Glow-type chemiluminescence sensor based on ABEI / Co2 + / GO-coated DNA hydrogel and application of glow-type chemiluminescence sensor

Abstract

The invention discloses a glow-type chemiluminescence sensor based on ABEI / Co < 2 + > / GO-coated DNA hydrogel and application of the glow-type chemiluminescence sensor. The glow type chemiluminescence sensor disclosed by the invention comprises an aptamer-primer compound, an annular template, a secondary primer, a Phi29 DNA polymerase, dNTPs, ABEI / Co < 2 + > / GO and a target object. According to the present invention, the formation of the DNA hydrogel is accelerated through the ABEI / Co < 2 + > / GO, and the diffusion of the reactant is delayed by using the DNA hydrogel so as to prolong the chemiluminescence reaction time, such that the glow-type chemiluminescence sensor is prepared, the flash-type chemiluminescence defect is effectively overcome, and the convenience is provided for the rapid on-site detection of the salmonella typhimurium.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a method based on ABEI / Co 2+ Glow-type chemiluminescent sensor based on / GO@DNA hydrogel and its application. Background Art

[0002] Salmonella typhimurium, as a ubiquitous foodborne pathogen, poses a serious threat to public health and can potentially cause serious diseases such as acute gastroenteritis and even sepsis. In view of this, it is particularly important to achieve efficient, accurate and rapid detection of this species in food. Although the microbial culture method has been established as a detection method specified in my country's national standards due to its intuitive principle, mature technology and high detection accuracy, its significant limitation is that the detection cycle is long, usually taking 3-5 days to complete, which is obviously lagging behind the demand for rapid response to foodborne pathogens in modern food safety management. Therefore, exploring and developing new technologies with high sensitivity, high accuracy and the ability to complete detection in a shorter time is of urgent significance for improving the efficiency of food safety detection and protecting public health.

[0003] Based on the limitations of existing detection methods, chemiluminescent biosensors have attracted much attention in the field of foodborne pathogen detection due to their advantages such as fast response speed and no need for external light source excitation. However, most chemiluminescent substrates, such as luminol and acridinium ester, exhibit flash chemiluminescence, that is, luminescence and annihilation are completed within a few seconds. The instantaneous luminescence characteristics often lead to poor detection accuracy and reproducibility.

[0004] DNA hydrogel is a three-dimensional network structure formed by highly cross-linked DNA chains in solution. Its dense network structure can effectively delay the diffusion of chemiluminescent reactants, thereby prolonging the chemiluminescence time and converting flash-type chemiluminescence into glow-type chemiluminescence. At present, there have been reports of glow-type chemiluminescence sensors based on DNA hydrogels. However, these DNA hydrogels mostly rely on the complementary base pairing between a large number of short DNA chains to form. This chemically synthesized DNA requires high-purity raw materials and professional instruments and equipment, and the resulting high cost of DNA synthesis limits its further promotion and application in the field of analysis and detection.

[0005] Rolling circle amplification (RCA) technology is an isothermal nucleic acid amplification technology. With its flexibility in DNA sequence design and constant temperature of reaction conditions, it has become an ideal platform for constructing DNA hydrogels. The ssDNA chains generated by this technology are rich in repeating units composed of RCA template complementary sequences in series, which endows DNA hydrogels with functionality and personalized customization potential. According to the aptamer sequence of the detection object, the corresponding primers and RCA reaction template sequences are designed. By utilizing the competitive binding between the aptamer, primer and target, DNA hydrogels can be generated under the induction of the target, supplemented by the corresponding detection signal output, thereby realizing sensitive detection of the target. However, the formation of RCA product DNA hydrogel usually takes 24 to 72 hours. The long gelation time brings many inconveniences to on-site rapid detection and cannot meet the on-site rapid detection of Salmonella typhimurium. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a 2+ / GO@DNA hydrogel glow-type chemiluminescent sensor and its application. 2+ / GO accelerates the formation of DNA hydrogel, and then uses DNA hydrogel to delay the diffusion of reactants to prolong the chemiluminescence reaction time, and prepares a glow-type chemiluminescence sensor, which effectively overcomes the defects of flash-type chemiluminescence and provides many conveniences for on-site rapid detection of Salmonella typhimurium.

