Primer for selectively detecting bacillus and preparation method thereof

Abstract

The invention discloses a primer for selectively detecting bacillus and a preparation method of the primer. The primer comprises noble metal nanoparticles of a bipyramid structure and a metal organic framework doped with lanthanide metal ions, and the metal organic framework doped with lanthanide metal ions grows in situ in a bipyramid tip area of the noble metal nanoparticles. According to the primer for selectively detecting the bacillus, disclosed by the invention, a marker picolinic acid of the bacillus can be selectively adsorbed in a complex sample, so that interference of environmental factors on detection is effectively avoided, and the detection accuracy of an SERS spectrum on the bacillus in a complex environment is greatly improved; besides, when the primer selectively adsorbs the marker picolinic acid, the Raman signal of the picolinic acid can be effectively enhanced by the electromagnetic fields generated by the two tips of the bipyramidal noble metal nanoparticles, so that the ultra-sensitive detection of the marker is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to a primer for selectively detecting Bacillus, and a preparation method of the primer. BACKGROUND

[0002] Bacillus is a common gram-positive bacteria belonging to Firmicutes. They are widely distributed in soil, water, air and in and outside of animals and plants. Bacillus has strong environmental adaptability and can produce endospores in complex environments. The endospores have good stability, can tolerate 60℃ high temperature for a long time, can survive for 20min at 120℃, and can maintain activity in gastric acid environment. The endospores of Bacillus are composed of more than 10% dry mass of 2,6-pyridinedicarboxylic acid (DPA), so the substance can be used as a marker molecule for detecting Bacillus. As one of Bacillus, Bacillus cereus often exists in milk, starch food, cooked food and food production lines. The enterotoxin secreted by Bacillus cereus will cause symptoms such as vomiting and diarrhea. Therefore, it is necessary to selectively detect Bacillus cereus in food.

[0003] Under light irradiation, photons interact with matter to produce scattering. Among them, the phenomenon that the frequency of scattered light is shifted is called Raman scattering. Raman scattering signals are derived from molecular vibrations and can characterize molecular bonds and chemical structure information. However, the Raman scattering cross-section is small, and the signal is usually extremely weak, which is difficult to effectively detect. The collective oscillation of free electrons on the surface of noble metals (surface plasmons) can be excited by laser, which can produce a localized enhanced electromagnetic field on the surface of the material, significantly amplifying the Raman signal intensity. The enhanced spectrum obtained in this way is called surface-enhanced Raman scattering (SERS).

[0004] As a rapid detection technique, SERS spectrum has wide application potential in the fields of food safety, environmental monitoring and material science. SERS spectrum can obtain the chemical bond position information of the detected molecule by adsorbing the detected molecule to the surface of the enhanced substrate, so as to realize the ultra-sensitive detection of trace substances. This characteristic can be used to analyze the composition of the substance, or to avoid potential safety hazards. However, in actual application, there are many non-target substances in the detected sample, which will also be adsorbed to the surface of the nano substrate and produce Raman signals, thereby interfering with the Raman signals of the target molecule. Although there are many sample pretreatment methods to remove the main interfering substances, these methods are often time-consuming and still leave a small amount of non-target substances. Due to the ultra-sensitive characteristic, the Raman signals of a small amount of non-target substances still exist in the surface enhanced Raman spectrum. In the prior art, most of the nano substrates have excellent Raman signal enhancement performance, but lack the ability of selective adsorption. In this case, most of the nano substrates can only detect a single substance, and the accuracy of detecting complex substances is low, and it is difficult to identify the target molecule.

[0005] Due to the interference of starch, fat, inorganic matter and other factors in food, the noble metal nano substrate with good Raman enhancement performance lacks the selective adsorption ability of the Bacillus marker pyridinedicarboxylic acid, so the detection accuracy of the surface enhanced Raman spectrum for the Bacillus marker pyridinedicarboxylic acid is low, and the Bacillus cannot be selectively detected in the actual sample. SUMMARY

[0006] The purpose of the present application is to provide a primer capable of selectively adsorbing the Bacillus marker pyridinedicarboxylic acid, while effectively enhancing the Raman signal of pyridinedicarboxylic acid, so as to realize the specific ultra-sensitive detection of Bacillus in the actual sample by SERS; another purpose of the present application is to provide a preparation method of the primer.

[0007] Technical scheme: The primer for selectively detecting Bacillus provided by the present application comprises noble metal nanoparticles in a double cone structure and metal organic framework doped with lanthanide metal ions, and the metal organic framework doped with lanthanide metal ions is in-situ grown on the double cone tip region of the noble metal nanoparticles.

