Preparation and application of a 5-aminolevulinic acid biosensor

Abstract

The invention discloses the preparation and application of a 5-aminolevulinic acid biosensor, relates to the technical field of biosensors, and comprises the following steps: step one: design and synthesize an RNA aptamer sequence with high affinity and specificity for 5-ALA; step two: combine the RNA aptamer sequence with the expression regulation region of a fluorescent reporter gene to construct a riboswitch structure; step three: clone the riboswitch structure into an expression vector, transform Escherichia coli or yeast; step four: screen and optimize the engineering strains for efficient expression; step five: immobilize the engineering strains on a biocompatible material to prepare a biosensor chip. In the present invention, an RNA aptamer capable of specifically recognizing 5-ALA is designed and combined with the expression regulation of a fluorescent reporter gene to achieve rapid and sensitive detection of 5-ALA.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, in particular to the preparation and application of a 5-aminolevulinic acid biosensor. Background Art

[0002] 5-Aminolevulinic acid (5-ALA) is an important bioactive molecule with wide applications in many fields. In the medical field, 5-ALA is used as a photosensitizer for photodynamic diagnosis and therapy, especially in the diagnosis and treatment of brain tumors such as malignant gliomas. In the agricultural field, 5-ALA, as a plant growth regulator, can promote crop growth, increase yield and stress resistance. In the field of biotechnology, 5-ALA is an important precursor for the synthesis of porphyrin compounds;

[0003] At present, the detection of 5-ALA mainly relies on instrumental analysis methods such as high performance liquid chromatography (HPLC) and mass spectrometry (MS). Although these methods are highly accurate, they have the following limitations: 1. The instruments are expensive, the operation is complicated, and professional personnel are required; 2. The sample pretreatment is cumbersome and the detection cycle is long; 3. It is difficult to achieve real-time and in-situ detection; 4. It is not suitable for rapid screening and high-throughput analysis. Therefore, it is of great significance to develop a simple, rapid and sensitive method for 5-ALA detection.

[0004] In recent years, the rapid development of biosensor technology has provided new ideas for solving this problem. Among them, riboswitch-based biosensors have shown great potential in the field of metabolite detection due to their high specificity and rapid response.

[0005] Riboswitches are a class of RNA elements that can specifically bind to small molecule ligands and regulate gene expression. They are usually composed of two functional domains: an aptamer domain that can specifically bind to ligands and an expression platform that controls gene expression. When a specific ligand is present, the riboswitch undergoes a conformational change, thereby regulating the transcription or translation of downstream genes. This feature makes riboswitches an ideal choice for designing biosensors.

[0006] The present invention aims to develop a new biosensor for 5-ALA detection using riboswitch technology. By designing an RNA aptamer that can specifically recognize 5-ALA and combining it with the expression regulation of a fluorescent reporter gene, rapid and sensitive detection of 5-ALA can be achieved. This method is expected to overcome the limitations of traditional detection methods and provide new technical support for the detection and application research of 5-ALA. Summary of the invention

[0007] The present invention aims to solve the following technical problems: 1. Develop a highly sensitive and highly specific 5-ALA biosensor; 2. Realize rapid and real-time detection of 5-ALA; 3. Reduce detection costs and simplify operation procedures; 4. Improve detection stability and repeatability; 5. Realize the possibility of portable and in-situ detection.

[0008] To achieve the above object, the present invention provides the following technical solution: a method for preparing a 5-aminolevulinic acid biosensor, comprising the following steps:

[0009] Step 1: Design and synthesize RNA aptamer sequences with high affinity and specificity for 5-ALA;

[0010] Step 2: combining the RNA aptamer sequence with the expression regulatory region of the fluorescent reporter gene to construct a riboswitch structure;

[0011] Step 3: Clone the riboswitch construct into an expression vector and transform E. coli or yeast;

[0012] Step 4: Screening and optimizing to obtain highly efficient expression engineering strains;

[0013] Step 5: Immobilize the engineered strain on a biocompatible material to prepare a biosensor chip.

