A cholesteric molecular tweezers artificial receptor and a method for detecting zinc ions in food

Abstract

The present invention provides a novel molecular tweezers D5. Based on D5, a novel method for rapid zinc ion detection is also provided, which is applied to the detection of actual food samples, opening up an efficient rapid zinc ion detection technology in the field of food science.

Description

Technical Field

[0001] The present invention relates to natural compound derivatives, as well as the fields of food detection and metal detection. Background Art

[0002] As we all know, metal ions are used everywhere in our daily lives, especially in the fields of food, environment, and medicine. Among the many metal ions, zinc is an essential trace element in the human body, second only to iron. It is mainly obtained through dietary fiber supplementation and plays an important role in the biological system. Studies have shown that Zn 2+ It is an important component of many enzymes and transcription factors in the body, and is directly involved in important life metabolic processes such as cell growth and development, gene regulation, metalloenzyme catalysis, neural signal transmission and immune function. 2+ Zinc ions play a key role in various fundamental biological processes in the human body. Appropriate amounts of zinc ions not only prevent dental caries and osteoporosis but also remove cholesterol and prevent atherosclerosis. In recent years, with the widespread use of metallic zinc and its compounds in industrial processes such as machining, electroplating corrosion protection, and battery electrodes, large amounts of zinc ions have been released into the environment. These ions accumulate in the human body through the food chain, causing numerous neurological diseases, including Alzheimer's disease, Parkinson's disease, and epilepsy. The World Health Organization (WHO) has reportedly established a safe range for Zn2+ in drinking water of no more than 45.9 μM. Therefore, the development of a low-cost, highly selective new method for detecting Zn2+ is crucial for diagnosing human diseases and monitoring environmental pollution.

[0003] In recent years, traditional methods such as flame atomic absorption spectrometry, inductively coupled plasma, and electrochemical methods have been frequently used for the detection of zinc ions. However, these methods are subject to many limitations in practical applications, such as expensive equipment, cumbersome operation, and high instrument precision requirements. Compared with traditional analytical methods, chemical sensors have attracted much attention due to their advantages such as high sensitivity, good selectivity, convenient operation, and low cost. In the past few decades, with the vigorous development of supramolecular chemical sensors, a variety of new receptor models have emerged, such as rhodamine, naphthalene imide, Schiff base, coumarin, etc. Although these macrocyclic compounds have special rigid structures, their binding sites are limited. Even if they are structurally modified, it is difficult to achieve interaction with more types of objects.

[0004] Cholesteric molecular tweezers are a new type of artificial receptor sensor based on cholesterol or its derivatives. Cholesterol, due to its rigid concave structure and inherent asymmetry, is an ideal building block for molecular tweezer receptors. Unlike conventional chemical sensors, the hydroxyl groups pointing toward the center of the concave surface and the carboxyl groups on the side chains in the backbone structure of cholesteric molecular tweezers are considered key sites in the design of molecular tweezer artificial receptors. These functional groups can be converted into a variety of functional groups through chemical modification, thereby constructing different molecular tweezer artificial receptors. Furthermore, cholesteric molecular tweezers, due to their specific structural features (such as cleft size and recognition site), can form a complementary microenvironment with substrate molecules, which facilitates the enhancement of interactions with the guest molecule, such as hydrogen bonding, electrostatic interactions, and hydrophobic interactions, thereby achieving efficient recognition and selective binding of specific substrate molecules. Furthermore, their synthesis methods are more convenient and efficient than those of most cyclic compounds. Therefore, cholesteric molecular tweezers have attracted great interest among researchers due to their advantages such as simple preparation, mild reaction conditions, and flexible and multifunctional recognition sites. Kim et al. used deoxycholic acid as the molecular skeleton and modified its structure by introducing dithioamino groups to design and synthesize a cholesteric molecular clamp receptor. They used the potential evaluation method to investigate the interaction between the receptor and the guest cation Ni. 2+ 、Fe 2+ 、Co 2+ 、Ag + 、Zn 2+ and Cu 2+ The recognition and coordination properties of the molecular clamp receptors were found to be + It has excellent affinity and selectivity. In addition, the inventors previously synthesized a cholesteric molecular tweezers artificial receptor L5 using deoxycholic acid as a raw material and microwave radiation technology. Through ultraviolet and fluorescence spectroscopy analysis, they concluded that it has the ability to recognize I-. Using nuclear magnetic titration and molecular simulation, they speculated on the possible recognition mechanism between the host and the guest. They established a method for rapid quantitative detection of iodide ions using chiral molecular tweezers and successfully applied it to the detection of I- in food samples (22. Lixia, L., Ying, Y., Zhe, L., & Jiangtao, L. (2023). Rapid detection of I- in food samples by cholesteric chiral artificial receptor L5. Food Chemistry, 409, 135194.).

