GO-AgNPs-Eu 3+ Fluorescent probe, paper-based sensor, preparation method and application thereof

Abstract

The present invention provides a GO-AgNPs-Eu<supgt;3+< / supgt> fluorescent probe, a paper-based sensor, and a preparation method and application thereof. The paper-based sensor is a paper-based sensor developed based on a GO-AgNPs-Eu<supgt;3+< / supgt> fluorescent probe loaded on a polyvinylidene fluoride (PVDF) microporous membrane in combination with a smartphone. The GO-AgNPs-Eu<supgt;3+< / supgt> fluorescent probe prepared by the present invention has high selectivity for DPA, strong anti-interference ability, and low detection limits for DPA and spores. The paper-based sensor realizes the quantitative detection of DPA and spores through the RGB change of the smartphone, and is verified by the actual spore amounts in milk and water. It realizes the rapid detection of foodborne spores on the dual platforms of fluorescence based on the GO-AgNPs-Eu<supgt;3+< / supgt> fluorescent probe and the paper-based sensor.

Description

Technical Field

[0001] The present invention relates to GO-AgNPs-Eu 3+ the preparation of a fluorescent probe and the development of a GO-AgNPs-Eu 3+ fluorescent probe-loaded polyvinylidene fluoride (PVDF) microfiltration membrane combined with a smartphone paper-based sensor, and specifically relates to the preparation of a fluorescent probe and the development of a paper-based sensor for the detection application of foodborne bacilli spores, belonging to the fields of nanomaterials and food safety detection. Background Art

[0002] Under environmental stress, bacilli form a round or oval dormant body - spore with extremely strong stress resistance through a series of temporal and spatial gene expressions to resist adverse environments. They can survive in a dormant state for several years and constantly sense environmental changes. Once suitable for growth, the spores can germinate into vegetative bodies with normal physiological activities and produce toxins, causing food spoilage and even foodborne diseases, bringing great economic losses to the food industry, and being highly pathogenic and even lethal, posing a great threat to human health and the development of enterprises. Therefore, developing a rapid, sensitive, highly reproducible, timely and effective detection technology is the key to preventing and controlling foodborne bacilli spores.

[0003] Spores are mainly composed of DNA, different receptor proteins, peptidoglycan, and pyridine dicarboxylic acid (2,6-dipicolinic acid, DPA), etc. Among them, DPA is one of the main components of spores (accounting for 15% of the dry weight of spores), exists in the spore cytoplasm, is its unique substance, and can be used as a biomarker for identifying spores. In order to timely evaluate the risk of spore contamination, currently, different technologies have been developed for the highly sensitive detection of spores, such as polymerase chain reaction, immunoassay, mass spectrometry, electrochemistry, and surface-enhanced Raman spectroscopy, etc., but they all have some inevitable defects. Among the reported technologies for the fluorescent detection of spore DPA, lanthanide ions have a strong specific chelation affinity with the spore biomarker DPA, and show fluorescence at a unique wavelength through the absorption energy transfer emission effect, enabling the rapid detection of DPA. Moreover, lanthanide-based fluorescent probes have a long fluorescence lifetime and a narrow linear emission band, and these characteristics help to effectively eliminate interference from background signals and improve the accuracy of fluorescence detection.

[0004] Therefore, the present invention utilizes the characteristics of lanthanide elements and GO-AgNPs to innovatively construct GO-AgNPs-Eu 3+ nanomaterials as a fluorescent probe for detecting the spore biomarker DPA, and loads a polyvinylidene fluoride (PVDF) microfiltration membrane combined with a smartphone to develop a GO-AgNPs-Eu-based 3+A paper-based sensor for nanomaterials, used for the rapid visual detection of spore DPA, and verified for spores in milk and water based on fluorescence and a paper-based sensor platform, so as to achieve the rapid on-site quantification and real-time online analysis of spores. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a GO-AgNPs-Eu 3+ fluorescent probe, a paper-based sensor and a preparation method thereof, and an application in the detection of foodborne spores. The GO-AgNPs-Eu 3+ fluorescent probe prepared by the present invention has high selectivity for DPA, strong anti-interference ability, low detection limit for DPA and spores. The fluorescence color of the paper-based sensor changes from blue to red with the increase of DPA concentration, and the quantitative detection of DPA and spores is realized through the RGB change of a smartphone, and the actual spore amount in milk and water is used for verification. The rapid detection of foodborne spores is realized based on the fluorescence of the GO-AgNPs-Eu 3+ fluorescent probe and the dual platforms of the paper-based sensor.

