Preparation method and application of supramolecular fluorescence sensing array

Abstract

The invention discloses a preparation method and application of a supramolecular fluorescence sensing array. The supramolecular fluorescence sensing array is a cucurbituril-based supramolecular fluorescence sensing array constructed by taking a TPE-P (at) Q [7] supramolecular fluorescence probe and a C7 (at) Q [8] supramolecular fluorescence probe as sensing units. The supramolecular fluorescence sensing array constructed by the invention not only can be used for qualitatively detecting four anticholine drugs, namely AS, RA, SH and IB, but also can be used for distinguishing different concentration gradients of the same drug. In addition, interference experiments prove that the sensing array has good anti-interference performance, and artificial urine, milk, pork and herbal tea samples prove that the constructed array also has the potential of simultaneously analyzing four anti-choline drugs in a complex system. The invention provides a novel method for detecting hyoscyamine anticholine drugs, and the method is more visual and convenient compared with a traditional detection technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and specifically relates to a preparation method of a supramolecular fluorescence sensor array and its application for the rapid identification and detection of four tropane anticholinergic drugs: atropine sulfate (AS), racemic anisodamine (RA), scopolamine hydrobromide (SH), and ipratropium bromide (IB). Background Art

[0002] Atropine sulfate, racemic anisodamine, and scopolamine hydrobromide, the active ingredients of atropine sulfate, racemic anisodamine, and scopolamine hydrobromide, are common tropane alkaloids. Aside from optical activity, the three differ only slightly in their structures. Scopolamine and anisodamine, respectively, are derived from the tropane of atropine by introducing an epoxy group and a hydroxyl group. Clinically, these three antagonists muscarinic (M) acetylcholine receptors, inhibiting parasympathetic nerves and widely used as anticholinergic drugs in humans and animals. However, excessive consumption of these drugs can be significantly toxic, leading to severe toxic reactions such as blurred vision, urinary retention, fever and dizziness, tachycardia, cognitive impairment, hallucinations, amnesia, and cognitive impairment. Furthermore, these drugs are often illegally abused as veterinary drugs, leading to the contamination of animal-derived foods and posing a potential risk to human health. Ipratropium bromide is a synthetic anticholinergic drug that is very similar in structure to atropine. Due to its quaternary amine structure, it becomes a safer compound than atropine. However, as an anticholinergic drug, it may also cause side effects such as tachycardia and decreased salivation.

[0003] Several analytical techniques have been used to detect atropine, anisodamine, scopolamine, and ipratropium bromide, such as chromatography, capillary electrophoresis, and surface-enhanced Raman spectroscopy. While these methods offer advantages in sensitivity and accuracy, they are often expensive and time-consuming. Newer methods have also been developed, such as carbon nanosensors, organic field-effect transistors, and electrophoresis microfluidic chips, but these methods primarily detect single drugs. Therefore, developing an efficient and sensitive sensing method for the simultaneous analysis of this class of structurally similar anticholinergic drugs is of great importance. Summary of the Invention

[0004] The present invention aims to provide a method for preparing a supramolecular fluorescence sensor array and a method for rapidly identifying and detecting four tropane anticholinergic drugs, namely atropine sulfate (AS), racemic anisodamine (RA), scopolamine hydrobromide (SH), and ipratropium bromide (IB). The constructed supramolecular sensor array is characterized in that a synthetic tetraphenylethylene molecule (TPE-P) and a commercially available dye coumarin 7 (C7) are selected as guest molecules ( Figure 1) were constructed with seven-membered cucurbitacin (Q[7]) and eight-membered cucurbitacin (Q[8]) to form two supramolecular fluorescent probes, TPE-P@Q[7] and C7@Q[8], respectively. These two probes were used as sensing units to construct a cucurbitacin-based supramolecular fluorescent sensor array. The sensor array was used to detect the cross-fluorescence response signals of four tropane anticholinergic drugs, and the four tropane anticholinergic drugs were qualitatively and quantitatively detected by linear discriminant analysis. The method is simple, sensitive, rapid and effective.

[0005] The present invention adopts the following technical solutions to achieve the invention objectives:

[0006] A method for preparing a supramolecular fluorescent sensor array is disclosed. The supramolecular fluorescent sensor array is constructed by using TPE-P@Q[7] supramolecular fluorescent probes and C7@Q[8] supramolecular fluorescent probes as sensing units to obtain a cucurbitacin-based supramolecular fluorescent sensor array. The constructed cucurbitacin-based supramolecular fluorescent sensor array is used to distinguish and detect tropane anticholinergic drugs atropine sulfate, racemic anisodamine, scopolamine hydrobromide, and ipratropium bromide.

[0007] The preparation method of the supramolecular fluorescence sensor array is carried out according to the following steps:

[0008] (1) Preparation of TPE-P@Q[7] supramolecular fluorescent probe:

[0009] S1: Dissolve 0.2-0.5 mmol of TPE and 1-5 mmol of 1-bromopropane in 5-15 mL of DMF solution, reflux at 90-110°C for 40-60 hours, cool after the reaction, gradually add acetone dropwise to extract the precipitate, wash the precipitate repeatedly with acetone, and dry at 60-80°C for 5-8 hours to obtain a white solid, namely TPE-P;

[0010] The specific synthesis reaction formula is as follows:

[0011]

[0012] S2: Dissolve 100-130 mg of seven-membered cucurbitacin in ultrapure water, sonicate for 5-15 min, transfer to a volumetric flask and adjust to a concentration of 1×10 -3 mol / L solution to obtain solution A, which is ready for use;

[0013] S3: Take 60-90 mg of TPE-P, dissolve it in ultrapure water, transfer it to a volumetric flask and make up to a concentration of 1×10 -3 mol / L solution to obtain solution B, which is ready for use;

[0014] S4: Take the solution A and solution B in the above step and mix them in a volumetric flask. The molar ratio of TPE-P and Q[7] is 1:10. Use ultrapure water to make up the volume, and the concentration is 1×10 -5 mol / L TPE-P@Q[7] supramolecular fluorescent probe;

[0015] (2) Preparation of C7@Q[8] supramolecular fluorescent probe:

[0016] SS1: Dissolve 10-20 mg of eight-membered cucurbitacin ring in ultrapure water, sonicate for 5-15 min, transfer to a volumetric flask and adjust to a concentration of 1×10 -4 mol / L solution to obtain solution C, which is ready for use;

[0017] SS2: Dissolve 30-40 mg of coumarin 7 in ultrapure water, transfer to a volumetric flask and dilute to a concentration of 1 × 10 -3 mol / L solution to obtain solution D, which is ready for use;

[0018] SS3: Take the above solution C and solution D and mix them in a volumetric flask. The molar ratio of Q[8] and C7 is 3:2. Use ultrapure water to make up the volume to obtain a concentration of 1×10 -5 mol / L of C7@Q[8] supramolecular fluorescent probe;

[0019] (3) Preparation of drug standard solution:

[0020] SSS1: Accurately weigh the analytically pure standards of the required drugs atropine sulfate, racemic anisodamine, scopolamine hydrobromide, and ipratropium bromide, dissolve them in ultrapure water, and prepare a molar concentration of 1×10 -2 mol / L atropine sulfate standard solution, 1×10 -2 mol / L racemic anisodamine standard solution, 1×10 -2 mol / L scopolamine hydrobromide standard solution, 1×10 -2 mol / L ipratropium bromide standard solution;

[0021] SSS2: Take 1×10 -2 mol / L atropine sulfate standard solution was added to 3 mL of TPE-P@Q[7] supramolecular fluorescent probe to prepare atropine sulfate-TPE-P@Q[7] mixed sample solution 1 with an atropine sulfate concentration of 20 μM;

[0022] Take 1×10 -2 mol / L atropine sulfate standard solution was added to 300 μL of TPE-P@Q[7] supramolecular fluorescent probe to prepare atropine sulfate-TPE-P@Q[7] mixed sample solution 2 with an atropine sulfate concentration of 20 μM;

