Cytosine repeat oligonucleotide and its application in improving peroxidase-like activity of fe3o4@au

Abstract

The application provides cytosine-rich ssDNA (C-rich ssDNA) and application thereof in improving peroxidase-like activity of Fe3O4@Au, and belongs to the technical field of nanocomposites; the C-rich ssDNA is obtained by connecting 20 continuous cytosine nucleotides to an aptamer; the sequence of the 20 continuous cytosine nucleotides is shown in SEQ ID NO. 1. Fe3O4@Au shows peroxidase-like activity, and after the C-rich ssDNA is used to modify Fe3O4@Au, the peroxidase-like activity is significantly enhanced, and then the detection limit of a sensing method based on Fe3O4@Au is reduced.

Description

Technical Field

[0001] This invention belongs to the field of nanocomposite materials technology, specifically relating to cytosine repeat oligonucleotides (C-richssDNA) and their application in improving the peroxidase-like activity of Fe3O4@Au. Background Technology

[0002] Salmonella is a major foodborne pathogen, reportedly causing over 350,000 deaths globally each year. *Salmonella typhimurium* is one of the major pathogenic serotypes of Salmonella and is frequently reported to contaminate various foods, including poultry, beef, milk, and lettuce. Developing a rapid, accurate, and sensitive detection method for *Salmonella typhimurium* is of great significance for food safety.

[0003] Nanozymes are functional nanomaterials with enzyme-like catalytic activity. Compared with natural enzymes, nanozymes have unique advantages such as high cost-effectiveness, good stability, and ease of modification. Currently, many nanomaterials, such as metal oxide nanoparticles, carbon nanomaterials, noble metals, and two-dimensional transition metal halides, have been reported to have peroxidase-like activities. Among various nanozymes, iron(III) oxide nanoparticles (Fe3O4NPs) have attracted much attention due to their mature synthesis technology, low cost, and high efficiency. Compared with single-component nanozymes, composite nanozymes can utilize the synergistic effect between components to improve catalytic activity, such as MoS2 / Fe3O4, Fe3O4 / rGO, and Fe3O4@graphitic. Au is one of the best materials for preparing composite nanozymes because its reduction preparation is simple, its chemical stability is reliable, and the reduced noble metal nanoparticles can be firmly fixed on the surface of the nanozyme, providing catalytic centers. Therefore, the synthesis and preparation of Fe3O4@Au is an economical and feasible approach to obtain highly catalytically active composite nanozymes.

[0004] Specificity is a key parameter for evaluating the performance of colorimetric methods. To improve the specificity of colorimetric methods, biorecognition elements—nucleic acid aptamers—are introduced. Nucleic acid aptamers are functional single-stranded oligonucleotide sequences that can specifically bind to targets. Compared with antibodies, nucleic acid aptamers have unique advantages such as small size, high affinity, strong specificity, high stability, and controllable modification, and are widely used in the construction of biosensors. Among various nanozyme-based nucleic acid aptamer sensors, the emerging nucleic acid aptamer-enhanced nanozyme activity sensing platform has been widely welcomed and applied to food detection. It is worth noting that different nucleotides in the nucleic acid aptamer have different enhancing abilities for nanozyme peroxidase activity. In addition, the length limitation of the nucleic acid aptamer restricts its ability to enhance the activity of nanozyme peroxidase, ultimately leading to a high limit of detection (LOD) for this sensing method. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide C-richssDNA and its application in improving the peroxidase-like activity of Fe3O4@Au. The C-richssDNA of the present invention can significantly improve the peroxidase-like activity of Fe3O4@Au, thereby reducing the detection limit of Fe3O4@Au-based sensing methods.

[0006] The present invention provides a cytosine repeat oligonucleotide C-richssDNA, which is obtained by linking 20 consecutive cytosine nucleotides to a nucleic acid aptamer; the sequence of the 20 consecutive cytosine nucleotides is shown in SEQ ID NO.1.

[0007] Preferably, the nucleotide sequence of the C-richssDNA is shown in SEQ ID NO.3.

[0008] This invention also provides the application of the C-richssDNA described above in enhancing the peroxidase-like activity of Fe3O4@Au.

[0009] This invention also provides the application of the C-richssDNA combined with Fe3O4@Au described above in the preparation of products for detecting Salmonella typhimurium.

[0010] The present invention also provides a kit for detecting Salmonella typhimurium, comprising C-richssDNA, Fe3O4@Au, H2O2, TMB and HAc-NaAc buffer as described in the above scheme.

[0011] The present invention also provides a method for detecting Salmonella typhimurium for non-diagnostic purposes based on the kit described above, comprising the following steps:

[0012] The sample to be tested was mixed with C-richssDNA aqueous solution and incubated for the first time. The product of the first incubation was centrifuged for the first time, and the supernatant after the first centrifugation was collected.

[0013] The supernatant was mixed with Fe3O4@Au aqueous solution for a second incubation.

[0014] H2O2, TMB and HAc-NaAc buffer were added to the second incubated solution to carry out the reaction in the dark. The reaction was terminated after the reaction in the dark and the absorbance of the reaction product at 452 nm was measured.

[0015] The absorbance of the reaction product at 452 nm is denoted as A, and the absorbance of the negative control at 452 nm is denoted as A0.

[0016] If the value of A0-A is equal to zero, it is determined that the sample to be tested does not contain Salmonella typhimurium;

[0017] If the value of A0-A is greater than zero, the sample to be tested is determined to contain Salmonella typhimurium.

[0018] Preferably, after measuring the absorbance of the reaction product at 452 nm and obtaining the absorbance value, the method further includes calculating the concentration of Salmonella typhimurium in the sample to be tested according to the formula shown in Formula 1, Formula 2 or Formula 3.

[0019] y = 0.1343x - 0.0298, correlation coefficient R 2 Equation 1 is 0.9960;

[0020] y = 0.0766x - 0.0389, correlation coefficient R 2 Equation 2 is 0.9825;

[0021] y = 0.0665x - 0.0231, correlation coefficient R 2 Equation 3 is 0.9875

[0022] The formula shown in Equation 1 is applicable when the sample to be tested is a pure bacterial sample;

[0023] The formula shown in Equation 2 is applicable when the sample to be tested is a lettuce sample;

[0024] The formula shown in Equation 3 is applicable when the sample to be tested is a milk sample;

[0025] In Equations 1 to 3, y is the absorbance value and x is the logarithmic value of the concentration of Salmonella typhimurium.

