CN113960008B - Fluorescent probe for rapidly and ultrasensitively detecting oxytetracycline and preparation method and application thereof - Google Patents

Abstract

The invention discloses a fluorescent probe for quickly and super-sensitively detecting oxytetracycline, a preparation method and application thereof, relates to the technical field of antibiotic primary detection, and discloses a fluorescent probe for quickly and super-sensitively detecting oxytetracyclineUsing OTC and Ca²⁺The special optical property is combined to prepare the dual-emission fluorescent probe, the red luminous CdTe QDs are used as an internal reference, and Ca on the surface of the CdTe QDs is²⁺The chelate is used as a receptor and a specificity recognition unit, after the OTC is added, the fluorescence emission of the formed Ca/CdTe-OTC complex is obviously enhanced, and the fluorescence emission of CdTe QDs remains unchanged, so that the visual detection of the OTC can be carried out within 0.1s, the detection limit is 1.31nM and is far lower than the maximum residual limit of 0.1mg/kg in animal-derived food specified by the world health organization, and the chelate is suitable for the detection of the OTC in water and meat products.

Description

Fluorescent probe for rapidly and ultrasensitively detecting oxytetracycline and preparation method and application thereof

The technical field is as follows:

the invention relates to the technical field of antibiotic initial detection, in particular to a fluorescent probe for quickly and super-sensitively detecting oxytetracycline and a preparation method and application thereof.

Background art:

it is well known that rapid response and ultrasensitivity are critical for probes, particularly in food safety, clinical diagnostics, and public screening for explosives or other contraband. The probe with high response speed can be widely applied to the field of public monitoring under the condition that stations, airports, public places and the like need to wait for the inspection result in a queue for a long time.

Compared with the traditional laboratory technology, the portable intelligent detection equipment can meet the requirement of detecting various residues on site and has satisfactory accuracy and sensitivity. Meanwhile, the cost is lower, and the introduction of sensitive materials such as miniaturized aptamer, monoclonal antibody, fluorescent nano-material and the like is an important way for the development of analytical devices. Paper-based probes have been applied to many fields as a simple detection device without additional devices, and have been expanded with the advent of microfluidic technology and colorimetry. Meanwhile, the flexible probe is paid much attention due to low cost, simple manufacturing process and excellent performance, and is applied to the construction of intelligent wearable medical diagnosis equipment.

Food safety issues caused by antibiotic abuse have attracted considerable attention over the past few decades. Oxytetracycline (OTC) is a broad-spectrum tetracycline antibiotic that has been widely used in human therapy, aquaculture and animal husbandry due to its good antibacterial and bactericidal effects. However, improper use of OTC drugs can result in high levels of residue in animal food, ground water and wastewater, and health threats such as drug resistance and allergic reactions through the food chain. The conventional analysis method for the oxytetracycline residue mainly comprises an ultraviolet spectrophotometry method, a fluorescence spectrophotometry method, a liquid chromatography-mass spectrometry method, a liquid chromatography method, a chemiluminescence method, a capillary electrophoresis method, an electrochemical probe method and the like. Therefore, the rapid, reliable, low-cost and sensitive in-situ detection method has important significance in environmental monitoring and food safety. To date, several convenient probes have been developed for the detection of OTC. The fluorescent probe based on Si NPs can respond to OTC, and Liu and the like construct the fluorescent probe by utilizing a molecular imprinting technology and are used for sensitively and selectively detecting tetracycline. However, these probes have difficulty meeting the portable and user-friendly standard of field detection, and therefore, it is very important to develop a sensitive and rapid visual quantitative detection method.

The invention content is as follows:

the invention aims to provide a fluorescent probe which can sensitively and accurately detect oxytetracycline without complex pretreatment.