[0023] Take 1×10 -2 mol / L racemic anisodamine standard solution was added to 3 mL of TPE-P@Q[7] supramolecular fluorescent probe to prepare racemic anisodamine-TPE-P@Q[7] mixed sample solution 1 with a racemic anisodamine concentration of 20 μM;

[0024] Take 1×10 -2 mol / L racemic anisodamine standard solution was added to 300 μL of TPE-P@Q[7] supramolecular fluorescent probe to prepare racemic anisodamine-TPE-P@Q[7] mixed sample solution 2 with a racemic anisodamine concentration of 20 μM;

[0025] Take 1×10 -2 mol / L scopolamine hydrobromide standard solution was added to 3 mL of TPE-P@Q[7] supramolecular fluorescent probe to prepare scopolamine hydrobromide-TPE-P@Q[7] mixed sample solution 1 with a scopolamine hydrobromide concentration of 20 μM;

[0026] Take 1×10 -2 mol / L scopolamine hydrobromide standard solution was added to 300 μL of TPE-P@Q[7] supramolecular fluorescent probe to prepare scopolamine hydrobromide-TPE-P@Q[7] mixed sample solution 2 with a scopolamine hydrobromide concentration of 20 μM;

[0027] Take 1×10 -2 mol / L ipratropium bromide standard solution was added with 3 mL of TPE-P@Q[7] supramolecular fluorescent probe to prepare ipratropium bromide-TPE-P@Q[7] mixed sample solution 1 with an ipratropium bromide concentration of 20 μM;

[0028] Take 1×10 -2 mol / L ipratropium bromide standard solution was added with 300 μL of TPE-P@Q[7] supramolecular fluorescent probe to prepare ipratropium bromide-TPE-P@Q[7] mixed sample solution 2 with an ipratropium bromide concentration of 20 μM;

[0029] Take 1×10 -2 mol / L atropine sulfate standard solution was added to 3 mL of C7@Q[8] supramolecular fluorescent probe to prepare atropine sulfate-C7@Q[8] mixed sample solution 1 with an atropine sulfate concentration of 20 μM;

[0030] Take 1×10 -2 mol / L atropine sulfate standard solution was added to 300 μL of C7@Q[8] supramolecular fluorescent probe to prepare atropine sulfate-C7@Q[8] mixed sample solution 2 with an atropine sulfate concentration of 20 μM;

[0031] Take 1×10 -2 mol / L racemic anisodamine standard solution was added to 3 mL of C7@Q[8] supramolecular fluorescent probe to prepare racemic anisodamine-C7@Q[8] mixed sample solution 1 with a racemic anisodamine concentration of 20 μM;

[0032] Take 1×10 -2 mol / L racemic anisodamine standard solution was added to 300 μL of C7@Q[8] supramolecular fluorescent probe to prepare racemic anisodamine-C7@Q[8] mixed sample solution 2 with a racemic anisodamine concentration of 20 μM;

[0033] Take 1×10 -2 mol / L scopolamine hydrobromide standard solution was added to 3 mL of C7@Q[8] supramolecular fluorescent probe to prepare scopolamine hydrobromide-C7@Q[8] mixed sample solution 1 with a scopolamine hydrobromide concentration of 20 μM;

[0034] Take 1×10 -2 mol / L scopolamine hydrobromide standard solution was added to 300 μL of C7@Q[8] supramolecular fluorescent probe to prepare scopolamine hydrobromide-C7@Q[8] mixed sample solution 2 with a scopolamine hydrobromide concentration of 20 μM;

[0035] Take 1×10 -2 mol / L ipratropium bromide standard solution was added with 3 mL of C7@Q[8] supramolecular fluorescent probe to prepare ipratropium bromide-C7@Q[8] mixed sample solution 1 with an ipratropium bromide concentration of 20 μM;

[0036] Take 1×10 -2 mol / L ipratropium bromide standard solution was added with 300 μL of C7@Q[8] supramolecular fluorescent probe to prepare ipratropium bromide-C7@Q[8] mixed sample solution 2 with an ipratropium bromide concentration of 20 μM;

[0037] (4) Fluorescence emission spectrum detection:

[0038] Respectively detecting the fluorescence emission spectra of the atropine sulfate-TPE-P@Q[7] mixed sample solution 1, the racemic hyoscine-TPE-P@Q[7] mixed sample solution 1, the scopolamine hydrobromide-TPE-P@Q[7] mixed sample solution 1, the ipratropium bromide-TPE-P@Q[7] mixed sample solution 1, the atropine sulfate-C7@Q[8] mixed sample solution 1, the racemic hyoscine-C7@Q[8] mixed sample solution 1, the scopolamine hydrobromide-C7@Q[8] mixed sample solution 1 and the ipratropium bromide-C7@Q[8] mixed sample solution 1 in step (3) to obtain the fluorescence wavelengths corresponding to the four drug standard solutions;

[0039] (5) Preparation of supramolecular fluorescence sensing array:

[0040] According to the fluorescence wavelengths corresponding to the four obtained drug standard solutions, the fluorescence intensities of the atropine sulfate-TPE-P@Q[7] mixed sample solution 2, the racemic hyoscine-TPE-P@Q[7] mixed sample solution 2, the scopolamine hydrobromide-TPE-P@Q[7] mixed sample solution 2, the ipratropium bromide-TPE-P@Q[7] mixed sample solution 2, the atropine sulfate-C7@Q[8] mixed sample solution 2, the racemic hyoscine-C7@Q[8] mixed sample solution 2, the scopolamine hydrobromide-C7@Q[8] mixed sample solution 2 and the ipratropium bromide-C7@Q[8] mixed sample solution 2 in step (3) are respectively detected;

[0041] Five parallel sets of data were collected for each mixed sample solution. The fluorescence intensity of the samples at the detection wavelength was detected sequentially on a full-function microplate reader to obtain a data matrix consisting of 2 sensor units × 4 scopolamine drugs × 5 parallel experiments. SPSS 22.0 software was used to perform linear discriminant analysis on the above data matrix.

[0042] In the above step (1), the preparation of TPE-P@Q[7] supramolecular fluorescent probe is as follows:

[0043] S1: 0.33 mmol of TPE and 2.67 mmol of 1-bromopropane were dissolved in 10 mL of DMF solution and refluxed at 100°C for 48 h. After the reaction was completed, the mixture was cooled and acetone was gradually added dropwise to extract the precipitate. The precipitate was washed repeatedly with acetone 3-5 times and dried at 70°C for 6 h to obtain a white solid, namely TPE-P.

[0044] S2: Dissolve 116.30 mg of the seven-membered cucurbit ring in ultrapure water, sonicate for 10 min, transfer to a volumetric flask and adjust the volume to a concentration of 1 × 10 -3 mol / L solution to obtain solution A, which is ready for use;

[0045] S3: Take 76.87 mg of TPE-P, dissolve it in ultrapure water, transfer it to a volumetric flask and make up to a concentration of 1×10 -3 mol / L solution to obtain solution B, which is ready for use;

[0046] S4: Take the solution A and solution B in the above step and mix them in a volumetric flask. The molar ratio of TPE-P and Q[7] is 1:10. Use ultrapure water to make up the volume, and the concentration is 1×10 -5 mol / L TPE-P@Q[7] supramolecular fluorescent probe.

[0047] In the above step (2), the preparation of C7@Q[8] supramolecular fluorescent probe:

[0048] SS1: Take 13.28 mg of eight-membered cucurbitacin ring, dissolve it in ultrapure water, sonicate for 10 min, transfer it to a volumetric flask and adjust the volume to a concentration of 1×10 -4 mol / L solution to obtain solution C, which is ready for use;

[0049] SS2: Dissolve 33.34 mg of coumarin 7 in ultrapure water, transfer to a volumetric flask and dilute to a concentration of 1 × 10 -3 mol / L solution to obtain solution D, which is ready for use;

[0050] SS3: Take the above solution C and solution D and mix them in a volumetric flask. The molar ratio of Q[8] and C7 is 3:2. Use ultrapure water to make up the volume to obtain a concentration of 1×10 -5 mol / L C7@Q[8] supramolecular fluorescent probe.