[0008] The noble metal nanoparticles are gold nanoparticles or silver nanoparticles, the length of the noble metal nanoparticles is 50-110 nm, and the diameter is 20-35 nm; the lanthanide metal is at least one of terbium (Tb) or europium (Eu); and the metal organic framework is ZIF-8 (zeolite imidazolate framework-8).

[0009] The doping amount of the lanthanide metal ion on the metal organic framework is 4.9% of the mass of the metal organic framework; and on the noble metal nanoparticle, the thickness of the metal organic framework coated on the tip of the noble metal nanoparticle is 20 nm, that is, the coating thickness of the MOF on the tip of the noble metal nanoparticle is 20 nm, and the coating thickness is used to represent the coating amount.

[0010] The preparation method of the primer comprises the following steps:

[0011] (1) The noble metal nanoparticles in a bipyramid structure are prepared by using a seed growth method;

[0012] (2) The metal organic framework is grown on the surface of the noble metal nanoparticles;

[0013] (3) The lanthanide metal ion is doped into the metal organic framework by immersion to obtain the primer for selectively detecting bacillus.

[0014] In step (2), specifically, cetyltrimethylammonium bromide, methyl imidazole and water are mixed to obtain solution A; the noble metal nanoparticle dispersion liquid and cetyltrimethylammonium bromide are mixed to obtain solution B; zinc nitrate and solution B are added to solution A respectively, and after reaction and centrifugation, the composite material with the metal organic framework selectively grown on the tip of the bipyramid of the noble metal nanoparticles is obtained; the molar ratio of the noble metal nanoparticles, methyl imidazole and zinc nitrate is 1:36-80:4.5-8.

[0015] In step (3), specifically, the composite material is soaked in a soluble salt solution of the lanthanide metal, and after soaking, the primer for selectively detecting bacillus is obtained; the molar ratio of the composite material and the lanthanide metal salt is 415:1.

[0016] The primer for selectively detecting bacillus is obtained by preparing the bipyramid noble metal nanoparticles with high uniformity in morphology by a wet chemical method, then selectively growing the metal organic framework material on the tip of the bipyramid of the noble metal nanoparticles by using a surfactant, and finally doping the lanthanide metal into the metal organic framework material by immersion. The metal organic framework compound doped with the lanthanide metal can selectively adsorb the marker pyridine dicarboxylic acid of bacillus, so that the pyridine dicarboxylic acid is gathered near the bipyramid, under the action of an external light field, a strong local electromagnetic field is generated near the tip of the bipyramid, so that the Raman signal of the marker pyridine dicarboxylic acid is greatly enhanced. The primer prepared in the application can selectively adsorb pyridine dicarboxylic acid, and effectively enhance the Raman signal of pyridine dicarboxylic acid by using the electromagnetic field generated by the bipyramid, so as to realize specific and ultra-sensitive detection of bacillus in a complex environment by using SERS spectrum.

[0017] Beneficial effects: compared with the prior art, the present application has the following remarkable advantages: the primer of the present application can selectively adsorb the marker pyridinedicarboxylic acid of bacillus in a complex sample, thereby effectively avoiding the interference of environmental factors on detection, and further greatly improving the detection accuracy of SERS spectrum on bacillus in a complex environment; in addition, while the primer of the present application selectively adsorbs the marker pyridinedicarboxylic acid, the electromagnetic field generated by the two tips of the bipyramidal noble metal nanoparticle can effectively enhance the Raman signal of pyridinedicarboxylic acid, realizing the super-sensitive detection of the marker; the present application solves the problems of time-consuming and environmental interference of traditional detection methods; the primer for selectively detecting bacillus of the present application can be applied to the rapid detection of bacillus in food and environmental monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The preparation flow chart and detection principle diagram of the primer for selectively detecting bacillus (Au@MOF-Tb) of the present application;

[0019] Figure 2 The transmission electron microscope (TEM) diagram of the gold nanobipyramid (Au);

[0020] Figure 3 The TEM diagram of the metal organic framework material (MOF) ZIF-8;

[0021] Figure 4 The TEM diagram of the primer for selectively detecting bacillus (Au@MOF-Tb) prepared in Example 1;

[0022] Figure 5 The TEM diagram of the primer for selectively detecting bacillus (Au@MOF-Tb) prepared in Example 5, at this time the metal organic framework material has been completely wrapped on the surface of the gold bipyramid;

[0023] Figure 6 The near-field electromagnetic field simulation diagram of the gold nanobipyramid;

[0024] Figure 7 The Raman spectrum detection effect of pyridinedicarboxylic acid by the gold nanobipyramid (Au), the metal organic framework material (MOF), the nanocomposite (Au@MOF) and the primer for selectively detecting bacillus (Au@MOF-Tb) prepared in Example 1;