[0007] The technical solution of the present invention is as follows:

[0008] ABEI / Co 2+ / GO@DNA hydrogel glow-type chemiluminescent sensor, the glow-type chemiluminescent sensor comprises an aptamer-primer complex, a circular template, a secondary primer, Phi29 DNA polymerase, dNTPs, ABEI / Co 2+ / GO and target;

[0009] The aptamer sequence in the aptamer-primer complex is shown in SEQ No. 1, and the primer sequence is shown in SEQ No. 2;

[0010] The template sequence is shown in SEQ No.3;

[0011] The secondary primer sequence is shown in SEQ No.4.

[0012] Furthermore, the ABEI / Co 2+ The preparation method of / GO is:

[0013] (1) ABEI solution and GO solution were mixed and incubated at 25°C and 1000 rpm for 12 h in the dark to obtain ABEI / GO;

[0014] (2) Then, cobalt chloride solution was added, and the mixture was incubated at 25°C and 1000 rpm in the dark for 3 h. The mixture was centrifuged at 12500 rpm for 20 min, the supernatant was discarded, and the mixture was washed with ultrapure water and redissolved to obtain ABEI / Co 2+ / GO.

[0015] Furthermore, in step (1), the ABEI solution is prepared by dissolving 27.633 mg of ABEI in 10 mL of 0.1 mol / L NaOH solution; the GO solution is prepared by dissolving 0.1 mg of GO in 1 mL of pure water and ultrasonically treating at 400 W for 1 h; the volume ratio of the ABEI solution to the GO solution is 0.1:1.

[0016] Furthermore, in step (2), the cobalt chloride solution is prepared by dissolving 237.9 mg of cobalt chloride hexahydrate in 100 mL of pure water; the volume ratio of the cobalt chloride solution to the GO solution is 0.25:1.

[0017] Furthermore, the preparation method of the aptamer-primer complex is:

[0018] (1) Mix 20 μL of 10 μmol / L aptamer and 20 μL of 10 μmol / L primer in a PCR tube, place in a PCR instrument, anneal at 95°C for 10 min, and then slowly cool to obtain an aptamer-primer complex;

[0019] (2) 14 μL of 5 U / μL exonuclease I was added thereto to enzymatically digest the unbound single-stranded DNA to prepare a purified aptamer-primer complex.

[0020] Furthermore, the preparation method of the circular template is as follows:

[0021] (1) 30 μL of 20 μmol / L circular primer and the chain template are mixed evenly, and hybridized by gradient cooling method to obtain the circular DNA precursor of the template;

[0022] The loop primer sequence is shown in SEQ No.5;

[0023] (2) Add 7.5 μL of 400 U / μL T 4DNA ligase was incubated at 37°C for 2 h and incubated at 65°C for 10 min to inactivate the enzyme; then 10 μL of 5U / μL ExoⅠ enzyme and 5 μL of 200U / μL ExoⅢ enzyme were added in sequence, first incubated at 37°C for 1 h, and then incubated at 80°C for 20 min to obtain a purified circular template.

[0024] An application of the glow type chemiluminescent sensor, wherein the glow type chemiluminescent sensor is used to detect Salmonella typhimurium.

[0025] Furthermore, the method for detecting Salmonella typhimurium using the glow-type chemiluminescent sensor comprises the following steps:

[0026] (1) Preparation of ABEI / Co 2+ / GO@DNA hydrogel;

[0027] (2) To ABEI / Co 2+ 100 μL of 0.01 mol / L H 2 O 2 After reacting in the solution for 1 min, the luminescent image was taken, and its RGB value was analyzed using Image J software. The RGB value was substituted into the standard curve to obtain the concentration of the target Salmonella typhimurium in the sample to be tested.