[0051] In the above step (4), the fluorescence emission spectrum detection conditions of the probe TPE-P@Q[7] are an excitation wavelength of 255 nm, a voltage of 490 V, and a slit of 10 nm; the fluorescence emission spectrum detection conditions of the probe C7@Q[8] are an excitation wavelength of 467 nm, a voltage of 500 V, and a slit of 10 nm.

[0052] The above-mentioned supramolecular fluorescence sensor array is used in the detection of tropane anticholinergic drugs. The application is to detect and distinguish four tropane anticholinergic drugs, namely atropine sulfate, racemic anisodamine, scopolamine hydrobromide and ipratropium bromide, by the supramolecular fluorescence sensor array. The detection method is to take the standard solution of the tropane anticholinergic drug to be detected, add it to the two supramolecular fluorescent probes TPE-P@Q[7] and C7@Q[8] respectively, detect the fluorescence intensity, input the detection value into the SPSS22.0 software, and perform LDA analysis.

[0053] The supramolecular fluorescence sensing array is not affected by 17 interfering substances, including K + 、Na + , Ca 2+ Mg 2+ 、Cu 2+ 、HCO3 - 、Cl - , alanine, glycine, leucine, methionine, valine, serine, proline, urea, glucose, glutathione.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1. This paper proposes a cucurbitacin-based supramolecular fluorescence sensor array constructed from two cucurbitacin-based supramolecular probes, TPE-P@Q[7] and C7@Q[8]. The array utilizes the differential response of each sensor unit to different tropane anticholinergic drugs to identify and detect four drugs (AS, RA, SH, and IB).

[0056] 2. The present invention selected a synthesized tetraphenylethylene molecule (TPE-P) and a commercially available dye coumarin 7 (C7) as guest molecules, and constructed two supramolecular fluorescent probes, TPE-P@Q[7] and C7@Q[8], with a seven-membered cucurbitacin (Q[7]) and an eight-membered cucurbitacin (Q[8]), respectively. The two probes were used as sensing units to construct a cucurbitacin-based supramolecular fluorescent sensor array. The sensor array responded differently to different tropane anticholinergic drugs. Combined with linear discriminant analysis, the four tropane anticholinergic drugs, AS, RA, SH and IB, could be identified and detected. The method is simple, sensitive, rapid and effective.

[0057] 3. The present invention is based on a supramolecular probe formed by two guest molecules and a cucurbit ring. The two probes have different fluorescence emission wavelengths. The fluorescence responses of the four drugs on the sensor array are pattern recognized by the LDA statistical algorithm, and the response of the sensor array to the sample to be tested is evaluated, thereby achieving detection of four tropane anticholinergic drugs (AS, RA, SH and IB) in aqueous solution. By adding common ions and active molecules in biological systems, it is evaluated that the array has good anti-interference ability. In addition, the array can also identify four tropane anticholinergic drugs in actual systems (artificial urine, milk, pork and herbal tea).

[0058] 4. The detection method of the four tropane anticholinergic drugs (AS, RA, SH and IB) provided by the present invention is more intuitive and convenient than traditional detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is the chemical structure of Q[7], Q[8] and the guest molecule TPE-P, C7;

[0060] Figure 2 It is TPE-P 1 H NMR spectrum;

[0061] Figure 3 It is TPE-P 1 H- 1 H COSY spectrum;

[0062] Figure 4 Is TPE-P (1 × 10 -5 mol / L) when the concentration of Q[7] is increased (a); Fluorescence intensity and the amount of substance ratio I505nm -N Q[7] / N TPE-P Relationship curve (b); in C7 (1×10 -5 mol / L) when the concentration of Q[8] is increased (c); Fluorescence intensity and the amount of substance ratio I 515nm -N Q[8] / N C7 Relationship curve (d);

[0063] Figure 5 The fluorescence emission spectra of probes TPE-P@Q[7] and C7@Q[8] in response to four drugs (20 μM) (a is probe TPE-P@Q[7]; b is probe C7@Q[8]);

[0064] Figure 6 is the fluorescence spectrum of (10 μM) TPE-P@Q[7] with the increase of different drug (AS, RA, SH and IB) concentrations;

[0065] Figure 7 is the I of (10 μM) TPE-P@Q[7] with increasing concentrations of different drugs (AS, RA, SH and IB) max ~N 药物 / N TPE-P@Q[7] picture;

[0066] Figure 8 is the fluorescence spectrum of (10 μM) C7@Q[8] with the increase of different drug (AS, RA, SH and IB) concentrations;

[0067] Figure 9 is the I of (10 μM) C7@Q[8] with increasing concentrations of different drugs (AS, RA, SH and IB) max～ N 药物 / N C7@Q[8] picture;

[0068] Figure 10 The fluorescence response fingerprints (a), heat maps (b) and radar maps (c) of TPE-P@Q[7] and C7@Q[8] to four drugs;

[0069] Figure 11 LDA score graphs of the fluorescence response of the supramolecular fluorescence sensing array to the four drugs at concentrations of 20 μM, 2 μM, and 80 μM (a is 20 μM; c is 2 μM; d is 20 μM) and HCA graph of the four drugs at a concentration of 20 μM (b);

[0070] Figure 12 This is the LDA score graph of the fluorescence response of the supramolecular fluorescence sensing array to four drugs at different concentrations in ultrapure water;

[0071] Figure 13 This is the linear relationship diagram of Factor 1 of the supramolecular fluorescence sensing array to different concentrations of four drugs;

[0072] Figure 14 This is a typical LDA score plot of the supramolecular sensing array for binary mixtures of AS / RA, AS / SH, SH / RA at different ratios, and ternary mixture of AS / RA / SH (total drug mixture concentration is 20 μM);

[0073] Figure 15 is the LDA score graph of the supramolecular sensing array’s response to four drugs (20 μM) and other interfering substances (100 μM);

[0074] Figure 16 is the LDA score graph of the supramolecular sensing array's response to four drugs (20 μM) in artificial urine, milk, pork, and herbal tea samples;

[0075] Figure 17 (a) is Q[7] 1 H NMR spectrum; (b) is TPE-P 1 HNMR spectrum; (c) is Q[7] / TPE-P (5:1) 1 HNMR spectrum; (d) is Q[7] / TPE-P / AS (5:1:2) 1 H NMR spectrum; (e) is Q[7] / AS (1:2) 1 H NMR spectrum; (f) is AS 1 H NMR spectrum. DETAILED DESCRIPTION

[0076] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention.

[0077] Example 1

[0078] Synthesis of guest molecule TPE-P:

[0079] 0.33 mmol of TPE (tetra(3-imidazoylphenyl)ethylene) and 2.67 mmol of 1-bromopropane were dissolved in 10 mL of DMF solution, reacted at 100°C, and refluxed for 48 h. After the reaction was completed, the solution was cooled and acetone was gradually added dropwise to extract the precipitate. The precipitate was repeatedly washed with acetone and dried at 70°C for 6 h to obtain 53.3% of white solid TPE-P.

[0080] Example 2

[0081] Preparation of TPE-P@Q[7] supramolecular fluorescent probe standard solution:

[0082] (1) Take 116.30 mg of Q[7], dissolve it in ultrapure water, sonicate for 10 min, transfer it to a 100 mL volumetric flask and adjust the volume to a concentration of 1 × 10 -3 mol / L solution to obtain solution A, which is ready for use;

[0083] (2) Take 76.87 mg of TPE-P, dissolve it in ultrapure water, transfer it to a 100 mL volumetric flask and dilute it to a concentration of 1×10 -3 mol / L solution to obtain solution B, which is ready for use;

[0084] (3) Take 10 mL of the above solution A and 1 mL of solution B in a 100 mL volumetric flask, mix them, and dilute to volume with ultrapure water to obtain a molar ratio of TPE-P and Q[7] of 1:10 and a concentration of 1×10 -5 mol / L TPE-P@Q[7] supramolecular fluorescent probe.