[0006] To solve the above technical problems, the present invention takes DPA and the spore biomarker DPA as the research objects, explores the interaction mechanism between the GO-AgNPs-Eu 3+ fluorescent probe and DPA, the selectivity and anti-interference ability of DPA, and designs a smartphone-assisted portable GO-AgNPs-Eu 3+ nano-visual paper-based sensor for the rapid on-site quantification and real-time online analysis of spores.

[0007] Specifically, the following technical solutions are adopted:

[0008] A preparation method of a GO-AgNPs-Eu 3+ fluorescent probe, comprising the following steps:

[0009] (1) Prepare graphene oxide (GO);

[0010] (2) Prepare EDTAD-modified GO-AgNPs nanoparticles by using graphene oxide (GO);

[0011] (3) Prepare GO-AgNPs-Eu 3+ fluorescent probe: Disperse the EDTAD-modified GO-AgNPs nanoparticles in ultrapure water, ultrasonically add an aqueous solution of Eu(NO 3 ) 3 ·6H 2 O and stir for a period of time; finally, centrifuge to collect the product, wash it several times with deionized water, and resuspend it in ultrapure water to obtain a GO-AgNPs-Eu 3+ fluorescent probe solution.

[0012] Further, the preparation method of graphene oxide (GO) in step (1) is as follows: Take 0.5 g of graphite flakes and 0.25 g of sodium nitrate and add them to 50 mL of concentrated sulfuric acid. After cooling the mixture to 0 °C, add potassium permanganate, stir at room temperature for 30 min, add 25 mL of ultrapure water and 2 mL of 30% hydrogen peroxide at 98 °C and keep for 15 min. Finally, wash with 0.1 M hydrochloric acid and water in sequence, and vacuum dry at 60 °C for 12 h to obtain graphene oxide.

[0013] Further, the preparation method of EDTAD-modified GO-AgNPs nanoparticles in step (2) is as follows: Add 200 μL of 0.5 mg / mL graphene oxide aqueous solution to 19 mL of ultrapure water and continuously stir (500 r / min) for 15 min (pH = 9), then add 0.3 mL of 10 mM silver nitrate solution and continuously stir for 30 min; then slowly add 50 μL of 0.01 M sodium borohydride solution and continuously stir for 4 h; subsequently, raise the solution temperature to 70 °C, then add 250 mL of ultrapure water, 25 mL of 1,2-bis(2-aminoethoxy)ethane solution and 500 mg of potassium hydroxide in sequence and stir vigorously (2000 r / min) for 24 h; then add 50 mL of 0.5 M sodium borohydride solution and keep reacting at 70 °C for 2 h. Then, collect by centrifugation and wash thoroughly with water to obtain a precipitate; Take 10 mg of the precipitate and ultrasonically disperse it in 5 mL of sodium bicarbonate buffer solution (pH = 9.6, 0.1 M), add 80 mg of EDTA dianhydride and stir for 2 h, then centrifuge and separate the prepared nanoparticles, then wash 4 times with sodium bicarbonate buffer solution and 2 times with deionized water, and vacuum dry at 60 °C for 12 h to obtain EDTAD-modified GO-AgNPs nanoparticles.

[0014] Further, in step (3), the aqueous solution concentration of Eu(NO 3 ) 3 ·6H 2 O is 0.01 M. Based on 10 mg of EDTAD-modified GO-AgNPs nanoparticles, 5 mL of ultrapure water and 5 mL of aqueous solution of Eu(NO 3 ) 3 ·6H 2 O are required, and the stirring time is 3 h. The concentration of the obtained GO-AgNPs-Eu 3+ fluorescent probe solution is 2 mg / mL.

[0015] The present invention also provides a GO-AgNPs-Eu 3+ fluorescent probe prepared by using the above preparation method.

[0016] Preparation method of a paper-based sensor: Immerse a PVDF microporous membrane as a substrate into the Tris buffer solution of the GO-AgNPs-Eu 3+ fluorescent probe described in claim 5, incubate for a period of time, and after taking it out, naturally dry it in the air to obtain a PVDF microporous membrane paper-based sensor.

[0017] Furthermore, the concentration of the Tris buffer solution is 10 mM, pH = 7.0, and the concentration of the GO-AgNPs-Eu 3+ fluorescent probe in the Tris buffer solution is 5 mg / mL, and the incubation time is 20 min.