[0026] Preferably, the working concentration of the C-richssDNA is 0.05 nM; the working concentration of the Fe3O4@Au is 0.2 μg / mL; the working concentration of the H2O2 is 15 mM; the working concentration of the TMB is 2 mM; the working concentration of the HAc-NaAc buffer is 10 mM; and the pH value of the HAc-NaAc buffer is 4.

[0027] Preferably, the incubation temperature is 25°C.

[0028] Preferably, the reaction temperature of the system is 37°C.

[0029] Preferably, the light-avoidance reaction time is 20 minutes.

[0030] This invention provides a cytosine repeat oligonucleotide C-richssDNA, obtained by linking 20 consecutive cytosine nucleotides to a nucleic acid aptamer; the sequence of the 20 consecutive cytosine nucleotides is shown in SEQ ID NO.1. The C-richssDNA of this invention can significantly improve the peroxidase-like activity of Fe3O4@Au, thereby reducing the detection limit of Fe3O4@Au-based sensing methods. Attached Figure Description

[0031] Figure 1 A schematic diagram of the colorimetric detection of Salmonella typhimurium based on C-richssDNA-enhanced Fe3O4@Au nanozyme;

[0032] Figure 2 The images show the characterization of the nanozymes, where (A) is the SEM of Fe3O4NPs, (B) is the SEM of Fe3O4@Au, (C) is the UV-Vis absorption spectrum of Fe3O4 and Fe3O4@Au, (D) is the Fourier transform infrared spectrum (FT-IR) of Fe3O4NPs and Fe3O4@Au, and (E) is the XRD pattern of Fe3O4NPs and Fe3O4@Au.

[0033] Figure 3 The absorbance values ​​are for solutions containing different reactants; (a) TMB + H2O2, (b) Nucleic acid aptamer + TMB + H2O2, (c) Fe3O4NPs + TMB + H2O2, (d) Fe3O4@Au + TMB + H2O2, (e) Fe3O4@Au + Nucleic acid aptamer + TMB + H2O2; (f) Fe3O4@Au + C-richssDNA + TMB + H2O2, (g) Fe3O4@Au + Salmonella Typhimurium + C-richssDNA + TMB + H2O2;

[0034] Figure 4 The results are for steady-state kinetic analysis, including (A) steady-state kinetic analysis results of Fe3O4NPs, Fe3O4@Au, nucleic acid aptamer / Fe3O4@Au and C-richssDNA / Fe3O4@Au with fixed TMB concentration; (B) steady-state kinetic analysis results of Fe3O4, Fe3O4@Au, nucleic acid aptamer / Fe3O4@Au and C-richssDNA / Fe3O4@Au with fixed H2O2 concentration; (C) double reciprocal plot with fixed TMB concentration; and (D) double reciprocal plot with fixed H2O2 concentration.

[0035] Figure 5 The zeta potential values ​​are for Fe3O4NPs, Fe3O4@Au, nucleic acid aptamers, C-richssDNA, nucleic acid aptamer / Fe3O4@Au, and C-richssDNA / Fe3O4@Au.

[0036] Figure 6The results show the changes in absorbance at 452 nm UV-Vis after DNA reacts with Fe3O4@Au. (A) shows the changes in absorbance at 452 nm UV-Vis after DNA with different base compositions reacts with Fe3O4@Au. (B) shows the changes in absorbance at 452 nm UV-Vis after DNA with different sequence lengths reacts with Fe3O4@Au. (C) shows the changes in absorbance at 452 nm UV-Vis after Fe3O4@Au is ligated with C-richssDNA and the concentration of C-richssDNA is optimized.

[0037] Figure 7 The effects of different factors on the activities of Fe3O4@Au and C-richssDNA / Fe3O4@Au peroxidases were investigated. Among them, (A) was the effect of H2O2 concentration, (B) was the effect of TMB concentration, (C) was the effect of reaction temperature, (D) was the effect of HAc-NaAc buffer pH, (E) was the effect of HAc-NaAc buffer concentration, and (F) was the effect of reaction time.

[0038] Figure 8 The results show the detection results of bacteria from low to high concentrations, where (A) represents the reaction products and full-wavelength detection results, and (B) represents the linear standard curve for the detection of Salmonella Typhimurium.

[0039] Figure 9 The results show the specific detection of the C-richssDNA / Fe3O4@Au biosensor.

[0040] Figure 10 The linear detection standard curves are shown, where (A) is the linear detection standard curve in lettuce and (B) is the linear detection standard curve in milk. Detailed Implementation

[0041] This invention provides a cytosine repeat oligonucleotide C-richssDNA, which is obtained by linking 20 consecutive cytosine nucleotides to a nucleic acid aptamer; the sequence of the 20 consecutive cytosine nucleotides is shown in SEQ ID NO.1, specifically: 5′-CCCCCCCCCCCCCCCCCCCC-3′.

[0042] In this invention, the nucleotide sequence of the nucleic acid aptamer is as shown in SEQ ID NO.2, specifically: 5′-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3′ ，See (Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella entericaserovars) 。

[0043] In this invention, the 20 consecutive cytosine nucleotides are linked to the 5′ end of the nucleic acid aptamer; the nucleotide sequence of the C-richssDNA is shown in SEQ ID NO.3, specifically: 5′-CCCCCCCCCCCCCCCCCCCCTATGGCGGCGTCACCCGACGGGGACTT GACATTATGACAG-3′.

[0044] In this invention, the 20 cytosine nucleotides and nucleic acid aptamers are linked together by phosphodiester bonds to form C-richssDNA.

[0045] This invention also provides the application of the C-richssDNA described above in enhancing the peroxidase-like activity of Fe3O4@Au.

[0046] This invention also provides the application of the C-richssDNA combined with Fe3O4@Au described above in the preparation of products for detecting Salmonella typhimurium.

[0047] The C-richssDNA of this invention can bind to Salmonella typhimurium through a unique spatial conformation and has extremely high selectivity, resulting in more reliable results.