[0028] Further, in step (1), the ABEI / Co 2+ The preparation method of / GO@DNA hydrogel is:

[0029] (1) Take 54 μL of the prepared aptamer-primer complex, add 246 μL of the target to be detected, and incubate at 37°C and 600 rpm for 45 min to allow the aptamer to recognize and bind to the target. Then filter through a 0.22 μm filter membrane and collect the filtrate to obtain free primers;

[0030] (2) Take 30 μL of free primer, mix it with 15 μL of 3 μmol / L secondary primer, 3 μL of circular template, 2.5 μL of 25 mmol / L dNTPs, 1 μL of pure water, 6 μL of 10× phi29 DNA polymerase reaction buffer and 2.5 μL of 10 U / μL phi29 DNA polymerase, and incubate it at 37°C, 800 rpm for 25 min; then add 15 μL of ABEI / Co 2+ / GO, and incubated at 37 °C, 1050 rpm for 10 min to obtain ABEI / Co 2+ / GO@DNA hydrogel.

[0031] Furthermore, the standard curve is: Y=49.24+1.34*X, R2 =0.9942, wherein Y represents the RGB value, and X represents the logarithmic value of the concentration of Salmonella typhimurium.

[0032] The beneficial technical effects of the present invention are:

[0033] The present invention utilizes the complementary sequence of the aptamer of Salmonella typhimurium and the competitive binding of Salmonella typhimurium to its aptamer to release the free complementary sequence. The free complementary sequence in the system serves as the primer of RCA, triggers the RCA reaction, and generates a large amount of long-chain DNA under the action of the secondary primer. Therefore, only in the presence of Salmonella typhimurium will the RCA reaction be triggered to generate sufficient long-chain DNA for subsequent self-assembly into DNA hydrogel. By adding ABEI / Co to the RCA system 2+ / GO, the long-chain DNA in the system quickly gelled under the effects of electrostatic attraction, π-π stacking, coordination and physical entanglement. 2+ / GO is wrapped inside the hydrogel. When the concentration of target Salmonella typhimurium in the test sample is higher, the DNA hydrogel is larger and the captured ABEI / Co 2+ The more / GO there is, the stronger the chemiluminescence intensity is. Therefore, the chemiluminescent image can be taken by a smartphone, and the RGB value can be analyzed by Image J software to determine the concentration of Salmonella typhimurium. In addition, the sensor was also used for actual sample detection, and there was no significant difference between the detection results and the plate count results, which proved the excellent performance of the sensor. The constructed detection method has the advantages of simple operation, high selectivity and specificity, and is of great significance for practical application in the field of food safety.

[0034] The present invention adopts ABEI / Co 2+ The addition of / GO into the system significantly accelerates the formation of DNA hydrogel and can effectively shorten the detection time.

[0035] The present invention utilizes the ability of DNA hydrogel to delay the diffusion of reactants to convert flash-type chemiluminescence into glow-type chemiluminescence. No precise and expensive chemiluminescence instrument is required. The chemiluminescence intensity can be measured by taking a photo with a smartphone and analyzing the RGB value with Image J software, which can meet the needs of on-site rapid detection of Salmonella typhimurium. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Based on ABEI / Co 2+ Schematic diagram of the glow-type chemiluminescent sensor based on / GO@DNA hydrogel for detecting Salmonella typhimurium.

[0037] Figure 2 ABEI / Co prepared in Example 1 2+ / Characterization diagram of GO.

[0038] In the figure: A, is ABEI / Co 2+ UV-visible absorption spectrum of ABEI / Co 2+ Fourier transform infrared spectrum of ABEI / Co 2+ X-ray photoelectron spectrum of ABEI / Co 2+ Co 2p peak diagram of ABEI / Co 2+ / GO; F, ABEI / Co 2+ / Zeta potential of GO.

[0039] Figure 3 ABEI / Co prepared in Example 2 2+ Characterization diagram of / GO@DNA hydrogel.

[0040] In the figure: A, is ABEI / Co 2+ / GO@DNA hydrogel; B, ABEI / Co 2+ / GO@DNA hydrogel and GO@DNA hydrogel; C, ABEI / Co 2+ Chemiluminescence kinetic curve of / GO@DNA hydrogel.