[0005] To date, although researchers have reported numerous molecular tweezers for detecting anions and cations, there have been no reports of using cholesteric molecular tweezers for zinc ion detection. Therefore, in the latest advances in molecular recognition, scholars are committed to developing simple, efficient, highly sensitive, and highly selective new zinc ion sensors, aiming to promote their application in various fields such as food science, life science, and environmental science.

[0006] According to previous studies, even molecular tweezers receptors with similar structures have different abilities to bind to metal ions. How to find a molecular tweezers receptor with specific detection capabilities for certain specific metal ions has been the focus of research for technicians in this field. Summary of the Invention

[0007] To address these issues, the present invention synthesized a cholesteric molecular tweezers artificial receptor, D5, using deoxycholic acid as a raw material and microwave radiation technology through a series of chemical reactions. The selective recognition of cations by the molecular tweezers artificial receptor D5 was studied using ultraviolet absorption spectroscopy and fluorescence spectroscopy. The recognition mechanism between the molecular tweezers D5 and zinc ions was systematically investigated using scanning electron microscopy, Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and computer molecular simulation. The present invention aims to develop a novel method for rapid zinc ion detection based on the molecular tweezers D5 and apply it to the detection of actual food samples, hoping to develop an efficient rapid zinc ion detection technology for the field of food science.

[0008] Specifically, the present invention provides a cholesteric molecular tweezers artificial receptor, the structural formula of which is as follows:

[0009]

[0010] The present invention also provides a method for detecting zinc ions in food. The digested sample to be tested is mixed with the cholesteric molecular tweezers artificial receptor shown as D5, and quantitative or qualitative detection is performed using ultraviolet absorption spectroscopy, fluorescence spectroscopy or infrared spectroscopy.

[0011] The digestion method includes: taking a sample to be tested, reacting it at 340-450° C. for more than 40-60 minutes in the presence of sulfate and sulfuric acid.

[0012] Wherein, the sulfate includes copper sulfate and potassium sulfate.

[0013] The detection wavelength of the ultraviolet absorption spectrum is 260 nm.

[0014] When ultraviolet absorption spectroscopy is used for quantitative detection, a standard curve equation is prepared based on the rise or fall of the ultraviolet absorption peak at 260 nm to calculate the zinc ion content.

[0015] Among them, when using ultraviolet absorption spectroscopy for detection, the detection limit of zinc ions is 0.78μmol·L -1 .

[0016] Among them, when using infrared spectroscopy for detection, at 3420cm -1 、2925cm -1 、1647cm -1 The presence of characteristic peaks indicates the presence of zinc ions.

[0017] The wavelength used for fluorescence spectroscopy detection is 350 nm.

[0018] Among them, fluorescence titration method is used for quantitative detection. According to the change of fluorescence intensity at 350nm, a standard curve equation is made to calculate the zinc ion content.

[0019] In the present invention, the cholesteric molecular tweezers artificial receptor D5 is generally dissolved in an organic solvent, such as dimethyl sulfoxide.

[0020] Beneficial effects of the present invention:

[0021] (1) This study synthesized a cholesteric molecular tweezers artificial receptor, D5, and investigated its recognition of different metal ions using UV-visible absorption and fluorescence spectroscopy. A method for rapid detection of Zn2+ using molecular tweezers D5 was established. By measuring Zn2+ in actual samples, quantitative and specific detection of Zn2+ in food samples was achieved.

[0022] (2) In dimethyl sulfoxide solution, molecular tweezers D5 can selectively recognize Zn2+, other anions and cations (Al 3+ 、Na + Mg 2+ 、Ag + , Pb 2+ 、Fe 3+ 、Mn 2+ , Ca 2+ , K + Br - 、Cl - 、F - , I - 、NO3-、HSO4 - 、H2PO4 - 、CH3COO - ) has little effect on the Zn2+ recognition process and can avoid interference of other metal ions on the detection results.