[0048] The present invention also provides a kit for detecting Salmonella typhimurium, comprising C-richssDNA, Fe3O4@Au, H2O2, TMB and HAc-NaAc buffer as described in the above scheme.

[0049] In this invention, the concentration of C-richssDNA in the kit is preferably 0.25 nM.

[0050] In this invention, the concentration of Fe3O4@Au in the kit is preferably 10 μg / mL.

[0051] In this invention, the concentration of H2O2 in the kit is preferably 300 mM.

[0052] In this invention, the concentration of TMB in the kit is preferably 60 mM.

[0053] In this invention, the concentration of HAc-NaAc buffer in the kit is preferably 20 mM; the pH value of the HAc-NaAc buffer is preferably 4.

[0054] The kit of the present invention preferably also includes H2SO4; the concentration of H2SO4 is preferably 9M, and the H2SO4 is used to terminate the light-protected reaction.

[0055] The present invention also provides a method for detecting Salmonella typhimurium for non-diagnostic purposes based on the kit described above, comprising the following steps:

[0056] The sample to be tested was mixed with C-richssDNA aqueous solution and incubated for the first time. The product of the first incubation was centrifuged for the first time, and the supernatant after the first centrifugation was collected.

[0057] The supernatant was mixed with Fe3O4@Au aqueous solution for a second incubation.

[0058] H2O2, TMB and HAc-NaAc buffer were added to the second incubated solution to carry out the reaction in the dark. The reaction was terminated after the reaction in the dark and the absorbance of the reaction product at 452 nm was measured.

[0059] The absorbance of the reaction product at 452 nm is denoted as A, and the absorbance of the negative control at 452 nm is denoted as A0.

[0060] If the value of A0-A is equal to zero, it is determined that the sample to be tested does not contain Salmonella typhimurium;

[0061] If the value of A0-A is greater than zero, the sample to be tested is determined to contain Salmonella typhimurium.

[0062] The present invention first mixes the sample to be tested with an aqueous solution of C-richssDNA and performs a first incubation. The product of the first incubation is then centrifuged, and the supernatant after the first centrifugation is collected.

[0063] In this invention, the sample to be tested is preferably a pure bacterial sample; when the sample to be tested is a bacterial suspension, it is preferable to further perform a second centrifugation and wash the precipitate after the second centrifugation before mixing, and use the washed precipitate as the sample to be tested and mix it with the C-richssDNA aqueous solution. In this invention, the speed of the second centrifugation is preferably 12000 rpm; the time of the second centrifugation is preferably 10 min; and the washing reagent is preferably 0.9% NaCl.

[0064] In this invention, the working concentration of the C-richssDNA is preferably 0.05 nM.

[0065] In this invention, the mixing temperature is preferably 25°C; the mixing process is preferably accompanied by stirring, the stirring speed is preferably 200 rpm; and the mixing time is preferably 1 hour.

[0066] In this invention, the rotation speed of the first centrifuge is preferably 5000 rpm; the centrifugation time is preferably 10 min.

[0067] After obtaining the supernatant from the first centrifugation, the present invention mixes the supernatant with an aqueous solution of Fe3O4@Au and performs a second incubation. In this invention, the working concentration of Fe3O4@Au is preferably 10 μg / mL. In this invention, the incubation temperature is preferably 25°C; the incubation process is accompanied by stirring; the stirring speed is preferably 200 rpm; and the incubation time is preferably 30 min.

[0068] In this invention, H2O2, TMB and HAc-NaAc buffer solution are added to the second incubated solution to carry out a light-protected reaction. The reaction is terminated after the light-protected reaction, and the absorbance of the reaction product at 452 nm is measured.

[0069] The absorbance of the reaction product at 452 nm is denoted as A, and the absorbance of the negative control at 452 nm is denoted as A0.

[0070] If the value of A0-A is equal to zero, it is determined that the sample to be tested does not contain Salmonella typhimurium;

[0071] If the value of A0-A is greater than zero, the sample to be tested is determined to contain Salmonella typhimurium.

[0072] In this invention, the working concentration of H2O2 is preferably 15 mM; the working concentration of TMB is preferably 2 mM; and the working concentration of HAc-NaAc buffer is preferably 10 mM.

[0073] In this invention, the preferred temperature for the light-protected reaction is 37°C; and the preferred time for the light-protected reaction is 20 min.

[0074] In this invention, the preferred method for terminating the light-avoidance reaction is to add H2SO4 to the reaction system to terminate the light-avoidance reaction.

[0075] In this invention, the determination of the absorbance of the reaction product at 452 nm preferably includes measuring the absorbance value at 452 nm using a SpectraMax i3x reader, the specifications of which are: MolecularDevices, Inc., Silicon Valley, CA.

[0076] After measuring the absorbance of the reaction product at 452 nm and obtaining the absorbance value, the present invention preferably further includes calculating the concentration of Salmonella typhimurium in the sample to be tested according to the formula shown in Formula 1, Formula 2 or Formula 3.

[0077] y = 0.1343x - 0.0298, correlation coefficient R 2 Equation 1 is 0.9960;

[0078] y = 0.0766x - 0.0389, correlation coefficient R 2 Equation 2 is 0.9825;

[0079] y = 0.0665x - 0.0231, correlation coefficient R 2 Equation 3 is 0.9875;

[0080] The formula shown in Equation 1 is applicable when the sample to be tested is a pure bacterial sample;

[0081] The formula shown in Equation 2 is applicable when the sample to be tested is a lettuce sample;

[0082] The formula shown in Equation 3 is applicable when the sample to be tested is a milk sample;

[0083] In Equations 1 to 3, y is the absorbance value and x is the logarithmic value of the concentration of Salmonella typhimurium.