[0018] The present invention also provides the application of the GO-AgNPs-Eu 3+ fluorescent probe in the rapid detection of foodborne bacilli and the application of the PVDF microporous membrane paper-based sensor prepared by the described preparation method in the rapid detection of foodborne bacilli. Specifically: Based on the GO-AgNPs-Eu 3+ nanomaterial-modified PVDF microporous membrane sensor, combined with a smartphone for on-site visual detection of bacillus DPA, and verification of bacilli in milk and water based on the fluorescence and paper-based sensor platform.

[0019] The GO-AgNPs-Eu 3+ fluorescent probe and PVDF microporous membrane paper-based sensor of the present invention in the application of foodborne bacilli. Based on the above fluorescent probe, it is applied to the following three aspects. (1) Research on the selectivity, anti-interference ability and interaction between the GO-AgNPs-Eu 3+ fluorescent probe and DPA; (2) Detection of DPA and the biomarker DPA of four representative bacilli (C. sporogenes spores, B. subtilis spores, B. cereus spores, B. thuringiensis spores); (3) Development of a polyvinylidene fluoride (PVDF) microporous membrane sensor based on GO-AgNPs-Eu 3+ nanomaterial modification, combined with a smartphone for on-site visual detection of bacillus DPA, and verification of bacilli in milk and water based on the fluorescence and paper-based sensor platform.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The present invention provides a preparation method of a GO-AgNPs-Eu 3+ fluorescent probe, and provides a GO-AgNPs-Eu 3+Development of a Fluorescent Probe-Loaded Polyvinylidene Fluoride (PVDF) Microporous Membrane Combined with a Smartphone Paper-Based Sensor. The GO-AgNPs-Eu prepared in the present invention 3+ The fluorescent probe has a high selectivity for DPA, strong anti-interference ability, and low detection limits for DPA and spores. The paper-based sensor realizes the quantitative detection of DPA and spores through the RGB change of the smartphone, and is verified using the actual spore amounts in milk and water. Realize the rapid detection of foodborne spores based on the fluorescence of the GO-AgNPs-Eu 3+ fluorescent probe and the dual platforms of the paper-based sensor. Description of the Drawings

[0022] Figure 1 For GO-AgNPs-Eu 3+ Characterization of the nanomaterials. A is the ultraviolet-visible spectrogram; B is the XRD characterization of the GO-AgNPs-Eu 3+ nanomaterials; C is the full-scan XPS of the GO-AgNPs-Eu 3+ nanomaterials; D is the XPS spectrum of C1s; E is the XPS spectrum of Ag3d; F is the XPS spectrum of Eu3d; G and H are the SEM images of the GO-AgNPs-Eu 3+ nanomaterials; I, J, K, and L are the EDS elemental mapping diagrams of the GO-AgNPs-Eu 3+ nanomaterials.

[0023] Figure 2 A is the fluorescence spectrogram of the GO-AgNPs-Eu 3+ nanomaterials; B is the comparison diagram of the fluorescence intensity of the GO-AgNPs-Eu 3+ nanomaterials at 616 nm; C is the fluorescence spectrum of the GO-AgNPs-Eu 3+ nanomaterials after adding different concentrations of DPA (0, 0.25, 0.5, 1.0, 2.0, 5.0, 7.5, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0 μM); D is the standard curve of the fluorescence intensity vs. DPA concentration based on the GO-AgNPs-Eu 3+ nanomaterials; E is the selectivity and anti-interference experiment of the GO-AgNPs-Eu 3+ nanomaterials (the gray bars show 200 μL of anti-interference solution in the GO-AgNPs-Eu 3+ nanomaterials, and the black bars show the subsequent addition of 30 μL of DPA); F shows the fluorescence color photos of DPA and various interfering compounds under 254 nm ultraviolet irradiation.

[0024] Figure 3A, C, E, and G are the fluorescence spectra of different concentrations of C. sporogenes spores, B. subtilis spores, B. cereus spores, and B. thuringiensis spores detected by fluorescence, respectively; B, D, F, and H are the fluorescence standard curves of different concentrations of C. sporogenes spores, B. subtilis spores, B. cereus spores, and B. thuringiensis spores detected by fluorescence, respectively.