[0025] Figure 8 The Raman spectrum detection effect of pyridinedicarboxylic acid of different concentrations by the primer for selectively detecting bacillus (Au@MOF-Tb) prepared in Example 1, the concentrations of pyridinedicarboxylic acid from top to bottom are 10 -6 , 10 -7 , 10 -8 , 10 -9,10 -10 ,10 -11 M;

[0026] Figure 9 The effect of the primer for detecting Bacillus prepared in Comparative Examples 4 and 5 on the Raman spectrum detection of pyridinedicarboxylic acid. DETAILED DESCRIPTION

[0027] Example 1

[0028] The present application relates to a method for preparing a primer for selectively detecting Bacillus, comprising the following steps:

[0029] (1) A freshly prepared sodium borohydride solution (0.01 M, 0.15 mL) was added to a solution composed of chloroauric acid (0.01 M, 0.125 mL), trisodium citrate (0.01 M, 0.25 mL) and water (9.625 mL) to obtain a seed solution under vigorous stirring, and the obtained seed solution was stored at room temperature for 2 hours before use;

[0030] (2) A seed solution (200 μL, 1.2 x 10 -4 M) of step (1) was added to the above growth solution, and the reaction was carried out at room temperature for 6 hours;

[0031] (3) The sample after the reaction of step (2) was centrifuged, and the precipitate (0.008 g) was dispersed in a cetyltrimethylammonium chloride solution (0.08 M, 30 mL), and then silver nitrate solution (0.01 M, 6 mL) and ascorbic acid solution (0.1 M, 3 mL) were added thereto, and the reaction was carried out in an oven for 4 hours;

[0032] (4) The solution after the reaction of step (3) was centrifuged, and the precipitate (0.014 g) after centrifugation was added to a cetyltrimethylammonium bromide solution (0.05 M, 30 mL), and was left to stand overnight, and then the supernatant was removed, and the remaining precipitate was re-dispersed in water; Subsequently, ammonia water (30 wt%, 0.4 mL) and hydrogen peroxide (0.1 M, 0.3 mL) were added to the solution, and the reaction was carried out for 4 hours; and then, the solution after the completion of the reaction was centrifuged, and the precipitate was re-dispersed in water to obtain a gold nanobipyramid dispersion solution;

[0033] (5) mixed cetyltrimethylammonium bromide solution (1 mM, 150 μL), methylimidazole (0.3 M, 600 μL) and water (680 μL) to obtain solution A; mixed gold nanobipyramid dispersion (0.005 M, 1 mL), cetyltrimethylammonium bromide solution (5 mM, 100 μL) and water (1 mL) to obtain solution B; added zinc nitrate solution (0.03 M, 750 μL) and solution B into solution A respectively, centrifuged after reaction for 25 min to obtain nanocomposites with metal organic framework selectively grown on the tips of gold nanobipyramids;

[0034] (6) immersed the nanocomposites (0.017 g) in terbium nitrate hexahydrate solution (0.5 mL, 10 mM) and waited for 10 min to obtain the primer (Au@MOF-Tb) for selectively detecting bacillus.

[0035] The primer prepared in Example 1 is composed of gold nanobipyramids and zeolitic imidazolate framework-8 (ZIF-8) doped with terbium ions, the ZIF-8 doped with terbium ions is selectively grown on the tip region of the gold nanobipyramids, and the thickness of the metal organic framework coated on the tip of the noble metal nanoparticles is 20 nm.

[0036] The detection method of the primer for selectively detecting bacillus prepared in Example 1 is as follows: mixed the primer (Au@MOF-Tb) (0.025 M, 40 μL) with the sample solution (10 μL) and dropped onto the solid surface, after the solution was dried, the Raman spectrometer was used for detection.

[0037] The dried solid surface was irradiated with a laser with a wavelength of 785 nm, and the SERS spectrum was collected. As shown in the detection results shown in Figure 7 , the bipyramid gold nanoparticles with metal organic framework doped with lanthanide metal can selectively adsorb and detect the marker pyridinedicarboxylic acid of bacillus. Compared with Comparative Example 1, Comparative Example 2 and Comparative Example 3, the primer (Au@MOF-Tb) has high selectivity and high detection sensitivity for the marker pyridinedicarboxylic acid of bacillus, as shown in Figure 8 , the lowest detection concentration is as low as 10 -11 M. While the detection lower limit of the marker pyridinedicarboxylic acid of bacillus in the prior art is in the range of 10 -7 M to 10 -9 M, the detection sensitivity is not as good as the bipyramid gold nanoparticles with metal organic framework doped with lanthanide metal prepared in Example 1. In addition, the marker pyridinedicarboxylic acid of bacillus can be selectively adsorbed in complex samples, thereby effectively avoiding the interference of complex environmental factors on detection and improving the detection accuracy.

