Electrochemical detection method for escherichia coli based on deoxyribozyme

Abstract

The invention discloses an electrochemical detection method for escherichia coli based on deoxyribozyme, and belongs to the technical field of analysis and detection. The method comprises the following steps: reducing sulfydryl of a deoxyribozyme-substrate connector, dropwise adding the reduced sulfydryl to a working electrode in a silk-screen printing gold electrode to immobilize deoxyribozyme, and dropwise adding 6-mercaptohexanol on the surface of the working electrode to seal unincubated sites on the surface of the gold electrode; dropping an escherichia coli intracellular mixture CIM prepared by cell disruption onto the silk-screen printing gold electrode, enabling the mixed solution to cover the working electrode, the counter electrode and the reference electrode, connecting an electrochemical workstation, and detecting the peak position and intensity. When deoxyribozyme is combined with an escherichia coli target, RNA in a deoxyribozyme-substrate connector is subjected to enzyme digestion, a substrate with methylene blue falls off, a methylene blue group is far away from the silk-screen printing gold electrode, an electric signal disappears subsequently, and rapid detection of escherichia coli in the environment is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and in particular relates to an electrochemical detection method for Escherichia coli based on deoxyribozyme. Background Art

[0002] E. coli is one of the common harmful bacteria in the intestines of humans and animals. Infection with E. coli in people with low immunity can cause symptoms such as urinary tract infection and gastrointestinal damage. The National Health Statistics Report shows that urinary tract infection is the second most common infection in the human body, with about 80,000 visits to the doctor each year. The most common infectious strain is the E. coli strain, which accounts for about 80% of urinary tract infection cases in outpatient clinics. Pathogenic strains such as E. coli O157:H7 produce toxins that damage the intestinal lining, causing anemia, stomach cramps and bloody diarrhea.

[0003] At present, E. coli detection mainly includes culture method and molecular biology method. Using culture method to count bacterial colonies is a common means for E. coli detection. This method can obtain accurate and effective results, but it has limitations for field applications. For example, this method requires a long time of culture to form colonies, which is difficult to meet field needs. Molecular biology method is also one of the common methods for E. coli detection. It has fast detection speed, good selectivity and low detection limit, but due to the expensive experimental instruments and complicated operation, it is still difficult to meet immediate needs. Therefore, although these methods are very effective in conventional indoor analysis, they are difficult to apply in daily field use due to complex operation, expensive instruments, and time-consuming detection. Therefore, it is necessary to develop a simple and rapid method for detecting E. coli.

[0004] With the continuous development of modern molecular biology, researchers have discovered that in addition to the classic Watson-Crick base pairing, some nucleic acid molecules also have the function of specifically binding or catalyzing specific target molecules. Deoxyribozymes (DNAzymes) are nucleic acid fragments with recognition, catalysis and regulation functions obtained through in vitro screening technology. For example, in a research result published in 2023, the research group of Professor Liu Meng of Dalian University of Technology obtained the deoxyribozymes (acidic RNA-cleaving DNAzymes) that cut RNA at pH 5.3 for the first time through in vitro screening, and named them aRCD-EC1 (In Vitro Selection of M 2+-Independent, Fast-Responding Acidic Deoxyribozymes for Bacterial Detection;Qinbin Zhou, Guangxiao Zhang, Yunping Wu, Qiang Zhang, Yi Liu, Yangyang Chang, and Meng Liu;Journal of the American Chemical Society 2023 145(39), 21370-21377;DOI:10.1021 / jacs.3c06155). The enzyme can be activated by Escherichia coli (E. coli) at pH 5.3. Based on the recognition and catalytic function of deoxyribozymes, a series of fluorescent, colorimetric and electrochemical sensors can be developed for the detection of pathogens in urine, feces and other environments.

[0005] Electrochemical biosensors are mainly composed of biorecognition elements, signal transduction devices, and signal output devices. The biorecognition element is fixed on the working electrode, and the biorecognition molecule specifically binds to the target. The signal conversion element can be used to obtain electrical signals, thereby achieving low detection limits and high sensitivity detection of the bound target. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide an electrochemical detection method for Escherichia coli based on DNAzyme. On the basis of the screened Escherichia coli DNAzyme-substrate connector, the present invention modifies the thiol group at the 3' end of the sequence so that the DNAzyme can be fixed on the screen-printed gold electrode, and modifies the methylene blue electrochemical group (Methylene Blue) at the 5' end. The methylene blue group can produce an obvious oxidation peak at about -0.2V. When the DNAzyme binds to the Escherichia coli target, the RNA in the DNAzyme-substrate connector is cleaved, the substrate with methylene blue falls off, the methylene blue group moves away from the screen-printed gold electrode, and the electrical signal disappears subsequently, thereby realizing rapid detection of Escherichia coli in the environment.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a DNAzyme-based electrochemical detection method for Escherichia coli, comprising the following steps:

[0009] (1) Preparation of E. coli intracellular mixture CIM: centrifuge the sample solution to be tested at 6000-10000 rpm, discard the supernatant, resuspend the obtained precipitate in the detection auxiliary solution, ultrasonically disrupt, centrifuge the obtained bacterial suspension at 2000-4000 rpm, take the supernatant, and heat it at 80-95°C for 2-10 min to obtain;

[0010] (2) Incubation and fixation of DNAzyme: The thiol group of the DNAzyme-substrate linker is reduced and added to the working electrode in the screen-printed gold electrode to fix the DNAzyme. 6-Mercaptohexanol (MCH) is added to the surface of the working electrode to block the unincubated sites on the gold electrode surface.

[0011] (3) Signal detection: The E. coli intracellular mixture CIM prepared in step (1) is dropped onto the screen-printed gold electrode obtained in step (2) so that the mixed solution covers the working electrode, the counter electrode and the reference electrode. The electrochemical workstation is connected to detect the peak position and intensity.

[0012] In the above technical solution, further, the detection auxiliary solution in step (1) is composed of: 50mM NaAc, 20mM EDTA, 150mM NaCl, 50mM KCl and 0.01% Tween 20, and the balance is water.

[0013] In the above technical solution, further, the pH of the detection auxiliary liquid described in step (1) is controlled at 5-6.

[0014] In the above technical solution, further, the specific process of the ultrasonic crushing in step (1) is: ultrasonic crushing for 20 to 40 seconds, freezing in ice for 1 to 3 minutes, and repeating this operation 5 to 10 times.

[0015] In the above technical solution, further, the centrifugation time in step (1) is controlled within 5 to 30 minutes.

[0016] In the above technical solution, further, the structure of the DNAzyme-substrate connector in step (2) is as follows: MB- TTTTTTCTATGAACTGACT / rA / TGACCTCACTACCAAG CAGGTCCATCGAGTGGTAGGATGCGGCGGTCAGTCGCACTGCTCCT-SH, the underlined nucleotide sequence part is the substrate chain, and the other nucleotide sequence parts are deoxyribozymes. The 5' end is modified with MB methylene blue electrochemical group, and the 3' end is modified with thiol. The substrate chain contains a deoxyribozyme cleavage site RNA—rA.

[0017] In the above technical solution, further, the specific process of the reduction described in step (2) is to mix the deoxyribozyme-substrate linker with tri(2-carboxyethyl)phosphine (TCEP) in the dark and at room temperature for reduction, the molar ratio of the deoxyribozyme-substrate linker to TCEP is 1:50-200, the volume of the mixed liquid added to the working electrode is 5-30 μL, and the volume of the 6-mercaptohexanol added is 5-30 μL.

[0018] In the above technical solution, further, the volume of the Escherichia coli intracellular mixture CIM added dropwise in step (3) is 100-200 μL.

[0019] In the above technical solution, further, in step (3), the experimental group to which the detection auxiliary liquid is added is used as a control.

[0020] In the above technical solution, further, the detection conditions of the electrochemical workstation in step (3) are:

[0021] Working mode: Square wave voltammetry,

[0022] Starting voltage: -0.6~-0.4V,

[0023] End voltage: 0~0.2V,

[0024] Potential increment: 0.001~0.005V,

[0025] Amplitude: 0.010~0.040V,

[0026] Frequency: 10~30Hz,

[0027] Standing time: 1 to 5 seconds,

[0028] Sensitivity: 0.0000001~0.0001.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The detection method of the present invention has a simple detection process, the equipment used is inexpensive and easy to carry, the reagents are safe and non-toxic, and Escherichia coli can be detected quickly, sensitively and highly selectively, effectively reducing the detection cost, being easy to promote and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0032] Figure 1 Schematic diagram of the screen-printed gold electrode used in Example 1.

[0033] Figure 2 It is a schematic diagram of the principle of the detection method of the present invention.

[0034] Figure 3 This is a diagram showing the feasibility test results of the detection method in Example 1.

[0035] Figure 4 This is a diagram of the selectivity test results of the detection method in Example 2. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0037] Unless otherwise specified, the materials and reagents in the examples can be obtained from commercial sources.

[0038] The detection auxiliary solution in the embodiment: 50 mM NaAc, 20 mM EDTA, 150 mM NaCl, 50 mM KCl and 0.01% Tween 20 were dissolved in 50 mL sterile water, dissolved by ultrasonication, adjusted to pH 5.3, and stored at 4°C.