The technical problem to be solved by the invention is realized by adopting the following technical scheme:

the invention aims to provide a preparation method of a fluorescent probe for quickly and ultrasensitively detecting oxytetracycline, which comprises the following steps:

(1) synthesis of CdTe QDs: adding Cd (CH)₃CO₂)₂·2H₂Dissolving O and 3-mercaptopropionic acid in deionized water, adjusting pH to alkalescence with NaOH aqueous solution, performing ultrasonic treatment, and adding K₂TeO₃Stirring with deionized water, and adding NaBH₄Carrying out ultrasonic treatment, heating the obtained mixture to boiling, carrying out reflux reaction, drying the obtained CdTe QDs after the reaction is finished, collecting dry powder, and storing the dry powder in a refrigerator for later use;

(2) synthesis of Ca/CdTe Probe: uniformly stirring distilled water and the CdTe QDs powder prepared in the step (1), and then adding CaCl₂·2H₂And O, stirring at room temperature, carrying out ultrasonic treatment, drying the obtained Ca/CdTe after the reaction is finished, collecting dry powder, and storing the dry powder in a refrigerator for later use.

The slightly basic pH is 8-12.

The Cd (CH)₃CO₂)₂·2H₂The molar ratio of O to 3-mercaptopropionic acid is 4:1 to 8: 1.

Said K₂TeO₃And Cd (CH)₃CO₂)₂·2H₂The molar ratio of O is 1:5-1: 10.

The NaBH₄And Cd (CH)₃CO₂)₂·2H₂The molar ratio of O is 3:1-6: 1.

The CdTe QDs powder and CaCl₂·2H₂The molar ratio of O is 1:1-1: 2.

The second purpose of the invention is to provide a fluorescent probe for rapidly and ultrasensitively detecting oxytetracycline, which is prepared by the preparation method.

The third purpose of the invention is to provide an application of the fluorescent probe in detecting the content of oxytetracycline and the oxytetracycline in water or meat products.

The ratio fluorescence system sensing mechanism of the fluorescent probe of the invention is as follows:

two stages are provided, as shown in FIG. 1.

Stage 1: ca due to specific strong chelating reaction with carboxyl and water molecules of 3-mercaptopropionic acid (MPA) -terminated CdTe QDs²⁺Is easy to be fixed on the surface of CdTe QDs, and a relatively stable Ca/CdTe mixed nano probe is successfully formed in the solution. In the absence of OTC, the formation of Ca from CdTeQDs²⁺The red emission of CdTe QDs at about 627nm is in direct contact with Ca²⁺Binding is slightly reduced, at which point the Ca/CdTe probe exhibits red fluorescence.

Stage 2: after contact with OTC, due to OTC and Ca²⁺Has higher coordination strength, and OTC can replace water molecules and Ca²⁺Coordination, the left and right of the atom orbit are divided into two bonds, the bridging Ca atom forms a bond to connect two complexes, and the bond of the connecting atom has two connecting points, so that coordinated water molecules can be easily connected. Due to oxytetracycline and Ca²⁺The stronger the bond between, the attractive force will change the electron distribution of the Ca atom, resulting in the Ca atom assuming a divalent state. Furthermore, because of the central ion Ca²⁺At d⁰Electronic valence state, no electrons on the orbit, and crystal field activation energy CFAE value was 0. There is no CFAE loss whether it is strong or weak, so the reaction proceeds very quickly. In addition, the combination of the central ion and the ligand is of an internal orbital type, and the energy is low, the binding force is strong, the spin is high, and Ca is enabled to be in a Ca state²⁺The binding with OTC is extremely fast, the Ca/CdTe-OTC formed shows green emission fluorescence at about 511nm, while the red fluorescence of CdTe QDs remains almost unchanged. With the continued addition of OTC, a clear change in fluorescence color from red to bright green can be observed, so that Ca/CdTe shows a visual fluorescence response to OTC, with Ca²⁺By combining the two fluorescent behaviors, the specific identification cell that is OTC forms a green emitting Ca/CdTe-OTC, while the red emitting CdTe QDs acts as an internal reference signal, a dual emission ratio fluorescent sensing system is successfully implemented, and these effects together result in the "shutter" speed response of the fluorescent probe.