[0085] Example 3:

[0086] Preparation of C7@Q[8] supramolecular fluorescent probe standard solution:

[0087] (1) Take 13.28 mg of Q[8], dissolve it in ultrapure water, sonicate for 10 min, transfer it to a 100 mL volumetric flask and adjust the volume to a concentration of 1×10 -4 mol / L solution to obtain solution C, which is ready for use;

[0088] (2) Take 33.34 mg of C7, dissolve it in ultrapure water, transfer it to a 100 mL volumetric flask and dilute it to a concentration of 1 × 10 -3 mol / L solution to obtain solution D, which is ready for use;

[0089] (3) Take 15 mL of the above solution C and 1 mL of solution D in a 100 mL volumetric flask, mix them, and make up to volume with ultrapure water. The molar ratio of Q[8] and C7 is 3:2, and the concentration is 1×10 -5 mol / L C7@Q[8] supramolecular fluorescent probe.

[0090] Example 4

[0091] Preparation of scopolamine drug standard solution:

[0092] Accurately weigh the analytically pure standard products of the required drugs atropine sulfate, racemic anisodamine, scopolamine hydrobromide and ipratropium bromide, dissolve them in ultrapure water, and prepare the molar concentration of 1×10 -2 mol / L atropine sulfate standard solution, 1×10 -2 mol / L racemic anisodamine standard solution, 1×10 -2mol / L scopolamine hydrobromide standard solution, 1×10 - 2 mol / L ipratropium bromide standard solution.

[0093] Example 5

[0094] Preparation of supramolecular fluorescence sensing array:

[0095] (1) The AS, RA, SH and IB standard solutions prepared in Example 4 were added to 3 mL of the TPE-P@Q[7] supramolecular fluorescent probe solution prepared in Example 2 to prepare a 20 μM mixed sample solution (the molar ratio of supramolecular probe to each drug was 1:2). The solution was allowed to stand for 10 min, and the fluorescence emission spectra of the solutions were measured.

[0096] The fluorescence of the probe TPE-P@Q[7] itself is bright blue. After adding drugs, the fluorescence at 478nm is quenched to varying degrees. The quenching degree of the four drugs is: AS=SH>IB>RA.

[0097] (2) The AS, RA, SH and IB standard solutions prepared in Example 4 were added to 3 mL of the C7@Q[8] supramolecular fluorescent probe solution prepared in Example 3 to prepare a 20 μM mixed sample solution (the molar ratio of supramolecular probe to each drug was 1:2). The solution was allowed to stand for 10 min, and the fluorescence emission spectra of the solutions were measured.

[0098] The fluorescence of the probe C7@Q[8] itself is deep yellow, and a significant blue shift occurs after the addition of drugs.

[0099] (3) Two supramolecular fluorescent probes, TPE-P@Q[7] and C7@Q[8] (both at a concentration of 10 μM), were prepared in ultrapure water. 300 μL of the above probe solutions, TPE-P@Q[7] and C7@Q[8], and mixed sample solutions of four tropane anticholinergic drugs (20 μM) were added to each well using a 96-well all-black sterile microtiter plate. Five sets of data were collected for each mixed sample. The fluorescence intensity of the samples at the detection wavelength was detected in sequence on a full-function microplate reader. This generated a data matrix consisting of 2 sensing units × 4 tropane drugs × 5 parallel experiments. The data matrix was then subjected to linear discriminant analysis (LDA) using SPSS version 22.0:

[0100] Each of the four drugs formed four independent data clusters, which were distinct from each other, and the replicate results for each drug were closely clustered. This data indicates that the sensor array can detect and identify four different drugs: AS, RA, SH, and IB.

[0101] Example 6

[0102] Statistical data analysis of four scopolamine drugs at different concentrations using the supramolecular fluorescence sensing array:

[0103] Using the method of Example 5, the fluorescence responses of AS, RA, SH, and IB at 10 different concentrations (0 μM, 2 μM, 4 μM, 6 μM, 10 μM, 15 μM, 20 μM, 30 μM, 50 μM, and 80 μM) were measured. A data matrix consisting of 2 sensing units × 10 concentrations × 5 parallel experiments was obtained and analyzed using LDA:

[0104] In the LDA plot, AS, RA, and SH concentrations (0 μM, 2 μM, 4 μM, 6 μM, 10 μM, 15 μM, 20 μM, 30 μM, 50 μM, and 80 μM) exhibit a series of distinct, independent clusters, with their distribution gradually transitioning from positive to negative factors along the x-axis. IB concentrations (0 μM, 2 μM, 4 μM, 6 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 50 μM) are distinct from each other, while replicate samples of the same concentration cluster together. These data demonstrate that the sensor array is capable of discriminating tropane anticholinergic drug concentrations to a certain extent.

[0105] Example 7

[0106] Statistical data analysis of scopolamine drugs at different mixing ratios using supramolecular fluorescence sensing array:

[0107] Using the method of Example 5, AS, RA, and SH were selected as analytes in this example. Two drug mixtures at five different molar ratios (10:0, 7:3, 5:5, 3:7, and 0:10) or three drug mixtures (10:0:0, 6:2:2, 2:6:2, 2:2:6, 0:10:0, and 0:0:10) were mixed with the sensing solution to form a data matrix of 2 sensing units × 5 mixing ratios × 5 replicates or 2 sensing units × 6 mixing ratios × 5 replicates. LDA was used for data analysis:

[0108] The four tropane anticholinergic drugs in binary and ternary mixtures all showed non-overlapping clusters. From these data results, it can be seen that the sensor array can identify and distinguish mixtures of tropane anticholinergic drugs.

[0109] Example 8

[0110] Study on the anti-interference performance of supramolecular fluorescence sensing array:

[0111] (1) Accurately weigh K + 、Na + , Ca 2+ Mg 2+ 、Cu2+ 、HCO3 - 、Cl - (where the cations are all perchlorates, Cl - Use tetrabutylammonium chloride, HCO3 - Analytical grade standards of alanine, glycine, leucine, methionine, valine, serine, proline, urea, glucose, and glutathione were dissolved in ultrapure water and prepared into 1×10 -2 mol / L standard solution.

[0112] (2) Using the method of Example 5, on a 96-well plate, first, the 17 standard solutions prepared in step (1) and the four tropane anticholinergic drugs (AS, RA, SH and IB standard solutions) prepared in Example 4 were added to the probe TPE-P@Q[7], and the fluorescence responses of the 17 interfering substances and the four tropane anticholinergic drugs were measured respectively; then, another 17 standard solutions prepared in step (1) and the four tropane anticholinergic drugs (AS, RA, SH and IB standard solutions) prepared in Example 4 were added to C7@Q[8], and the fluorescence responses of the 17 interfering substances and the four tropane anticholinergic drugs were measured respectively; finally, the two fluorescence intensity data of the two probes were extracted and imported into SPSS software for linear discriminant analysis (LDA).

[0113] The 17 common interfering substances in biological systems clustered with drugs in different areas and did not affect the recognition of tropane anticholinergic drugs.

[0114] Example 9

[0115] Statistical analysis of scopolamine in four real samples (artificial urine, milk, pork, and herbal tea) using a supramolecular fluorescence sensing array:

[0116] (1) Artificial urine and herbal tea were filtered through a 0.22 μm filter. The milk sample was prepared by mixing 10 mL of pure milk with 5 mL of 10% trichloroacetic acid solution, ultrasonicating for 2 minutes, centrifuging at 4000 rpm for 10 minutes, and filtering the supernatant through a 0.22 μm pore nylon filter. The pretreatment of pork samples followed the method of the "National Food Safety Standard of the People's Republic of China" (GB31658.19-2022). Briefly, 2 g of pork sample was taken into a centrifuge tube, 20 mL of 0.1 mol / L potassium dihydrogen phosphate buffer was added, shaken for 10 minutes, and centrifuged at 10000 r / min for 10 minutes at 4°C. Accurately transfer 10 mL of supernatant to another centrifuge tube, add 5 mL of 0.2 mol / L potassium dihydrogen phosphate solution, mix, and filter through a 0.22 μm filter.