[0084] In this invention, Fe3O4@Au exhibits peroxidase-like activity, and this activity is enhanced after Fe3O4@Au modifies the C-richssDNA of this invention. Compared with nucleic acid aptamers, C-richssDNA significantly enhances the peroxidase-like activity of Fe3O4@Au, corresponding to a dark yellow signal. Based on this, this invention constructs a rapid detection method for Salmonella Typhimurium. When Salmonella Typhimurium is present in the sample, the nucleic acid aptamer fragment in the C-richssDNA specifically binds to Salmonella Typhimurium to form a C-richssDNA / Salmonella Typhimurium complex. After centrifugation, the supernatant is incubated with Fe3O4@Au. Due to the reduction of C-richssDNA in the supernatant, the peroxidase-like activity of Fe3O4@Au cannot be significantly increased. In the presence of the substrate (TMB-H2O2), the supernatant turns pale yellow. When Salmonella Typhimurium is absent from the sample, a large amount of C-rich ssDNA in the supernatant adsorbs onto the surface of Fe3O4@Au, increasing the peroxidase-like activity of Fe3O4@Au and causing the solution to turn deep yellow. Therefore, by enhancing the peroxidase-like activity of Fe3O4@Au with C-rich ssDNA, highly sensitive detection of Salmonella Typhimurium can be achieved.

[0085] The method of this invention can improve the sensitivity of detecting Salmonella Typhimurium. Furthermore, the C-richssDNA of this invention does not affect the affinity of the nucleic acid aptamer for Salmonella Typhimurium, and can selectively recognize Salmonella Typhimurium, thereby ensuring the specificity of Salmonella Typhimurium detection.

[0086] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0087] Preparation method of Fe3O4NPs: 1.08 g FeCl3·6H2O, 1.2 g PSSMA, 3.0 g NaAc and 40 mL ethylene glycol were added to a round-bottom flask and stirred for 1 h at 50 °C with a magnetic stirrer to form a homogeneous mixture. Then 0.6 g NaOH was added and stirring was continued for 1 h until the NaOH was completely dissolved. Subsequently, the mixture was transferred to a stainless steel autoclave lined with Teflon and heated at 200 °C for 9 h. The reaction product was washed three times with 30 mL of 50% (v / v) ethanol aqueous solution and 30 mL of ultrapure water, respectively. Finally, Fe3O4 NPs were dried in a vacuum oven at 50 °C for 12 h.

[0088] Preparation method of Fe3O4@Au nanozyme: 100 mg of Fe3O4 NPs were added to 200 mL of 0.01% (m / v) HAuCl4·3H2O and stirred for 1 h at 280 rpm. Then, 1.5 g of NaBH4 was added and mixed thoroughly at 220 rpm for 30 min. The reaction product was magnetically separated and washed three times with 30 mL of 50% (v / v) ethanol aqueous solution and 30 mL of ultrapure water, respectively. Finally, the Fe3O4@Au nanozyme was dried overnight in a vacuum drying oven at 50 °C.

[0089] Typical colorimetric assay for Salmonella typhimurium (see schematic diagram) Figure 1 The reaction was as follows: 1 mL of bacteria was centrifuged at 12000 rpm for 10 min and washed with 0.9% NaCl. The mixture was resuspended in 200 μL of 0.25 nM C-rich ssDNA and incubated at 200 rpm for 1 h. After centrifugation at 5000 rpm for 10 min, the supernatant was incubated with 20 μL of 10 μg / mL Fe3O4@Au solution. After incubation for 30 min, 50 μL of 300 mM H2O2, 30 μL of 60 mM TMB, and 550 μL of 20 mM HAc-NaAc buffer (pH = 4.0) were added. The reaction was carried out in the dark for 20 min, and then 100 μL of 9 M H2SO4 was added to terminate the reaction. The absorbance of the reaction product at 452 nm was measured using a SpectraMax i3x reader.

[0090] Reagents used in the test:

[0091]

[0092]

[0093] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0094] The strain information used in the embodiments of this invention is as follows:

[0095]

[0096] a ATCC: American Culture Collection; United States

[0097] b CMCC: China Center for Medical Bacterial Culture Collection; China

[0098] Example 1

[0099] 1. Characterization

[0100] Experimental procedure:

[0101] When taking SEM and XRD images, the samples are sent to the Scientific Compass platform for processing.

[0102] SEM

[0103] Instrument model: German ZEISS Sigma300; Spray sputtering target: gold-palladium alloy / pure gold / pure platinum;

[0104] Main technical parameters:

[0105] Electron gun: Schottky field emission electron gun; resolution: 1.0nm@15kV, 1.6nm@1kV; electron beam path: electron beam without cross-beam path inside the lens barrel; accelerating voltage: 0.02-30kV; continuously adjustable in 10V steps.

[0106] XRD: Instrument model: Rigaku Smart Lab;

[0107] FT-IR: Mix an appropriate amount of nanozyme and potassium bromide, then grind until no large particles are visible. Press the powder into thin tablets using a tablet press, at a density of 400–4000 cm⁻¹. -1 The image was obtained by scanning in the specified band.

[0108] UV-vis: Add an appropriate volume of 1 mg / mL Fe3O4 NPs and Fe3O4@Au to a cuvette, and record the full wavelength using a SpectraMax i3x reader (Molecular Devices; Inc., Silicon Valley; CA) to obtain the image.

