CN113866146A

CN113866146A - Construction of graphene oxide-based aptamer sensor, method for detecting fumonisin B1 and application

Info

Publication number: CN113866146A
Application number: CN202111150140.3A
Authority: CN
Inventors: 郭晓东; 张梦柯; 梁双林; 韦阳; 谢明树; 张晓伟
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2021-12-31

Abstract

The invention provides a method for constructing a graphene oxide-based aptamer sensor and detecting fumonisin B1 and application thereof, wherein the construction process comprises the following steps: designing and synthesizing a nucleic acid aptamer fluorescent probe; carrying out fluorescence quenching by utilizing the conjugated adsorption effect of the graphene oxide and the aptamer; binding a target fumonisin B1 with an aptamer, changing the spatial conformation of the aptamer and recovering a fluorescent signal; cutting an aptamer by endonuclease DNase I, releasing fumonisin B1 as a target, diluting and preparing a standard solution, and measuring a fluorescence signal standard curve; pretreating an actual sample to be detected, and determining the content of fumonisin B1 by using a fluorescence aptamer sensor detection platform according to a linear model; the detection method has the advantages of high sensitivity and high specificity, can quickly detect the content of fumonisin B1 in wheat flour, has higher conformity with an ELISA detection kit, and has important practical application value in food safety detection.

Description

Construction of graphene oxide-based aptamer sensor, method for detecting fumonisin B1 and application

Technical Field

The invention belongs to the technical field of rapid detection of fumonisin B1, and particularly relates to construction of an oxidized graphene-based aptamer sensor based on enzyme signal amplification, and a method and application for detecting fumonisin B1(FB 1).

Background

Fumonisins are one of the most toxic mycotoxins, with four subtypes (A, B, C and P), of which FB1 is the most toxic and most prevalent, accounting for about 70%. In 2002, FB1 was recognized as a grade 2B carcinogen by the world health organization International agency for research on cancer. The U.S. Food and Drug Administration (FDA) has defined that the total amount of fumonisins (FB1, FB2, and FB3) in a corn product should not exceed 2mg kg^-1. Therefore, the development of rapid, highly sensitive and highly specific FB1 detection technology is very important to the food safety field.

The traditional FB1 detection technology is mainly based on a detection platform of an instrument method, and comprises a high performance liquid chromatography, a gas chromatography-mass spectrometry tandem method, a high performance liquid chromatography-mass spectrometry tandem method and the like, however, the detection steps of the method are complex, the requirement on sample processing is high, the time consumption is long, and professional instruments and operators are needed. In contrast, immunoassays have the advantages of being rapid, simple, convenient, and the like, including enzyme-linked immunosorbent assays (ELISAs), immunochromatography, and immunosensor methods. However, the flexible application of the method is limited by the problems that the preparation of the antibody is not easy and the stability of the antibody is difficult to ensure.

Aptamers, also known as "chemical antibodies", are single-stranded DNA or RNA oligonucleotides screened by the ligand system evolution technique of exponential enrichment (SELEX). Compared with an antibody, the nucleic acid aptamer has the advantages of in vitro synthesis, low cost, easy modification, high stability, high specificity and the like. Aptamers have been used to develop aptamer sensors using fluorescent, electrochemical, array technologies to detect FB1 as target recognition elements. However, these methods generally require a copolymerization reaction of the aptamer with the probe, a time-consuming detection process. Graphene Oxide (GO) is a two-dimensional nanomaterial, and is widely used for the construction of sensing platforms in recent years due to its unique photoelectric properties. GO can be used as a fluorescence quencher for quenching fluorescence generated by the fluorescence-labeled aptamer, and can be used as a protective agent for protecting the aptamer from digestion by endonuclease. Relevant studies on the detection of FB1 by GO-based aptamer sensors based on enzyme signal amplification have not been reported.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a construction method of a GO-based nucleic acid aptamer sensor based on enzyme signal amplification and a method and application for detecting FB 1.

In order to achieve the above purpose, the solution of the invention is as follows:

as a first aspect, the present invention provides a method for constructing a GO-based nucleic acid aptamer sensor, comprising the steps of:

(1) modifying carboxyl-X-Rhodamine (ROX) fluorescent group at the 5' end of the aptamer to obtain the fluorescence-labeled aptamer;

(2) and carrying out fluorescence quenching by combining the GO with the pi-pi conjugated adsorption effect of the fluorescence-labeled aptamer to obtain the GO-based aptamer sensor.