[0041] Figure 4 The relationship diagram between the concentration of Salmonella typhimurium and the RGB value and its specificity diagram when the glow-type chemiluminescent sensor prepared in Example 3 is used for detection.

[0042] In the figure: A, RGB value spectrum measured at different concentrations of Salmonella typhimurium; B, standard curve diagram of glow-type chemiluminescent sensor detecting Salmonella typhimurium concentration; C, specificity diagram. DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0044] The term "dNTPs" in the present invention refers to a mixture of four monodeoxyribonucleotides: dATP, dGTP, dTTP and dCTP.

[0045] The T used in the following examples 4 DNA ligase, exonuclease I (Exo I), exonuclease III (Exo III), and phi29 DNA polymerase can all be purchased through commercial channels.

[0046] In the following examples, the preparation methods of ABEI solution, GO solution and cobalt chloride solution are as follows:

[0047] The preparation method of ABEI solution is as follows: 27.633 mg of ABEI is dissolved in 10 mL of 0.1 mol / L NaOH solution;

[0048] The GO solution was prepared by dissolving 0.1 mg GO in 1 mL pure water and ultrasonically treating it at 400 W for 1 h;

[0049] The cobalt chloride solution is prepared by dissolving 237.9 mg of cobalt chloride hexahydrate in 100 mL of pure water.

[0050] The nucleotide sequences in the following examples are as follows:

[0051]

[0052]

[0053] Figure 1 The present invention is based on ABEI / Co 2+ Schematic diagram of the glow-type chemiluminescent sensor based on / GO@DNA hydrogel for detecting Salmonella Typhimurium. First, a complementary sequence (complementary chain / primer) was designed, which is not only partially complementary to the aptamer sequence of Salmonella Typhimurium, but also can be used as a primer to trigger the RCA reaction. First, the aptamer-primer hybridization product is prepared. When Salmonella Typhimurium is present in the system, the aptamer tends to bind to Salmonella Typhimurium, causing the aptamer-primer double strand to be opened and the primer to be released. The released primer binds to the circular template (Circ-DNA) through the action of base complementary pairing, thereby inducing an RCA reaction to generate long ssDNA1. At this time, the secondary primer in the system will hybridize with the long ssDNA1, and under the catalysis of phi29DNA polymerase, a new RCA reaction will be carried out with the long ssDNA1 as a template to generate long ssDNA2. Then, ABEI / Co 2+ / GO, the long ssDNA in the system will quickly gel through electrostatic attraction, π-π stacking, coordination and physical entanglement. 2+ / GO is wrapped inside the hydrogel. Finally, the hydrogel is transferred to H 2 O 2 The more Salmonella typhimurium there is in the system, the more long ssDNA is produced by the RCA reaction, and the ABEI / Co captured by the hydrogel 2+ The more / GO, the stronger the chemiluminescence intensity. Therefore, the measured chemiluminescence intensity is positively correlated with the content of Salmonella typhimurium in the system. By taking chemiluminescent images with a smartphone and analyzing their RGB values ​​with Image J software, Salmonella typhimurium can be quantitatively detected.

[0054] Example 1 ABEI / Co 2+ Preparation of GO

[0055] (1) 100 μL of 0.01 mol / L ABEI solution was mixed with 1 mL of 0.1 mg / mL GO solution, placed in a constant temperature mixer, and incubated at 25°C and 1000 rpm in the dark for 12 h to prepare ABEI / GO.

[0056] (2) Add 250 μL of 10 mmol / L cobalt chloride solution, place in a constant temperature mixer, incubate at 25°C, 1000 rpm, in the dark for 3 h, then centrifuge at 12500 rpm for 20 min, discard the supernatant, and wash twice with pure water to remove unbound ABEI and Co 2+ The resulting precipitate is ABEI / Co 2+ / GO, and disperse the precipitate in 50 μL pure water for later use.