[0038] Example 2

[0039] The present application relates to a method for preparing primers for selectively detecting Bacillus, comprising the following steps:

[0040] (1) A freshly prepared sodium borohydride solution (0.01M, 0.15mL) was added to a mixture of chloroauric acid solution (0.01M, 0.125mL), trisodium citrate solution (0.01M, 0.25mL) and water (9.625mL) to obtain a seed solution under vigorous stirring, which was stored at room temperature for 2 hours before use;

[0041] (2) A growth solution was prepared by sequentially adding chloroauric acid solution (0.01M, 2mL), silver nitrate solution (0.01M, 0.4mL), hydrochloric acid (1M, 0.8mL) and ascorbic acid solution (0.1M, 0.32mL) into a cetyltrimethylammonium bromide solution (0.1M, 40mL); then the seed solution (200μL, 1.2×10 -4 M) of step (1) was added to the above growth solution, and the reaction was carried out at room temperature for 6 hours;

[0042] (3) The sample after the reaction of step (2) was centrifuged, and the precipitate (0.008g) was dispersed in a cetyltrimethylammonium chloride solution (0.08M, 30mL), followed by adding silver nitrate solution (0.01M, 6mL) and ascorbic acid solution (0.1M, 3mL) thereto, and the reaction was carried out in an oven for 4 hours;

[0043] (4) The solution after the reaction of step (3) was centrifuged, and the precipitate (0.014g) after centrifugation was added to a cetyltrimethylammonium bromide solution (0.05M, 30mL), and was allowed to stand overnight, followed by removing the supernatant, and the remaining precipitate was re-dispersed in water; then, ammonia water (30wt%, 0.4mL) and hydrogen peroxide (0.1M, 0.3mL) were added to the solution, and the reaction was carried out for 4 hours; then, the solution after the reaction was centrifuged, and the precipitate was re-dispersed in water to obtain a gold nanobipyramid dispersion solution;

[0044] (5) A solution A was prepared by mixing a cetyltrimethylammonium bromide solution (1mM, 100μL), methyl imidazole (0.4M, 1000μL) and water (350μL) and stirring for 10 minutes; a solution B was prepared by mixing the gold nanobipyramid dispersion solution (0.005M, 1mL), a cetyltrimethylammonium bromide solution (5mM, 150μL) and water (1mL) and stirring for 10 minutes; a metal-organic framework material selectively grown on the tip of the gold nanobipyramid was obtained by adding a zinc nitrate solution (0.04M, 1000μL) and the solution B to the solution A, respectively, centrifuging after the reaction for 25 minutes;

[0045] (6) The nanocomposite (about 0.017 g) was immersed in a terbium nitrate hexahydrate solution (0.5 mL, 10 mM) for 10 minutes to obtain a primer (Au@MOF-Tb) for selective detection of Bacillus.

[0046] The primer for selective detection of Bacillus prepared in Example 2 has a thicker MOF layer, and the specific SERS detection method is as follows: the primer (0.025 M, 40 μL) is mixed with a test solution (10 μL) and then dropped onto a solid surface, and after the solution is dried, a Raman spectrometer is used for detection. A laser with a wavelength of 785 nm is used to irradiate the dried solid surface, and the SERS spectrum is collected. The detection performance of the primer obtained in Example 2 is basically the same as that of Example 1.

[0047] Example 3

[0048] The preparation method of the primer for selective detection of Bacillus according to the present application comprises the following steps:

[0049] (1) Freshly prepared sodium borohydride solution (0.01 M, 0.15 mL) is added to a mixture of chloroauric acid solution (0.01 M, 0.125 mL), trisodium citrate solution (0.01 M, 0.25 mL) and water (9.625 mL) to obtain a seed solution under vigorous stirring, and the obtained seed solution is stored at room temperature for 2 hours before use;

[0050] (2) Chloroauric acid solution (0.01 M, 2 mL), silver nitrate solution (0.01 M, 0.4 mL), hydrochloric acid (1 M, 0.8 mL) and ascorbic acid solution (0.1 M, 0.32 mL) are sequentially added to a hexadecyltrimethylammonium bromide solution (0.1 M, 40 mL) to prepare a growth solution; then the seed solution (200 μL, 1.2 × 10 -4 M) of step (1) is added to the above growth solution, and the reaction is carried out at room temperature for 6 hours;

[0051] (3) The sample after step (2) is centrifuged, and the precipitate (0.008 g) is dispersed in a hexadecyltrimethylammonium chloride solution (0.08 M, 30 mL), and then silver nitrate solution (0.01 M, 6 mL) and ascorbic acid solution (0.1 M, 3 mL) are added thereto, and the reaction is carried out in an oven for 4 hours;

