Self-calibration microbial sensor based on DNA self-assembly

Abstract

The invention relates to a self-assembled three-dimensional ball-flower type bimodal DNA sensor based on a rolling ring amplification technology, which is used for detecting target DNA of microorganisms in an early fouling stage. The sensor consists of a lock ring probe, target DNA (Deoxyribose Nucleic Acid) and a fluorescent nanogold probe. When the target DNA exists, the locked loop probe specifically recognizes the target DNA, rolling circle amplification is carried out under the action of T4DNA ligase and phi29 DNA polymerase, and a stable nano flower ball structure is formed through self-assembly. One end of the fluorescent signal probe modifies the fluorescent nanogold, and the other end of the fluorescent signal probe is complementary with a rolling circle amplification product to form a complete sensor after combination. The fluorescence spectrophotometer is used for measuring a fluorescence signal, the intensity of the fluorescence signal is positively correlated with the concentration of the target DNA, and qualitative and quantitative detection of the target DNA is realized. The nanometer flower ball structure enables a fluorescence signal to be enhanced and a color signal to be weakened, and self-calibration is achieved through comparison of the fluorescence signal and the color signal. The specific sequence of the sulfate reducing bacteria is taken as the target gene, so that the kit has high selectivity and anti-interference capability, and is suitable for rapid detection of early-stage fouling microorganisms.

Description

Technical Field

[0001] The present invention relates to the technical fields of biosensors and marine biofouling detection, and particularly to a self-calibrating microbial sensor based on DNA self-assembly. Background Art

[0002] Marine biofouling refers to the phenomenon that marine organisms attach and grow on the surfaces of marine facilities such as ships, offshore platforms, and pipelines. This phenomenon not only increases the navigation resistance of ships, reduces the navigation efficiency, but also causes corrosion and damage to marine facilities, increases the maintenance cost, and even has a negative impact on the marine ecological environment. Therefore, timely detection and control of marine biofouling have important economic and environmental significance.

[0003] Microbial attachment is the starting point of marine biofouling, and its early attachment behavior will trigger the attachment and growth of subsequent large marine organisms. The detection of early fouling microorganisms is of great significance for the prevention and control of marine biofouling. By detecting the presence and species of microorganisms at an early stage, measures can be taken in advance to reduce the occurrence and spread of fouling.

[0004] At present, although conventional gene sequencing technologies are widely used in microbial detection, they have some limitations. For example, gene sequencing requires complex laboratory equipment, cannot provide results quickly on-site, and requires professional analysts for operation and data analysis. These factors limit its wide application in the early detection of marine biofouling. Therefore, it is particularly important to develop a new type of detection method that is fast, sensitive, and operable on-site.

[0005] The present invention aims to provide a self-calibrating DNA sensor based on DNA self-assembled nanostructures, which uses rolling circle amplification technology and fluorescent nanogold probes to achieve rapid and sensitive detection of the nucleic acid sequences of early fouling microorganisms. The sensor has the characteristics of high selectivity, high sensitivity, and strong anti-interference ability, and can quickly detect the presence of microorganisms on-site, providing an effective technical means for the early prevention and control of marine biofouling. Summary of the Invention

[0006] The present invention aims to provide a self-calibrating DNA sensor based on DNA self-assembled nanostructures for rapid and sensitive detection of the nucleic acid sequences of early fouling microorganisms. The sensor can overcome the deficiencies of existing gene sequencing technologies, achieve rapid on-site detection, improve the detection efficiency and accuracy, and provide an efficient technical means for the early prevention and control of marine biofouling.

[0007] The present invention is achieved through the following technical solutions:

[0008] 1. The sensor of the present invention is composed of the following components: (1) Locked nucleic acid probe: used to specifically recognize the nucleic acid sequence (target DNA) of the target microorganism; (2) Target DNA: specific nucleic acid sequence from early fouling microorganisms, such as the nucleic acid sequence of sulfate-reducing bacteria (SRB) or other microorganisms; (3) Fluorescent gold nanoprobe: one end is modified with a fluorescent gold nanoparticle signal marker, and the other end is complementary to the rolling circle amplification product sequence.

[0009] 2. In the presence of target DNA, the two-arm sequences of the locked nucleic acid probe can specifically bind to the target DNA and form a circular structure under the action of T4 DNA ligase. Subsequently, under the action of phi29 DNA polymerase, the locked nucleic acid probe undergoes a rolling circle amplification reaction and self-assembles into a stable nanoflower spherical DNA structure. The fluorescent gold nanoprobe binds to the nanoflower through complementary sequences, and the intensity of the fluorescent signal is positively correlated with the concentration of target DNA. The target DNA is qualitatively and quantitatively detected by measuring the fluorescent signal with a fluorescence spectrophotometer.

[0010] 3. The sensor of the present invention also designs a self-calibrating fluorescent DNA probe for correcting the deviation of the detection signal. The nanoflower structure results in an enhanced fluorescent signal and a weakened color signal, and the detection signal is corrected by comparing the fluorescent and color signals, further improving the accuracy and reliability of the detection.

