

FIG. 3A shows the results of optimizing the concentration of the triblock probe, FIG. 3B shows the results of optimizing the assembly temperature of the triblock probe, FIG. 3C shows the results of optimizing the hybridization temperature, and FIG. 3D shows Na in the hybridization buffer ⁺ Concentration optimization results, FIG. 3E shows the Mg in hybridization buffer ²⁺ Concentration optimization results, FIG. 3F shows the results of 0, 10, 20% DMSO concentration optimization at 1nM target concentration, FIG. 3G shows the results of further optimization at 6%, 8%, 10%, 12% DMSO concentration, and FIG. 3H shows the results of 6%, 8%, 10%, 12% DMSO concentration optimization at 100pM target concentration;

FIG. 4 shows the results of actual sample detection.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

TABLE 1 sequence information

Example 1 preparation of transgenic double-stranded RNA

The transgenic double-stranded RNA prepared in this example is used as a detection target of an electrochemical sensor, and comprises the following steps:

(1) Synthesizing a segment of DNA fragment with the length of 852bp and containing 261bp inverted repeat sequence and 98bp spacer sequence, and cloning the fragment into a pET-28a (+) vector with a T7 promoter;

(2) Amplifying a DNA fragment containing target RNA and a T7 promoter and having a length of 1034bp from the recombinant plasmid by using PCR;

(3) After purification, using 1034bp DNA fragment as template, under the action of RNA polymerase, in vitro transcription is carried out for 1 hour at 37 ℃, and target RNA is synthesized;

(4) The synthesized target RNA is purified and placed in-80 ℃ for standby.

Example 2 preparation of electrochemical sensor

Polishing the gold electrode by alumina, and respectively placing the polished gold electrode in ethanol and water for ultrasonic treatment for 2min; then the solution is connected into an electrochemical workstation circuit, and is subjected to electrochemical cleaning by using 0.5M sulfuric acid aqueous solution;

the gold electrode after electrochemical cleaning is washed by water and dried by nitrogen, 3 mu M of triblock probe-1, triblock probe-2 or triblock probe-3 is dripped, and incubated overnight at 45 ℃;

the electrode was blocked with 0.1mM Mercaptoethanol (MCH) at room temperature for 1h to prepare an electrochemical sensor modified with a triblock probe, which was stored at 4℃for use.

Example 3 electrochemical detection

The electrochemical detection principle is shown in figure 1, a triblock probe modified on the surface of a gold electrode is used as a capture probe to carry out specific complementary hybridization with a target transgenic double-stranded RNA, after a biotin labeling detection probe and a spacer probe are combined with the target transgenic double-stranded RNA, HPR is labeled on a sandwich structure of the triblock probe and the target transgenic double-stranded RNA-detection probe by utilizing the strong affinity of biotin-avidin, and TMB or H is added ₂ O ₂ Detection of the transgenic double-stranded RNA is achieved by the electrochemical signal generated.

The method comprises the following steps:

(1) 10nM of the target transgenic double-stranded RNA prepared in example 1 was hybridized with 100nM of biotin-labeled detection probes (SEQ ID NOS: 4-20) and spacer probes (SEQ ID NOS: 21-22) in hybridization buffer (50 mM Na) ₂ HPO ₄ 、50mM NaH ₂ PO ₄ 5M NaCl) and heating at 90 ℃ for 10min for denaturation; subsequently, the mixture was placed on ice and cooled for 20min;

(2) Dropping the denatured solution on the surface of the electrochemical sensor prepared in the example 2, and reacting for 1h at 50 ℃; after cleaning, 5U/mL streptavidin-HRP is dripped, and the mixture is incubated for 20min at room temperature;

(3) After thorough cleaning, the electrochemical sensor is transferred to an electrochemical cell filled with TMB, the electrochemical detection is carried out by a connecting circuit, the scanning speed of the cyclic voltammetry is 100mV/s, the scanning speed is in the range of 0-0.7V, and the current change in 100s at 100mV is recorded by a current time curve test.

FIG. 2A is a cyclic voltammogram at a scan rate of 100mV/s, with electron transfer between TMB and gold, and after hybridization of the electrode to 10nM target double-stranded RNA, HRP-catalyzed electrochemical reaction occurs; the current signal in steady state is shown in fig. 2B.