[0023] (3) The coordination ratio of D5 to zinc ion is 1:1, and the detection limit is 0.78umol / L, which is far lower than the maximum allowable concentration of zinc ion in drinking water (45.9uM) set by the World Health Organization. Through nuclear magnetic titration, Fourier transform infrared spectroscopy, field emission scanning electron microscopy and computer theoretical simulation, it was determined that the nitrogen atom on 3α-OCONH in molecular tweezers D5, the carbonyl oxygen atom in NO2-PhCONH and the oxygen atom on the 12β position interact with Zn through the CHEF effect. 2+ Complexation mechanism by which coordination occurs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 . Ultraviolet absorption spectra and fluorescence emission spectra of cholesteric molecular tweezers D5 in the presence of different cations; Among them, Figure 1 A. Cholesteric molecular tweezers D5 (1×10 -4 mol / L) in different cations (1×10 -3 mol / L) in the presence of UV-visible absorption spectrum; Figure 1 B. Cholesteric molecular tweezers D5 (1×10 -4 mol / L) in different cations (1×10 -3 mol / L) in the presence of fluorescence emission spectra

[0025] Figure 2 .Cholesteric molecular tweezers D5 (1×10 -4 mol / L) were added with Zn2+ and other interfering ions (1×10 -3 mol / L) fluorescence intensity change histogram

[0026] Figure 3 .Cholesteric molecular tweezers D5 (1×10 -4 mol / L) were added with Zn2+ and other interfering ions (1×10 -3 mol / L) UV-visible absorption spectrum

[0027] Figure 4 .Spectral changes of cholesteric molecular tweezers D5 and different concentrations of zinc ions; Figure 4 A. Cholesteric molecular tweezers D5 (1×10 -4 mol / L) and different concentrations of Zn 2+ UV-visible titration spectroscopy; Figure 4 B. Cholesteric molecular tweezers D5 (1×10 -4 mol / L) and different concentrations of Zn 2+ Fluorescence titration spectroscopy

[0028] Figure 5 . Coordination map of cholesteric molecular tweezers D5; Figure 5 A. Cholesteric molecular tweezers D5 and Zn2+ Plot 1 / [G0] versus 1 / ΔA to form the complex. Figure 5 B. Cholesteric molecular tweezers D5 and Zn 2+ Job curve of coordination

[0029] Figure 6 .Cholesteric molecular tweezers D5 and Zn 2+ Microstructural observation and energy spectrum imaging analysis of the complex; (A) FESEM image of the cholesteric molecular tweezers D5 (×3000, 5kv). (B) FESEM image of the cholesteric molecular tweezers D5 in the presence of Zn2+ (×6000, 5kv). (C) FESEM image of the cholesteric molecular tweezers D5 in the presence of Zn2+ (×20000, 5kv). (D) Energy spectrum imaging of the cholesteric molecular tweezers D5 in the presence of Zn2+ (×20000, 5kv)

[0030] Figure 7 .Cholesteric molecular tweezers D5 and complex D5-Zn 2+ Infrared spectrum of

[0031] Figure 8 .Cholesteric molecular tweezers D5 in DMSO-d6 with Zn2+ 1 H NMR spectra: (A) +0 equiv; (B) +1 equiv; (*, NO2-PhCONH; ◆3α-OCONH; ●12β-H)

[0032] Figure 9 .Cholesteric molecular tweezers D5 (1×10 -4 mol / L) for Zn 2+ (1×10 -3 mol / L) quantitative detection standard curve DETAILED DESCRIPTION

[0033] Example 1

[0034] 1.1 Materials and Instruments

[0035] Chemical reagents used in this experiment were obtained from Sigma-Aldrich and used without further purification. Metal ions were generally prepared using nitrates. Rice, wheat flour, spinach, banana, cucumber, and laver were purchased from Xining, Qinghai Province. Fluorescence emission spectra were measured using an RF-6000 fluorometer (Kyoto, Japan); UV absorption spectra were measured using a UV-2600 UV-visible spectrophotometer (Kyoto, Japan); mass spectrometry analysis was performed using a Thermo Q Exactive ultrahigh-resolution liquid chromatography-mass spectrometer (Shanghai, China); chemical reactions were performed using an MCR-3 microwave chemical reactor (Zhengzhou, China); nuclear magnetic resonance spectra were recorded using an AVANCE NEO nuclear magnetic resonance spectrometer (Rheinstetten, Germany); microscopic images were observed using a JSM-7900F field-emission scanning electron microscope (Kyoto, Japan); and infrared spectra were recorded using a Nicolet 6700 Fourier transform infrared spectrometer (Shanghai, China).