[0052] (4) centrifuging the solution after the reaction of step (3), adding the precipitate (0.014 g) after centrifugation to a cetyltrimethylammonium bromide solution (0.05 M, 30 mL), standing overnight, then removing the supernatant and redispersing the remaining precipitate in water; then, adding ammonia water (30 wt%, 0.4 mL) and hydrogen peroxide (0.1 M, 0.3 mL) to the solution, and reacting for 4 hours; then, centrifuging the solution after the reaction is complete, redispersing the precipitate in water, and obtaining a gold nanobipyramid dispersion;

[0053] (5) mixing a cetyltrimethylammonium bromide solution (1 mM, 100 μL), methylimidazole (0.5 M, 600 μL), and water (650 μL) to obtain solution A, mixing a gold nanobipyramid dispersion (0.005 M, 1 mL), a cetyltrimethylammonium bromide solution (5 mM, 100 μL), and water (1 mL) to obtain solution B, adding a zinc nitrate solution (0.05 M, 750 μL) and solution B to solution A, respectively, and centrifuging after reacting for 25 minutes to obtain a nanocomposite in which metal-organic framework materials selectively grow on the tips of the gold nanobipyramids;

[0054] (6) immersing the nanocomposite (about 0.017 g) in a terbium nitrate hexahydrate solution (0.5 mL, 10 mM) and waiting for 10 minutes to obtain a primer (Au@MOF-Tb) for selectively detecting bacillus.

[0055] The detection method of the primer for selectively detecting bacillus prepared in Example 3 is as follows: after mixing the primer (0.025 M, 40 μL) with a test solution (10 μL), the mixture is added dropwise to a solid surface, the solution is dried, and then a Raman spectrometer is used for detection. A laser with a wavelength of 785 nm is used to irradiate the dried solid surface, and SERS spectra are collected. The SERS detection performance of the primer obtained in Example 3 is basically the same as that of Example 1.

[0056] Comparative Example 1

[0057] A method for preparing gold nanobipyramids, comprising the following steps:

[0058] (1) adding a freshly prepared sodium borohydride solution (0.01 M, 0.15 mL) to a mixture of chloroauric acid solution (0.01 M, 0.125 mL), trisodium citrate solution (0.01 M, 0.25 mL), and water (9.625 mL) to obtain a seed solution under vigorous stirring, and storing the obtained seed solution at room temperature for 2 hours before use;

[0059] (2) A growth solution was prepared by sequentially adding chloroauric acid solution (0.01 M, 2 mL), silver nitrate solution (0.01 M, 0.4 mL), hydrochloric acid (1 M, 0.8 mL), and ascorbic acid solution (0.1 M, 0.32 mL) into a hexadecyltrimethylammonium bromide solution (0.1 M, 40 mL); then the seed solution (200 μL, 1.2 x 10 -4 M) of step (1) was added into the above growth solution, and the reaction was carried out at room temperature for 6 hours;

[0060] (3) The sample after the reaction of step (2) was centrifuged, and the precipitate (0.008 g) was dispersed into a hexadecyltrimethylammonium chloride solution (0.08 M, 30 mL), and then silver nitrate solution (0.01 M, 6 mL) and ascorbic acid solution (0.1 M, 3 mL) were added thereinto, and the reaction was carried out in an oven for 4 hours;

[0061] (4) The solution after the reaction of step (3) was centrifuged, and the precipitate (0.014 g) after centrifugation was added into a hexadecyltrimethylammonium bromide solution (0.05 M, 30 mL), and was left overnight, and then the supernatant was removed, and the remaining precipitate was re-dispersed into water; subsequently, ammonia water (30 wt%, 0.4 mL) and hydrogen peroxide (0.1 M, 0.3 mL) were added into the solution, and the reaction was carried out for 4 hours; then, the solution after the reaction was centrifuged, and the precipitate was re-dispersed in water, to obtain a gold nanobipyramid dispersion.

[0062] The method for detecting the Bacillus marker by using the gold nanobipyramids prepared in Comparative Example 1 was as follows: the gold nanobipyramid dispersion (0.025 M, 40 μL) was mixed with the solution (10 μL) to be detected, and then was dropped onto a solid surface, and after the solution was dried, a Raman spectrometer was used for detection. The dried solid surface was irradiated with a laser with a wavelength of 785 nm, and the SERS spectrum was collected, and the detection result was as shown in Figure 7 .

[0063] Comparative Example 2

[0064] A preparation method of a metal organic framework material was as follows: a hexadecyltrimethylammonium bromide solution (1 mM, 100 μL), a methyl imidazole (0.5 M, 600 μL), and water (650 μL) were mixed, and were stirred for 10 minutes, to obtain a solution A; a zinc nitrate solution (0.05 M, 750 μL) and a hexadecyltrimethylammonium bromide solution (5 mM, 100 μL) were added into the solution A, and the reaction was carried out for 25 minutes, and then was centrifuged, to obtain the metal organic framework material.