Preferably, in the step (1), the excitation wavelength of the fluorescence-labeled aptamer is 575-595nm, the emission wavelength is 595-615nm, and the color of the emitted light is orange-red; aptamer sequences such as: 5 '-ROX-ATACCAGCTTATTCAATTAATCGCATTACCTTATACCAGCTTATTCAATTACGTCTGCACATACCAGCTTATTCAATTAGATAGTAAGTGCAATCT-3' (SEQ ID NO. 1).

Preferably, in step (2), the fluorescence intensity of the fluorescence-labeled aptamer is 758.34-928.61, and after GO is bound, the fluorescence is quenched, and the fluorescence intensity is 4.77-39.97.

As a second aspect, the present invention provides a GO-based nucleic acid aptamer sensor obtained by the above-described construction method.

As a third aspect, the present invention provides a method of validating the GO-based nucleic acid aptamer sensor described above, comprising the steps of:

(1) reacting the GO-based nucleic acid aptamer sensor (i.e., aptamer/GO complex) with the target FB 1;

(2) and endonuclease DNase I enzyme digestion aptamer, releasing target FB1, and realizing target circulation signal amplification.

Preferably, in the step (1), the reaction time is 5-15 min; the nucleic acid aptamer in the GO-based nucleic acid aptamer sensor is combined with FB1, the spatial conformation is changed, the nucleic acid aptamer is separated from GO, the fluorescence signal is recovered, and the fluorescence intensity is 55.23-305.21.

Preferably, in the step (2), the reaction time is 25-35 min; the fluorescence intensity of the aptamer is 136.83-756.12.

As a fourth aspect, the present invention provides a method for detecting FB1 using the GO-based nucleic acid aptamer sensor described above, comprising the steps of:

(1) preparing FB1 standard solutions with different concentration gradients, analyzing by using a GO-based nucleic acid aptamer sensor, detecting the intensity of a fluorescence signal and a spectral curve, and fitting a standard curve;

(2) and pretreating an actual sample to be detected, detecting by using a GO-based nucleic acid aptamer sensor, and determining the content of FB1 according to a linear model of a standard curve.

Preferably, in step (1), the concentration of the FB1 standard solution is 0ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 10ng/mL and 20ng/mL respectively.

Preferably, in the step (2), the pretreatment process is as follows: adding a scalar quantity of FB1 into an actual sample to be detected, then performing target extraction, filtering, and collecting filtrate;

preferably, the addition amount of FB1 is 0ng/mL, 1.5ng/mL, 8ng/mL, 15ng/mL, respectively.

As a fifth aspect, the invention provides a method for detecting FB1 by using a GO-based nucleic acid aptamer sensor, which can be used for quickly detecting the FB1 content in a wheat flour sample and has important practical application value in food safety detection.

Due to the adoption of the scheme, the invention has the beneficial effects that:

firstly, the invention researches and applies the GO-based nucleic acid aptamer sensor with enzyme signal amplification to detect FB1 for the first time, the enzyme signal amplification does not need professional instruments, the operation is simple and convenient, the sensitivity is high, the sensor is combined with the aptamer sensor, the high-sensitivity rapid detection of FB1 without the assistance of professional instruments can be realized, and the detection limit is far lower than the international limit.

Secondly, the GO-based nucleic acid aptamer sensor disclosed by the invention is high in accuracy when used for detecting FB1, and has higher conformity with an ELISA detection kit, thereby indicating that the detection technology has higher market potential.

Thirdly, the invention provides a new idea for constructing the aptamer sensor with high sensitivity, high specificity and simple preparation process, and the preparation method is novel and can be applied to the food detection industry.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

Fig. 1 is a schematic diagram of a GO-based Aptamer sensor according to the present invention (Graphene oxide is Graphene oxide, Aptamer is an Aptamer).

FIG. 2 shows the Fluorescence emission spectra of comparative FB1 with DNase I in the presence of example 2 according to the invention (Wavelength on the abscissa, Fluorescence Intensity on the ordinate).

FIG. 3 is a fluorescence spectrum of FB1 detected by the GO-based nucleic acid aptamer sensor in example 2 of the present invention.

FIG. 4 is a fluorescence plot of the GO-based nucleic acid aptamer sensor detecting FB1 in example 2 of the present invention.

FIG. 5 is a schematic diagram of the specific analysis of the GO-based nucleic acid aptamer sensor detection method in example 3 of the present invention.

Detailed Description

The invention provides a construction method of a GO-based nucleic acid aptamer sensor, a method for detecting FB1 and application. Specifically, the nucleic acid aptamer is used as a molecular recognition element, GO is used as a fluorescence quenching substance, and a GO-based nucleic acid aptamer sensor detection platform is constructed by combining endonuclease digestion and target circulating signal amplification functions.