[0117] (2) The four filtered solutions (artificial urine, milk, pork, and herbal tea) were diluted by half and replaced with ultrapure water. The methods of Examples 2, 3, and 4 were used to prepare TPE-P@Q[7] supramolecular fluorescent probe solution, C7@Q[8] supramolecular fluorescent probe solution, and tropane anticholinergic drug standard solution.

[0118] (3) Using the method of Example 5, the fluorescence responses of four samples (artificial urine, milk, pork, and herbal tea) were measured, and LDA analysis was performed on the fluorescence data:

[0119] The four tropane anticholinergic drugs in all four samples showed five non-overlapping clusters. This data shows that the sensor array can still identify and distinguish the four tropane anticholinergic drugs in a practical system.

[0120] In order to obtain the solution of the present invention and verify the technical effect of the present invention, the inventors conducted a large number of experimental studies, some of which are recorded as follows:

[0121] 1 Experimental part

[0122] 1.1 Experimental Reagents

[0123] Coumarin 7 (C7), atropine sulfate (AS), racemic anisodamine (RA), scopolamine hydrobromide (SH), ipratropium bromide (IB), and other experimental reagents were purchased from Beijing Inokai Technology Co., Ltd. Artificial urine was purchased from Beijing Solaibao Technology Co., Ltd. Herbal tea, milk, and pork were purchased from a local supermarket. Ultrapure water was used in the experiments, and all experimental reagents were of analytical grade.

[0124] 1.2 Experimental instruments

[0125] Fluorescence spectrophotometer (Varian Cary Eclipse), multifunctional microplate reader (Thermo Fisher Varioskan Flash). Nuclear magnetic resonance: Bruker AVANCE NEO-400 MHz nuclear magnetic resonance spectrometer.

[0126] 2 Experimental methods

[0127] 2.1 Preparation of each reagent

[0128] (1) Synthesis of guest molecule TPE-P

[0129] 0.33 mmol of TPE (tetra(3-imidazoylphenyl)ethylene) and 2.67 mmol of 1-bromopropane were dissolved in 10 mL of DMF solution, reacted at 100°C, and refluxed for 48 h. After the reaction was completed, the solution was cooled and acetone was gradually added dropwise to extract the precipitate. The precipitate was repeatedly washed with acetone and dried at 70°C for 6 h to obtain 53.30% of white solid TPE-P.

[0130] (2) Preparation of Q[7], Q[8], TPE-P, and C7 standard solutions

[0131] Q[7]: Take 116.30 mg of Q[7], dissolve it in ultrapure water, and use a 100 mL volumetric flask to make up to volume with ultrapure water to obtain solution A, the concentration of which is 1×10 -3 mol / L, for future use.

[0132] TPE-P: Take 76.87 mg of TPE-P, dissolve it in ultrapure water, and use a 100 mL volumetric flask to make up to volume with ultrapure water to obtain solution B, the concentration of which is 1×10 -3 mol / L, for future use.

[0133] Q[8]: 13.28 mg of Q[8] was dissolved in ultrapure water, ultrasonicated for 10 min, and diluted to volume with a 100 mL volumetric flask to obtain solution C, whose concentration was 1×10 -4 mol / L, for future use.

[0134] C7: Dissolve 33.34 mg of C7 in ultrapure water and dilute to volume with ultrapure water in a 100 mL volumetric flask to obtain solution D, the concentration of which is 1 × 10 -3 mol / L, for future use.

[0135] (3) Preparation of hyoscyamine drug standard solution

[0136] Accurately weigh the analytically pure standard products of the required drugs atropine sulfate, racemic anisodamine, scopolamine hydrobromide and ipratropium bromide, dissolve them in ultrapure water, and prepare the molar concentration of 1×10 -2 mol / L atropine sulfate standard solution, 1×10 -2 mol / L racemic anisodamine standard solution, 1×10 -2 mol / L scopolamine hydrobromide standard solution, 1×10 - 2 mol / L ipratropium bromide standard solution.

[0137] (4) Preparation of interfering substance standard solution

[0138] (1) Accurately weigh K + 、Na + , Ca2+ Mg 2+ 、Cu 2+ 、HCO3 - 、Cl - (where the cations are all perchlorates, Cl - Use tetrabutylammonium chloride, HCO3 - Analytical grade standards of alanine, glycine, leucine, methionine, valine, serine, proline, urea, glucose, and glutathione were dissolved in ultrapure water and prepared into 1×10 -2 mol / L standard solutions, a total of 17 standard solutions.

[0139] 2.2 Fluorescence spectroscopy experiment

[0140] All tropane anticholinergic drugs were prepared as 10 mM solutions with ultrapure water for use, Q[7], TPE-P and C7 were prepared as 1 mM solutions with ultrapure water for use, and Q[8] was prepared as 0.1 mM solution for use.

[0141] The fluorescence spectrum of TPE-P titrated by Q[7] was recorded using a fluorescence spectrophotometer under the following conditions: x =255nm, the slits are all 10nm, and the voltage is 490V. The fluorescence spectrum detection conditions for Q[8] titration of C7 are: E x =467 nm, the slits were all 10 nm, and the voltage was 500 V. Tropane anticholinergic drugs were added to the prepared 10 μM concentration of TPE-P@Q[7] (10 μM: 100 μM) and C7@Q[8] (10 μM: 15 μM) solutions to make the final drug concentration 20 μM. The fluorescence spectra of the two probe solutions to the drugs were scanned under the above conditions.

[0142] Different concentrations of drugs were added to the two sensing solutions and the fluorescence spectra were scanned. The following SternVolmer equation was used to fit I max ~N 药物 / N 传感溶液 Linear data, calculate the binding constant of the sensing solution and the drug:

[0143]

[0144] I0 = initial fluorescence intensity of the probe, I final = Final fluorescence intensity of the probe, I q = fluorescence intensity at a given drug concentration, [S] = probe concentration, [Q] = total concentration after drug addition, K SV =Stern-Volmer constant.

[0145] 2.3 Sensor array experiment

[0146] Preparation of TPE-P@Q[7] supramolecular fluorescent probe standard solution: 116.30 mg of Q[7] was dissolved in ultrapure water, ultrasonicated, transferred to a 100 mL volumetric flask and fixed to volume to a concentration of 1×10 -3 mol / L solution for later use; take 76.87 mg of TPE-P, dissolve it in ultrapure water, transfer it to a 100 mL volumetric flask and dilute it to a concentration of 1×10 -3 mol / L solution for later use; take 10 mL of the above Q[7] solution A and 1 mL of TPE-P solution B in a 100 mL volumetric flask, mix them, and dilute to volume with ultrapure water to obtain a molar ratio of TPE-P and Q[7] of 1:10 and a concentration of 1×10 -5 mol / L TPE-P@Q[7] supramolecular fluorescent probe.

[0147] Preparation of C7@Q[8] supramolecular fluorescent probe standard solution: 13.28 mg of Q[8] was dissolved in ultrapure water, ultrasonicated, transferred to a 100 mL volumetric flask and fixed to a concentration of 1 × 10 -4 mol / L solution for later use; take 33.34 mg of C7, dissolve it in ultrapure water, transfer it to a 100 mL volumetric flask and dilute it to a concentration of 1×10 -3 mol / L solution for later use; 15 mL of the above Q[8] solution and 1 mL of C7 solution were mixed in a 100 mL volumetric flask, and the volume was fixed with ultrapure water to obtain a Q[8] and C7 molar ratio of 3:2 and a concentration of 1×10 -5 mol / L C7@Q[8] supramolecular fluorescent probe.