Example 4 Condition optimization

(1) Optimization of triblock probe concentration

Three-block probes with different concentrations (0.5 mu M, 1 mu M, 2 mu M, 3 mu M and 5 mu M) are dripped on the surface of the pretreated gold electrode, electrochemical sensors with different numbers of three-block probes modified on the surface are prepared, and transgenic double-stranded RNA is detected.

As a result, as shown in FIG. 3A, in the range of 0.5 to 3. Mu.M, the electric signal value increases as the concentration of the triblock probe increases, and when the concentration of the triblock probe is 3. Mu.M, the maximum electric signal value is obtained, and the signal to noise ratio S/N is 82.64 at the maximum, and as the concentration of the triblock probe further increases to 5. Mu.M, both the electric signal value and the signal to noise ratio decrease, probably because the triblock probe on the gold electrode surface is excessively dense, and the binding of the triblock probe to the transgenic double-stranded RNA is affected.

In the subsequent experiments, 3 μm triblock probe-modified gold electrodes were used to prepare electrochemical sensors.

(2) Optimization of assembly temperature for triblock probes

The 3. Mu.M triblock probe was added dropwise to the pretreated gold electrode surface and incubated overnight at 37℃or 45℃to prepare different electrochemical sensors for detection of transgenic double stranded RNA.

As shown in fig. 3B, compared with the incubation temperature of 37 ℃, the electrochemical sensor prepared by using the incubation temperature of 45 ℃ has better electrochemical performance, and the electrical signal value and the signal to noise ratio are high, probably because the electrochemical sensor prepared by using the incubation temperature of 45 ℃ has stronger adsorption binding force between the triblock probe and the gold electrode, and the electrochemical sensor has stable performance.

In a subsequent experiment, 3 μm triblock probes were added dropwise to the pretreated gold electrode surface and incubated overnight at 45 ℃ to prepare an electrochemical sensor.

(3) Optimization of hybridization temperature

The modified product of the transgenic RNA combined with the detection probe and the spacer probe is dripped on the surface of an electrochemical sensor, and hybridized with the triblock probe at 37 ℃, 45 ℃ or 50 ℃ to detect the transgenic double-stranded RNA.

As a result, as shown in FIG. 3C, when the hybridization temperature was 45℃or 50℃the obtained electric signal value was higher than that when the hybridization temperature was 37℃and the signal-to-noise ratio was higher at 50℃than at 45 ℃.

Therefore, in order to improve the detection sensitivity of the electrochemical sensor, the hybridization temperature was set to 50℃in the subsequent experiments.

(4) Na in hybridization buffer ⁺ 、Mg ²⁺ Optimization of concentration

The present example also explores hybridization buffer formulations in order to optimize the detection performance of the electrochemical sensor for transgenic double-stranded RNA.

As shown in FIG. 3D, when Na is contained in the hybridization buffer ⁺ When the concentration of (2) is 5M, the maximum electric signal value and signal to noise ratio can be obtained; as shown in FIG. 3E, when the hybridization buffer does not contain Mg ²⁺ When the maximum electric signal value and signal-to-noise ratio are obtained.

In subsequent experiments, a formulation of 50mM Na was used ₂ HPO ₄ 、50mM NaH ₂ PO ₄ Hybridization reaction was performed with hybridization buffer of 5M NaCl.

(5) Optimization of DMSO concentration

Since the transgenic double-stranded RNA has an inverted repeat sequence inside and has a hairpin structure, the secondary structure of the double-stranded RNA must be opened before hybridization with the triblock probe and the detection probe. In the embodiment, DMSO is added into the system to assist in opening the secondary structure of the double-stranded RNA at high temperature, and prevent the reformation of the secondary structure of the dsRNA, so that the sensitivity of the electrochemical sensor is improved.

When the concentration of the transgenic double-stranded RNA was 1nM, as shown in FIG. 3F, 10% DMSO helps to increase the electrical signal value and decrease the background signal value compared to the control group without DMSO and the experimental group with 20% DMSO; further limiting the amount of DMSO added to a range of 6% to 12%, as shown in fig. 3G, the highest electrical signal value was obtained when 6% DMSO was added to the system, but the highest signal to noise ratio S/n=5.07 was obtained when 8% DMSO was added.