As shown in fig. 1, the method for constructing the GO-based nucleic acid aptamer sensor of the present invention comprises the steps of:

(1) designing and synthesizing a nucleic acid aptamer fluorescent probe: modifying ROX fluorescent group at the 5' end of the aptamer to obtain fluorescence-labeled aptamer;

(2) fluorescence quenching of GO: namely, the GO is combined with the pi-pi conjugated adsorption effect of the fluorescence labeled aptamer for fluorescence quenching to obtain the GO-based aptamer sensor.

Wherein, in the step (1), the excitation wavelength of the fluorescence labeled aptamer can be 575-595nm, preferably 585nm, the emission wavelength can be 595-615nm, preferably 605nm, and the color of the emitted light is orange red; the aptamer sequence is 5 '-ROX-ATACCAGCTTATTCAATTAATCGCATTACCTTATACCAGCTTATTCAATTACGTCTGCACATACCAGCTTATTCAATTAGATAGTAAGTGCAATCT-3' (SEQ ID NO. 1).

In the step (2), the specific process of fluorescence quenching is as follows: ROX fluorophore-labeled aptamer was diluted to 100nmol/L in Tris buffer and 20. mu.g mL^-1The GO is fully mixed at room temperature to form an aptamer/GO compound, and the fluorescence signal of the aptamer is obviously weakened due to quenching.

Wherein, the highest fluorescence intensity of the fluorescence-labeled aptamer can be 758.34-928.61, preferably 928.61, and after GO is combined, the fluorescence is quenched, and the fluorescence intensity can be 4.77-39.97, preferably 4.77.

The GO-based nucleic acid aptamer sensor is obtained by the construction method.

The method for verifying the GO-based nucleic acid aptamer sensor comprises the following steps of:

(1) the recognition action mechanism of the aptamer molecules is as follows: i.e. reaction of GO-based nucleic acid aptamer sensor with target FB 1: 10ng mL^-1FB1 in the aptamer/GO complex is added to react, the aptamer binds to the target FB1, and the spatial conformation of the aptamer changesSeparating the aptamer from GO and recovering the fluorescence signal;

(2) the function of endonuclease DNase I, the amplification of target cycle signals: endonuclease DNase I cleaves the aptamer: adding 100U of endonuclease DNase I into the aptamer for reaction, and carrying out enzyme digestion on the aptamer to release a target FB1, thereby realizing target circulation signal amplification.

Wherein, in the step (1), the reaction time can be 5-15min, preferably 10 min; the fluorescence intensity may be 55.23 to 305.21, preferably 178.61.

In the step (2), the reaction time can be 25-35min, preferably 30 min; the fluorescence intensity of the aptamer may be 136.83-756.12, preferably 442.48.

The method for detecting FB1 by using the GO-based nucleic acid aptamer sensor comprises the following steps:

(1) FB1 standard curve fitting: preparing FB1 standard solutions with different concentration gradients, analyzing by using a GO-based nucleic acid aptamer sensor, detecting the intensity of a fluorescence signal and a spectral curve by using a spectrophotometer, and determining a fitting standard curve;

(2) and measuring the content of FB1 in the actual sample wheat flour: and (3) pretreating an actual sample to be detected, detecting by using a GO-based nucleic acid aptamer sensor, and determining the content of FB1 according to a linear model of a standard curve.

Wherein, in the step (1), the process of determining the fitted standard curve is as follows: the FB1 standard solution was diluted to different concentrations, 0ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, and 20ng/mL, and the standard solution and 100U of endonuclease DNase I were added to the aptamer/GO complex simultaneously for a reaction time of 30min, an excitation wavelength of 585nm, an emission wavelength of 605nm, a slit width for both excitation light and emission light was set to 10nm, and the emission color was orange-red. And (4) measuring the fluorescence signal intensity and a spectral curve, and fitting a standard curve.

In the step (2), the content of the FB1 is determined as follows: wheat flour samples were pre-treated, 2g (2.00 + -0.05 g) of each sample was accurately weighed, the target amounts of FB1 were 0ng/mL, 1.5ng/mL, 8ng/mL and 15ng/mL, respectively, 2mL of 50% methanol aqueous extract was added for target extraction, and the resulting mixture was filtered three times. And finally, collecting the filtrate, and detecting by using a GO-based nucleic acid aptamer sensor.

The present invention will be further described with reference to the following examples.