[0065] The method for detecting Bacillus sp. markers using the metal organic framework material prepared in Comparative Example 2 is as follows: the metal organic framework material dispersion (0.025 M, 40 μL) is mixed with the test solution (10 μL), and then added dropwise to the solid surface. After the solution is dried, a Raman spectrometer is used for detection. A laser with a wavelength of 785 nm is used to irradiate the dried solid surface, and the SERS spectrum is collected. The detection results are shown in Figure 7

[0066] Comparative Example 3

[0067] A method for preparing a nanocomposite (Au@MOF) includes the following steps:

[0068] (1) Freshly prepared sodium borohydride solution (0.01 M, 0.15 mL) is added to a mixture of chloroauric acid solution (0.01 M, 0.125 mL), trisodium citrate solution (0.01 M, 0.25 mL) and water (9.625 mL) to obtain a seed solution. The seed solution is stored at room temperature for 2 hours and then used;

[0069] (2) Chloroauric acid solution (0.01 M, 2 mL), silver nitrate solution (0.01 M, 0.4 mL), hydrochloric acid (1 M, 0.8 mL) and ascorbic acid solution (0.1 M, 0.32 mL) are sequentially added to the cetyltrimethylammonium bromide solution (0.1 M, 40 mL) to prepare a growth solution. Then, the seed solution (200 μL, 1.2 × 10 -4 M) of step (1) is added to the above growth solution, and the reaction is carried out at room temperature for 6 hours;

[0070] (3) The sample after step (2) is centrifuged, and the precipitate (0.008 g) is dispersed in the cetyltrimethylammonium chloride solution (0.08 M, 30 mL). Then, silver nitrate solution (0.01 M, 6 mL) and ascorbic acid solution (0.1 M, 3 mL) are added, and the reaction is carried out in an oven for 4 hours;

[0071] (4) The solution after step (3) is centrifuged, and the precipitate (0.014 g) after centrifugation is added to the cetyltrimethylammonium bromide solution (0.05 M, 30 mL), and is left overnight. Then, the supernatant is removed, and the remaining precipitate is re-dispersed in water. Subsequently, ammonia water (30 wt%, 0.4 mL) and hydrogen peroxide (0.1 M, 0.3 mL) are added to the solution, and the reaction is carried out for 4 hours. Then, the solution after the reaction is completed is centrifuged, and the precipitate is re-dispersed in water to obtain a gold nanopyramid dispersion;

[0072] ​(5) mixed cetyltrimethylammonium bromide solution (1 mM, 100 μL), methylimidazole (0.3 M, 600 μL) and water (650 μL) to obtain solution A; mixed gold nanobipyramid dispersion (0.005 M, 1 mL), cetyltrimethylammonium bromide solution (5 mM, 100 μL) and water (1 mL) to obtain solution B; added zinc nitrate solution (0.03 M, 750 μL) and solution B to solution A respectively, centrifuged after reaction for 25 min to obtain nanocomposites with metal-organic framework selectively grown on the tips of gold nanobipyramids.

[0073] The method for detecting Bacillus markers by using the nanocomposites (Au@MOF) prepared in Comparative Example 3 is as follows: mixed the nanocomposite dispersion (0.025 M, 40 μL) with the sample solution (10 μL) and then dropped onto the solid surface, after the solution was dried, the Raman spectrometer was used for detection. The dried solid surface was irradiated with a laser of 785 nm wavelength, and the SERS spectrum was collected. The detection results are shown in Fig. 2. Figure 7 Figure 7 In the above method, the concentration of the pyridinedicarboxylic acid is 10 -6 M, and the detection effect from strong to weak is in the order of primer for selectively detecting Bacillus (Au@MOF-Tb), nanocomposite (Au@MOF), gold nanobipyramid (Au) and metal-organic framework (MOF).

[0074] Comparative Example 4

[0075] A preparation method of a primer for detecting Bacillus, comprising the following steps:

[0076] (1) added freshly prepared sodium borohydride solution (0.01 M, 0.15 mL) to a solution composed of chloroauric acid (0.01 M, 0.125 mL), trisodium citrate (0.01 M, 0.25 mL) and water (9.625 mL) to obtain a seed solution under vigorous stirring, and the obtained seed solution was stored at room temperature for 2 hours before use;

[0077] (2) added chloroauric acid solution (0.01 M, 2 mL), silver nitrate solution (0.01 M, 0.4 mL), hydrochloric acid (1 M, 0.8 mL) and ascorbic acid solution (0.1 M, 0.32 mL) to cetyltrimethylammonium bromide solution (0.1 M, 40 mL) in sequence to prepare a growth solution; then added the seed solution (200 μL, 1.2 × 10 -4 M) of step (1) to the above growth solution, and reacted at room temperature for 6 hours;

