Ion regulation G-quadruplex sensitized DNA optical fiber sensing detection device and detection method

Abstract

The application belongs to the technical field of micro-nano optical fiber sensing, and provides an ion-regulated G-quadruplex sensitized DNA optical fiber sensing detection device and a detection method, which comprises a transmission optical fiber, an optical fiber DNA sensing probe, a microfluidic chip and a spectrum analyzer; DNA aptamer is fixed on the surface of a uniform area of a modified tapered micro-nano optical fiber to form an optical fiber DNA sensing probe; the optical fiber DNA sensing probe is immersed in a solution containing a target DNA to be detected; a broadband light source inputs laser to the optical fiber DNA sensing probe; the target DNA is detected by using the sensitive characteristics of the evanescent wave on the surface of the probe to the change of the external environment and the conformation change of the aptamer caused by the specific combination of the DNA aptamer and the target DNA; the target DNA is specifically combined with extended DNA; the conformation change of the extended DNA is regulated by ions to realize the sensitized sensing detection of the target DNA; and the application has the advantages of high detection precision and short response time for DNA detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of micro-nano optical fiber sensing, in particular to an ion-regulated G-quadruplex sensitized DNA optical fiber sensing detection device and a detection method. BACKGROUND

[0002] DNA (deoxyribonucleic acid) is a nucleic acid that carries the genetic information necessary for synthesizing RNA and proteins in biological cells, and is an essential biological macromolecule for the development and normal operation of organisms. DNA is formed by two anti-parallel deoxyribonucleotide chains spiraling through base pairing principles, and the arrangement order of bases carries a large amount of genetic information. Through DNA detection, the arrangement order of bases can be obtained to obtain genetic information, which can effectively promote the rapid development of fields such as gene analysis, drug research and development, genetic disease prevention, disease diagnosis and treatment, environmental engineering and biomedical engineering. Therefore, DNA detection plays an important role and significance in modern medical diagnosis, treatment and health care.

[0003] Existing DNA detection methods include molecular hybridization, agarose gel electrophoresis, fluorescence detection, polymerase chain reaction, and optical density measurement. The above methods generally have limitations such as complicated operation, low sensitivity, and unsatisfactory detection limit, which cannot meet the needs of modern medicine in molecular diagnosis, prevention, and disease treatment. Therefore, it is necessary to develop a DNA sensor with simple structure, convenient operation, excellent detection performance, and flexibility.

[0004] The invention patent with publication number CN109557051A discloses a sensitized microRNA optical fiber sensing device and a manufacturing and measuring method. It uses a nanometer copper sulfide layer and a graphene oxide spacer layer to form an interface layer with a localized plasmon resonance energy enhancement effect. The evanescent wave of the tapered micro-nano optical fiber interferometer is sensitive to changes in the external environment. The specific binding of microRNA and single-stranded DNA probes causes changes in the refractive index, which can detect a limit detection concentration of 9.64 x 10 -21 However, this method does not use a microfluidic chip, cannot achieve low-dose detection, and is prone to errors in operation. SUMMARY

[0005] The present application aims to provide an ion-regulated G-quadruplex sensitized DNA optical fiber sensing detection device and a detection method, which has a simple structure, high measurement accuracy, and short response time, and can meet the needs of modern medicine in molecular diagnosis, prevention, and disease treatment.

[0006] To achieve the above purpose, the technical solution adopted by the present application is as follows:

[0007] The ion-regulated G-quadruplex-sensitized DNA optical fiber sensing detection device comprises a transmission optical fiber, an optical fiber DNA sensing probe, a microfluidic chip, a spectrum analyzer and an intelligent peristaltic pump; the optical fiber DNA sensing probe is embedded in the groove of the microfluidic chip, the input end of the optical fiber DNA sensing probe is connected to an external light source through a connecting transmission optical fiber, and the output end of the optical fiber DNA sensing probe is connected to the input end of the spectrum analyzer through a transmission optical fiber; the intelligent peristaltic pump is connected to the liquid inlet end of the microfluidic chip through a microfluidic tube, and is used for pumping the target DNA solution to be detected, the extended DNA solution and the ion solution into the groove of the microfluidic chip; the optical fiber DNA sensing probe is obtained by the following steps:

[0008] (1) The ordinary optical fiber is prepared into a tapered micro-nano optical fiber by a fusion heating tapering method; the tapered micro-nano optical fiber is composed of a transition zone and a uniform zone, and the transition zone and the uniform zone form an interference structure;

[0009] (2) The tapered micro-nano optical fiber is soaked in an aiptasia solution to generate a large number of silicon hydroxyl groups on the surface of the tapered micro-nano optical fiber, and then is soaked in a silane coupling agent to generate a large number of amino groups on the surface of the tapered micro-nano optical fiber; then the tapered micro-nano optical fiber is soaked in a glutaraldehyde solution to obtain a surface-modified tapered micro-nano optical fiber;

[0010] (3) The surface-modified tapered micro-nano optical fiber is soaked in a DNA aptamer solution, and the DNA aptamer is combined with the target DNA molecule through non-covalent bonds to form a biological sensitive film on the surface of the tapered micro-nano optical fiber, so that the refractive index of the tapered micro-nano optical fiber is changed, and the optical fiber DNA sensing probe is obtained.

[0011] In the application, preferably, the external light source is a broadband light source, and the broadband light source is a shock-resistant broadband laser.

[0012] In the application, preferably, the diameter of the uniform zone of the tapered micro-nano optical fiber is less than 10 microns.

[0013] In the application, preferably, the diameter of the groove of the microfluidic chip is 300-600 microns.

[0014] In the application, preferably, the wavelength range of the spectrum analyzer is 600-1700 nm.

[0015] In the application, preferably, the flow range of the intelligent peristaltic pump is 0.01-1000 ml / min.

[0016] The application further provides a DNA detection method using the ion-regulated G-quadruplex-sensitized DNA optical fiber sensing detection device, comprising the following steps:

[0017] The target DNA solution to be detected is injected into the groove of the microfluidic chip by using the intelligent peristaltic pump, so that the optical fiber DNA sensing probe is immersed in the solution containing the target DNA to be detected; an external light source inputs incident laser to the optical fiber DNA sensing probe, and forms an evanescent wave on the surface of the optical fiber DNA sensing probe; after a period of reaction, the optical fiber DNA sensing probe binds the target DNA, and then the extension DNA solution is added by using the intelligent peristaltic pump, so that the target DNA specifically binds with the extension DNA; then, the potassium ion solution with a molar concentration of 3M is added by using the intelligent peristaltic pump, the conformation change of the extension DNA is regulated by ions, the evanescent wave on the surface of the optical fiber DNA sensing probe is sensitive to the change of the external environment, the wavelength change of the evanescent wave resonance peak is detected by using the spectrum analyzer, the correlation between the concentration of the target DNA and the change of the evanescent wave is studied, the linear relationship between the concentration of the target DNA and the wavelength of the resonance peak is established, and the sensitive sensing detection of the target DNA is realized.

[0018] The linear relationship between the concentration of the target DNA and the wavelength of the resonance peak is as follows: y = 0.0001x + 0.0001, wherein y is the wavelength of the resonance peak, and x is the concentration of the target DNA.

[0019] As described above, due to the adoption of the technical solutions, the present application has the following beneficial effects:

[0020] The present application constructs the optical fiber DNA sensing probe by using the tapered micro-nano optical fiber, utilizes the characteristics that the evanescent wave on the surface of the probe is sensitive to the change of the external environment, and realizes the detection of the target DNA caused by the specific binding between the DNA sensing probe and the target DNA and the conformation change of the aptamer.

[0021] The detection device of the present application is small, flexible, anti-electromagnetic radiation, and easy to operate, and can well avoid the complex operation and interference of other components of the commonly used method.