Example 1:

the construction method of the GO-based nucleic acid aptamer sensor of the embodiment includes the following steps:

(1) modifying ROX fluorescent group at the 5' end of the aptamer to obtain fluorescence-labeled aptamer; the excitation wavelength is 585nm, the emission wavelength is 605nm, and the color of emitted light is orange red; aptamer sequences such as: 5 '-ROX-ATACCAGCTTATTCAATTAATCGCATTACCTTATACCAGCTTATTCAATTACGTCTGCACATACCAGCTTATTCAATTAGATAGTAAGTGCAATCT-3' (SEQ ID NO. 1).

(2) ROX fluorophore-labeled aptamer was diluted to 100nmol/L in Tris buffer and 20. mu.g mL^-1The GO is fully mixed at room temperature to form an aptamer/GO compound, and the fluorescence signal of the aptamer is obviously weakened due to quenching.

Example 2:

as shown in fig. 2, the fluorescence emission spectrum curve and the standard curve were prepared:

10ng mL^-1and the FB1 is added into the aptamer/GO complex to react for 10min, the aptamer is combined with a target FB1, the spatial conformation of the aptamer is changed, the aptamer is separated from GO, and a fluorescent signal is recovered.

Adding 100U endonuclease DNase I into the aptamer for reaction for 30min, and carrying out enzyme digestion on the aptamer to release a target FB1, thereby realizing target circulation signal amplification.

As shown in FIGS. 3 and 4, the FB1 standard solution was diluted to different concentrations, respectively, 0ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, and 20ng/mL (the concentrations decreased from top to bottom in the line in FIG. 3), and the standard solution and 100U of endonuclease DNase I were simultaneously added to the aptamer/GO complex for a reaction time of 30min, an excitation wavelength of 585nm, an emission wavelength of 605nm, a slit width of both excitation light and emission light set to 10nm, and the emission light was orange-red in color. And (4) measuring the fluorescence signal intensity and a spectral curve, and fitting a standard curve.

Example 3:

measuring the content of FB1 in the actual sample wheat flour:

pretreating wheat flour samples, accurately weighing 2g of each sample, adding 8ng/mL of FB1 standard solution, adding 2mL of 50% methanol water extract for target extraction, and filtering the obtained mixture three times. And finally, collecting the filtrate, and detecting by using a GO-based nucleic acid aptamer sensor. According to the standard curve measured in example 2, the content of FB1 in the test sample is determined to be 7.93 ng/mL. The specific determination result of the method is shown in fig. 5, the GO-based nucleic acid aptamer sensor is constructed, and the fluorescence intensity recovery conditions in the presence of different targets are contrastively analyzed, wherein the fluorescence intensity recovery conditions comprise aflatoxin B1(AFB1), aflatoxin M1(AFM1), ochratoxin A (OTA) and a control group. The concentration of all target toxins was 5 ng/mL.

As can be seen in fig. 1, the aptamer modified the ROX fluorophore; when GO exists, a nucleic acid aptamer/GO compound is formed, and the fluorescence signal of the nucleic acid aptamer is obviously weakened due to the quenching effect; when FB1 exists, the aptamer molecule recognizes a target FB1, the aptamer spatial conformation changes, the aptamer is separated from GO, and the fluorescence signal is recovered; when the endonuclease DNase I exists, the aptamer is cut by enzyme, the target FB1 is released, and target circulation signal amplification is realized.

As can be seen from FIG. 2, when 20. mu.g mL^-1In the presence of GO (bottom line of fig. 2), the fluorescence signal of the aptamer decreases significantly; when 10ng mL^-1In the presence of FB1 (middle line in fig. 2), the aptamer molecule recognizes FB1 and the fluorescent signal is restored; when FB1 was present with endonuclease DNase I (top line of fig. 2), the fluorescence signal was significantly enhanced, confirming the amplification of the target cycle signal caused by the presence of endonuclease.

As can be seen from FIGS. 3 and 4, the concentration of FB1 ranged from 0.5ng mL to 20ng mL^-1Then, the fluorescence signal intensity is in positive correlation with the concentration of FB1, and the correlation coefficient R²＝0.995。

As can be seen from fig. 5, the fluorescence intensities detected in the presence of the other three mycotoxins (including aflatoxin B1(AFB1), aflatoxin M1(AFM1), ochratoxin a (ota)) were not significantly different from the control group and were significantly reduced from the fluorescence intensities detected in the presence of FB1 under the same FB1 detection conditions. The detection method of the invention is shown to have high specificity.