[0014] Preferably, in the step 1, an RNA aptamer sequence with high affinity and specificity for 5-ALA is designed and synthesized by a combination of computer-aided design and in vitro screening, wherein the computer-aided design screens out sequences that can specifically bind to 5-ALA from a random RNA library by systematic evolution ligand index enrichment technology, and during the screening process, gradient elution and competitive binding strategies are used to obtain aptamers with high affinity and high specificity.

[0015] Preferably, in step 2, the secondary structure of RNA is designed, and the fluorescent reporter gene is GFP or luciferase.

[0016] Preferably, in step 2, RNA folding prediction software and an in vitro transcription-translation system are used to optimize the performance of the riboswitch, wherein the RNA folding prediction software includes MXfold2 and RNAfold web server, and the in vitro transcription-translation system uses DNA containing RNA polymerase as a template to synthesize RNA through the principle of complementary base pairing to simulate the transcription process in vivo.

[0017] Preferably, in step three, the optimized riboswitch structure is cloned into an appropriate expression vector, wherein for the E. coli expression system, a pET series vector is selected, and for the yeast expression system, a pPICZ series vector is selected.

[0018] Preferably, in step 4, the constructed expression vector is transformed into the screened host strain, and for Escherichia coli, the BL21 (DE3) strain is selected, and for the yeast expression system, the Pichia pastoris X-33 strain is selected.

[0019] Preferably, the screening process includes antibiotic screening, monoclonal isolation, and small-scale induced expression testing.

[0020] Preferably, the culture conditions are optimized, including parameters such as culture medium composition, temperature, pH value, and induction time.

[0021] Preferably, in step five, the immobilization process adopts a cross-linking fixation method, using glutaraldehyde as a cross-linking agent to fix the bacteria on a porous silica gel or agarose gel carrier.

[0022] Preferably, a method for preparing a 5-aminolevulinic acid biosensor is used, and the 5-aminolevulinic acid biosensor is used as a photosensitizer for photodynamic diagnosis and treatment in the medical field, a plant growth regulator in the agricultural field, and the detection and analysis of porphyrin compound precursors in the biotechnology field.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] High sensitivity: The biosensor can detect 5-ALA at the nanomolar level, with a sensitivity superior to traditional detection methods. By optimizing the RNA aptamer sequence and riboswitch structure, accurate detection of low-concentration 5-ALA is achieved, making early diagnosis and trace analysis possible;

[0025] High specificity: RNA aptamer sequences that have undergone multiple rounds of screening and optimization have extremely high selectivity for 5-ALA. Even in complex biological samples, they can effectively distinguish 5-ALA from structural analogs, greatly reducing the false positive rate and improving the reliability of detection;

[0026] Rapid detection: Compared with traditional chromatography and mass spectrometry methods, this biosensor can complete the quantitative analysis of 5-ALA within minutes. The fast-response riboswitch system reduces the detection time from hours to minutes, greatly improving the detection efficiency;

[0027] Low cost: This method does not require expensive instruments and professional operators, and can complete the analysis by simple fluorescence detection. The mass production of biosensor chips further reduces the cost of a single test, making it an economical and affordable 5-ALA detection solution;

[0028] Real-time monitoring: The dynamic response characteristics of the riboswitch enable the sensor to achieve continuous and real-time monitoring of 5-ALA concentration. This feature has important application value in the fields of metabolic engineering and fermentation process control, and can be used to optimize the production process of 5-ALA;

[0029] Portability: The miniaturized design of the sensor chip makes it easy to integrate into portable devices. Combined with microfluidics technology, a handheld 5-ALA detection instrument can be developed to facilitate rapid on-site detection and immediate diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the overall preparation process of the present invention. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0032] An embodiment provided by the present invention is a method for preparing a 5-aminolevulinic acid biosensor, comprising the following steps:

[0033] Step 1: Design and synthesize RNA aptamer sequences with high affinity and specificity for 5-ALA;

[0034] RNA aptamer sequences with high affinity and specificity for 5-ALA were designed and synthesized by combining computer-aided design and in vitro screening. Sequences that can specifically bind to 5-ALA were screened from a random RNA library by systematic evolution ligand index enrichment technology. During the screening process, gradient elution and competitive binding strategies were used to obtain aptamers with high affinity and specificity.