[0109] Au was reduced using the reduction method. + The reduction occurs on the surface of Fe3O4NPs to enhance their catalytic activity. Figure 2 The scanning electron microscope images A and B in the figure show that Fe3O4NPs and Fe3O4@Au exhibit a uniform spherical morphology. Figure 2 A in the figure shows that Fe3O4NPs are quasi-spherical with an average diameter of approximately 207 nm. For example... Figure 2 As shown in Figure B, Au NPs are dispersed in a granular manner on the spherical surface of Fe3O4 NPs, forming a partial envelope structure. The Fe3O4 NPs and Fe3O4@Au were characterized using UV-Vis spectroscopy, as shown in Figure B. Figure 2 As shown in Figure C, the UV-Vis spectrum of Fe3O4NPs shows no obvious absorption peak in the 450–700 nm range, while Fe3O4@Au exhibits a maximum absorption peak at 550 nm, indicating the formation of the composite nanozyme Fe3O4@Au. A possible reason for this phenomenon is that once Au coats Fe3O4NPs, the dielectric effect between the metallic Au and Fe3O4NPs may be suppressed. This conjugated structure of Au and Fe3O4NPs is confirmed by FT-IR spectroscopy, as shown in Figure C. Figure 2 As shown in D, the FT-IR spectrum of Fe3O4NPs at 3430 cm⁻¹ -1 1623cm -1 1390cm -1 These peaks correspond to the stretching vibrations of the OH, C=O, and CO bonds on the carboxyl group, respectively. At 590 cm⁻¹ -1 The peak observed at [location] belongs to the Fe-O stretching vibration of Fe3O4NPs. Meanwhile, a new peak appears at 474 cm⁻¹ in the FT-IR spectrum of Fe3O4@Au. -1 This belongs to the Au-O tensile vibration. The above results indicate the successful synthesis of Fe3O4@Au, consistent with the UV-vis characterization results. X-ray diffraction spectra of the Fe3O4@Au nanocomposite and Fe3O4 NPs were measured. Figure 2As shown in Figure E, Fe3O4NPs exhibited typical XRD diffraction peaks at 2θ values ​​of 30.1°, 35.4°, 43°, 53.3°, 57.0°, and 62.3°, corresponding to (220), (311), (400), (422), (511), and (440), respectively. These peaks matched well with the XRD patterns of the corresponding crystallographic planes (JCPDS 19-0629). The high intensity and sharp peak shapes of the diffraction peaks indicated good crystallinity of the product. Additional diffraction peaks were detected in Fe3O4@Au, which could be indexed to the (200), (220), and (311) planes of Au, exhibiting a cubic structure (JCPDS04-0784), indicating the presence of Au in the sample. The diffraction peak intensities of Fe3O4NPs were significantly reduced, almost disappearing, indirectly indicating that Au seeds had been adsorbed onto the surface of Fe3O4NPs. These results are consistent with those of Fe3O4@Au, indicating that the heterostructure was successfully formed.

[0110] 2. Verify the feasibility of the experiment

[0111] Experimental procedure:

[0112] There are 7 groups, and each group contains the following substances:

[0113] (a)TMB + H2O2;

[0114] (b) Nucleic acid aptamer + TMB + H2O2;

[0115] (c)Fe3O4+TMB+H2O2;

[0116] (d)Fe3O4@Au+TMB+H2O2;

[0117] (e)Fe3O4@Au+nucleic acid aptamer+TMB+H2O2;

[0118] (f)Fe3O4@Au+C-richssDNA+TMB+H2O2;

[0119] (g)Fe3O4@Au+Salmonella typhimurium+C-richssDNA+TMB+H2O2.

[0120] First, 200 μL of 0.25 nM C-richss DNA or nucleic acid aptamers were incubated with Salmonella Typhimurium. After centrifugation, the supernatant was incubated with 20 μL of 10 μg / mL Fe3O4@Au at 25℃ and 200 rpm for 30 min. Then, sodium acetate buffer at pH 4 and other substances in the above system were added, and the reaction was carried out in the dark for 20 min. The full wavelength was measured using SpectraMaxi3xreader.

[0121] like Figure 3 As shown, when only H2O2 and TMB are present in the system, there is almost no absorption at 452 nm (curve a), indicating that no oxidation reaction occurs. The difference is not significant when nucleic acid aptamers are present in the H2O2-TMB system (curve b), indicating that the presence of nucleic acid aptamers alone does not cause an oxidation reaction. When Fe3O4 nanoparticles are present in the H2O2-TMB system, a slight absorption peak appears at 452 nm (curve c), indicating that Fe3O4NPs have peroxidase-like activity. After adding Fe3O4@Au, a significant absorption peak appears at 452 nm (curve d), indicating that the synergistic effect of Au enhances the peroxidase-like activity of Fe3O4NPs. When nucleic acid aptamer / Fe3O4@Au is present in the system (curve e), the absorption peak at 452 nm is higher than that of Fe3O4@Au, indicating that the nucleic acid aptamer adsorbs on the Fe3O4@Au surface, enhancing the peroxidase-like activity of Fe3O4@Au. When Fe3O4@Au was incubated with the same concentration of C-rich ssDNA (curve f) instead of the nucleic acid aptamer, the absorbance at 452 nm was significantly enhanced, indicating that C-rich ssDNA had a stronger ability to enhance the activity of Fe3O4@Au-type peroxidases than the nucleic acid aptamer. When Fe3O4@Au, C-rich ssDNA, and Salmonella typhimurium were added to the system, the absorbance at 452 nm decreased significantly (curve g), indicating that the specific binding of C-rich ssDNA to Salmonella typhimurium led to the separation of C-rich ssDNA from the Fe3O4@Au surface. These results demonstrate the feasibility of this sensing method, enabling the detection of Salmonella typhimurium by visual inspection or instruments.

[0122] 3. Enzyme kinetics studies

[0123] Experimental procedure:

[0124] 200 μL of 10 nM nucleic acid aptamer and C-rich ssDNA were incubated with 20 μL of 1.25 μg / mL Fe3O4@Au solution at room temperature for 30 min to prepare mixtures of nucleic acid aptamer / Fe3O4@Au and C-rich ssDNA / Fe3O4@Au. Subsequently, steady-state kinetics of 15 μg / mL Fe3O4, 1.25 μg / mL Fe3O4@Au, nucleic acid aptamer / Fe3O4@Au, and C-rich ssDNA / Fe3O4@Au were determined in 0.01 M sodium acetate buffer (pH=4). Furthermore, a typical experiment was conducted by varying the concentration of one substrate while keeping the concentration of another constant. The absorbance at 652 nm was recorded every 30 seconds for 10 minutes using a SpectraMax i3x reader (Molecular Devices, Inc., Silicon Valley, CA). Catalytic kinetic parameters were calculated according to the Michaelis-Menten equation.

[0125] ν=ν max ×[S] / (K m +[S])

[0126] In the formula, ν is the initial velocity, ν max [S] represents the maximum reaction rate, [S] represents the substrate concentration, and K represents the maximum reaction rate. m This is the Michaelis constant.