[0057] ABEI / Co 2+ UV-visible absorption spectrum, Fourier transform infrared spectrum, X-ray photoelectron spectroscopy, transmission electron microscopy image and Zeta potential of / GO Figure 2 As shown;

[0058] The successful assembly of ABEI was verified by UV-visible absorption spectroscopy, such as Figure 2 As shown in Figure A, ABEI has two characteristic absorption peaks at 291nm and 223nm, and GO has a characteristic absorption peak at 230nm. 2+ After that, the maximum absorption peak blue-shifted from 230 nm to 222 nm, and a new absorption peak appeared at 290 nm (i.e., the characteristic absorption peak of ABEI), indicating that ABEI has been successfully loaded on GO.

[0059] By analyzing GO, ABEI / GO and ABEI / Co 2+ The Fourier transform infrared spectroscopy results of / GO can further determine whether ABEI is successfully loaded, such as Figure 2 As shown in Figure B, except for 2885-3715cm -1 In addition to the broad and strong stretching vibration of OH near 1723.51cm -1 The peak at 1614.27 is the stretching vibration peak of C=O, 1226.32 is the vibration peak of epoxy group (COC), and 1050.45 is the vibration peak of CO. 2+ / GO are both at 1379.63cm -1 and 833.35cm-1 A new vibration peak appears at , which is due to the amide bond in the ABEI molecule, indicating that ABEI in ABEI / Co 2+ / GO. In addition, with the successful assembly of ABEI and Co 2+ The peaks around 1723.51, 1226.32, and 1050.45 decreased or disappeared with the addition of 2+ It plays an important role in assembly.

[0060] The composition of the material was further investigated using X-ray photoelectron spectroscopy, such as Figure 2 As shown in Figure C, after loading ABEI, N1s component appeared at 399.08 eV, and the N / O ratio gradually increased (GO, ABEI / GO and ABEI / Co 2+ / GO N / O ratios are 0.043, 0.080 and 0.14, respectively, which strongly proves that ABEI has been functionalized onto the GO surface. 2+ After that, the characteristic peak of Co appeared at 770-810 eV, indicating that Co 2+ Successful modification. Figure 2 Figure D is the peak distribution of Co 2p. The peaks at 802.28eV and 783.68eV correspond to Co in Co-N, and the peaks at 796.18eV and 780.28eV correspond to Co in Co-O, indicating that Co 2+ It can be assembled by coordinating with atoms such as O and N of GO or ABEI.

[0061] ABEI / Co was characterized by transmission electron microscopy. 2+ / GO was characterized, such as Figure 2 As shown in Figure E, ABEI / Co 2+ / GO still presents a two-dimensional sheet structure, but compared with GO, the wrinkles of the sheet structure are reduced, which further indicates that the oxygen-containing functional groups in ABEI and Co 2+ In addition, the ABEI / Co 2+ / GO's Zeta potential, such as Figure 2 As shown in Figure F, with the ABEI and Co 2+ Gradual loading of ABEI / Co 2+ / GO's charge shifted 51 mV to the positive direction, and the charge was reversed to positive. This may be due to the fact that ABEI / Co 2+ This is the main reason why GO has such superior accelerated gelation performance.

[0062] Example 2 ABEI / Co 2+ Preparation of GO@DNA hydrogel

[0063] (1) Preparation of aptamer-primer complex: Accurately pipette 20 μL of 10 μmol / L aptamer and 20 μL of 10 μmol / L primer into a PCR tube and mix well. Place in a PCR instrument and anneal at 95°C for 10 min. Then slowly cool to room temperature. Add 14 μL of exonuclease I (5 U / μL) and 6 μL of matching buffer (10×). Mix well. Use the PCR instrument to run under the set program to remove free single strands and prepare the purified aptamer-primer complex. The specific procedure is: incubate at 37°C for 3 h, then heat at 80°C for 15 min.