[0148] All experiments were performed in black 96-well plates. 1×10 -5 The drug was qualitatively identified by mixing the drug under investigation with the sensing solution to final concentrations of 2 μM, 20 μM, and 80 μM. The fluorescence signal of the mixed solution was measured and the experiment was repeated five times under the same conditions. The fluorescence response pattern was calculated using the formula F = (I - I0) / (I1 - I0), where I and I1 are the fluorescence intensities of the sensing solution with and without drug, respectively, and I0 is the fluorescence intensity of TPE-P or C7. Linear discriminant analysis (LDA) results were obtained by processing 40 matrix data (2 sensing units × 4 drugs × 5 replicates) using SPSS software. The limit of detection (LOD) of the drug was calculated according to the formula LOD = 3σ / k, where σ is the standard deviation of the five blank samples and k is the slope of the calibration curve between factor 1 and drug concentration.

[0149] 2.4 Preparation of actual samples

[0150] Artificial urine and herbal tea were filtered through a 0.22 μm filter. Milk samples were prepared by mixing 10 mL of pure milk with 5 mL of 10% trichloroacetic acid solution, sonicating for 2 minutes, centrifuging at 4000 rpm for 10 minutes, and filtering the supernatant through a 0.22 μm nylon filter. The specific processing method for pork samples was to take 2 g of pork sample into a centrifuge tube, add 20 mL of 0.1 mol / L potassium dihydrogen phosphate buffer, shake and mix for 10 minutes, and centrifuge at 10000 rpm for 10 minutes at 4°C. Accurately transfer 10 mL of the supernatant to another centrifuge tube, add 5 mL of 0.2 mol / L potassium dihydrogen phosphate solution, mix, and filter through a 0.22 μm filter. The above filtered solution was used instead of ultrapure water to prepare the probe solution and drug to prepare the real sample.

[0151] 3 Results and Discussion

[0152] 3.1 Construction of the probe

[0153] Studies have shown that tetraphenylethylene-type aggregation-induced emission molecules and coumarin can assemble with cucurbitacin to form supramolecular complexes with good photophysical properties and can respond to compounds with similar structures. This part first synthesized tetrakis(1-imidazolephenyl)ethylene derivatives (TPE-P), whose chemical structure is shown by 1 H NMR spectroscopy and 1 H- 1 H COSY was characterized ( Figure 2 、 3 ).

[0154] Then, the synthesized tetraphenylethylene molecule (TPE-P) and dye coumarin 7 (C7) were selected as guest molecules, Q[7] and Q[8] were used as host molecules, and the binding behavior of TPE-P / C7 and Q[7] / Q[8] in aqueous solution was studied by fluorescence spectroscopy. The fluorescence titration spectrum of TPE-P and Q[7] is shown in Figure 2. Figure 4 As shown in Figure a, with the increase of Q[7] concentration (0-300 μM), the fluorescence intensity of TPE-P gradually increased and slightly red-shifted. When the amount of Q[7] was NQ[7] / NTPE-P=10, the fluorescence intensity tended to be stable, and the spectrum red-shifted from 485 nm to 505 nm. Figure 4 b is the molar ratio diagram of TPE-P and Q[7]. Figure 4 c) As the concentration of Q[8] increases, C7 red shifts, the fluorescence intensity at 515nm gradually decreases, and the fluorescence intensity at 597nm gradually increases. The relationship between the fluorescence intensity at the maximum wavelength and NQ[8] / NC7 is plotted ( Figure 4 d), the results show that the action ratio of C7 to Q[8] is 2:3.

[0155] 3.2 Response of probe to tropane anticholinergic drugs

[0156] Preparation of hyoscyamine drug standard solution: accurately weigh the analytically pure standard products of the required drugs atropine sulfate, racemic anisodamine, scopolamine hydrobromide and ipratropium bromide, dissolve them in ultrapure water, and prepare a molar concentration of 1×10 -2 mol / L atropine sulfate standard solution, 1×10 -2 mol / L racemic anisodamine standard solution, 1×10 -2 mol / L scopolamine hydrobromide standard solution, 1×10 -2 mol / L ipratropium bromide standard solution.

[0157] Based on the above research, we constructed two probes TPE-P@Q[7] (10μM: 100μM) and C7@Q[8] (10μM: 15μM) (hereinafter referred to as S1 and S2). By adding four structurally similar tropane anticholinergic drugs (AS, RA, SH and IB) into the probe S1 and S2 solutions, we explored the fluorescence response of the two probes to the four drugs. When the four drugs (20μM) were added to the two probe solutions (10μM), the four drugs had different degrees of influence on the fluorescence of each probe. Figure 5 a. When the four drugs were added to S1, the fluorescence of S1 at 478 nm was quenched to varying degrees. AS and SH quenched the fluorescence to a greater extent. The quenching degree of the four drugs was: AS = SH > IB > RA. When the four tropane anticholinergic drugs were added to the sensing solution S2, a significant blue shift occurred ( Figure 5 b) For example, the addition of RA and SH blue-shifted the maximum emission peak from 597 nm to 563 nm, while IB and AS blue-shifted the maximum emission peak of S2 to 521 nm. The order of fluorescence intensity response at 521 nm was IB > AS > RA > SH. These results indicate that S1 and S2 produce different fluorescence responses to the four drugs.

[0158] We explored the fluorescence changes of four drugs and two probes and their binding ability through the fluorescence titration experiment of drugs and probes. The four drugs were added to two sensing solutions respectively, and the following results were obtained: Figure 6 and Figure 8 The fluorescence spectrum shown in the figure and Figure 7 and Figure 9 As shown in I max ~N 药物 Figure 1. The spectra show that the degree of quenching or blue shift of each drug to the sensing solution is different. The binding constants (K) of the four drugs to the sensing solution were calculated using the modified Stern-Volmer equation. SV(Table 1). The results showed that the binding constants between each drug and the probe were different, indicating that the probes had different binding abilities for each drug, resulting in differential responses. These results further confirmed that the constructed probes could be used to construct a sensor array responsive to four structurally similar anticholinergic drugs.

[0159] Table 1 Binding constants of TPE-P@Q[7] and C7@Q[8] for four drugs

[0160]

[0161] Next, a microporous reader was used to monitor the fluorescence response value at a specific wavelength after the drug was added to S1 and S2 (the monitoring wavelength for S1 was 478 nm, and the monitoring wavelength for S2 was 521 nm). The characteristic fingerprint of each drug was calculated using the formula F = (I-I0) / (I1-I0) Figure 10 a), the results show that each drug can form a unique fingerprint. By converting the F value into a heat map and radar map ( Figure 10 b, c). The radius and color of each drug in the figure more intuitively show the differences between each drug, enabling visual differentiation of the four drugs. These results demonstrate that the designed probe has the potential to identify and differentiate four tropane anticholinergic drugs.

[0162] 3.3 Qualitative identification of drugs using supramolecular sensing arrays

[0163] Based on the above research, we used S1 and S2 as sensing units to construct a supramolecular fluorescence sensing array to identify and distinguish tropane anticholinergic drugs. First, we added a mixed solution of sensing units and drugs (10μM: 20μM) to a 96-well black plate, repeated the experiment 5 times for each mixed solution, and used a microwell reader to collect the fluorescence data of the 96-well plate. In this way, we constructed a data array of 2 sensing units × 4 drugs × 5 repeated experiments to identify four tropane anticholinergic drugs (AS, RA, SH and IB). The 40 data points were imported into SPSS22.0 software for LDA analysis, and a score graph containing two scoring factors was obtained ( Figure 11 a). The results show that the four drugs each form four independent data clusters, which are distinguishable from each other, and the repeated experimental results of each drug are closely clustered. Hierarchical clustering diagram ( Figure 11 b) shows the same differentiation results, with AS and IB grouped together, and RA and SH grouped together. The clustering results correlate with the similarity of the drug structures. The results indicate that the supramolecular fluorescence sensing array can achieve good qualitative discrimination of the four drugs.