[0127] By varying the concentration of a substrate (TMB or H2O2), typical Michaelis-Menten curves were obtained; see [link to relevant documentation]. Figure 4 Draw a Lineweaver-Burk diagram to obtain K. m and ν max K m K is a key parameter for determining the affinity of an enzyme for its substrate. m The lower the K0, the higher the affinity. As shown in Table 1, the K0 of Fe3O4@Au with TMB as the substrate... m The K value is higher than that of Fe3O4NPs, while that of Fe3O4@Au with H2O2 as the substrate is higher. m The Kc value is lower than that of Fe3O4NPs, indicating that the increased activity of Fe3O4@Au peroxidases may be due to the higher affinity of Fe3O4@Au for H2O2, resulting in the generation of more hydroxyl radicals (·OH), which accelerates the oxidation of TMB. After the addition of the nucleic acid aptamer, the Kc ratio of the nucleic acid aptamer with TMB as a substrate to Fe3O4@Au... mThe affinity was 1.5 times lower than that of Fe3O4@Au, indicating that the addition of the nucleic acid aptamer enhanced the affinity of Fe3O4@Au for TMB. The Kg of C-rich ssDNA / Fe3O4@Au with TMB as a substrate... m The value was lower than that of the nucleic acid aptamer / Fe3O4@Au, indicating that C-richssDNA can enhance the affinity of Fe3O4@Au for TMB more than the nucleic acid aptamer. The difference in ability between C-richssDNA and the nucleic acid aptamer may be due to the addition of 20 consecutive cytosine nucleotides at the 5' end of the nucleic acid aptamer.

[0128] Table 1 Comparison of kinetic parameters of different nanozymes

[0129]

[0130] Experimental procedure:

[0131] To further verify the mechanism by which C-richssDNA enhances the peroxidase-like activity of Fe3O4@Au, six groups were set up to explore the surface potential of different substances.

[0132] Add water to each of the following solutions to make up to 400 μL: 40 μL of 0.25 mg / mL Fe3O4 NPs, 40 μL of 0.25 mg / mL Fe3O4@Au, 360 μL of 500 nM nucleic acid aptamer, and 360 μL of 500 nM C-richssDNA. Mix 40 μL of 0.25 mg / mL Fe3O4@Au with either 360 μL of 500 nM nucleic acid aptamer or C-richssDNA. Incubate all six solutions at 25 °C and 200 rpm for 30 min. Add 600 μL of 10 mM HAc-NaAc buffer (pH = 4), and measure the potential value using a particle size analyzer.

[0133] like Figure 5As shown, the zeta potential of Fe3O4NPs is -26.2 eV, while that of Fe3O4@Au is +6.86 eV, indicating that Au is successfully anchored on the surface of Fe3O4NPs. These results are in good agreement with the steady-state kinetic data. TMB is positively charged in pH 4 buffer. Due to electrostatic adsorption, Fe3O4NPs have a high affinity for TMB; conversely, the electrostatic repulsion between Fe3O4@Au and TMB reduces the affinity between Fe3O4@Au and TMB. The zeta potentials of the nucleic acid aptamer and C-richssDNA are -3.47 eV and -5.45 eV, respectively. The difference in zeta potential originates from 20 consecutive cytosine nucleotides. This is because the zeta potential of C-rich ssDNA differs from that of the nucleic acid aptamer. The zeta potential of C-rich ssDNA / Fe3O4@Au is more negative than that of the nucleic acid aptamer / Fe3O4@Au, resulting in a higher affinity of C-rich ssDNA / Fe3O4@Au for TMB.

[0134] 4. Effects of oligonucleotides on the activity of Fe3O4@Au peroxidases

[0135] Experimental procedure:

[0136] Take 200 μL of 250 nM A 14 T 14 C 14 G 14 Incubate with 20 μL of 10 μg / mL Fe3O4@Au solution at 25℃ and 200 rpm for 30 min. Add 50 μL of 300 mM H2O2, 30 μL of 60 mM TMB, and 550 μL of 20 mM HAc-NaAc buffer (pH = 4.0), and react in the dark for 20 min. Then add 100 μL of 9 M H2SO4 to stop the reaction. Measure the absorbance of the reaction product at 452 nm using a SpectraMax i3xreader.

[0137] Take 200 μL of 250 nM C 10 C 20 C 30 C 40The sample was incubated with 20 μL of 10 μg / mL Fe3O4@Au solution at 25℃ and 200 rpm for 30 min. Then, 50 μL of 300 mM H2O2, 30 μL of 60 mM TMB, and 550 μL of 20 mM HAc-NaAc buffer (pH = 4.0) were added, and the reaction was carried out in the dark for 20 min. Finally, 100 μL of 9 M H2SO4 was added to stop the reaction. The absorbance at 452 nm was measured using a SpectraMax i3xreader (Molecular Devices, Inc., Silicon Valley, CA).

[0138] Take 200 μL of C-rich ssDNA of different concentrations and 20 μL of 10 μg / mL Fe3O4@Au solution and incubate at 25℃ and 200 rpm for 30 min. Add 50 μL of 300 mM H2O2, 30 μL of 60 mM TMB and 550 μL of 20 mM HAc-NaAc buffer (pH = 4.0), and react in the dark for 20 min. Then add 100 μL of 9 M H2SO4 to stop the reaction. The absorbance value at 452 nm is measured using a SpectraMax i3x reader (Molecular Devices, Inc., Silicon Valley, CA).

[0139] like Figure 6 As shown in A, all nucleotides can enhance the peroxidase-like activity of Fe3O4@Au. The order in which nucleotides enhance the catalytic activity of Fe3O4@Au is: C 14 >A 14 >T 14 >G 14 >No DNA. C 14 Modified Fe3O4@Au NPs exhibited the strongest activity among the four nucleotides. At buffer pH 4.0, cytosine nucleotides were protonated, reducing their negative charge, which facilitated surface charge neutralization of Fe3O4@Au NPs, decreasing the repulsion between DNA molecules, and thus allowing for the adsorption of more cytosine nucleotides. Therefore, we investigated the effects of different amounts of cytosine nucleotides on the peroxidase activity of Fe3O4@Au NPs, such as... Figure 6 As shown in B, the order in which the number of cytosine nucleotides increases the catalytic activity of nanozymes is: C. 20 >C 10 >C 30 >C 40>No DNA. The optimal sequence length is approximately 20 cytosine nucleotides. Longer ssDNA is more readily adsorbed by Fe3O4@Au because the interaction between Fe3O4@Au and longer ssDNA is stronger. When the number of cytosine nucleotides exceeds 20, an excessive number of cytosine nucleotides cannot bind effectively to the nanozyme surface, and the interaction weakens. Figure 6 As shown in Figure C, the effect of C-rich ssDNA concentration on nanozyme activity was investigated. With increasing C-rich ssDNA concentration, the absorbance at 452 nm first increased and then decreased. The absorbance reached its maximum at a C-rich ssDNA concentration of 0.05 nM. With further increases in C-rich ssDNA concentration, excess C-rich ssDNA competed with the Fe3O4@Au nanozyme for TMB adsorption, leading to inhibition of Fe3O4@Au peroxidase activity.