[0064] (2) Preparation of circular template: 30 μL of 20 μmol / L circular primer and 30 μL of 20 μmol / L template were mixed evenly, and hybridization was performed using a gradient cooling method (gradient cooling: heating at 95°C for 2 min, heating at 65°C for 2 min, incubating at 60°C for 6 min, then slowly cooling to 20°C at a rate of 0.1°C per second, and maintaining at 20°C for 30 s, and finally incubating at 4°C for 10 min) to obtain a circular DNA precursor; then 7.5 μL of T 4 DNA ligase (400U / μL) and matching buffer (10×) were incubated at 37°C for 2h, and incubated at 65°C for 10min to inactivate the enzyme to prepare a circular template; then 10μL ExoⅠ (5U / μL), 5μLExoⅢ (200U / μL) and 10μL 10×ExoⅠ reaction buffer were added, first incubated at 37°C for 1h, and then incubated at 80°C for 20min to obtain a purified circular template; the purified circular template was placed at 4°C for use.

[0065] (3) Preparation of free primers: Take 54 μL of the purified aptamer-primer complex obtained in step (1), add 246 μL of the target to be detected, and incubate at 37°C and 600 rpm for 45 min to allow the aptamer to recognize and bind to the target. Then filter through a 0.22 μm filter membrane, collect the filtrate, and obtain the free primers.

[0066] (4) Take 30 μL of the free primer obtained in step (3), add 15 μL of 3 μmol / L secondary primer, 3 μL of the purified circular template obtained in step (2), 2.5 μL of dNTPs (deoxynucleotides) (25 mmol / L), 1 μL of pure water and 2.5 μL of phi29 DNA polymerase (10 U / μL), mix well and incubate at 37°C, 800 rpm for 25 min; then add 15 μL of ABEI / Co prepared in Example 1 2+ / GO, and incubated at 37 °C and 1050 rpm for 10 min to obtain ABEI / Co 2+ / GO@DNA hydrogel. ABEI / Co 2+ The scanning electron microscopy test results of / GO@DNA hydrogel are shown in Figure 3 As shown in A.

[0067] like Figure 3 As shown in Figure A, the scanning electron microscopy results show that ABEI / Co 2+ / GO@DNA hydrogel still presents a porous structure, which provides a structural basis for the construction of glow-type chemiluminescent sensors.

[0068] Example 3 Preparation of glow type chemiluminescent sensor:

[0069] The ABEI / Co prepared in Example 2 2+ After washing twice, the GO@DNA hydrogel was transferred to an opaque 96-well plate and 100 μL of H 2 O 2 Solution (0.1 mol / L) was incubated in dark environment for 1 min, and then chemiluminescence images were taken with Huawei Nova 7 mobile phone in dark environment, and their RGB values ​​were analyzed with Image J software. By collecting and measuring the chemiluminescence signal over a period of time, the chemiluminescence kinetic curve was drawn. The results are shown in Figure 3 As shown in C.

[0070] like Figure 3 As shown in Figure C, the chemiluminescence kinetic curve shows that ABEI / Co 2+ / GO exhibited typical flash chemiluminescence characteristics, while ABEI / Co 2+ The chemiluminescence of ABEI / GO@DNA hydrogel entered a stable period after a growth period of about 50 seconds, indicating that 2+ / GO@DNA hydrogel can be used to construct glow-type chemiluminescent sensors.

[0071] The concentration of Salmonella typhimurium was changed (7.5 CFU / mL, 7.5×10 1 CFU / mL, 7.5×10 2 CFU / mL, 7.5×10 3 CFU / mL, 7.5×10 4 CFU / mL, 7.5×10 5 CFU / mL, 7.5×10 6 CFU / mL, 7.5×10 7 CFU / mL), repeat the above operation, the test results are as follows Figure 4 A and Figure 4As shown in B, as the concentration of Salmonella typhimurium increases, the RGB value becomes higher. The RGB value is positively correlated with the logarithmic value of the concentration of Salmonella typhimurium. The linear range is 7.5 CFU / mL-7.5×10 7 CFU / mL (Y = 49.24 + 1.34 * X, R 2 =0.9942, where Y represents the RGB value and X represents the logarithmic value of the concentration of Salmonella typhimurium). The detection limit of Salmonella typhimurium is 6.39 CFU / mL.