[0025] Figure 4 A is the complete release process of DPA after B. subtilis spores are treated at 121 °C for 30 min; B is the simplified flowchart of DPA detection based on the GO-AgNPs-Eu 3+ nanomaterials test strip and smartphone sensor; C is the fluorescence color image and linear relationship diagram (by plotting the linear relationship between R / B intensity and concentration) of different concentrations of DPA solution (0 - 400 μM) and spore solution (10 1 -10 7 cfu / mL) under the irradiation of a 254 nm handheld ultraviolet lamp by the designed paper visual sensor. Detailed implementation manners

[0026] The following specific examples will further illustrate the present invention in conjunction with the accompanying drawings. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the content of the above invention.

[0027] Example 1

[0028] As Figure 1 shown, the preparation and characterization of the GO-AgNPs-Eu 3+ fluorescent probe include the following steps:

[0029] (1) Preparation of graphene oxide (GO): Add 0.5 g of graphite flakes and 0.25 g of sodium nitrate to concentrated sulfuric acid. After cooling the mixture to 0 °C, add potassium permanganate, stir at room temperature for 30 min, add ultrapure water and 30% hydrogen peroxide at 98 °C and keep for 15 min. Finally, wash with 0.1 M hydrochloric acid and water in sequence, and vacuum dry at 60 °C for 12 h to obtain graphene oxide.

[0030] (2) Preparation of EDTAD-modified GO-AgNPs nanoparticles: Add 200 μL of 0.5 mg / mL graphene oxide aqueous solution to 19 mL of ultrapure water and stir continuously for 15 min (pH = 9), then add 0.3 mL of 10 mM silver nitrate solution and stir continuously for 30 min; then slowly add 50 μL of 0.01 M sodium borohydride solution and stir continuously for 4 h; subsequently, raise the solution temperature to 70 °C, then add 250 mL of ultrapure water, 25 mL of 1,2-bis(2-aminoethoxy)ethane solution and 500 mg of potassium hydroxide in sequence and stir vigorously for 24 h; then add 50 mL of 0.5 M sodium borohydride solution and keep reacting at 70 °C for 2 h. Then, collect by centrifugation and wash thoroughly with water to obtain a precipitate; take 10 mg of the precipitate and ultrasonically disperse it in 5 mL of sodium bicarbonate buffer solution (pH = 9.6, 0.1 M), add 80 mg of EDTA dianhydride and stir for 2 h. Then, centrifuge and separate the prepared nanoparticles, then wash 4 times with sodium bicarbonate buffer solution and 2 times with deionized water, and dry in vacuum at 60 °C for 12 h to obtain EDTAD-modified GO-AgNPs nanoparticles.

[0031] (3) Preparation of GO-AgNPs-Eu 3+ fluorescent probe: Disperse 10 mg of GO-AgNPs in 5 mL of water, ultrasonically add 5 mL of Eu(NO 3 ) 3 ·6H 2 0 (0.01 M) and stir for 3 h. Finally, collect the product by centrifugation, wash several times with deionized water, and resuspend in 5 mL of ultrapure water to obtain a 2 mg / mL GO-AgNPs-Eu 3+ fluorescent probe solution.

[0032] Characterize the GO-AgNPs-Eu 3+ fluorescent probe prepared in the above steps. As Figure 1 shown, A is the ultraviolet-visible spectrum; B is the XRD characterization of GO-AgNPs-Eu 3+ nanomaterials; C is the full-scan XPS of GO-AgNPs-Eu 3+ nanomaterials; D is the XPS spectrum of C1s; E is the XPS spectrum of Ag3d; F is the XPS spectrum of Eu3d; G and H are the SEM images of GO-AgNPs-Eu 3+ nanomaterials; I, J, K and L are the EDS element mapping diagrams of GO-AgNPs-Eu 3+ nanomaterials.

[0033] The results show that graphene oxide has a relatively broad plasmon resonance around 300 nm. When GO-AgNPs nanoparticles are synthesized by reduction with sodium borohydride, the oxygen functional groups of graphene oxide are masked, resulting in the disappearance of the absorption peak. However, a characteristic absorption peak caused by AgNPs is observed at 390 nm, and the color of the mixture turns yellow. The EDTA ligand on the GO-AgNPs nanoparticles binds to Eu 3+ After binding, the increase in the diameter of the nanoparticles leads to the broadening of the ionic resonance absorption peak, and it can be preliminarily judged that GO-AgNPs-Eu 3+ nanomaterials are formed. The AgNPs are well-dispersed on the surface of graphene. The particulate Ag element (red) is distributed on the surface of GO (green), and Eu (blue) is evenly dispersed on the surface of GO-AgNPs nanoparticles.