[0078] ​(3) centrifuging the sample after step (2) reaction, and dispersing the precipitate (0.008 g) into a cetyltrimethylammonium chloride solution (0.08 M, 30 mL), then adding a silver nitrate solution (0.01 M, 6 mL) and an ascorbic acid solution (0.1 M, 3 mL) into the solution, and reacting in an oven for 4 hours;

[0079] (4) centrifuging the solution after step (3) reaction, and adding the precipitate (0.014 g) after centrifugation into a cetyltrimethylammonium bromide solution (0.05 M, 30 mL), and standing overnight, then removing the supernatant, and re-dispersing the remaining precipitate into water; subsequently, adding ammonia water (30 wt%, 0.4 mL) and hydrogen peroxide (0.1 M, 0.3 mL) into the solution, and reacting for 4 hours; then, centrifuging the solution after reaction, and re-dispersing the precipitate into water to obtain a gold nanobipyramid dispersion solution;

[0080] (5) mixing a cetyltrimethylammonium bromide solution (1 mM, 150 μL), a methyl imidazole (0.3 M, 600 μL) and water (680 μL) to obtain solution A, and mixing a gold nanobipyramid dispersion solution (0.005 M, 1 mL), a cetyltrimethylammonium bromide solution (5 mM, 100 μL) and water (1 mL) to obtain solution B, and adding a zinc nitrate solution (0.03 M, 750 μL) and solution B into solution A respectively, and centrifuging after reacting for 25 minutes to obtain a nanocomposite with metal-organic framework materials selectively grown on the tips of the gold nanobipyramids;

[0081] (6) immersing the nanocomposite (0.017 g) into a terbium nitrate hexahydrate solution (0.5 mL, 20 mM) to obtain a primer (Au@MOF-Tb) for selectively detecting bacillus after waiting for 10 minutes.

[0082] The primer prepared in Comparative Example 4 is composed of gold nanobipyramids and zeolitic imidazolate framework-8 doped with terbium ions, and the zeolitic imidazolate framework-8 doped with terbium ions is selectively grown on the tip region of the gold nanobipyramids.

[0083] The detection method of the primer for selectively detecting bacillus prepared in Comparative Example 4 is as follows: mixing the primer (0.025 M, 40 μL) with a test solution (10 μL) and dropping onto a solid surface, and detecting using a Raman spectrometer after the solution is dried. A laser with a wavelength of 785 nm is used to irradiate the dried solid surface, and SERS spectrum is collected. The detection result is shown in FIG. 6, and the detection sensitivity of the primer is weaker than that of the primer described in Examples 1-3. Figure 9

[0084] Comparative Example 5

[0085] ​A method for preparing a primer for detecting bacillus, comprising the following steps:

[0086] (1) A freshly prepared solution of sodium borohydride (0.01 M, 0.15 mL) was added to a solution of chloroauric acid (0.01 M, 0.125 mL), trisodium citrate (0.01 M, 0.25 mL) and water (9.625 mL) to obtain a seed solution under vigorous stirring, and the obtained seed solution was stored at room temperature for 2 hours before use;

[0087] (2) A growth solution was prepared by sequentially adding a chloroauric acid solution (0.01 M, 2 mL), a silver nitrate solution (0.01 M, 0.4 mL), a hydrochloric acid solution (1 M, 0.8 mL) and an ascorbic acid solution (0.1 M, 0.32 mL) into a cetyltrimethylammonium bromide solution (0.1 M, 40 mL), and then the seed solution (200 μL, 1.2 × 10 -4 M) of step (1) was added to the above growth solution, and the reaction was carried out at room temperature for 6 hours;

[0088] (3) The sample after the reaction of step (2) was centrifuged, and the precipitate (0.008 g) was dispersed in a cetyltrimethylammonium chloride solution (0.08 M, 30 mL), and then a silver nitrate solution (0.01 M, 6 mL) and an ascorbic acid solution (0.1 M, 3 mL) were added thereto, and the reaction was carried out in an oven for 4 hours;

[0089] (4) The solution after the reaction of step (3) was centrifuged, and the precipitate (0.014 g) after centrifugation was added to a cetyltrimethylammonium bromide solution (0.05 M, 30 mL), and was left to stand overnight, and then the supernatant was removed, and the remaining precipitate was re-dispersed in water; then, an ammonia water (30 wt%, 0.4 mL) and a hydrogen peroxide solution (0.1 M, 0.3 mL) were added to the solution, and the reaction was carried out for 4 hours; then, the solution after the reaction was centrifuged, and the precipitate was re-dispersed in water to obtain a gold nanobipyramid dispersion solution;