[0072] Specificity is one of the important characteristics of biosensors. In order to verify the specificity of this experimental method, the response of the constructed glow-type chemiluminescent sensor to Staphylococcus aureus, Escherichia coli, Bacillus cereus and Listeria monocytogenes was measured. Figure 4 As shown in Figure C, only the Salmonella typhimurium system showed a significant increase in chemiluminescence intensity, while the chemiluminescence signals caused by the other four foodborne pathogens changed less, indicating that this method has good specificity for the detection of Salmonella typhimurium.

[0073] Comparative Example 1 Preparation of GO

[0074] The preparation method of Comparative Example 1 is basically the same as that of Example 1, except that the "100 μL of 0.01 mol / L ABEI solution" in step (1) of Example 1 is replaced by "100 μL of ultrapure water", and the "250 μL of 10 mmol / L cobalt chloride solution" is replaced by "250 μL of ultrapure water", and the rest of the contents remain unchanged.

[0075] Comparative Example 2 Preparation of GO@DNA hydrogel

[0076] The preparation method of Comparative Example 2 is basically the same as that of Example 2, except that 15 μL of ABEI / Co prepared in Example 1 is added to the 2+ / GO" is replaced by "add 15 μL of GO prepared in Comparative Example 1".

[0077] Test Case

[0078] (1)ABEI / Co 2+ Effect of / GO on the formation rate of hydrogel

[0079] Take ABEI / Co prepared in Example 2 of the present invention 2+ / GO@DNA hydrogel and GO@DNA hydrogel prepared in comparative example 2 were observed and photographed. The results are as follows Figure 3 As shown in B. Figure 3 As can be seen in Figure B, under the same experimental operation and time, ABEI / Co 2+ / GO@DNA hydrogel has been clearly gelled; GO@DNA hydrogel has hardly been gelled, and there is still a lot of dispersed GO in the solution. 2+ / GO has a more significant accelerating gelation effect, indicating that ABEI / Co 2+ / GO can effectively extend the gelation time of DNA hydrogel, thereby shortening the detection time.

[0080] (2) Actual sample testing

[0081] This method was used to determine the concentration of Salmonella typhimurium in commercial chicken samples, and then the plate count method was used to verify the method. The sample was pretreated as follows: first, 25g of commercial chicken was added to 225mL of PBS buffer for homogenization and sterilization. Then centrifuged at 1000rpm for 5min, the precipitate was discarded, and the supernatant was taken. Then 300μL of the supernatant was mixed with 600μL of Salmonella typhimurium bacterial solution of different concentrations to prepare the actual sample to be tested. The results are shown in Table 1. Through SPSS analysis, there was no significant difference between the test results of this method and the test results of the plate count method, indicating that the test results of this method are more reliable.

[0082] Table 1 Results of spiked chicken samples tested by this method and plate count method (n=3)

[0083]

[0084] Note: In the same row, the same letters indicate that there is no significant difference between the two test results (p>0.05, n=3), and different lowercase letters indicate significant difference.

[0085] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. ABEI / Co 2+ / GO@DNA hydrogel glow-type chemiluminescent sensor, characterized in that The glow type chemiluminescent sensor comprises an aptamer-primer complex, a circular template, a secondary primer, a Phi29 DNA polymerase, dNTPs, ABEI / Co 2+ / GO and target; The aptamer sequence in the aptamer-primer complex is shown in SEQ No. 1, and the primer sequence is shown in SEQ No. 2; The template sequence is shown in SEQ No.3; The secondary primer sequence is shown in SEQ No.

4.

2. The glow type chemiluminescent sensor according to claim 1, characterized in that: The ABEI / Co 2+ The preparation method of / GO is: (1) ABEI solution and GO solution were mixed and incubated at 25°C and 1000 rpm for 12 h in the dark to obtain ABEI / GO; (2) Then, cobalt chloride solution was added, and the mixture was incubated at 25°C and 1000 rpm in the dark for 3 h. The mixture was centrifuged at 12500 rpm for 20 min, the supernatant was discarded, and the mixture was washed with ultrapure water and redissolved to obtain ABEI / Co 2+ / GO.