[0034] In addition, the crystal form of GO-AgNPs-Eu 3+ nanomaterials is characterized by XRD. As shown in curve (a) of Figure B, a sharp diffraction peak (2θ) of GO is distributed at 10.7°. Curve (b) shows that the diffraction peaks (2θ) of GO-AgNPs are distributed at 10.7°, 38.24°, 44.4°, 64.5°, and 77.46°, corresponding to the (001) plane of GO, the (111), (200), (220), and (311) crystal planes of the silver cubic structure, respectively, which are all consistent with the powder diffraction file database card (JCPDS 75-1609). Curve (b) shows both the diffraction peak (2θ) of GO and the diffraction peak (2θ) of AgNPs. The deconvoluted XPS C1s region has three significant peaks at 284.1, 285.8, and 287.8 eV, corresponding to sp 2 carbon, O-C-O, and O-C=O bonds, respectively. The deconvolution of the XPS spectrum of Ag3d shows that the binding energies of Ag3d5 / 2 and Ag3d3 / 2 have two peaks at 267.8 and 373.8 eV, respectively. The formed AgNPs-Eu 3+ nanomaterials contain metallic state (Ag0). Two peaks can be observed at 1134.1 eV and 1163.7 eV, corresponding to Eu(II)3d5 / 2 and Eu(III)3d5 / 2, respectively. These characterizations can fully prove the successful synthesis of GO-AgNPs-Eu 3+ nanomaterials.

[0035] Example 2

[0036] Application of the GO-AgNPs-Eu 3+ fluorescent probe prepared in Example 1 in the detection of DPA.

[0037] Mix 200 μL of DPA sample and 2 μL of GO-AgNPs-Eu3+ The nanomaterials (at a concentration of 2 mg / mL) were added to 500 μL of Tris buffer (10 mM, pH = 7.0), and the emission spectra were collected using a Hitachi F4600 fluorescence spectrophotometer. All tests were carried out at room temperature, and the scanning rate was 1200 nm·min -1 , and the fluorescence intensity was measured at an excitation wavelength of 282 nm and an emission wavelength of 616 nm. The excitation and emission slits were fixed at 10 nm, and the fluorescence spectrum was scanned at a voltage of 700 V. The number of experimental repetitions n = 3.

[0038] Figure 2 As shown in A, GO, GO-AgNPs, and GO-AgNPs-Eu 3+ could not emit the characteristic fluorescence (red fluorescence) of Eu 3+ at 616 nm, and DPA was also a non-fluorescent compound. After adding DPA to the system, the characteristic fluorescence of Eu 3+ was emitted at 616 nm, and the red fluorescence of Eu 3+ was effectively enhanced through the antenna effect (AE), and the energy was transferred to the lanthanide ions (Eu 3+ ). By comparing the fluorescence intensities at 616 nm ([[]] Figure 2 B), the fluorescence intensity of the GO-AgNPs-Eu 3+ nanomaterials was much higher than that of the single lanthanide ion (Eu 3+ ). The GO-AgNPs nanoparticles had the effect of enhancing the fluorescence effect. The GO-AgNPs nanoparticles would hinder the coordination of water molecules, and the Eu 3+ ions could also coordinate with the groups on the surface of the GO-AgNPs nanoparticles, reducing the coordination with water molecules to avoid energy loss and enhancing the fluorescence intensity.

[0039] After adding different concentrations of DPA to the GO-AgNPs-Eu 3+ nanomaterials, the fluorescence emission spectra were observed ([[]] Figure 2 C). When the DPA concentration was between 0 μM and 45 μM, the fluorescence emission peak at 616 nm increased with the increase of the DPA concentration. Figure 2 As shown in D, the fluorescence intensity of the GO-AgNPs-Eu 3+ nanomaterials showed a good linear correlation with the DPA concentration in the range of 0 μM to 45 μM. The linear regression equation was y = 691.09x - 907.78, and R 2 = 0.99. And according to the LOD = 3σ / k formula calculation, the LOD was 4.62 nM.