[0164] We further investigated the ability of the sensor array to discriminate between the four drugs at lower and higher concentrations. Figure 11c, d) show that the sensor array can distinguish all four tropane anticholinergic drugs at both a low concentration of 2 μM and a high concentration of 80 μM, with clear intervals between each drug, demonstrating that the sensor array maintains reliable and sensitive discrimination capabilities at both low and high drug concentrations. Furthermore, the precision of the method was determined by analyzing 10 replicate samples (Table 2). The results demonstrate that the method has an RSD of <6%, indicating good precision.

[0165] Table 2 Precision of the sensor array for identifying four drugs

[0166]

[0167] 3.4 Quantitative Analysis of Drugs by Supramolecular Sensing Array

[0168] In order to verify the quantitative analysis capability of the sensor array for drugs, we added a series of concentrations (0μM, 2μM, 4μM, 6μM, 10μM, 15μM, 20μM, 30μM, 50μM, 80μM) of drugs to the sensor array for experiments. Figure 12 ) As can be seen, AS, RA and SH formed separate groups at 10 concentrations in the range of 0-80 μM, and IB could also achieve good distinction in the range of 0-50 μM, with each group separated from each other, while parallel samples with the same concentration were clustered in one group. Figure 13 The linear correlation between the lgC value of each drug and factor 1 is shown. Within a certain concentration range, the lgC value of each drug shows a good linear relationship with factor 1. The linear equation and detection limit of the sensor array for the four drugs were calculated, and the detection limit was as low as 10 -7 M (Table 3). The sensor array had a 100% accuracy rate in distinguishing the four drug concentrations. The results showed that the sensor array had good quantitative analysis capabilities for the four drugs.

[0169] Table 3 Linear relationship and detection limit of the sensor array for four drugs

[0170]

[0171] 3.5 Detection of mixed samples by supramolecular sensing array

[0172] The ability to identify mixtures in complex systems is an advantage of the sensor array, so we next evaluated the sensor array's ability to identify complex mixtures. AS, RA, and SH are common anticholinergic drugs and are also toxic tropane alkaloids derived from natural products. Some foods derived from natural products are often contaminated by mixtures of two or more of them, so we chose AS, RA, and SH as the analysis objects. A series of molar ratios (10:0, 7:3, 5:5, 3:7, 0:10) of two drug mixtures were mixed with the sensor solution to form a data matrix of 2 sensor units × 5 mixing ratios × 5 replicate samples. The following was obtained by LDA: Figure 14 The results showed that five replicates of the same ratio for each binary mixture were clustered into a single group, distinct from other ratios and without overlapping. The sensor array also achieved the same differentiation results for multiple ratios of the ternary mixture, with a cross-validation rate and accuracy of 100%.

[0173] 3.6 Sensor Array Anti-interference Experiment and Analysis of Actual Samples

[0174] Based on the detection effect of the sensor array on four drugs in water system, we further explored the practicality of the sensor array in actual sample detection. First, in order to better detect actual samples, we evaluated the selectivity of the sensor array. Common ions and active molecules were selected as interfering substances, including K + 、Na + , Ca 2+ Mg 2+ 、Cu 2+ 、HCO3 - 、Cl - , alanine, glycine, leucine, methionine, valine, serine, proline, urea, glucose, glutathione. 17 interfering substances (the concentration after adding the probe standard solution is 100 μM) and four anticholinergic drugs (the concentration after adding the probe standard solution is 20 μM) were added to the sensor array for detection, and the grouping scheme is shown in Table 4. Figure 15 It shows that the interfering substances and the four drugs are distributed in different areas ( Figure 15 , blue dotted box), most of the ions and amino acids were closely clustered with the blank control group and had almost no effect on the detection of the four drugs, while Na + Although leucine, methionine, glucose, and glutathione responded to the sensor array, they were clearly distinguished from the four drugs. This shows that the sensor array has good anti-interference performance and can be further used in the research of actual sample detection.

[0175] Table 4 Detection grouping scheme

[0176]

[0177]

[0178] Atropine sulfate, racemic anisodamine, and scopolamine hydrobromide—active ingredients in atropine sulfate, racemic anisodamine, and scopolamine hydrobromide—are secondary plant metabolites and widely used tropane anticholinergics. They are emerging plant toxins and can cause food contamination. Herbal teas and cereals, for example, are a major source of daily exposure to toxic alkaloids. They can also cause residues in humans and animals through drug abuse, posing a potential health hazard to both humans and animals. In particular, large doses of atropine are administered to slaughtered pork, causing urine retention and weight gain for profit. Scopolamine, a well-known addictive compound, is also illegally added to some beverages. Studies have shown that these drugs can be detected in a variety of matrices, highlighting the need for identifying these drugs in diverse matrices. In recent years, various techniques have been developed for the detection of tropane alkaloids in diverse real-world samples, including biological fluids, plant-derived foods, beverages, and animal-derived foods. Therefore, in order to study whether the proposed array has the potential for practical application, we selected biological fluids (artificial urine), animal-derived foods (milk and pork), and plant-derived foods (herbal tea) as actual systems. Three tropane alkaloids (20 μM) were added to the sensor array prepared using these real systems. Because ipratropium bromide is potentially toxic to the human body, and in order to strengthen the proof that the sensor array has the ability to identify drugs of this type with similar structures, we also added ipratropium bromide to the sensor array. The sensor array can produce different fluorescence responses to these four drugs, and the following fluorescence responses were obtained after LDA treatment. Figure 16 Results are shown. For artificial urine, the four drugs and the blank control were clustered into five non-overlapping groups. A leave-one-out crossover method verified that the sensor array correctly identified and differentiated the drugs 100% of the time. The sensor array also displayed unique array responses for milk, pork, and herbal tea samples, and the distributions of the four drugs were similar in these real-world systems. These results demonstrate the sensor array's excellent interference resistance and its potential for distinguishing these drugs in real-world samples.

[0179] 3.7 Discussion of possible mechanisms

[0180] In order to explore the interaction mechanism between the sensor array and the drug, the effect of the probe TPE-P@Q[7] on atropine sulfate (AS) was taken as an example. 1 H NMR studies the interaction between TPE-P@Q[7] and AS. First, for the TPE-P@Q[7] complex, the hydrogen proton resonance peak of Q[7] is as follows: Figure 17 As shown in (a), the hydrogen proton resonance peaks H1-8 on the free TPE-P are as follows Figure 17 (b) When 5 times the concentration of Q[7] is added to the TPE-P solution, the H1-8 proton signal on TPE-P moves to the high field ( Figure 17 (c)), it can be proved that Q[7] simultaneously encapsulates the benzene ring, imidazole and alkyl chain of TPE-P. For the AS@Q[7] complex, when the Q[7] solution is added to the free AS solution, the Hr, Hh and Hq protons of the tropane part in AS move to the high field, so that the azabicyclic ring (tropane) enters the Q[7] cavity; the Ha-c protons and He protons of the benzene ring move to the low field, indicating that the phenylpropionic acid part is located outside the cucurbitacin cavity, forming a host-guest complex of AS@Q[7] ( Figure 17 (ef)). When 2 times the equivalent of AS was added to the TPE-P@Q[7] complex, the Ha-r protons of AS did not change significantly compared with the AS@Q[7] complex, while the H6 protons on the imidazole ring, H7-8 protons on the benzene ring, and H1 protons on the alkyl chain of TPE-P showed downfield shifts compared with the TPE-P@Q[7] complex ( Figure 17 (d)). These results indicate that AS can competitively bind to Q[7] on TPE-P and occupy the cavity of Q[7]. Possible mode of action is as follows Figure 17 As shown in the figure on the right.