The invention has the beneficial effects that: the present invention utilizes OTC and Ca²⁺The special optical property is combined to prepare a dual-emission fluorescent probe, red-emitting CdTe QDs are used as internal reference, and Ca on the surface of the CdTe QDs is used as²⁺Chelation is used as a receptor and a specificity recognition unit, after the OTC is added, the fluorescence emission of the formed Ca/CdTe-OTC complex is obviously enhanced, and the fluorescence emission of CdTe QDs is kept unchanged, so that the OTC can be visually detected within 0.1s, the detection limit is 1.31nM and is far lower than the maximum residual limit of 0.1mg/kg in animal-derived food specified by the world health organization, and the method is suitable for the detection of the OTC in water and meat products and provides a simple and ultra-fast strategy for the visual field quantitative detection of the OTC.

Description of the drawings:

FIG. 1 is a diagram showing the mechanism of detection of OTC by fluorescent probe;

FIG. 2(A) a TEM image of CdTe; (B) TEM image of Ca/CdTe; (C) TEM image of Ca/CdTe-OTC; (D) UV-visible spectra of Ca/CdTe and Ca/CdTe-OTC; (E) FT-IR spectra of Ca/CdTe and Ca/CdTe-OTC; (F) XPS plots of Ca/CdTe-OTC;

FIG. 3 is an X-ray photoelectron spectrum of Ca/CdTe (A) Ca/CdTe, (B) Te 3D, (C) O1S, (D) Cd 3D, (E) N1S, (F) Ca 2p, (G) C1S, (H) S2 p;

FIG. 4(A) DLS data for CdTe; (B) particle size distribution map of CdTe QDs;

FIG. 5 is the Zeta potentials of CdTe, Ca/CdTe, OTC, Ca/CdTe/OTC;

FIG. 6(A) fluorescence excitation and emission spectra of CdTe, (B) fluorescence excitation and emission spectra of Ca/CdTe;

FIG. 7 shows Ca²⁺And optimal concentration ratio of OTC;

FIG. 8 is the effect of pH on fluorescent probes;

FIG. 9 is a graph of the effect of temperature on a fluorescent probe;

FIG. 10 shows the photostability of fluorescent probes within 360 min;

FIG. 11(A) fluorescence spectra of probe solutions exposed to different concentrations of OTC, inset is a corresponding fluorescence photograph under 365nm UV irradiation; (B) fluorescence intensity ratio I₅₁₁/I₆₂₇Linear relationship to OTC concentration;

FIG. 12 shows fluorescence spectra and corresponding fluorescence photographs of OTC detected at fluorescence intensity ratios of 1/1(A) and 3/1(B), respectively;

FIG. 13(A) fluorescence intensity ratio of probes when different interfering substances were added at 100nM (I)₅₁₁/I₆₂₇) The inset from the side is the corresponding fluorescent photograph under 365nm UV illumination; (B) testing the selectivity and the anti-interference of the probe;

FIG. 14 shows Ca in elimination test²⁺Induced experimental error;

FIG. 15 shows the content detection of oxytetracycline by G/R.

The specific implementation mode is as follows:

in order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easy to understand, the invention is further explained by combining the specific embodiments and the drawings.

Preparation of fluorescent probe:

(1) synthesis of CdTe QDs: 0.04g of Cd (CH)₃CO₂)₂·2H₂Dissolving O and 0.1g 3-mercaptopropionic acid in 100ml deionized water, adjusting pH to 9 with 0.5mol NaOH water solution, performing ultrasonic treatment for 30min, and adding 0.02g K₂TeO₃Stirred with 100ml of deionized water for 30min, then 0.12g of NaBH was added₄Ultrasonic treatment ofAnd treating for 30min, heating the obtained mixture to boil, performing reflux reaction for 24h, drying the obtained CdTe QDs in a vacuum oven at 60 ℃ after the reaction is finished, collecting dry powder, and storing in a refrigerator at 4 ℃ for later use.