[0036] 1.2 Synthesis of the Cholesterol Molecular Tweezer Artificial Receptor D5

[0037] The synthetic route for the cholesteric molecular tweezers artificial receptor is shown in Scheme 1. Intermediate 1 (p-methoxybenzoic acid hydrazide) and intermediate 3 (3α,12α-dihydroxy-5β-24-cholanoic acid methyl ester) were synthesized according to previously reported methods. Intermediate 3 (0.5 mmol), anhydrous CHCl₃ (10 mL), anhydrous pyridine (0.5 mL), and triphosgene (0.18 mmol) were placed in a 50 mL round-bottom flask and heated under reflux at 65°C for 6 h. After the reaction was complete, intermediate 1 (1 mmol) and anhydrous pyridine (0.5 mL) were added, and the reaction was continued under reflux at 65°C for 24 h. The reaction progress was monitored by TLC. After the reaction was complete, CHCl₃ was evaporated under reduced pressure. The residue was dissolved in ethyl acetate and the resulting solution was washed sequentially with saturated NaHCO₃ and saturated brine. The organic phase was collected and dried over anhydrous Na₂SO₄ overnight, filtered, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography [silica gel H eluent: petroleum ether: ethyl acetate = 1:1.5] to obtain a pale yellow solid D5 in a 90% yield. D5:C 33 H 47 N3O8,mp116-117℃,IR(KBr)(cm -1):3287,2941,2869,1727,1603,1529,1452,1348,1251,1039,853; δ:10.66(s,1 H,NO2-PhCONH),9.29(s,1H,3α-OCONH),8.34(t,J=2.0Hz,2H,ArH),8.08(d,J=8 .2,2H,ArH),4.69-4.58(m,1H,3β-H),4.40(s,1H,12β-H),3.56(s,3H,COOCH3), 0.99(d,J=6.3Hz,3H,21-CH3),0.88(s,3H,19-CH3),0.58(s,3H,18-CH3).ESI-MS m / z(%):636.24([M+Na] + ,100).Anal.Calcd.forC 33 H 47 N3O8: C, 64.58; H, 7.72; N, 6.85. Found: C, 64.50; H, 7.74; N, 6.83%.

[0038]

[0039] 1.3 Selective recognition of metal ions by cholesteric molecular tweezers D5

[0040] Cholesteric molecular tweezers D5 were dissolved in DMSO to prepare artificial receptor stock solution (1×10 -4 mol / L), prepare different types of nitrates (Zn 2+ 、Al 3+ 、Na + Mg 2+ 、Ag + , Pb 2+ 、Fe 3+ 、Mn 2+ , Ca 2+ , K + ) of metal ion stock solution (1×10 -3 mol / L). At room temperature, the artificial receptor D5 stock solution was pipetted into equal amounts of different metal ion solutions. UV absorption spectra were measured between 200 and 400 nm, and fluorescence spectra were measured between 200 and 600 nm. The fluorescence spectra were analyzed with an excitation slit width of 5 nm, an emission slit width of 5 nm, an excitation wavelength λ of 270 nm, and a sensitivity of 1.

[0041] 1.4 Effect of coexisting ions

[0042] In order to explore whether the cholesteric molecular tweezers D5 interferes with the selectivity for Zn2+ in the presence of other metal ions, an anti-interference experiment was conducted. -4 mol / L cholesteric molecular tweezers D5 solution was successively added with equal amounts of Zn 2+ The spectral changes of the solution and other single anions and cations (Al3+, Na+, Mg2+, Ag+, Pb2+, Fe3+, Mn2+, Ca2+, K+, Br-, Cl-, F-, I-, NO3-, HSO4-, H2PO4-, CH3COO-) were observed under the same fluorescence and UV conditions.

[0043] 1.5 UV and fluorescence spectroscopic titration analysis and Job experiment of Zn2+ recognition by cholesteric molecular tweezers D5

[0044] At a concentration of 1.0×10 -4 mol / L cholesteric molecular tweezers D5 solution was gradually added with Zn 2+ Solution (1.0×10 - 3 mol / L), adding 50μL each time, and observing the change of the spectrum under the same UV and fluorescence conditions. Plot the reciprocal of the zinc ion concentration in the complex (1 / G0) against the reciprocal of the change in the UV absorbance of the complex at 260nm (1 / △A) to calculate the effect of the cholesteric molecular tweezers D5 and Zn 2+ The complexation constant (Ka) and free energy change value (-△G0) of the product.