[0035] By designing and optimizing RNA aptamer sequences, accurate recognition and detection of 5-ALA can be achieved, detection sensitivity can be increased to the nanomolar level, and interference from other molecules can be minimized;

[0036] Step 2: combining the RNA aptamer sequence with the expression regulatory region of the fluorescent reporter gene to construct a riboswitch structure;

[0037] RNA folding prediction software and an in vitro transcription and translation system are used to optimize the performance of the riboswitch. The RNA folding prediction software includes MXfold2 and RNAfold web server. The in vitro transcription and translation system uses DNA containing RNA polymerase as a template to synthesize RNA through the principle of base complementary pairing, simulates the transcription process in vivo, and is used to design the secondary structure of RNA. Its fluorescent reporter gene is GFP or luciferase, ensuring that the riboswitch can effectively inhibit the expression of the reporter gene in the absence of 5-ALA, and in the presence of 5-ALA, the riboswitch conformation changes, thereby releasing the inhibition of the reporter gene expression.

[0038] By utilizing the rapid response characteristics of the riboswitch, the detection time is shortened from hours to minutes, thus achieving dynamic and continuous monitoring of 5-ALA concentration.

[0039] Step 3: Clone the riboswitch construct into an expression vector and transform E. coli or yeast;

[0040] The optimized riboswitch structure was cloned into an appropriate expression vector, wherein the pET series vectors were selected for the E. coli expression system, and the pPICZ series vectors were selected for the yeast expression system;

[0041] The pET vector uses the strong transcription and translation system of T7 bacteriophage to efficiently express the target gene in the host cell. T7 RNA polymerase synthesizes mRNA 5 times faster than E. coli RNA polymerase, making the expression level of the target gene very high, and almost all cell resources are used to express the target protein;

[0042] The pET vector is regulated by the T7 promoter, which is completely dependent on the activity of T7 RNA polymerase. In the absence of T7 RNA polymerase, the target gene is in a closed state and will not be expressed, thus avoiding the toxicity of the target gene expression protein to the host bacteria and ensuring the stability of the plasmid;

[0043] The pPICZ series vectors use the methanol-induced AOX1 promoter to achieve high-level expression of target genes in Pichia pastoris. This expression system has a strict regulatory mechanism to ensure efficient expression under methanol induction;

[0044] pPICZ series vectors contain multiple commonly used endonuclease sites to facilitate the cloning of different genes. In addition, the vector also contains the Zeocin resistance gene, which can be used for screening in Escherichia coli and Pichia pastoris;

[0045] The pPICZ series vectors are suitable for a variety of Pichia yeast strains, including X33, GS115, SMD1168H, KM71H, etc., which allows experimenters to choose the appropriate strain for experiments as needed;

[0046] The pPICZ series vectors have the characteristics of high copy number and can stably express the target gene in Pichia pastoris, ensuring the reliability and repeatability of experimental results;

[0047] By integrating the riboswitch and reporter gene system, no complicated instruments and equipment are required, which greatly reduces the detection cost and simplifies the operation steps, making it suitable for various laboratory environments;

[0048] Step 4: Screening and optimizing to obtain highly efficient expression engineering strains;

[0049] The constructed expression vector was transformed into the selected host strain. For E. coli, the BL21 (DE3) strain was selected, which lacks lon and ompT proteases and is conducive to the stable expression of foreign proteins. For the yeast expression system, the Pichiapastoris X-33 strain was selected, which has the characteristics of methanol-induced expression and is convenient for controlling the protein expression time.

[0050] The screening process includes antibiotic screening, monoclonal isolation, small-scale induced expression testing, and optimization of culture conditions, including parameters such as medium composition, temperature, pH, and induction time;

[0051] Optimize the structural design and expression system of riboswitches to improve the stability of biosensors and ensure good consistency and repeatability of test results in different batches and conditions;

[0052] Step 5: Immobilizing the engineered strain on a biocompatible material to prepare a biosensor chip;

[0053] The immobilization process adopts the cross-linking fixation method, using glutaraldehyde as a cross-linking agent to fix the bacteria on a porous silica gel or agarose gel carrier; this immobilization method not only ensures the activity of the bacteria, but also improves the stability and reusability of the sensor;