[0039] Example 1

[0040] This embodiment provides a DNAzyme-based electrochemical detection method for Escherichia coli and a process for detecting Escherichia coli, which specifically includes the following steps:

[0041] Step 1: Cultivation of E. coli: Add 10 μL of E. coli BL21 to 5 mL of LB medium and culture overnight. The temperature of the shaker is 37°C and the shaking speed is 150 rpm. After overnight culture, the concentration of E. coli is measured. When the OD 600 =1, remove the E. coli from the shaker.

[0042] Step 2: Preparation of E. coli CIMs: Take out 1 mL of E. coli suspension (OD 600 =1), centrifuge for 10 min (8000 rpm, 4°C), discard the supernatant, resuspend the cell mass deposited at the bottom in 300 μL reaction buffer (detection auxiliary liquid), break the suspension in an ultrasonic machine for 30 s, place it in ice and freeze it for 2 min, repeat this operation 6 times, place the suspension in a centrifuge and centrifuge it again for 10 min (3000 rpm, 4°C), aspirate the supernatant, place it at 90°C and heat it for 5 min to obtain the corresponding CIM-EC, and the prepared CIMs are stored in a -20°C refrigerator.

[0043] Step 3: Incubation and fixation of DNAzyme: DNAzyme-substrate linker and tri(2-carboxyethyl)phosphine (TCEP) were mixed at a molar ratio of 1:100 in the dark and at room temperature to reduce the thiol group, 10 μL of the mixture was added dropwise to the working electrode in the screen-printed gold electrode to fix the DNAzyme, and 10 μL of 6-mercaptohexanol (MCH) was added dropwise to the surface of the working electrode to block the unincubated sites on the gold electrode surface;

[0044] The structure of the DNAzyme-substrate connector is as follows: MB- TTTTTTCTATGAACTGACT / rA / TGACCTCACTACCAAG CAGGTCCATCGAGTG GTAGGATGCGGCGGTCAGTCGCACTGCTCCT-SH, the underlined nucleotide sequence part is the substrate chain, and the other nucleotide sequence parts are deoxyribozymes. The 5' end is modified with MB methylene blue electrochemical group, the 3' end is modified with thiol group, and the substrate chain contains a deoxyribozyme cleavage site RNA-rA.

[0045] Step 4: Signal detection: Set up experimental and control groups; in the experimental group, drop 100 μL of E. coli CIM onto the screen-printed gold electrode so that the mixed solution covers the working electrode, counter electrode and reference electrode, and connect the electrochemical workstation; in the blank control group, drop 100 μL of detection auxiliary liquid. Set SWV (square wave voltammetry) parameters for electrochemical detection: starting voltage: -0.4V, end voltage: 0V, potential increment: 0.004V, amplitude: 0.020V, frequency: 15Hz, standing time: 2s, sensitivity: 0.0000001.

[0046] The results are as follows Figure 3 As shown, in the experimental group, due to the binding of the DNAzyme to the Escherichia coli target, the substrate with methylene blue fell off, the methylene blue group was away from the screen-printed gold electrode, and the oxidation peak at the -0.2V position was lower than that of the blank group, indicating that this method is feasible.

[0047] Example 2 Selectivity of the detection method

[0048] Step 1: Bacterial culture: Add 10 μL of E. coli to 5 mL of LB medium and culture overnight. The temperature of the shaker is 37°C and the shaking speed is 150 rpm. After overnight culture, the concentration of E. coli is measured. When the OD 600 =1, remove the E. coli from the shaker.

[0049] The culture methods of Pseudomonas aeruginosa, Bacillus cereus, Porphyromonas gingivalis, and Streptococcus mutans are the same as the above-mentioned culture method of Escherichia coli.

[0050] Step 2: Preparation of CIMs: Take out 1 mL of E. coli suspension (OD 600=1), centrifuge for 10 min (8000 rpm, 4°C), discard the supernatant, resuspend the cell mass deposited at the bottom in 300 μL reaction buffer (detection auxiliary liquid), break the suspension in an ultrasonic machine for 30 s, place it in ice cubes and freeze it for 2 min, repeat this operation 6 times, place the suspension in a centrifuge and centrifuge it again for 10 min (3000 rpm, 4°C), absorb the supernatant, place it in 90°C and heat it for 5 min to obtain the corresponding CIM, and the obtained CIMs are stored in a -20°C refrigerator. The preparation method of CIM for Pseudomonas aeruginosa, Bacillus cereus, Porphyromonas gingivalis, and Streptococcus mutans is the same as above.

[0051] Step 3: Incubation and fixation of DNAzyme: The DNAzyme-substrate linker was mixed with TCEP at a molar ratio of 1:100 in the dark and at room temperature to reduce the thiol group. 10 μL of the mixture was added to the working electrode in the screen-printed gold electrode to fix the DNAzyme. 10 μL of 6-mercaptohexanol (MCH) was added to the surface of the working electrode to block the unincubated sites on the gold electrode surface.