[0022] The optical fiber DNA sensing probe and the signal transmission line are integrated in a single optical fiber, the detection precision is high, the response time is short, and the measurement of a small dose and a trace concentration of a given sequence of DNA can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a functional schematic diagram of an ion-regulated G-quadruplex sensitized DNA optical fiber sensing detection device according to an embodiment of the present application.

[0024] Figure 2 is a structural schematic diagram of an optical fiber DNA sensing probe according to the present application.

[0025] Figure 3is a schematic diagram of the wavelength change of a certain mode of surface plasmon wave interference peak with the concentration when the optical fiber DNA sensing probe is paired and combined with the target DNA;

[0026] Figure 4 is a schematic diagram of the wavelength change of a certain mode of surface plasmon wave interference peak with the concentration when the optical fiber DNA sensing probe is paired and combined with the target DNA, and then the target DNA is paired and combined with the extended DNA under the action of high concentration potassium ions.

[0027] In the drawings, 1 is a broadband light source; 2 is a tapered micro-nano optical fiber; 3 is a transmission optical fiber; 4 is a microfluidic chip; 5 is an intelligent peristaltic pump; 6 is a microfluidic tube; 7 is a spectrum analyzer; 8 is incident laser; 9 is plasmon wave; 10 is DNA aptamer; 11 is target DNA solution; 12 is extended DNA solution; 13 is ion solution. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0029] It should be noted that when a component is considered to be "connected" to another component, it can be directly connected to the other component or a middle component can exist at the same time.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more related listed items. EMBODIMENTS

[0031] As Figure 1The application discloses an ion-regulated G-quadruplex-sensitized DNA optical fiber sensing detection device, which comprises a transmission optical fiber 3, an optical fiber DNA sensing probe, a microfluidic chip 4, a spectrum analyzer 7 and an intelligent peristaltic pump 5; the optical fiber DNA sensing probe is arranged in a groove of the microfluidic chip 4, the diameter of the groove of the microfluidic chip 4 is 300-600 microns, and the requirement of small-dose detection can be met; the input end of the optical fiber DNA sensing probe is connected with an external light source through a connecting transmission optical fiber 3, and the output end of the optical fiber DNA sensing probe is connected with the input end of the spectrum analyzer 7 through the transmission optical fiber 3, wherein the transmission optical fiber 3 is a common single-mode optical fiber, the external light source is a broadband light source 1, the broadband light source 1 is an anti-vibration broadband laser, and the range covered by the interference spectrum can be met; the intelligent peristaltic pump 5 is connected with the liquid inlet end of the microfluidic chip 4 through a microfluidic tube 6, and the intelligent peristaltic pump 5 is used for pumping a target DNA solution 11 to be detected, an extended DNA solution 12 and an ion solution 13 into the groove of the microfluidic chip 4; referring to Figure 2 The optical fiber DNA sensing probe is obtained through the following steps:

[0032] (1) the common optical fiber is prepared into a tapered micro-nano optical fiber 2 through a fusion heating tapering method, and the operation is simple, convenient and fast; the tapered micro-nano optical fiber 2 is composed of a transition zone and a uniform zone, and the transition zone and the uniform zone form an interference structure; the diameter of the uniform zone of the tapered micro-nano optical fiber 2 is less than 10 microns, so that the surface of the sensing optical fiber has a strong evanescent field;

[0033] (2) the tapered micro-nano optical fiber 2 is soaked in an arowana solution, so that a large number of silicon hydroxyl groups are generated on the surface of the tapered micro-nano optical fiber 2, and then the tapered micro-nano optical fiber 2 is soaked in a silane coupling agent, so that a large number of amino groups are generated on the surface of the tapered micro-nano optical fiber 2; then the tapered micro-nano optical fiber 2 is soaked in a glutaraldehyde solution, so that a surface-modified tapered micro-nano optical fiber 2 is obtained;

[0034] (3) the surface-modified tapered micro-nano optical fiber 2 is soaked in a DNA aptamer 10 solution, the DNA aptamer 10 is arranged on the surface of the tapered micro-nano optical fiber 2 through non-covalent bond, so that a biological sensitive film which can be used for specific combination with a target DNA molecule and can cause the refractive index of the surface of the tapered micro-nano optical fiber 2 to change is formed, and the optical fiber DNA sensing probe is obtained.