(2) Synthesis of Ca/CdTe probe: mixing 100ml distilled water and 100mg CdTe QDs powder prepared in step (1), stirring, and adding equal molar amount of CaCl₂·2H₂And O, stirring at room temperature, carrying out ultrasonic treatment for 60min, after the reaction is finished, drying the obtained Ca/CdTe in a vacuum oven at 60 ℃, collecting dry powder, and storing the dry powder in a refrigerator at 4 ℃ for later use.

Structural characterization of the fluorescent probe:

considering that the detection sensitivity of the probe to the analyte is related to the property of the probe, the structural characteristics of CdTe QDs and Ca/CdTe probes are respectively researched by using FT-IR, UV-vis, XPS, Zeta potential and DLS particle size distribution, and the spectral characteristics of the CdTe QDs and the Ca/CdTe probes are also researched to find out the optimal emission.

The morphology and particle size of the CdTe quantum dots characterized by TEM are shown in FIG. 2A. CdTe quantum dots exhibit an almost monodisperse spherical morphology and a relatively uniform distribution with an average diameter of 2.5-3 nm. The sharp lattice fringes (inset in fig. 2A) at a single point in the HRTEM image suggest high crystallinity of the CdTe quantum dots, with a fringe spacing of about 0.22 nm. As can be seen from TEM (FIG. 2B), Ca²⁺Chelation with CdTe quantum dots, Ca/CdTe aggregation, was accompanied by a decrease in fluorescence intensity at 627nm (FIG. 7). Clear lattice fringes, with a fringe spacing of about 0.23nm, were also observed, indicating high crystallinity of Ca/CdTe. In FIG. 2C, the fringe spacing for the highly crystalline Ca/CdTe-OTC is estimated to be 0.23nm, with dispersion relatively the same as Ca/CdTe. These results clearly demonstrate the successful preparation of Ca/CdTe nanoprobes, and that OTC and Ca²⁺The reaction product of (a) has good chemical stability while maintaining a stable molecular state.

Furthermore, there was a clear peak at 273nm in the UV-vis data, indicating that the Ca/CdTe synthesis was successful (FIG. 2D). After addition of OTC, a new peak appeared at 384nm, ascribed to the UV absorption of oxytetracycline. FIG. 2E shows FTIR spectra of Ca/CdTe nanostructures. 3425cm^-1The broader absorption band belonging to the-OH extension of the carboxyl groupContraction vibration, -CH₂Can be attributed to 2935cm^-1At a small frequency band, symmetric and asymmetric vibrations of carboxyl groups (-COO-) appear at 1390cm^-1After addition of OTC, C ═ O bond 1621cm^-1The peak of (A) was shifted to 1568cm^-1Here, the bonding process is indicated.

To get a clearer understanding of the elements and structure of Ca/CdTe quantum dots, XPS spectra were further recorded. As shown in FIG. 2F, elemental peaks of C, O, Ca, Cd, and Te can be seen in the full scan spectrum, indicating the presence of Ca/CdTe and the corresponding elemental high resolution spectrum is shown in FIG. 3. The C1 s spectrum shows three peaks at 284.8, 285.7 and 288.2eV, respectively due to C C, C-N and C O bonds. High resolution O1s spectra revealed three peaks at 531.1, 532.4 and 535.5eV, belonging to C O, C-OH and C-O species respectively. These data are consistent with the results of infrared spectroscopy, further indicating the carboxyl groups on the surface of the quantum dots. High resolution spectra of Ca showed peaks of 347.2 and 350.9eV, assigned to Ca 2p_3/2And Ca 2p_1/2. Observed with Cd 3d_5/2The peaks at 405.2 and 405.8eV being relevant, while the peaks at 408.8 and 409.5eV correspond to Cd 3d_3/2。Te 3d_5/2The other peaks of (2) appear at 572.3, 572.6, 575.8 and 576.5eV and at 583.1 and 586.3eV correspond to Te 3d_3/2. These results indicate the presence and composition of Ca/CdTe.