To this end, the concentration of transgenic double stranded RNA was further reduced to 100pM, as shown in FIG. 3H, when 6% DMSO was added to the system, the highest signal to noise ratio was obtained.

Example 5 extraction of double-stranded RNA from transgenic maize

Crushing frozen transgenic corn leaf tissues into powder by using liquid nitrogen in a mortar, and then extracting total RNA by using an RNA extraction kit;

the purity and integrity of the total RNA are evaluated by a biological analyzer, and the concentration is measured by an ultraviolet-visible spectrophotometer;

the total RNA samples extracted were stored at-80℃as actual samples for electrochemical sensors.

As a result, as shown in FIG. 4, the constructed electrochemical sensor can detect double-stranded RNA in transgenic corns 11061-P, 11061-N, 11019-P and 11048-P (supplied by North Biotechnology Co., ltd. In Beijing) under optimal conditions.

EXAMPLE 6 digital PCR quantitative detection

After the extracted total RNA is reversely transcribed into cDNA, the cDNA is diluted to a proper concentration, and digital PCR quantitative detection is carried out to determine the content of the trans-RNA dsRNA in the total RNA sample to be about one ten thousandth.

In conclusion, the surface of the electrochemical sensor is modified with the triblock probe, two sections of capture probes of the triblock probe capture the transgenic double-stranded RNA simultaneously, and the stacking effect of the base is combined, so that the specific, sensitive and stable transgenic double-stranded RNA detection is realized, and the electrochemical sensor has wide application prospect in the RNAi transgenic double-stranded RNA detection field.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

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Claims

1. A kit for detecting transgenic double-stranded RNA, the kit comprising a triblock probe-based electrochemical sensor comprising a gold electrode and a triblock probe;

the triblock probe is modified on the surface of the gold electrode through the adsorption of the PolyA and the gold electrode;

the nucleic acid sequence of the triblock probe on the electrochemical sensor is shown as SEQ ID NO. 1-3.

2. The kit of claim 1, further comprising a detection probe labeled at the 3' end with biotin;

the kit further comprises a spacer probe;

the kit also comprises dimethyl sulfoxide, avidin labeled HRP, TMB or H ₂ O ₂ Any one or a combination of at least two of these.

3. The kit according to claim 2, wherein the detection probe comprises a nucleic acid sequence shown in SEQ ID NO. 4-20;

the interval probe comprises a nucleic acid sequence shown in SEQ ID NO. 21-22.

4. A method for detecting transgenic double-stranded RNA, wherein the method uses the kit of any one of claims 1 to 3 to detect the transgenic double-stranded RNA.

5. The method of detection according to claim 4, characterized in that the method of detection comprises the steps of:

(3) Adding an avidin-labeled HRP, and incubating;

(4) Addition of TMB and H ₂ O ₂ Electrochemical detection is performed.

6. The method according to claim 5, wherein the temperature of the co-incubation in step (1) is 85-95 ℃.

7. The method according to claim 5, wherein the incubation time in step (1) is 5 to 20min.

8. The method according to claim 5, wherein the time for placing on ice in the step (1) is 5 to 20 minutes.

9. The method according to claim 5, wherein dimethyl sulfoxide is added to the co-incubation system in step (1).

10. The method according to claim 5, wherein the hybridization reaction in step (2) is carried out at a temperature of 40 to 60 ℃.

11. The method according to claim 5, wherein the hybridization reaction in step (2) is performed for 40 to 60 minutes.

12. The method according to claim 5, wherein the incubation time in step (3) is 10 to 30 minutes.

13. The method according to claim 5, wherein the electrochemical detection in step (4) is performed by cyclic voltammetry.

14. The method according to claim 13, wherein the cyclic voltammetry has a scanning rate of 80 to 100mV/s.

15. The method according to claim 13, wherein the cyclic voltammetry has a scanning voltage of 0 to 0.7V.

16. Use of the kit of claim 1 for the preparation of an RNAi-based transgenic crop toxicity detection reagent.