[0035] In some preferred embodiments, the flow range of the intelligent peristaltic pump 5 is 0.01-1000 (ml / min), the diameter range of the microflow tube 6 is 1-3 mm, the speed and flow of the sample pumping are met, the wavelength range of the spectrum analyzer 7 is 600-1700 nm, and such a spectrum analyzer 7 has the advantages of large measurement range, superior performance and convenient operation. In the present application, the optical fiber DNA sensing probe is built in the microflow chip 4, the target DNA solution 11 to be measured, the extended DNA solution 12 and the ion solution 13 are pumped into the microflow chip 4 through the microflow tube 6 by the intelligent peristaltic pump 5, the speed and flow of the pumping can be controlled, and precise micro-sensing and detection are realized.

[0036] The ion-regulated G-quadruplex sensitized DNA optical fiber sensing detection device is used to realize target DNA sensitized sensing detection. Specifically, the following steps are included:

[0037] The intelligent peristaltic pump 5 is used to inject the target DNA solution 11 to be measured into the groove of the microflow chip 4, so that the optical fiber DNA sensing probe is immersed in the solution containing the target DNA to be measured. The external light source inputs incident laser 8 to the optical fiber DNA sensing probe, and forms evanescent wave 9 on the surface of the optical fiber DNA sensing probe. After a period of reaction, the optical fiber DNA sensing probe binds the target DNA, the intelligent peristaltic pump 5 is used to add the extended DNA solution 12, so that the target DNA specifically binds with the extended DNA, then the intelligent peristaltic pump 5 is used to add the potassium ion solution 13 with a molar concentration of 3M, the conformation change of the extended DNA is regulated by ions, the evanescent wave 9 on the surface of the optical fiber DNA sensing probe is sensitive to the change of the external environment, the spectrum analyzer 7 is used to detect the wavelength change of the evanescent wave 9 resonance peak, the correlation between the concentration of the target DNA and the change of the evanescent wave 9 is studied, the linear relationship between the concentration of the target DNA and the wavelength of the resonance peak is established, and the sensitized sensing detection of the target DNA is realized.

[0038] In the present application, the DNA aptamer 10 is fixed on the surface of the uniform area of the modified tapered micro-nano optical fiber 2 by non-covalent bond, and the optical fiber DNA sensing probe is formed. The optical fiber DNA sensing probe is immersed in the target DNA solution 11 to be measured, the broadband light source 1 inputs laser to the optical fiber DNA sensing probe, the evanescent wave 9 on the surface of the probe is sensitive to the change of the external environment, and the conformation change of the aptamer caused by the specific binding of the DNA aptamer 10 and the target DNA realizes the detection of the target DNA. The target DNA specifically binds with the extended DNA, the conformation change of the extended DNA is regulated by ions, and the sensitized sensing detection of the target DNA is realized.

[0039] The conformational change of the aptamer caused by the specific binding between the DNA aptamer and the target DNA in the application further manifests as a change in the refractive index of the surface of the tapered micro-nano optical fiber, and the relationship between the change in the interference spectrum wavelength and the refractive index is:

[0040] (1)

[0041] wherein, is the change in the interference spectrum wavelength, K is the refractive index sensitivity of the tapered micro-nano optical fiber, is the change in the refractive index of the surface of the tapered micro-nano optical fiber.

[0042] The target DNA is further specifically combined with the extended DNA, and the conformational change of the extended DNA is regulated by ions, which further promotes the change in the refractive index of the surface of the tapered micro-nano optical fiber 2, and realizes the sensitive sensing detection of the target DNA, and the relationship between the change in the interference spectrum wavelength and the refractive index is:

[0043] (2)

[0044] wherein, is the change in the interference spectrum wavelength, K is the refractive index sensitivity of the tapered micro-nano optical fiber, is the change in the refractive index of the surface of the tapered micro-nano optical fiber.