[0040] Different types of aromatic ligands, amino acids, and various common cations or anions were detected (3,5-DPA, BA, mPA, p-PA, o-PA, 2,4-DPA, 2,5-DPA, 3,4-DPA, Asa, Ala, Cys, Gly, K + , Fe 3+ , Mg 2+ , Na + , NO 3 - , Cl - , SO 4 2- ). Under the same detection conditions ( Figure 2 E), only DPA caused a signal response at 616 nm, and none of the interfering substances could cause obvious fluorescence changes. Figure 2 F also confirmed that under ultraviolet lamp (254 nm) irradiation, other interfering substances could not cause obvious color changes, and only the DPA solution emitted obvious red fluorescence. The GO-AgNPs-Eu 3+ nanomaterials had high selectivity due to the high coordination ability of DPA chelating with Eu 3+ in a tridentate chelation mode.

[0041] Example 3

[0042] Verification of the quantitative detection of DPA in spores by the GO-AgNPs-Eu 3+ fluorescent probe prepared in Example 1.

[0043] Preparation of Clostridium sporogenes (C. sporogenes) spores. The frozen C. sporogenes spores were streaked on NA medium with porcelain beads to obtain single colonies of C. sporogenes. Single colonies were selected and transferred to 20 mL of RCM medium for 6 hours of culture, and then 200 μL was inoculated into the RCM solid medium for 3 - 7 days of culture. The spores were collected by centrifugation (8000 rpm, 10 min, 4 °C), and washed 5 - 7 times with cold sterile deionized water to remove impurities such as vegetative cells in the spore suspension. After washing, microscopy was performed again. It could be used only when ≥95% of the spores in the field of view were translucent and there were no visible small impurities.

[0044] Preparation of Bacillus subtilis (B. subtilis) spores. The B. subtilis spores were streaked on an LB plate with porcelain beads and cultured overnight until single colonies grew. After three generations of activation, fresh single colonies were selected and transferred to 20 mL of LB liquid medium for overnight culture (200 rpm, 37 °C) until the OD 600 was 1.2 - 1.5, and then transferred to the spore-promoting growth medium DSM for culture (200 rpm, 37 °C) at a ratio of 1:100. The collection method was the same as above.

[0045] Preparation of Bacillus cereus (B. cereus) spores. Streak the B. cereus beads on an NA plate and incubate overnight until single colonies grow. Pick a single colony and transfer it to 20 mL of liquid nutrient broth medium (200 rpm, 37 °C) and culture for about 6 h. Then transfer it to a nutrient agar plate supplemented with 10.05 g / L manganese sulfate tetrahydrate at a ratio of 1:100 and culture for 2 - 7 days. The collection method is as above.

[0046] Preparation of Bacillus thuringiensis (B. thuringiensis) spores. Streak the B. thuringiensis beads on an LB plate and incubate overnight until single colonies grow. Pick a single colony and transfer it to 20 mL of LB liquid medium and culture overnight until the OD 600 is 1.2 - 1.5. Then inject 200 μL into the ICPM solid medium and culture for 3 - 7 days. The collection method is as above.

[0047] Preliminarily adjust the obtained spore concentration to OD 600 = 0.5, approximately 10 7 cfu / mL. Continuously dilute a certain volume of the spore suspension with sterile water, and inoculate the spore dilution into the medium at 37 °C. After culturing for 24 h, the specific concentration of the spores can be obtained by plate counting. And dilute the spores into different concentrations and treat them at 121 °C for 30 min to ensure complete release of DPA.

[0048] Add 200 μL of the spore suspension and 2 μL of GO-AgNPs-Eu 3+ nanomaterials (concentration 2 mg / mL) to 500 μL of Tris buffer (10 mM, pH = 7.0), and measure the fluorescence intensity after standing at room temperature for 5 min.

[0049] The fluorescence detection results of four foodborne spores are as Figure 3 shown. After adding spore suspensions with different concentrations, the characteristic fluorescence emission intensity of the GO-AgNPs-Eu 3+ nanomaterials increases with the increase of the spore concentration. In the concentration range of 3.18×10 1 cfu / mL - 3.18×10 7 cfu / mL, establish a linear relationship between the concentration of C. sporogenes spores and the fluorescence intensity. There is a good linear relationship for C. sporogenes spores between 10 4 - 10 7 . The linear equation is y = 2.93×10 -4 x + 96.56, R 2= 0.997. According to the detection limit calculation formula, the detection limit of C. sporogenes spores was calculated to be 2.37×10 4 cfu / mL. By the same method, B. subtilis spores (2.54×10 1 cfu / mL - 2.54×10 7 cfu / mL), B. cereus spores (2.63×10 1 cfu / mL - 2.63×10 7 cfu / mL) and B. thuringiensis spores (4.27×10 1 cfu / mL - 4.27×10 7 cfu / mL) were also detected, and the linear relationships between the different concentrations of the three types of spores and the fluorescence intensity were established respectively. The results showed that all three types of spores had good linear relationships between 10 4 -10 7 . The linear equations of the three types of spores were y = 2.96×10 -4 x + 74.92 (R 2 = 0.999), y = 3.06×10 -4 x + 79.92 (R 2 = 0.998) and y = 2.9×10 -4 x + 139.3 (R 2 = 0.998) respectively, and the detection limits LOD were 2.6×10 4 cfu / mL, 2.52×10 4 cfu / mL and 2.66×10 4 cfu / mL respectively.