[0011] 4. The detection method of the present invention includes the following steps: (1) Extract the DNA from the target sample; (2) Mix the extracted DNA with the locked nucleic acid probe and the fluorescent gold nanoprobe; (3) Perform a rolling circle amplification reaction under the action of T4 DNA ligase and phi29 DNA polymerase; (4) Measure the intensity of the fluorescent signal, and correct the detection signal by comparing the fluorescent and color signals; (5) Qualitatively and quantitatively detect the target DNA according to the correlation between the fluorescent signal intensity and the concentration of target DNA.

[0012] Compared with the prior art, the present invention has the following remarkable advantages: (1) High sensitivity and high specificity: The sensitivity and specificity of the detection are significantly improved by the rolling circle amplification technology and the fluorescent gold nanoprobe, and low-concentration target DNA can be detected. (2) Rapid detection: Without complex laboratory equipment and professional personnel, the detection can be quickly completed on-site, suitable for real-time monitoring in the marine environment. (3) Self-calibration function: Self-calibration is achieved by comparing the fluorescent and color signals, effectively correcting the deviation of the detection signal and improving the accuracy and reliability of the detection. (4) Universality: The sensor of the present invention is not only applicable to sulfate-reducing bacteria (SRB), but also can be extended to the detection of other microorganisms, with high universality. (5) Strong anti-interference ability: The nanoflower structure and the enhanced mechanism of the fluorescent signal enable it to have strong anti-interference ability in complex environments and are applicable to various marine fouling environments.

[0013] In summary, the present invention provides an efficient, sensitive and on-site operable detection method, which provides strong technical support for the early prevention and control of marine biofouling. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

[0015] Figure 1 : Schematic diagram of the detection principle of a self-calibrating DNA sensor based on a DNA self-assembled nanostructure.

[0016] Figure 2 : Electron microscopy and elemental characterization diagrams of the nanoflower structure.

[0017] Figure 3 : Result diagrams of the detection of different base mismatches by a self-calibrating DNA sensor based on a DNA self-assembled nanostructure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] The present invention will be described in detail below in conjunction with the drawings and specific embodiments. At the same time, it should be noted here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments. For some well-known technologies, those skilled in the art can also adopt other alternative methods for implementation; moreover, the drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0019] It should be pointed out that in the specification, the mention of "one embodiment", "embodiment", "exemplary embodiment", "some embodiments", etc. indicates that the described embodiment may include specific features, structures or characteristics, but not necessarily every embodiment includes such specific features, structures or characteristics. In addition, when combining embodiments to describe specific features, structures or characteristics, the implementation of such features, structures or characteristics in combination with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the relevant art.

[0020] Generally, the terms can be understood at least in part from their use in the context. For example, at least in part depending on the context, the term "one or more" used herein can be used to describe any feature, structure or characteristic in a singular sense, or can be used to describe a combination of features, structures or characteristics in a plural sense. In addition, the term "based on" can be understood as not necessarily intended to convey a set of exclusive factors, but rather, at least in part depending on the context, allowing for the existence of other factors that may not be explicitly described.

[0021] Refer toFigures 1 to 3

[0022] Example 1: Construction of Three-Dimensional Flower Ball-Type Nano DNA Structure

[0023] (1) Mix 10 μL of 5'-phosphorylated padlock probe (concentration: 1.0×10 -5 M) with 20 μL of target substrate in 1×T4 DNA ligase buffer, heat to 95 °C for 5 minutes, and then cool to room temperature for 3 hours.

[0024] (2) Add 10 μL of T4 DNA ligase and react overnight. Heat to 65 °C for 10 minutes to inactivate T4 DNA ligase.

[0025] (3) Add 5 μL of Exonuclease I (ExoI) and mix in 1×ExoI buffer (67 mM glycine KOH, 6.7 mM MgCl5, 1 mM DTT, pH 9.5), react at room temperature for 1.5 hours to remove unsuccessfully ligated template probes, and then heat to 80 °C for 15 minutes to inactivate ExoI.

[0026] (4) Conduct a rolling circle amplification (RCA) reaction in 100 μL of solution, including 50 μL of the above template primer mixture, 10 μL of phi29 DNA polymerase, 10 μL of dNTPs, 10 μL of 10×phi29 DNA polymerase buffer, and reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT).

[0027] (5) React at room temperature for 12 hours, and then heat to 65 °C for 10 minutes to terminate the reaction. Centrifuge and wash the reaction product three times with ultrapure water, and store the final solution in the dark at 4 °C for later use.

[0028] Example 2:

[0029] Binding of Nano Gold Probe to Three-Dimensional Flower Ball-Type Nano DNA Structure

[0030] (1) Dissolve 0.08 mmol of chloroauric acid (HAuCl4) in 25 mL of ultrapure water. After stirring evenly, add 0.01 mmol of trisodium citrate as a reducing agent, heat to boiling and keep stirring until the solution color turns wine red, indicating the formation of nano gold particles.

[0031] (2) After cooling the nano gold solution to room temperature, add 10 μL of DNA probe modified with thiol (concentration: 1.0×10 -5 M), and incubate at room temperature for 2 hours to allow the DNA probe to bind to the nano gold surface through thiol.