[0052] Step 4: Signal detection: In the experimental group, 100 μL of E. coli CIM was dropped onto the screen-printed gold electrode so that the mixed solution covered the working electrode, the counter electrode and the reference electrode, and then connected to the electrochemical workstation. The SWV (square wave voltammetry) parameters were set for electrochemical detection: starting voltage: -0.4 V, end voltage: 0 V, potential increment: 0.004 V, amplitude: 0.020 V, frequency: 15 Hz, standing time: 2 s, sensitivity: 0.0000001. The signal detection methods for Pseudomonas aeruginosa, Bacillus cereus, Porphyromonas gingivalis and Streptococcus mutans were the same as above.

[0053] The results are as follows Figure 4 As shown, the current density of the Escherichia coli experimental group is significantly higher than that of the Pseudomonas aeruginosa group, the Bacillus cereus group, the Porphyromonas gingivalis group and the Streptococcus mutans group, indicating that the detection method can specifically detect Escherichia coli, indicating that the detection method of the present invention has very excellent selectivity.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A DNAzyme-based electrochemical detection method for Escherichia coli, characterized in that: The following steps are involved: (1) Preparation of E. coli intracellular mixture CIM: centrifuge the sample solution to be tested at 6000-10000 rpm, discard the supernatant, resuspend the obtained precipitate in the detection auxiliary solution, ultrasonically disrupt, centrifuge the obtained bacterial suspension at 2000-4000 rpm, take the supernatant, and heat it at 80-95°C for 2-10 min to obtain; (2) Incubation and fixation of DNAzyme: The thiol group of the DNAzyme-substrate linker is reduced and added to the working electrode in the screen-printed gold electrode to fix the DNAzyme. 6-Mercaptohexanol (MCH) is added to the surface of the working electrode to block the unincubated sites on the gold electrode surface. (3) Signal detection: The E. coli intracellular mixture CIM prepared in step (1) is dropped onto the screen-printed gold electrode obtained in step (2) so that the mixed solution covers the working electrode, the counter electrode and the reference electrode. The electrochemical workstation is connected to detect the peak position and intensity.

2. The detection method according to claim 1, characterized in that: The detection auxiliary solution described in step (1) is composed of: 50mM NaAc, 20mM EDTA, 150mM NaCl, 50mM KCl and 0.01% Tween 20, and the balance is water. The pH of the detection auxiliary solution is controlled at 5-6.

3. The detection method according to claim 1, characterized in that: The specific process of the ultrasonic crushing in step (1) is: ultrasonic crushing for 20 to 40 seconds, freezing in ice for 1 to 3 minutes, and repeating this operation 5 to 10 times.

4. The detection method according to claim 1, characterized in that: The centrifugation time in step (1) is controlled within 5 to 30 minutes.

5. The detection method according to claim 1, characterized in that: The structure of the DNAzyme-substrate linker in step (2) is as follows: MB- TTTTTTCTATGAACTGACT / rA / TGACCTCA CTACCAAG CAGGTCCATCGAGTGGTAGGATGCGGCGGTCAGTCGCACTGCTCCT-SH, the underlined nucleotide sequence part is the substrate chain, and the other nucleotide sequence parts are deoxyribozymes. The 5' end is modified with MB methylene blue electrochemical group, the 3' end is modified with thiol group, and the substrate chain contains a deoxyribozyme cleavage site RNA-rA.

6. The detection method according to claim 1, characterized in that: The specific process of the reduction described in step (2) is to mix the deoxyribozyme-substrate linker with tri(2-carboxyethyl)phosphine (TCEP) in the dark and at room temperature for reduction, the molar ratio of the deoxyribozyme-substrate linker to TCEP is 1:50-200, the volume of the mixed liquid added to the working electrode is 5-30 μL, and the volume of 6-mercaptohexanol added is 5-30 μL.

7. The detection method according to claim 1, characterized in that: The volume of the E. coli intracellular mixture CIM added dropwise in step (3) is 100-200 μL.

8. The detection method according to claim 1, characterized in that: In step (3), the experimental group to which the detection auxiliary liquid was added was used as a control.

9. The detection method according to any one of claims 1 to 8, characterized in that: The detection conditions of the electrochemical workstation in step (3) are: Working mode: Square wave voltammetry, Starting voltage: -0.6~-0.4V, End voltage: 0~0.2V, Potential increment: 0.001~0.005V, Amplitude: 0.010~0.040V, Frequency: 10~30Hz, Standing time: 1 to 5 seconds, Sensitivity: 0.0000001~0.0001.

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