[0050] Example 4

[0051] As Figure 4 shown, the verification and application of the GO-AgNPs-Eu 3+ nano-vision paper-based sensor.

[0052] The obtained spore concentration was initially adjusted to OD 600 = 0.5, approximately 10 7 cfu / mL. A certain volume of the spore suspension was serially diluted with sterile water, and the spore diluent was inoculated onto the medium at 37°C. After culturing for 24 h, the specific concentration of the spores could be obtained by plate counting. The spores diluted to different concentrations were treated at 121°C for 30 min to ensure complete release of DPA. 200 μL of the spore suspension and 2 μL of GO-AgNPs-Eu 3+The nanomaterials (at a concentration of 2 mg / mL) were added to 500 μL of Tris buffer (10 mM, pH = 7.0), and the fluorescence intensity was measured after standing at room temperature for 5 min.

[0053] First, the purchased PVDF microporous membrane (purchased from Deli Filter Technology Co., hydrophilic organic filter membrane, pore size of 0.45 μM, diameter of 13 mm) was used as a substrate and immersed in the Tris buffer of GO-AgNPs-Eu 3+ nanomaterials (the concentration of Tris buffer was 10 mM, pH = 7.0, and the concentration of GO-AgNPs-Eu 3+ fluorescent probe in Tris buffer was 5 mg / mL), incubated for 20 min, and after taking out, it was naturally dried to obtain the PVDF microporous membrane paper-based sensor.

[0054] Then, 20 μL of DPA solutions with different concentrations were dropped onto the PVDF microporous membrane, photos were taken with a smartphone, and finally, visual analysis was carried out with a handheld ultraviolet lamp under irradiation at 254 nm. The results are as Figure 4 shown in B. As the concentration of DPA increased, the color of the test paper gradually changed from blue to purple and then to red. For digital quantification, through the color recognition APP installed on the smartphone, as a signal reader and analyzer, the color intensities of red, green, and blue (RGB) in the fluorescent color image were digitized, and the RGB change value was calculated to perform semi-quantitative analysis.

[0055] 20 μL of DPA solution (0 - 400 μM) was dropped onto the PVDF microporous membrane paper-based sensor and dried. As Figure 4 shown in C, under irradiation with 254 nm ultraviolet light, as the concentration of DPA increased, the fluorescence color on the PVDF microporous membrane gradually changed from blue to purple and then to red, and the concentration of DPA (0 - 400 μM) could be reflected by the ratio of R to B values (R / B). There was a good linear relationship between the concentration of DPA and G / B (R 2 = 0.99). The corresponding linear regression equation was y(R / B) = 0.00019x(C DPA ), and the detection limit LOD was 13.1 μM.

[0056] Taking B. subtilis spores as an example, different concentrations of B. subtilis spores (2.54×10 1 cfu / mL~2.54×10 7 cfu / mL) were treated at 121 °C for 30 min to completely release DPA. The flow chart of DPA release from B. subtilis spores is as Figure 4As shown in A. Using GO-AgNPs-Eu 3+ The nano-material paper-based sensor was used to test DPA in spore metabolites at different concentrations, as Figure 4 shown in C. As the concentration of B. subtilis spores increased, the fluorescence color on the PVDF microporous membrane gradually changed from blue to purple under ultraviolet light irradiation, indicating that the designed paper-based sensor has the ability to visually detect real spores. Moreover, there is a good linear relationship between the logarithm of the spore concentration and G / B (R 2 = 0.99). The corresponding linear regression equation is y(R / B) = 0.0312x(lgC spores ), and the detection limit LOD is 6.3 cfu / mL.