[0045] The Job method was used to further determine the relationship between the cholesteric molecular tweezers D5 and Zn 2+ Always keep the cholesteric molecular tweezers D5 and Zn 2+ The total concentration is 5×10 -4 mol / L, prepare cholesteric molecular tweezers D5 and Zn 2+ The absorbance values ​​of a series of mixed solutions with molar ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 were measured. The absorbance was plotted against the molar ratio of the main compound to obtain the Job dot diagram of the complex and determine the composition ratio of the complex.

[0046] 1.6 Cholesteric molecular tweezers D5 and Zn 2+ Study on the mechanism of action

[0047] In order to more intuitively observe the cholesteric molecular tweezers D5 and Zn 2+The interaction and coordination mechanism of Zn2+ in cholesteric molecular tweezers D5 were studied by field emission scanning electron microscopy (FESEM) for microstructure observation and energy spectrum imaging analysis. The changes of functional groups before and after the addition of Zn2+ to cholesteric molecular tweezers D5 were observed by Fourier transform infrared spectroscopy, and the possible binding sites of the two were preliminarily inferred. 2+ The changes in proton chemical shifts on different functional groups in the nuclear magnetic hydrogen spectra before and after further determined the binding site of the molecular tweezers D5 and Zn2+ and the possible driving force of the recognition interaction; finally, with the help of computer molecular simulation programs, density functional theory (DFT) optimization calculations and molecular structure simulations of the cholesteric molecular tweezers D5 and its complexes were performed at the B3LYP / 6-31G(d) level.

[0048] 1.7 Zn in food samples 2+ Quantitative detection of

[0049] The concentration of cholesteric molecular tweezers D5 was 1.0×10 -4 mol / L solution was gradually added with Zn 2+ , when Zn 2+ The concentration is 0~4×10 -4 mol / L range, the Zn 2+ The concentration was plotted against the UV-visible absorbance value to obtain the standard curve equation. Then, according to the formula LOD = 3σ / k, the effect of artificial receptor D5 on Zn 2+ The detection limit is σ, where σ is the standard deviation and k is the slope of the linear fit. 2 g of rice, wheat flour, spinach, banana, cucumber and seaweed were randomly selected and placed in a dry digestion tube. 0.2 g of copper sulfate, 6 g of potassium sulfate and 10 mL of sulfuric acid were added respectively. The samples were digested at 400 ° C for 1 hour and then cooled to room temperature. The digested samples were diluted and used as the ion test samples. 1 mL of cholesteric molecular tweezers D5 solution (1×10 -4 mol / L) was mixed with 1 mL of the sample solution in a test tube and the absorbance at 260 nm was measured. Each experiment was repeated three times and the average value was taken. The zinc ion content in each sample was calculated according to the above standard curve equation.

[0050] Results and Discussion

[0051] 2.1 Cholesteric molecular tweezers D5 on Zn 2+ Selective recognition

[0052] In order to study the selective recognition of different ions by the artificial receptor D5, different metal ion solutions were added to the cholesteric molecular tweezers D5 solution, and the changes in the UV absorption spectrum and fluorescence spectrum were observed. The results are as follows: Figure 1 Before adding cations, the UV absorption spectrum of the cholesteric molecular tweezers D5 showed a strong absorption peak at 285nm ( Figure 1A). When different metal ions were added to the cholesteric molecular tweezers D5 solution, only the addition of Zn2+ showed a special change different from that of other ions. Not only did the intensity of the characteristic UV absorption peak decrease significantly, but the position of the characteristic absorption peak also shifted significantly to 260nm, indicating that the cholesteric molecular tweezers D5 may have the ability to selectively recognize Zn2+. Furthermore, fluorescence spectroscopy was used to explore the emission properties under the same conditions, such as Figure 1 As shown in Figure B, the cholesteric molecular tweezers D5 and each complex exhibited a fluorescence emission peak at 350 nm, with a significant fluorescence enhancement effect observed upon the addition of Zn2+. This is likely due to the torsion of the receptor molecule during coordination in the presence of Zn2+, which increases its rigidity and reduces rotation and vibration of the receptor molecule, resulting in less energy loss and, consequently, enhanced fluorescence. In summary, the cholesteric molecular tweezers D5 binds to Zn2+ with good specificity, enabling highly selective recognition.