3. The glow type chemiluminescent sensor according to claim 2, characterized in that: In step (1), the ABEI solution is prepared by dissolving 27.633 mg of ABEI in 10 mL of 0.1 mol / L NaOH solution; the GO solution is prepared by dissolving 0.1 mg of GO in 1 mL of pure water and ultrasonically treating the solution at 400 W for 1 h; the volume ratio of the ABEI solution to the GO solution is 0.1:

1.

4. The glow type chemiluminescent sensor according to claim 2, characterized in that: In step (2), the cobalt chloride solution is prepared by dissolving 237.9 mg of cobalt chloride hexahydrate in 100 mL of pure water; the volume ratio of the cobalt chloride solution to the GO solution is 0.25:

1.

5. The glow type chemiluminescent sensor according to claim 1, characterized in that: The preparation method of the aptamer-primer complex is: (1) Mix 20 μL of 10 μmol / L aptamer and 20 μL of 10 μmol / L primer in a PCR tube, place in a PCR instrument, anneal at 95°C for 10 min, and then slowly cool to obtain an aptamer-primer complex; (2) 14 μL of 5 U / μL exonuclease I was added thereto to enzymatically digest the unbound single-stranded DNA to prepare a purified aptamer-primer complex.

6. The glow type chemiluminescent sensor according to claim 1, characterized in that: The method for preparing the circular template is as follows: (1) 30 μL of 20 μmol / L circular primer and the chain template are mixed evenly, and hybridized by gradient cooling method to obtain the circular DNA precursor of the template; The loop primer sequence is shown in SEQ No.5; (2) Add 7.5 μL of 400 U / μL T4 DNA ligase, incubate at 37°C for 2 h, and incubate at 65°C for 10 min to inactivate the enzyme; then add 10 μL of 5 U / μL ExoⅠ enzyme and 5 μL of 200 U / μL ExoⅢ enzyme in sequence, incubate at 37°C for 1 h, and then incubate at 80°C for 20 min to obtain the purified circular template.

7. An application of the glow type chemiluminescent sensor according to any one of claims 1 to 6, characterized in that: The glow type chemiluminescent sensor is used to detect Salmonella typhimurium.

8. The use according to claim 7, characterized in that: The method for detecting Salmonella typhimurium using the glow-type chemiluminescent sensor comprises the following steps: (1) Preparation of ABEI / Co 2+ / GO@DNA hydrogel; (2) To ABEI / Co 2+ After adding 100 μL of 0.01 mol / L H2O2 solution to the / GO@DNA hydrogel and reacting for 1 min, the luminescence image was taken and its RGB value was analyzed using Image J software. The RGB value was substituted into the standard curve to obtain the concentration of the target Salmonella typhimurium in the sample to be tested.

9. The use according to claim 8, characterized in that: In step (1), the ABEI / Co 2+ The preparation method of / GO@DNA hydrogel is: (1) Take 54 μL of the prepared aptamer-primer complex, add 246 μL of the target to be detected, and incubate at 37°C and 600 rpm for 45 min to allow the aptamer to recognize and bind to the target. Then filter through a 0.22 μm filter membrane and collect the filtrate to obtain free primers; (2) Take 30 μL of free primer, mix it with 15 μL of 3 μmol / L secondary primer, 3 μL of circular template, 2.5 μL of 25 mmol / L dNTPs, 1 μL of pure water, 6 μL of 10× phi29 DNA polymerase reaction buffer and 2.5 μL of 10 U / μL phi29 DNA polymerase, and incubate it at 37°C, 800 rpm for 25 min; then add 15 μL of ABEI / Co 2+ / GO, and incubated at 37 °C, 1050 rpm for 10 min to obtain ABEI / Co 2+ / GO@DNA hydrogel.

10. The use according to claim 8, characterized in that: The standard curve is: Y=49.24+1.34*X, R 2 =0.9942, wherein Y represents the RGB value, and X represents the logarithmic value of the concentration of Salmonella typhimurium.

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