Furthermore, DLS data show size distributions of CdTe QDs around 1.5-4.0nm (fig. 4A), similar to that in TEM. As shown in FIG. 4B, the Zeta potential of Ca/CdT obtained showed a decrease in absolute value compared to CdTe quantum dots (FIG. 5). This is due to the addition of Ca²⁺The resulting quantum dots cluster together, which can also be demonstrated in TEM. In addition, the excitation and emission spectra of Ca/CdTe and Ca/CdTe-OTC are shown in FIG. 6, with the optimum excitation wavelength fixed at 365 nm.

Ca²⁺Effect of concentration, pH and temperature on fluorescent probes:

Ca²⁺the concentration also affects Ca²⁺Chelation with OTC, we found that the optimal chelation ratio was 1:1, as shown in fig. 7. The pH value and the temperature have certain influence on the fluorescence intensity of the probe and also influence Ca²⁺Chelate with OTCKey factors of the synthesis. As shown in FIG. 8, the fluorescence spectrum of the probe was hardly affected by the change of pH 4 to 9. As shown in FIG. 9, the fluorescence intensity ratio I was measured at a temperature of 4 to 40 deg.C₅₁₁/I₆₂₇There was no significant change. After addition of OTC, after 360min I₅₁₁/I₆₂₇The ratio remained stable (as shown in fig. 10). These results verify the stability of the probe and lay the foundation for practical application.

Influence of the fluorescent Probe ratio:

the sensitivity and the practical application value of the sensing system are influenced by the chromaticity change in the visualization. Therefore, by adjusting Ca before exploring the effect of the assay²⁺And the concentration ratio of CdTe-QDs in the sensing system to obtain the fluorescence intensity ratio I₅₁₁/I₆₂₇Changes are the best visualization effect represented. When the reaction of OTC with Ca/CdTe is complete, Ca can be observed over a wide range of detectable concentrations²⁺The color change is most pronounced with a 2:1 ratio of green to red peaks inside the/CdTe probe, so 2:1 is selected as the optimal ratio for constructing the probe, see FIGS. 11 and 12.

Selectivity and interference rejection of fluorescent probes:

for an excellent fluorescence sensing system, it is critical that the fluorescence sensor has good selectivity for oxytetracycline, and that the selective response to oxytetracycline be maintained even in the presence of interferents. Therefore, a series of experiments were performed to explore the selectivity and anti-interference ability of fluorescent probes in the detection of oxytetracycline. Under the same conditions, pesticides (thiram, chlorpyrifos) and heavy metals (Pb) are used²⁺、Cr³⁺、Hg²⁺) And part of antibiotics (tetracycline, chlortetracycline, amoxicillin, gentamicin, norfloxacin and chloramphenicol) are used for carrying out experiments on the fluorescent probe. See in particular fig. 13.

In order to objectively reflect the application advantages of the fluorescent probe of the present invention, the results are shown in Table 1, compared with other methods reported in the prior art.

TABLE 1

Materials	Linear range	Response time	LOD	Ref.
					BCD	0.1-100μM	30s	410nM		1
AuNCs@OVA	0.1-5μM	30s	0.09mg/mL	2
					kappa-Carr C-CDs	0-100μM	120s	50nM		3
GQDs/cdTe@MIPs	0.1-5μM	600s	3.5nM	4
					S，N-CDs	0.5-1000μM	30s	260nM		5
CD@AMP/Eu NCPs	1-100μM	180s	25nM								6
					Liquid chromatography	0.06-0.4μM	937.5s	60nM	7
silicon-based nanoparticles	0.2-20μM	120s	180nM							8
					Ca/CdTe	0-367nM	＜0.1s	1.31nM	This work

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6.Chen，Lili；Xu，Hui；Wang，Li.MICROCHIMICA ACTA.187，(2020).

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Table 1 can comparatively clearly demonstrate that the present probe has an ultra-short response time, as low as 0.1s, compared to previous probes; lower detection limit, as low as 1.31 nM. Ultra-fast response speeds may provide insight into the strategy of designing fast and sensitive portable detection devices in a wider range of applications.