[0053] 2.2 Interference of coexisting ions

[0054] In order to verify the selectivity of the synthesized cholesteric molecular tweezers D5 for Zn2+, this study conducted an anti-interference experiment. Other anions and cations with the same amount of substance as zinc ions were added to the system where the artificial receptor D5 coexists with zinc ions, and the interference effect of the coexisting ions was investigated using fluorescence and ultraviolet spectroscopy. Figure 2 As shown in Figure 2, when other anions and cations coexist with Zn2+, the fluorescence intensity of the cholesteric molecular tweezers D5 does not show significant difference from that when only zinc ions are present. Figure 3 As shown in the results of UV absorption spectroscopy, the addition of other interfering ions to the coexistence system of cholesteric molecular tweezers D5 and zinc ions did not change the peak position, shape, or number of the UV absorption spectrum, indicating that other interfering ions do not affect the interaction system between cholesteric molecular tweezers D5 and zinc ions. This is consistent with the results of fluorescence signal analysis. Therefore, cholesteric molecular tweezers D5 have a certain degree of anti-interference ability in Zn2+ recognition and can be used as an artificial receptor sensor with good selectivity.

[0055] 2.3 Spectrometric titration analysis and determination of coordination ratio

[0056] In order to study the recognition sensitivity of the cholesteric molecular tweezers D5 to Zn2+, UV and fluorescence titration experiments were carried out in DMSO solution. Figure 4 As shown in A, with the increase of Zn2+(1.0×10 -3 mol / L) was added continuously, and the UV absorbance of the solution at 260nm showed a regular downward trend. The binding characteristics of the artificial receptor D5 and Zn2+ were further verified by fluorescence titration experiments. Figure 4As shown in B, with the increase of Zn2+ concentration, the fluorescence intensity response value at 350nm gradually increases, and the fluorescence curve shows a regular upward trend. It is preliminarily speculated that the complexation between the cholesteric molecular tweezers D5 and Zn2+ may occur, and the guest Zn 2+ Entering the molecular cleft of the cholesteric molecular tweezers D5 leads to intramolecular charge transfer, which causes the fluorescence intensity and ultraviolet absorbance to show a regular upward or downward trend. The results show that the cholesteric molecular tweezers D5 binds to Zn2+ to form a stable complex.

[0057] In order to further determine the complexation between cholesteric molecular tweezers D5 and Zn2+, the UV-visible absorption spectrum was used as the research object. The results showed that with the increase of Zn 2+ When the concentration increases, the UV absorbance of the solution shows a regular downward trend. 2+ The concentration is 2×10 -5 ~2×10 -4 In the range of mol / L, according to the Hildebrand--Benes equation, 1 / [G0] is plotted against 1 / ΔA ( Figure 5 A), a straight line (R 2 =0.995), indicating that the cholesteric molecular tweezers D5 and Zn 2+ A 1:1 complex was formed. Based on the intercept 1 / a and the slope 1 / a·Ka of the straight line, the binding constant Ka of the complex was calculated to be 3.43×10 3 L / mol, the free energy change -△G0 is 20.17KJ / mol, indicating that the molecular tweezers D5 and Zn2+ can form a stable complex. In addition, the equimolar ratio continuous change method is another method to determine the coordination ratio. Figure 5 B knows that

[0058] [D5] / [D5+Zn 2+ ] is 0.5, the UV absorbance reaches the maximum value, which also proves that the cholesteric molecular tweezers D5 and Zn 2+ The coordination ratio between them is 1:1.

[0059] 2.4 Cholesteric molecular tweezers D5 and Zn 2+ Microstructure observation and energy spectrum imaging analysis of the complex

[0060] Field emission scanning electron microscopy of the complex formed by cholesteric molecular tweezers D5 and Zn2+ Figure 6 As shown in the figure, without adding Zn2+( Figure 6 A), Cholesteric molecular tweezers D5 is a dense block structure with a relatively smooth surface; when it forms a complex with zinc ions ( Figure 6B, 6C), the morphology of the cholesteric molecular tweezers D5 changed significantly, forming an irregular wrinkled cross-linked structure, and the zinc ions were wrapped in the center of the specific active site of the cholesteric molecular tweezers, which was conducive to the formation of a stable polymer. To further verify whether Zn2+ was embedded in the molecular tweezers cleft, energy spectrum imaging analysis was performed using a JSM 7900F high-resolution transmission scanning electron microscope at an accelerating voltage of 5.0kV ( Figure 6 D), the green part in the figure is Zn2+, and the red part is the carbon skeleton of the cholesteric molecular tweezers D5, indicating that most of the zinc ions enter the host structure and are wrapped by the molecular tweezers D5 to form a stable complex.