[0057] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. Preparation method of a GO-AgNPs-Eu 3+ fluorescent probe It is characterized in that It includes the following steps: (1) Prepare graphene oxide (GO); (2) Prepare EDTAD-modified GO-AgNPs nanoparticles using graphene oxide (GO); (3) Preparation of GO-AgNPs-Eu 3+ Fluorescent probe: Dispersed the EDTAD-modified GO-AgNPs nanoparticles in ultrapure water, and ultrasonically added an aqueous solution of Eu(NO 3 ) 3 ·6H 2 O and stirred for a period of time; Finally, centrifuged to collect the product, washed several times with deionized water, and resuspended in ultrapure water to obtain the GO-AgNPs-Eu 3+ fluorescent probe solution; The preparation method of the EDTAD-modified GO-AgNPs nanoparticles in the step (2) is as follows: Add 200 μL of 0.5 mg / mL graphene oxide aqueous solution to 19 mL of ultrapure water and stir continuously for 15 min, then add 0.3 mL of 10 mM silver nitrate solution and stir continuously for 30 min; then slowly add 50 μL of 0.01 M sodium borohydride solution and stir continuously for 4 h; subsequently, raise the solution temperature to 70 °C, then add 250 mL of ultrapure water, 25 mL of 1,2-bis(2-aminoethoxy)ethane solution and 500 mg of potassium hydroxide in sequence and stir vigorously for 24 h; then add 50 mL of 0.5 M sodium borohydride solution and keep reacting at 70 °C for 2 h, then collect by centrifugation and wash thoroughly with water to obtain a precipitate; take 10 mg of the precipitate, ultrasonically disperse it in 5 mL of sodium bicarbonate buffer solution, add 80 mg of EDTA dianhydride and stir for 2 h, then centrifuge, wash and dry the prepared nanoparticles to obtain EDTAD-modified GO-AgNPs nanoparticles. μ ​ 2. The preparation method of the GO-AgNPs-Eu 3+ fluorescent probe It is characterized in that The preparation method of graphene oxide (GO) in step (1) is as follows: Take 0.5 g of graphite flakes and 0.25 g of sodium nitrate and add them to 50 mL of concentrated sulfuric acid. After cooling the mixture to 0 °C, add potassium permanganate, stir at room temperature for 30 min, add 25 mL of ultrapure water and 2 mL of 30% hydrogen peroxide by mass fraction at 98 °C and keep for 15 min. Finally, wash with 0.1 M hydrochloric acid and water in sequence, and vacuum dry at 60 °C for 12 h to obtain graphene oxide.

3. The preparation method of the GO-AgNPs-Eu 3+ fluorescent probe It is characterized in that In step (3), the aqueous solution concentration of Eu(NO 3 ) 3 ·6H 2 O is 0.01 M. Based on 10 mg of GO-AgNPs nanoparticles modified with EDTA, 5 mL of ultrapure water is required, and 5 mL of the aqueous solution of Eu(NO 3 ) 3 ·6H 2 O. The stirring time is 3 h, and the concentration of the obtained GO-AgNPs-Eu 3+ fluorescent probe solution is 2 mg / mL.

4. GO-AgNPs-Eu fluorescent probe prepared by the preparation method according to any one of claims 1-3 3+ ​ 5. A preparation method of a paper-based sensor It is characterized in that: Immerse the PVDF microporous membrane as a substrate into the Tris buffer solution of the GO-AgNPs-Eu 3+ fluorescent probe, incubate for a period of time, take it out and naturally dry it in the air to obtain the PVDF microporous membrane paper-based sensor.

6. The preparation method of the paper-based sensor according to claim 5 It is characterized in that: The concentration of Tris buffer is 10 mM, pH = 7.0, GO-AgNPs-Eu 3+ The concentration of the fluorescent probe in Tris buffer is 5 mg / mL, and the incubation time is 20 min.

7. Use of the GO-AgNPs-Eu 3+ fluorescent probe in the rapid detection of foodborne bacilli.

8. Application of the PVDF microporous filter membrane paper-based sensor prepared by the preparation method according to claim 5 or 6 in the rapid detection of foodborne bacilli.

9. The application according to claim 8 It is characterized in that: GO-AgNPs-Eu-Based 3+ A PVDF microporous membrane sensor modified with nanomaterials, combined with a smartphone for on-site visual detection of spore DPA, and verification of spores in milk and water based on a fluorescence and paper-based sensor platform.

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