[0032] (3) Wash the gold nanoprobe three times by centrifugation (12,000 rpm, 15 minutes) to remove unbound DNA probes, and finally resuspend the product in ultrapure water and store it in the dark at 4 °C for later use.

[0033] (4) Mix 10 μL of the three-dimensional flower-like nano DNA structure with 200 μL of the gold nanoprobe and incubate at room temperature for 6 hours.

[0034] (5) Centrifuge and wash the reaction product three times with ultrapure water, and resuspend the product in ultrapure water. Use a fluorescence spectrometer to detect the fluorescence curve, repeat the detection at least three times, and record the fluorescence intensity.

[0035] Example 3:

[0036] Bacterial culture and DNA amplification

[0037] (1) Use a modified Postgate medium (complete medium): Add 0.5 g of Na2SO4, 0.5 g of K2HPO4, 1 g of NH4Cl, 0.1 g of CaCl2, 2 g of MgSO4, 1 g of yeast powder, and 4 mL of sodium lactate to 1 L of filtered seawater at natural pH. After autoclaving, add L-cysteine with a final concentration of 0.5 g / L by filter sterilization. Inoculate the strain at an inoculation amount of 1% and culture at 30 °C.

[0038] (2) Use PCR to amplify the target region to generate a large amount of single-stranded target DNA. The specific steps are as follows: template DNA, 2 μL; 2×EasyTaq PCR SuperMix, 50 μL; ultrapure water, 41.5 μL; dNTPs, 2 μL; forward primer, 2.5 μL; reverse primer, 2 μL (either the forward or reverse primer is in relative excess). Thermal cycling conditions: Pre-denature at 95 °C for 5 minutes, then denature at 95 °C for 30 seconds, anneal at 55 °C for 30 seconds, for a total of 30 cycles, and finally extend at 72 °C for 5 minutes.

[0039] The present invention covers any substitutions, modifications, equivalent methods, and schemes made within the essence and scope of the present invention. For the public to have a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, and those skilled in the art can fully understand the present invention without these detailed descriptions. In addition, well-known methods, processes, procedures, components, and circuits are not described in detail to avoid unnecessary confusion to the essence of the present invention.

[0040] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A self-assembled three-dimensional flower ball-shaped dual-mode DNA sensor based on rolling circle amplification technology for rapid determination of early fouling microorganisms, characterized in that, Comprising: A padlock probe for specifically recognizing target DNA; Target DNA for detecting specific fouling microorganisms; A fluorescent nanogold probe, with a fluorescent nanogold signal marker modified at one end and complementary to the rolling circle amplification product sequence at the other end; a nanoflower spherical DNA sensor self-assembled through rolling circle amplification reaction under the action of T4 DNA ligase and phi29 DNA polymerase for detecting target DNA.

2. The DNA sensor according to claim 1, wherein The fluorescent nanogold probe forms a stable nanoflower structure by binding to the complementary sequence of the rolling circle amplification product.

3. The DNA sensor according to claim 1, characterized in that, The intensity of the fluorescent signal is positively correlated with the concentration of target DNA, and the fluorescent signal is measured by a fluorescence spectrophotometer to achieve qualitative and quantitative detection of target DNA.

4. The DNA sensor according to claim 1, characterized in that, The nanoflower structure causes an increase in fluorescent signal and a decrease in color signal, and the detection signal is corrected by comparing the fluorescent and color signals.

5. The DNA sensor according to claim 1, wherein The target DNA is a specific nucleic acid sequence of microorganisms, including but not limited to the nucleic acid sequence of sulfate-reducing bacteria (SRB), and can be used for rapid detection of common fouling microorganisms.

6. The DNA sensor according to claim 1, wherein The two-arm sequence of the padlock probe can specifically bind to target DNA and form a circular structure under the action of T4 DNA ligase.

7. The DNA sensor according to claim 1, wherein The fluorescent nanogold signal marker is quantum dot fluorescent nanogold, which has high fluorescence stability and high sensitivity.

8. The DNA sensor according to claim 1, wherein The detection platform of the sensor further includes a self-calibrating fluorescent DNA probe for correcting the deviation of the detection signal.

9. The DNA sensor according to claim 1, characterized in that, The detection method of the sensor includes the following steps: S1. Extract DNA from the target sample; S2. Mix the extracted DNA with the padlock probe and the fluorescent nanogold probe; S3. Perform a rolling circle amplification reaction under the action of T4 DNA ligase and phi29 DNA polymerase; S4. Measure the intensity of the fluorescent signal, and correct the detection signal by comparing the fluorescent and color signals; S5. Qualitatively and quantitatively detect target DNA according to the correlation between the fluorescent signal intensity and the concentration of target DNA.

10. The DNA sensor according to claim 1, wherein The sensor is used for detecting early fouling microorganisms, has high universality, and can be applied to the detection of specific nucleic acid sequences of various fouling microorganisms, including but not limited to sulfate-reducing bacteria (SRB).

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