[0045] After introducing the sensitivity mechanism of regulating the conformational change of the extended DNA by ions, the change in the interference spectrum wavelength is further increased, that is, is greater than , and therefore the sensitive sensing detection of the target DNA can be realized.

[0046] As shown in Figure 3 , the schematic diagram of the change in the wavelength of a certain mode of the surface evanescent wave resonance peak of the optical fiber DNA sensing probe with the concentration when the optical fiber DNA sensing probe is combined with the target DNA. In the range of 10 -12 ~ 10 -8 M, with the increase of the concentration of the target DNA, the wavelength of the resonance peak presents a linear change, and the linear relationship can be expressed as:

[0047] (3)

[0048] After the optical fiber DNA sensing probe is combined with the target DNA, the target DNA is further specifically combined with the extended DNA, and the conformational change of the extended DNA is regulated by high-concentration potassium ions (concentration, 3 M), so as to realize the sensitive sensing detection of the target DNA.

[0049] As shown in Figure 4The diagram illustrates the wavelength variation of a certain mode of the evanescent wave resonance peak on the surface of the fiber optic DNA sensing probe as a function of concentration when the target DNA pairs and binds to extended DNA, under the influence of high-concentration potassium ions. Under the sensitization-enhancing effect of high-concentration potassium ions, the target DNA concentration is 10... -15 ~10 -8 Within the range M, as the concentration of target DNA increases, the wavelength of the resonance peak exhibits a linear change, which can be expressed as:

[0050] (4)

[0051] pass Figure 3 and Figure 4 In comparison, under the sensitization-enhancing effect of high-concentration potassium ions, the detection range of the target DNA was significantly improved, from 10... -12 ~10 -8 M becomes 10 -15 ~10 -8 M has reached three orders of magnitude.

[0052] In this invention, a high concentration of potassium ions (concentration, 3M) is required for ion enhancement. When the ion concentration is low, the ion regulation enhancement effect is not obvious.

[0053] The working principle of this invention is as follows:

[0054] Laser light emitted from broadband light source 1 is injected into tapered micro / nano fiber 2 to form an interference spectrum. This interference spectrum is input into a spectrometer 7, and the change in refractive index on the surface of the tapered micro / nano fiber 2 can be measured by monitoring and analyzing the wavelength of the interference spectrum. Specifically, DNA aptamers 10 are fixed to the surface of a uniform region of the modified tapered micro / nano fiber 2 through non-covalent bonding, forming a fiber-optic DNA sensing probe. The conformational change of the aptamer caused by the specific binding of DNA aptamer 10 to the target DNA promotes the change in refractive index on the surface of the tapered micro / nano fiber 2, thus realizing the detection of the target DNA. The target DNA then specifically binds to extended DNA, and the conformational change of the extended DNA is ion-regulated, further promoting the change in refractive index on the surface of the tapered micro / nano fiber 2, thus realizing the enhanced sensing detection of the target DNA.

[0055] In summary, the present invention provides an ion-regulated G quadruple-sensitized DNA fiber optic sensing and detection device and method. Through wavelength demodulation, the detection process is label-free for the sample and has the advantages of simplicity, speed, high detection accuracy and short response time. It can realize the measurement of small doses and trace concentrations of a given DNA sequence.

[0056] The above description is directed to the preferred embodiment of the present application. It is to be understood that other variants and modifications will be apparent to the skilled in the art in view of the foregoing description, which are intended to fall within the scope of the application.