[0090] (5) A cetyltrimethylammonium bromide solution (1 mM, 150 μL), a methyl imidazole (1.5 M, 600 μL) and water (680 μL) were mixed to obtain solution A, and a gold nanobipyramid dispersion solution (0.005 M, 1 mL), a cetyltrimethylammonium bromide solution (5 mM, 100 μL) and water (1 mL) were mixed to obtain solution B, and a zinc nitrate solution (0.15 M, 750 μL) and solution B were added to solution A, respectively, and the reaction was carried out for 25 minutes, and then centrifugation was carried out to obtain a nanocomposite in which a metal-organic framework material was selectively grown on the tip of a gold nanobipyramid;

[0091] (6) The nanocomposite (0.017 g) was immersed in a terbium nitrate hexahydrate solution (0.5 mL, 10 mM) for 10 minutes to obtain a primer (Au@MOF-Tb) for selective detection of Bacillus.

[0092] The primer prepared in Comparative Example 5 is composed of a gold nanobipyramid and a zeolitic imidazolate framework-8 doped with terbium ions, and the zeolitic imidazolate framework-8 doped with terbium ions completely wraps the surface of the gold nanobipyramid.

[0093] The detection method of the primer for selective detection of Bacillus prepared in Comparative Example 5 is as follows: the primer (0.025 M, 40 μL) is mixed with a test solution (10 μL) and then dropped onto a solid surface, and after the solution is dried, a Raman spectrometer is used for detection, a laser with a wavelength of 785 nm is used to irradiate the dried solid surface, and SERS spectra are collected, and the detection results are shown in FIG. 6. Figure 9 As shown in FIG. 6, the detection sensitivity of the primer is weaker than that of the primers described in Examples 1-3 and Comparative Example 4. This is because the thick MOF makes Au@MOF-Tb have no SERS performance, because the adsorbed DPA molecules are too far away from Au to enhance Raman.

[0094] The primer of the present application can selectively adsorb the marker pyridinedicarboxylic acid of Bacillus in a complex sample, effectively avoiding the interference of complex environmental factors on detection; in addition, after selectively adsorbing the marker pyridinedicarboxylic acid, the primer of the present application can generate a local electromagnetic field to enhance the Raman signal of the specifically adsorbed pyridinedicarboxylic acid, thereby realizing specific and ultra-sensitive detection of Bacillus (wherein Bacillus is Bacillus cereus).

Claims

1. A primer for selective detection of Bacillus subtilis, characterized in that: It includes noble metal nanoparticles with a bipyramidal structure and metal-organic framework materials doped with lanthanide metal ions, with the metal-organic frameworks doped with lanthanide metal ions growing in situ at the tip region of the bipyramidal structure of the noble metal nanoparticles.

2. The primers for selective detection of Bacillus according to claim 1, characterized in that: The noble metal nanoparticles are gold nanoparticles or silver nanoparticles, and the length of the noble metal nanoparticles is 50-110 nm and the diameter is 20-35 nm.

3. The primers for selective detection of Bacillus according to claim 1, characterized in that: The lanthanide metal is at least one of terbium or europium.

4. The primers for selective detection of Bacillus according to claim 1, characterized in that: The metal-organic framework is ZIF-8.

5. The primers for selective detection of Bacillus according to claim 1, characterized in that: The doping amount of lanthanide metal ions in the metal-organic framework is 4.9% to 5% of the mass of the metal-organic framework.

6. The primers for selective detection of Bacillus according to claim 5, characterized in that: On the noble metal nanoparticles, the metal-organic framework is coated at the tip of the noble metal nanoparticles with a thickness of not less than 20 nm.

7. The method for preparing the primers according to claim 1, characterized in that, Includes the following steps: (1) Noble metal nanoparticles with a bipyramidal structure were prepared by seed growth method; (2) Growing metal-organic frameworks on the surface of noble metal nanoparticles; (3) Lanthanide metal ions were doped into the metal-organic framework by soaking to obtain primers for selective detection of Bacillus.

8. The preparation method according to claim 7, characterized in that: In step (2), specifically: hexadecyltrimethylammonium bromide, methylimidazole and water are mixed and stirred thoroughly to obtain solution A; the noble metal nanoparticle dispersion and hexadecyltrimethylammonium bromide are mixed and stirred thoroughly to obtain solution B; zinc nitrate and solution B are added to solution A respectively, and centrifuged after reaction to obtain a composite material in which metal-organic frameworks are selectively grown on the bipyramidal tips of noble metal nanoparticles.

9. The preparation method according to claim 8, characterized in that: The molar ratio of precious metal nanoparticles, methylimidazole, and zinc nitrate is 1:36-80:4.5-8.

10. The preparation method according to claim 7, characterized in that: In step (3), specifically, the composite material is immersed in a soluble salt solution of lanthanide metals, and after immersion, primers for selective detection of Bacillus are obtained.

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