[0061] 2.5 Cholesteric molecular tweezers D5 and Zn 2+ Infrared spectroscopy analysis of the effect

[0062] In order to further clarify the mechanism of action of cholesteric molecular tweezers D5 and Zn2+, infrared spectroscopy was performed. Figure 7 It can be seen that 3431cm -1 、2936cm -1 、1577cm -1 The peaks at are attributed to the vibration peaks of amide NH, methyl CH, and C=O of the cholesteric molecular tweezers D5. 2+ After binding, the stretching vibration peak of amide NH blue shifted to 3420 cm -1 At the same time, the stretching vibration peak of methyl CH blue shifted to 2925cm -1 The stretching vibration peak of carbonyl group red-shifted to 1647 cm -1 Based on this, it can be inferred that amide NH, methyl CH, and C=O may directly or indirectly participate in the coordination of Zn2+, thereby causing charge transfer and thus leading to changes in the infrared spectrum.

[0063] 2.6 NMR titration results and analysis

[0064] The H NMR spectroscopy further confirmed the relationship between the cholesteric molecular tweezers D5 and Zn 2+ The results are as follows: Figure 8 As shown, no Zn was added 2+ When 1.0 equiv of Zn was added, the chemical shifts of hydrogen protons in the cholesteric molecular tweezers D5 were 10.66, 9.29 and 4.40 ppm, respectively, for NO2-PhCONH, 3α-OCONH and 12β-H. 2+After that, the hydrogen proton signal peaks in NO2-PhCONH and 3α-OCONH in the molecular tweezers D5 disappeared, and the hydrogen proton chemical shift of the 12β-H group moved to the low field region by 0.28ppm. It is speculated that the nitrogen atom on 3α-OCONH, the carbonyl oxygen atom in NO2-PhCONH and the oxygen atom on the 12β position in the molecular tweezers D5 may be transferred to Zn 2+ Provide electrons, resulting in changes in the hydrogen proton signal peak. 2+ When coordinated with the nitrogen and oxygen atoms in the molecular tweezers D5, the free rotation of the benzene ring connected to the cholesteric arm of D5 is inhibited, and the π conjugated system is enhanced, thereby triggering the chelation enhanced fluorescence effect (CHEF). This result effectively explains the selective effect of the molecular tweezers D5 and Zn 2+ The fluorescence intensity of the mixture was significantly higher than that of other ion mixtures. It was inferred that the nitrogen atom on the 3α-OCONH in the molecular tweezers D5, the carbonyl oxygen atom in NO2-PhCONH, and the oxygen atom at the 12β position may have coordinated with the zinc ion, and CHEF is considered to be the main mechanism.

[0065] 2.8 Zn in food samples 2+ Detection

[0066] Based on the above mechanism, it is shown that the cholesteric molecular tweezers D5 has a good recognition effect on zinc ions. In order to verify the detection ability of cholesteric molecular tweezers D5 in actual samples, rice, wheat flour, spinach, banana, cucumber and seaweed were randomly selected as experimental samples to quantitatively detect the content of zinc ions. Based on the standard curve and detection limit test of the quantitative detection of zinc ions by cholesteric molecular tweezers D5, the zinc ion concentration was 0-4.0×10 -4 mol / L, the concentration of zinc ions in the process of complex formation is plotted against the UV-visible absorbance value ( Figure 9 ), the standard curve equation was obtained by fitting y = -0.0013x + 0.9207 (R 2 =0.9992), according to the formula LOD = 3σ / k, the cholesteric molecular tweezers D5 to Zn 2+ The detection limit is 0.78 μmol / L, which is far lower than the maximum allowable concentration of Zn2+ in drinking water stipulated by the World Health Organization (WHO) (45.9 μM). To better compare the recognition performance of artificial receptors for zinc ions with those reported in other literature, the detection performance of the cholesteric molecular tweezers D5 prepared in this paper and some representative other types of metal ions for Zn2+ are listed in Table 1. The comparison of the results in the table shows that the cholesteric molecular tweezers D5 has the advantages of low detection limit, wide detection range, and simple synthesis route. It can achieve highly selective recognition of Zn2+ at low concentrations and has broad application prospects in the field of rapid identification and detection of metal ions.