Claims

1. A DNA fiber optic sensing and detection device with ion-regulated G quadruple-sensitized properties, characterized in that: The device comprises a transmission optical fiber, a fiber DNA sensing probe, a microfluidic chip, a spectrum analyzer and an intelligent peristaltic pump; the fiber DNA sensing probe is built in a groove of the microfluidic chip, an input end of the fiber DNA sensing probe is connected to an external light source through a connecting transmission optical fiber, and an output end of the fiber DNA sensing probe is connected to an input end of the spectrum analyzer through a transmission optical fiber; the intelligent peristaltic pump is connected to a liquid inlet end of the microfluidic chip through a microfluidic tube, and is used for pumping a target DNA solution to be detected, an extended DNA solution and a 3M potassium ion solution into the groove of the microfluidic chip; the fiber DNA sensing probe is obtained through the following steps: (1) a common optical fiber is prepared into a tapered micro-nano optical fiber through a fusion heating tapering method; the tapered micro-nano optical fiber is composed of a transition zone and a uniform zone, and the transition zone and the uniform zone form an interference structure; (2) the tapered micro-nano optical fiber is soaked in an aiptasia solution to generate a large number of silicon hydroxyl groups on the surface of the tapered micro-nano optical fiber, and then is soaked in a silane coupling agent to generate a large number of amino groups on the surface of the tapered micro-nano optical fiber; then the tapered micro-nano optical fiber is soaked in a glutaraldehyde solution to obtain a surface-modified tapered micro-nano optical fiber; (3) the surface-modified tapered micro-nano optical fiber is soaked in a DNA aptamer solution, the DNA aptamer is combined with the target DNA molecule through non-covalent bonds to form a biological sensitive film on the surface of the tapered micro-nano optical fiber, and the biological sensitive film can cause a change in the refractive index of the surface of the tapered micro-nano optical fiber, thereby obtaining the fiber DNA sensing probe.

2. The ion-regulated G-quadruplex-sensitized DNA optical fiber sensing and detecting device according to claim 1, characterized in that: The external light source is a broadband light source, and the broadband light source is a shock-resistant broadband laser. 3.The ion-regulated G-quadruplex-sensitized DNA optical fiber sensing and detecting device according to claim 1, characterized in that: The diameter of the uniform zone of the tapered micro-nano optical fiber is less than 10 microns.

4. The ion-regulated G-quadruplex-sensitized DNA optical fiber sensing and detecting device according to claim 1, characterized in that: The diameter of the groove of the microfluidic chip is 300-600 microns.

5. The ion-regulated G-quadruplex sensitized DNA optical fiber sensing and detecting device according to claim 1, characterized in that: The wavelength range of the spectrum analyzer is 600-1700 nm.

6. The ion-regulated G-quadruplex sensitized DNA optical fiber sensing and detecting device according to claim 2, characterized in that: The flow range of the intelligent peristaltic pump is 0.01-1000 ml / min.

7. The method for DNA detection using the ion-regulated G-quadruplex- sensitized DNA optical fiber sensing detection device according to any one of claims 1-6, characterized in that, The device comprises the following steps: a target DNA solution to be detected is injected into the groove of the microfluidic chip by using the intelligent peristaltic pump, so that the fiber DNA sensing probe is immersed in the solution containing the target DNA to be detected; an external light source inputs incident laser to the fiber DNA sensing probe, and forms an evanescent wave on the surface of the fiber DNA sensing probe; after a period of reaction, the fiber DNA sensing probe is combined with the target DNA, and then an extended DNA solution is added by using the intelligent peristaltic pump, so that the target DNA is specifically combined with the extended DNA; then a 3M potassium ion solution is added by using the intelligent peristaltic pump, so that the conformation of the extended DNA is changed by ion regulation; the wavelength change of the evanescent wave resonance peak is detected by using the spectrum analyzer, the correlation between the concentration of the target DNA and the change of the evanescent wave is studied, a linear relationship between the concentration of the target DNA and the wavelength of the resonance peak is established, and the sensitive sensing detection of the target DNA is realized.

8. The method of claim 7, wherein: The linear relationship between the target DNA concentration and the wavelength of the resonance peak is as follows: where y is the wavelength of the resonance peak and x is the target DNA concentration.

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