[0140] 5. Optimization of sensing conditions

[0141] Experimental procedure:

[0142] Experimental group: 1 mL of bacteria was centrifuged at 12,000 rpm for 10 min, washed with 0.9% NaCl, resuspended in 200 μL of 0.25 nM C-rich ssDNA, incubated at 200 rpm for 1 h, centrifuged at 5000 rpm for 10 min, and the supernatant was incubated with 20 μL of 10 μg / mL Fe3O4@Au solution for 30 min. 50 μL of H2O2, 30 μL of TMB, and 550 μL of HAc-NaAc buffer (pH = 4.0) were added to the system, and the reaction was carried out in the dark for a period of time. Then, 100 μL of 9M H2SO4 was added to stop the reaction. The absorbance at 452 nm was measured using a SpectraMax i3xreader (Molecular Devices, Inc., Silicon Valley, CA). Control group: 1 mL of 0.9% NaCl was used instead of 1 mL of bacterial culture.

[0143] The ability of C-rich ssDNA to enhance the activity of Fe3O4@Au peroxidases is crucial for achieving the high performance of the proposed method, which depends on the adsorption affinity between C-rich ssDNA and Fe3O4@Au. Parameters such as H2O2 concentration, TMB, HAc-NaAc buffer, pH, reaction temperature, and reaction time were optimized. A0 and A represent the absorbance of the detection system at 452 nm in the absence and presence of Salmonella Typhimurium, respectively. First, we optimized the H2O2 concentration, such as... Figure 7As shown in Figure A, the absorbance value A0-A first increases and then decreases with increasing H2O2 concentration. The A0-A value reaches its maximum when the H2O2 concentration is 15 mM, indicating that 15 mM H2O2 is the optimal concentration for C-rich ssDNA to enhance the activity of Fe3O4@Au nanozymes. Next, the concentration of TMB will be optimized, as follows... Figure 7 As shown in Figure B, the absorbance value first increases and then decreases with increasing TMB concentration. The A0-A value reaches its maximum when the TMB concentration is 2 mM, indicating that 2 mM is the optimal concentration for C-rich ssDNA to enhance the activity of Fe3O4@Au nanozymes. Figure 7 As shown in Figure C, we investigated the enzymatic reaction elevation at three common temperatures, finding the optimal temperature to be 37℃. This indicates that 37℃ is the optimal reaction temperature for nanozyme adsorption of C-rich ssDNA. Then, the pH of the HAc-NaAC buffer was optimized, as shown in Figure C. Figure 7 As shown in D, A0-A reaches its maximum value when the pH of the HAc-NaAc buffer is 4, indicating that pH 4 of the HAc-NaAc buffer is the optimal pH for nanozyme adsorption of C-rich ssDNA. The catalytic activity of the reaction system without Salmonella typhimurium in the HAc-NaAc buffer is higher than that of the reaction system with Salmonella typhimurium. When the solution pH is further increased, the A0-A value decreases instead. Figure 7 As shown in E, A0-A first increases and then decreases with increasing HAc-NaAc buffer concentration. A0-A reaches its maximum value when the HAc-NaAc buffer concentration is 10 mM, indicating that 10 mM HAc-NaAc buffer is the optimal concentration for nanozyme adsorption of C-rich ssDNA. Finally, the reaction time was optimized, as shown in... Figure 7 As shown in F, A0-A first increases and then decreases. When the reaction time is 20 min, A0-A reaches its maximum value, indicating that 20 min is the optimal reaction time for nanozyme adsorption of C-rich ssDNA.

[0144] 6. Sensitivity

[0145] Experimental procedure:

[0146] Centrifuge 1 mL of bacteria at different concentration gradients at 12000 rpm for 10 min, wash with 0.9% NaCl, resuspend in 200 μL of 0.25 nM C-rich ssDNA, and incubate at 200 rpm for 1 h. After centrifugation at 5000 rpm for 10 min, incubate the supernatant with 20 μL of 10 μg / mL Fe3O4@Au solution for 30 min. Add 50 μL of 300 mM H2O2, 30 μL of 60 mM TMB, and 550 μL of 20 mM HAc-NaAc buffer (pH = 4.0) to the system, react in the dark for 20 min, and then add 100 μL of 9 M H2SO4 to stop the reaction. Measure the absorbance at 452 nm using a SpectraMax i3x reader (Molecular Devices; Inc., Silicon Valley; CA).

[0147] Under optimal conditions, use 2.8 × 10 1 ~2.8×10 6 The sensitivity of this method for determining the concentration of Salmonella typhimurium in CFU / mL. For example... Figure 8 As shown in Figure A, the solution color changes from dark yellow to pale yellow as the concentration of Salmonella typhimurium increases. Figure 8 From B, we can see that A-A0 has a good linear relationship with the logarithm of the concentration of Salmonella typhimurium, y = 0.1343x - 0.0298, and the correlation coefficient R is [missing value]. 2 The limit of detection was 0.9960. Based on 3σ / S, the limit of detection was 1.8 CFU / mL, where σ is the standard deviation of the blank sample and S is the slope of the linear equation. Importantly, the colorimetric method involving Fe3O4@Au recorded an ultra-low LOD and a convenient operating temperature (37°C). The high sensitivity can be attributed to two factors: (1) The special C-rich ssDNA has a dual enhancing effect, which can improve the peroxidase-like activity of Fe3O4@Au. The C-rich ssDNA consists of two DNA sequences. One of them is an oligonucleotide sequence with the same base composition, which has been shown to enhance the peroxidase-like activity of Fe3O4@Au. (2) Fe3O4@Au has high peroxidase-like activity. These characteristics ensure the high quality of Fe3O4@Au nanoparticles as signal detection probes, which also reveals the advantages of our proposed tagless C-rich ssDNA / Fe3O4@Au, such as high sensitivity, time saving, and simple operation. Therefore, this method has high sensitivity for the detection of Salmonella typhimurium.