[0067] Table 1. Comparison of different zinc ion recognition sensors

[0068]

[0069] To verify the practicality of this method for Zn2+ detection, the Zn2+ content in food samples was calculated based on the standard curve. The results are shown in Table 2. The Zn2+ content in rice, wheat flour, spinach, banana, cucumber, and laver samples was 17.258, 11.745, 21.534, 1.954, 3.626, and 23.501 μg / g, respectively. To further verify the accuracy of the results, the Zn2+ content in these food samples was tested for recovery using the standard addition method. Zn2+ content was calculated by adding different concentrations of Zn2+ standard solution (0 μg / g, 10 μg / g, and 20 μg / g). The results are shown in Table 3. The recovery ranged from 98.85% to 108.20%, and the relative standard deviation was within 5%. At the same time, the zinc content in six food samples was tested using the flame atomic absorption spectrometry method in the national standard GB / T.14-2017 "Determination of Zinc in Foods". After comparative analysis, the relative error between the test results of this experiment and the flame atomic absorption spectrometry method was less than 5%, indicating that this method has high accuracy and can quantitatively detect the zinc ion content in food samples.

[0070] Table 2. Determination of Zn2+ in food samples by cholesteric molecular tweezers D5

[0071]

[0072] Table 3. Zn in spiked food samples 2+ Determination of recovery rate (n=3)

[0073]

[0074] in conclusion

[0075] In this study, deoxycholic acid was used as the raw material to synthesize the cholesteric molecular tweezers artificial receptor D5 through a series of chemical reactions. Using UV and fluorescence spectroscopy analysis, it was found that the molecular tweezers D5 has a specific recognition effect on Zn2+ in DMSO solution, and the binding constant between the two is 3.43×10 3 L / mol, the free energy change -△G0 is 20.17KJ / mol, and the detection limit is 0.78μmol / L. Compared with other sensors, it has a lower detection limit and better anti-interference ability. The molecular tweezers D5 and Zn 2+In order to clarify the recognition mechanism between molecular tweezers D5 and Zn2+, the microstructural changes before and after the coordination of molecular tweezers D5 and Zn2+ were studied. It was found that Zn2+ could enter the crack of molecular tweezers D5 and form a stable complex. At the same time, further analysis was conducted using nuclear magnetic resonance hydrogen spectroscopy and molecular simulation. The nitrogen atom on 3α-OCONH in molecular tweezers D5, the carbonyl oxygen atom in NO2-PhCONH and the oxygen atom on the 12β position interacted with Zn2+ through the CHEF mechanism. 2+ The coordination reaction occurs, enabling efficient identification. Furthermore, by randomly measuring the zinc content in different food samples, a method for detecting zinc ions in real samples was established. This method offers significant advantages in terms of detection efficiency and cost, along with simplicity and reliability. Therefore, it is expected that a rapid Zn2+ detection kit will be developed for widespread application in Zn2+ determination in food, environmental, and other fields.

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Claims

1. A method for detecting zinc ions in food, characterized in that, The digested sample to be tested is mixed with the cholesteric molecular tweezers artificial receptor shown in D5, and is quantitatively or qualitatively detected using ultraviolet absorption spectroscopy, fluorescence spectroscopy, or infrared spectroscopy; The D5 is .

2. The method according to claim 1, characterized in that The digestion method includes: taking a sample to be tested, reacting it at 340-450° C. for more than 40-60 minutes in the presence of sulfate and sulfuric acid.

3. The method according to claim 2, characterized in that The sulfates include copper sulfate and potassium sulfate.

4. The method according to claim 1, wherein The detection wavelength of the UV absorption spectrum was 260 nm.

5. The method according to claim 1, wherein When using ultraviolet absorption spectroscopy for quantitative detection, a standard curve equation is prepared based on the rise or fall of the ultraviolet absorption peak at 260 nm to calculate the zinc ion content.

6. The method according to claim 5, characterized in that When using ultraviolet absorption spectroscopy for detection, the detection limit of zinc ions is 0.78 μmol·L -1 .

7. The method according to claim 1, characterized in that When using infrared spectroscopy for detection, at 3420 cm -1 、2925cm -1 、1647cm -1 The presence of characteristic peaks indicates the presence of zinc ions.

8. The method according to claim 1, characterized in that The wavelength for detection using fluorescence spectroscopy was 350 nm.

9. The method according to claim 1, characterized in that Fluorescence titration was used for quantitative detection. A standard curve equation was prepared based on the change in fluorescence intensity at 350 nm to calculate the zinc ion content.

Images