[0054] By combining the riboswitch system with microfluidics technology, a miniaturized and integrated detection device is developed to achieve portable detection and on-site analysis of 5-ALA and expand its application range;

[0055] Specifically, the core of the present invention is to design a 5-ALA-responsive riboswitch. When 5-ALA is present in the environment, the RNA aptamer binds to it, causing the conformation of the riboswitch to change, thereby activating the expression of the downstream fluorescent reporter gene. The fluorescence intensity is positively correlated with the 5-ALA concentration, and the quantitative analysis of 5-ALA can be achieved by detecting the fluorescence signal;

[0056] The 5-aminolevulinic acid biosensor obtained by the above preparation method can be applied to photosensitizers for photodynamic diagnosis and treatment in the medical field, plant growth regulators in the agricultural field, and detection and analysis of porphyrin compound precursors in the biotechnology field.

[0057] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for preparing a 5-aminolevulinic acid biosensor, comprising the following steps: Step 1: Design and synthesize RNA aptamer sequences with high affinity and specificity for 5-ALA; Step 2: combining the RNA aptamer sequence with the expression regulatory region of the fluorescent reporter gene to construct a riboswitch structure; Step 3: Clone the riboswitch construct into an expression vector and transform E. coli or yeast; Step 4: Screening and optimizing to obtain highly efficient expression engineering strains; Step 5: Immobilize the engineered strain on a biocompatible material to prepare a biosensor chip.

2. The method for preparing a 5-aminolevulinic acid biosensor according to claim 1, characterized in that: In the step 1, an RNA aptamer sequence with high affinity and specificity for 5-ALA is designed and synthesized by a method combining computer-aided design and in vitro screening, wherein the computer-aided design screens out sequences that can specifically bind to 5-ALA from a random RNA library by systematic evolution ligand index enrichment technology, and during the screening process, gradient elution and competitive binding strategies are used to obtain aptamers with high affinity and high specificity.

3. The method for preparing a 5-aminolevulinic acid biosensor according to claim 1, characterized in that: In the step 2, the secondary structure of RNA is designed, and the fluorescent reporter gene is GFP or luciferase.

4. The method for preparing a 5-aminolevulinic acid biosensor according to claim 2, characterized in that: In the step 2, RNA folding prediction software and an in vitro transcription and translation system are used to optimize the performance of the riboswitch. The RNA folding prediction software includes MXfold2 and RNAfoldweb server. The in vitro transcription and translation system uses DNA containing RNA polymerase as a template to synthesize RNA through the principle of base complementary pairing to simulate the transcription process in vivo.

5. The method for preparing a 5-aminolevulinic acid biosensor according to claim 1, characterized in that: In the step three, the optimized riboswitch structure is cloned into an appropriate expression vector, wherein for the E. coli expression system, a pET series vector is selected, and for the yeast expression system, a pPICZ series vector is selected.

6. The method for preparing a 5-aminolevulinic acid biosensor according to claim 1, characterized in that: In the step 4, the constructed expression vector is transformed into the screened host strain. For Escherichia coli, the BL21 (DE3) strain is selected, and for the yeast expression system, the Pichia pastoris X-33 strain is selected.

7. The method for preparing a 5-aminolevulinic acid biosensor according to claim 6, characterized in that: The screening process includes antibiotic screening, single clone isolation, and small-scale induced expression testing.

8. The method for preparing a 5-aminolevulinic acid biosensor according to claim 7, characterized in that: The culture conditions are optimized, including culture medium composition, temperature, pH value, and induction time parameters.

9. The method for preparing a 5-aminolevulinic acid biosensor according to claim 8, characterized in that: In the step 5, the immobilization process adopts a cross-linking fixation method, using glutaraldehyde as a cross-linking agent to fix the bacteria on a porous silica gel or agarose gel carrier.

10. Application of the 5-aminolevulinic acid biosensor obtained by the preparation method according to claim 1, characterized in that: The 5-aminolevulinic acid biosensor is used in the detection and analysis of photosensitizers for photodynamic diagnosis and treatment in the medical field, plant growth regulators in the agricultural field, and porphyrin compound precursors in the biotechnology field.

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