The method for detecting the content of the terramycin comprises the following steps:

drawing a standard curve: dissolving the fluorescent probe in 2ml of distilled water in a 3ml clean cuvette, adding different amounts of terramycin solution into the Ca/CdTe probe solution to enable the final concentration range of the terramycin to be 0-367nM, and respectively measuring the fluorescence intensity of the fluorescent probe before and after adding the standard terramycin solution. And establishing a linear formula according to the change value of the fluorescence intensity at the emission peak of the system before and after the oxytetracycline is added and the concentration of the oxytetracycline, and drawing a standard curve. The linear formula established is as follows: y-0.435 x +0.289 (R)²And 0.998), wherein y is the reduction value of the fluorescence intensity of the system before and after the oxytetracycline is added, namely x is the concentration of the oxytetracycline added.

To evaluate the feasibility and reproducibility in practical applications, Ca/CdTe nanoprobes were used to detect oxytetracycline in practical samples by standard addition methods. Pork and pig urine were obtained from local pig farms, lake water from local rivers, and Ca was also eliminated before testing²⁺The experimental error caused is shown in fig. 14.

TABLE 2 determination of OTC spiked recovery in real samples (water, pork, minced meat)

As shown in Table 2, good recovery estimates for spiked 10, 50 and 100nM OTC in real samples ranged from 93-104% with Relative Standard Deviation (RSD) ranging from 0.45% to 3.41%, indicating good accuracy and reliability. The results show that the probe of the invention is suitable for practical application in water and meat products.

Meanwhile, the content of the oxytetracycline measured is directly read by a G/B value method, as shown in FIG. 15. And (3) measuring the content of the oxytetracycline in the sample, and performing RGB digitization on the color after fluorescent irradiation to obtain a result. Making a standard curve, and establishing a linear formula: y is 0.0036x +0.241 (R)²0.997), where y is the ratio of the green value to the red value read on the fluorogram and x is the measured oxytetracycline concentration. And (5) comparing with a formula, and calculating the content of the oxytetracycline in the sample solution.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (2)

1. The application of the fluorescent probe in detecting the oxytetracycline content of water bodies or meat products is characterized in that: the step of detecting the content of the oxytetracycline comprises the establishment of a fluorescence intensity ratio I₅₁₁/I₆₂₇Linear relationship with oxytetracycline concentration;

the preparation method of the fluorescent probe comprises the following steps:

(1) synthesis of CdTe QDs: adding Cd (CH)₃CO₂)₂·2H₂O and 3-mercaptopropionic acidAdding NaOH aqueous solution to adjust pH to alkalescence in deionized water, ultrasonic treating, and adding K₂TeO₃Stirring with deionized water, and adding NaBH₄Carrying out ultrasonic treatment, heating the obtained mixture to boiling, carrying out reflux reaction, drying the obtained CdTe QDs after the reaction is finished, collecting dry powder, and storing the dry powder in a refrigerator for later use;

(2) synthesis of Ca/CdTe Probe: uniformly stirring distilled water and the CdTe QDs powder prepared in the step (1), and then adding CaCl₂·2H₂O, stirring at room temperature, carrying out ultrasonic treatment, drying the obtained Ca/CdTe after the reaction is finished, collecting dry powder, and storing the dry powder in a refrigerator for later use;

the Cd (CH)₃CO₂)₂·2H₂The molar ratio of O to 3-mercaptopropionic acid is 4:1-8: 1;

said K₂TeO₃And Cd (CH)₃CO₂)₂·2H₂The molar ratio of O is 1:5-1: 10;

the NaBH₄And Cd (CH)₃CO₂)₂·2H₂The molar ratio of O is 3:1-6: 1;

the CdTe QDs powder and CaCl₂·2H₂The molar ratio of O is 1:1-1: 2.

2. Use according to claim 1, characterized in that: the slightly basic pH = 8-12.

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