In conclusion, the GO-based aptamer sensor based on enzyme signal amplification can detect FB1, and mainly based on molecular recognition of aptamers, fluorescence quenching of GO and enzyme digestion of endonuclease, a fluorescent nanoprobe with a fluorescence detection signal is constructed, a aptamer sensor detection platform is established, and rapid and high-sensitivity detection of FB1 in a wheat flour sample can be realized.

The aspects, embodiments, features of the present invention should be considered in all respects as illustrative and not restrictive, the scope of the invention being defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.

Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.

Unless specifically stated otherwise, use of the terms "comprising", "including", "having" or "having" is generally to be understood as open-ended and not limiting.

It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.

While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.

Sequence listing

<110> Shanghai university of transportation

<120> construction of graphene oxide-based aptamer sensor, method for detecting fumonisin B1 and application

<141> 2021-09-29

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 96

<212> DNA

<213> Artificial Sequence (Artficial Sequence)

<400> 1

ataccagctt attcaattaa tcgcattacc ttataccagc ttattcaatt acgtctgcac 60

ataccagctt attcaattag atagtaagtg caatct 96

Claims

1. A method for constructing a graphene oxide-based aptamer sensor is characterized by comprising the following steps: which comprises the following steps:

(1) modifying carboxyl-X-rhodamine fluorescent group at the 5' end of the aptamer to obtain the fluorescence-labeled aptamer;

(2) and carrying out fluorescence quenching by conjugated adsorption of the graphene oxide and the fluorescence-labeled aptamer to obtain the graphene oxide-based aptamer sensor.

2. The method for constructing a graphene oxide-based aptamer sensor according to claim 1, wherein: in the step (1), the excitation wavelength of the fluorescence-labeled aptamer is 575-595nm, the emission wavelength is 595-615nm, and the color of the emitted light is orange red; the sequence of the aptamer is shown as SEQ ID NO. 1.

3. The method for constructing a graphene oxide-based aptamer sensor according to claim 1, wherein: in the step (2), the highest fluorescence intensity of the fluorescence-labeled aptamer is 758.34-928.61, and after the fluorescence-labeled aptamer is combined with graphene oxide, the fluorescence is quenched, and the fluorescence intensity is 4.77-39.97.

4. A graphene oxide-based aptamer sensor, comprising: which is obtained by the method of construction according to any one of claims 1 to 3.

5. A method of validating the graphene oxide-based aptamer sensor of claim 4, wherein: which comprises the following steps:

(1) reacting the graphene oxide-based aptamer sensor with a target fumonisin B1;

(2) and carrying out endonuclease DNaseI enzyme digestion aptamer reaction to release a target fumonisin B1, thereby realizing target circulating signal amplification.

6. The method of validating a graphene oxide-based aptamer sensor according to claim 5, wherein: in the step (1), the reaction time is 5-15 min; in the graphene oxide-based aptamer sensor, an aptamer is combined with fumonisin B1, spatial conformation is changed, the aptamer is separated from graphene oxide, a fluorescence signal is recovered, and the fluorescence intensity is 55.23-305.21; and/or the presence of a gas in the gas,

in the step (2), the reaction time is 25-35 min; the fluorescence intensity of the aptamer is 136.83-756.12.

7. A method for detecting fumonisin B1 using the graphene oxide-based aptamer sensor according to claim 4, wherein: which comprises the following steps:

(1) preparing fumonisin B1 standard solutions with different concentration gradients, analyzing by using a graphene oxide-based aptamer sensor, detecting the intensity of a fluorescence signal and a spectrum curve, and fitting a standard curve;

(2) and pretreating an actual sample to be detected, detecting by using the graphene oxide-based aptamer sensor, and determining the content of fumonisin B1 according to a linear model of a standard curve.

8. The method for detecting fumonisin B1 by using the graphene oxide-based aptamer sensor according to claim 7, wherein the graphene oxide-based aptamer sensor comprises: in the step (1), the concentrations of the fumonisin B1 standard solution are respectively 0ng/mL, 0.5ng/mL, 2ng/mL, 5ng/mL, 10ng/mL and 20 ng/mL; and/or the presence of a gas in the gas,

in the step (2), the pretreatment process comprises the following steps: adding a scalar quantity of fumonisins B1 into an actual sample to be detected, then performing target extraction, filtering, and collecting filtrate;

preferably, the addition amount of fumonisin B1 is 0ng/mL, 1.5ng/mL, 8ng/mL, 15ng/mL respectively.

9. Use of the graphene oxide-based aptamer sensor according to claim 4 in food safety detection.

10. Use of the graphene oxide-based aptamer sensor according to claim 4 for detecting the content of fumonisin B1 in a wheat flour sample.