[0148] 7. Specificity

[0149] Experimental procedure:

[0150] Take 1 mL of a concentration of 10 5 Several pathogenic bacteria at CFU / mL (Salmonella Typhimurium, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Listeria monocytogenes, Escherichia coli O) 157 (H7, Salmonella enteritidis) were centrifuged at 12000 rpm for 10 min, washed with 0.9% NaCl, resuspended in 200 μL of 0.25 nM C-rich ssDNA, incubated at 200 rpm for 1 h, centrifuged at 5000 rpm for 10 min, and the supernatant was incubated with 20 μL of 10 μg / mL Fe3O4@Au solution for 30 min. 50 μL of 300 mM H2O2, 30 μL of 60 mM TMB, and 550 μL of 20 mM HAc-NaAc buffer (pH = 4.0) were added to the system, and the reaction was carried out in the dark for 20 min. Then, 100 μL of 9 M H2SO4 was added to stop the reaction. The absorbance at 452 nm was measured using a SpectraMax i3x reader (Molecular Devices, Inc., Silicon Valley, CA).

[0151] The specificity of this method was evaluated using six other pathogenic bacteria. For example... Figure 9 As shown, it is clear that only *Salmonella typhimurium* caused a sharp increase in signal compared to other bacteria. The results indicate that this method has good specificity for *Salmonella typhimurium*, which is due to the specific recognition ability of C-rich ssDNA for *Salmonella typhimurium*.

[0152] 8. Detection of Salmonella Typhimurium in actual samples

[0153] Experimental procedure:

[0154] Lettuce and milk were purchased from the local market. Before inoculation, conventional culture methods were used to confirm that the milk and lettuce were not contaminated with *Salmonella typhimurium*. To simulate food contamination, 1 mL of bacteria at different concentrations was inoculated into 24 mL of milk (or 24 g of lettuce), followed by the addition of 225 mL of 0.9% NaCl. After thorough mixing, 1 mL of the mixture was centrifuged at 5000 rpm for 10 min and washed twice with 1 mL of sterile 0.9% NaCl. Finally, the sediment was resuspended in 1 mL of sterile 0.9% NaCl, and *Salmonella typhimurium* was detected following the same procedure.

[0155] like Figure 10 As shown, the regression equations for the lettuce and milk samples are y = 0.0766x - 0.0389 (R²). 2 =0.9825) and y = 0.0665x - 0.0231(R) 2=0.9875). The slopes of both regression equations for food samples were smaller than those for pure cultures, indicating a decrease in the sensitivity of this method in food samples. This may be due to the increase in background signal caused by the non-specific binding of nanozymes to food components. Based on 3σ / S, the LODs of this method in lettuce and milk were 2.7 CFU / mL and 3.2 CFU / mL, respectively. These results validate the applicability and feasibility of the proposed method for detecting Salmonella typhimurium in real samples.

[0156] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of cytosine repeating oligonucleotide C-rich ssDNA combined with Fe3O4@Au in the preparation of products for detecting Salmonella typhimurium, characterized in that, The C-rich ssDNA is obtained by linking 20 consecutive cytosine nucleotides to a nucleic acid aptamer; the sequence of the 20 consecutive cytosine nucleotides is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the C-rich ssDNA is shown in SEQ ID NO.

3.

3. A kit for detecting Salmonella Typhimurium and a method for detecting Salmonella Typhimurium for non-diagnostic purposes, characterized in that, The kit for detecting Salmonella typhimurium includes cytosine repeat oligonucleotide C-rich ssDNA, Fe3O4@Au, H2O2, TMB, HAc-NaAc buffer, and a negative control; the C-rich ssDNA is obtained by linking 20 consecutive cytosine nucleotides to nucleic acid aptamers; the sequence of the 20 consecutive cytosine nucleotides is shown in SEQ ID NO.1; The method includes the following steps: The sample to be tested was mixed with C-rich ssDNA aqueous solution and incubated for the first time. The product of the first incubation was centrifuged for the first time, and the supernatant after the first centrifugation was collected. The supernatant was mixed with Fe3O4@Au aqueous solution for a second incubation. H2O2, TMB and HAc-NaAc buffer were added to the second incubated solution to carry out the reaction in the dark. The reaction was terminated after the reaction in the dark and the absorbance of the reaction product at 452 nm was measured. The absorbance of the reaction product at 452 nm is denoted as A, and the absorbance of the negative control at 452 nm is denoted as A0. If the value of A0-A is equal to zero, it is determined that the sample to be tested does not contain Salmonella typhimurium; If the value of A0-A is greater than zero, the sample to be tested is determined to contain Salmonella typhimurium.

4. The method according to claim 3, characterized in that, After measuring the absorbance of the reaction product at 452 nm and obtaining the absorbance value, the concentration of Salmonella typhimurium in the sample to be tested is calculated according to the formula shown in Formula 1, Formula 2 or Formula 3. y = 0.1343x - 0.0298, and the correlation coefficient R² is 0.9960 (Equation 1); y = 0.0766x - 0.0389, and the correlation coefficient R² is 0.9825 (Equation 2). y = 0.0665x - 0.0231, and the correlation coefficient R² is 0.9875 (Equation 3); The formula shown in Equation 1 is applicable when the sample to be tested is a pure bacterial sample; The formula shown in Equation 2 is applicable when the sample to be tested is a lettuce sample; The formula shown in Equation 3 is applicable when the sample to be tested is a milk sample; In Equations 1 to 3, y is the absorbance value and x is the logarithmic value of the concentration of Salmonella typhimurium.

5. The method according to claim 3, characterized in that, The working concentration of the C-rich ssDNA is 0.05 nM; the working concentration of the Fe3O4@Au is 0.2 μg / mL; the working concentration of the H2O2 is 15 mM; the working concentration of the TMB is 2 mM; the working concentration of the HAc-NaAc buffer is 10 mM; and the pH value of the HAc-NaAc buffer is 4.

6. The method according to claim 3, characterized in that, The incubation temperature was 25°C, and the reaction temperature was 37°C.

7. The method according to claim 3, characterized in that, The light-avoidance reaction time is 20 minutes.

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