[0181] 3.8 Summary

[0182] In this study, the synthesized tetraphenylethylene derivative TPE-P and the commercially available dye C7 were assembled with Q[7] and Q[8], respectively. The sensitivity and cross-responsiveness of these two probes to subtle differences in the structure of tropanes were used to construct a supramolecular fluorescence sensor array for the detection of four tropane anticholinergic drugs. The fluorescence F values ​​of the four drugs after the interaction with the sensor array were processed using linear discriminant analysis, and the four drugs at different concentrations in pure water (2, 20 and 80 μM) were successfully identified with good reproducibility. In addition, the sensor array can also identify different concentrations of the same drug, showing a good linear relationship in the concentration range of 2 to 80 μM, with a detection limit of ~10 -7 We also investigated the sensor array's ability to discriminate between mixtures and its detection performance in the presence of common interfering ions and reactive small molecules, achieving satisfactory results. Finally, we applied the sensor array to detect drugs in real samples, successfully identifying four tropane anticholinergics in artificial urine, herbal tea, milk, and pork, further demonstrating the value of sensor arrays for detecting anticholinergic drug residues in humans and food.

Claims

1. A method for preparing a supramolecular fluorescence sensor array, characterized in that: The supramolecular fluorescent sensing array is constructed by using TPE-P@Q[7] supramolecular fluorescent probe and C7@Q[8] supramolecular fluorescent probe as sensing units to obtain a cucurbitacin-based supramolecular fluorescent sensing array, which is used to distinguish and detect tropane anticholinergic drugs atropine sulfate, racemic anisodamine, scopolamine hydrobromide and ipratropium bromide.

2. The method for preparing a supramolecular fluorescence sensor array according to claim 1, wherein: The preparation method of the supramolecular fluorescence sensor array is carried out according to the following steps: (1) Preparation of TPE-P@Q[7] supramolecular fluorescent probe: S1: Dissolve 0.2-0.5 mmol of TPE and 1-5 mmol of 1-bromopropane in 5-15 mL of DMF solution, reflux at 90-110°C for 40-60 hours, cool after the reaction, gradually add acetone dropwise to extract the precipitate, wash the precipitate repeatedly with acetone, and dry at 60-80°C for 5-8 hours to obtain a white solid, namely TPE-P; The specific synthesis reaction formula is as follows: S2: Dissolve 100-130 mg of seven-membered cucurbitacin in ultrapure water, sonicate for 5-15 min, transfer to a volumetric flask and adjust to a concentration of 1×10 -3 mol / L solution to obtain solution A, which is ready for use; S3: Take 60-90 mg of TPE-P, dissolve it in ultrapure water, transfer it to a volumetric flask and make up to a concentration of 1×10 - 3 mol / L solution to obtain solution B, which is ready for use; S4: Take the solution A and solution B in the above step and mix them in a volumetric flask. The molar ratio of TPE-P and Q[7] is 1:

10. Use ultrapure water to make up the volume, and the concentration is 1×10 -5 mol / L TPE-P@Q[7] supramolecular fluorescent probe; (2) Preparation of C7@Q[8] supramolecular fluorescent probe: SS1: Dissolve 10-20 mg of eight-membered cucurbitacin ring in ultrapure water, sonicate for 5-15 min, transfer to a volumetric flask and adjust to a concentration of 1×10 -4 mol / L solution to obtain solution C, which is ready for use; SS2: Dissolve 30-40 mg of coumarin 7 in ultrapure water, transfer to a volumetric flask and dilute to a concentration of 1 × 10 - 3 mol / L solution to obtain solution D, which is ready for use; SS3: Take the above solution C and solution D and mix them in a volumetric flask. The molar ratio of Q[8] and C7 is 3:

2. Use ultrapure water to make up the volume to obtain a concentration of 1×10 -5 mol / L of C7@Q[8] supramolecular fluorescent probe; (3) Preparation of drug standard solution: SSS1: Accurately weigh the analytically pure standards of the required drugs atropine sulfate, racemic anisodamine, scopolamine hydrobromide, and ipratropium bromide, dissolve them in ultrapure water, and prepare a molar concentration of 1×10 -2 mol / L atropine sulfate standard solution, 1×10 -2 mol / L racemic anisodamine standard solution, 1×10 -2 mol / L scopolamine hydrobromide standard solution, 1×10 - 2 mol / L ipratropium bromide standard solution; SSS2: Take 1×10 -2 mol / L atropine sulfate standard solution was added to 3 mL of TPE-P@Q[7] supramolecular fluorescent probe to prepare atropine sulfate-TPE-P@Q[7] mixed sample solution 1 with an atropine sulfate concentration of 20 μM; Take 1×10 -2 mol / L atropine sulfate standard solution was added to 300 μL of TPE-P@Q[7] supramolecular fluorescent probe to prepare atropine sulfate-TPE-P@Q[7] mixed sample solution 2 with an atropine sulfate concentration of 20 μM; Take 1×10 -2 mol / L racemic anisodamine standard solution was added to 3 mL of TPE-P@Q[7] supramolecular fluorescent probe to prepare racemic anisodamine-TPE-P@Q[7] mixed sample solution 1 with a racemic anisodamine concentration of 20 μM; Take 1×10 -2 mol / L racemic anisodamine standard solution was added to 300 μL of TPE-P@Q[7] supramolecular fluorescent probe to prepare racemic anisodamine-TPE-P@Q[7] mixed sample solution 2 with a racemic anisodamine concentration of 20 μM; Take 1×10 -2 mol / L scopolamine hydrobromide standard solution was added to 3 mL of TPE-P@Q[7] supramolecular fluorescent probe to prepare scopolamine hydrobromide-TPE-P@Q[7] mixed sample solution 1 with a scopolamine hydrobromide concentration of 20 μM; Take 1×10 -2 mol / L scopolamine hydrobromide standard solution was added to 300 μL of TPE-P@Q[7] supramolecular fluorescent probe to prepare scopolamine hydrobromide-TPE-P@Q[7] mixed sample solution 2 with a scopolamine hydrobromide concentration of 20 μM; Take 1×10 -2 mol / L ipratropium bromide standard solution was added with 3 mL of TPE-P@Q[7] supramolecular fluorescent probe to prepare ipratropium bromide-TPE-P@Q[7] mixed sample solution 1 with an ipratropium bromide concentration of 20 μM; Take 1×10 -2 mol / L ipratropium bromide standard solution was added with 300 μL of TPE-P@Q[7] supramolecular fluorescent probe to prepare ipratropium bromide-TPE-P@Q[7] mixed sample solution 2 with an ipratropium bromide concentration of 20 μM; Take 1×10 -2 mol / L atropine sulfate standard solution was added to 3 mL of C7@Q[8] supramolecular fluorescent probe to prepare atropine sulfate-C7@Q[8] mixed sample solution 1 with an atropine sulfate concentration of 20 μM; Take 1×10 -2 mol / L atropine sulfate standard solution was added to 300 μL of C7@Q[8] supramolecular fluorescent probe to prepare atropine sulfate-C7@Q[8] mixed sample solution 2 with an atropine sulfate concentration of 20 μM; Take 1×10 -2 mol / L racemic anisodamine standard solution was added to 3 mL of C7@Q[8] supramolecular fluorescent probe to prepare racemic anisodamine-C7@Q[8] mixed sample solution 1 with a racemic anisodamine concentration of 20 μM; Take 1×10 -2 mol / L racemic anisodamine standard solution was added to 300 μL of C7@Q[8] supramolecular fluorescent probe to prepare racemic anisodamine-C7@Q[8] mixed sample solution 2 with a racemic anisodamine concentration of 20 μM; Take 1×10 -2 mol / L scopolamine hydrobromide standard solution was added to 3 mL of C7@Q[8] supramolecular fluorescent probe to prepare scopolamine hydrobromide-C7@Q[8] mixed sample solution 1 with a scopolamine hydrobromide concentration of 20 μM; Take 1×10 -2 mol / L scopolamine hydrobromide standard solution was added to 300 μL of C7@Q[8] supramolecular fluorescent probe to prepare scopolamine hydrobromide-C7@Q[8] mixed sample solution 2 with a scopolamine hydrobromide concentration of 20 μM; Take 1×10 -2 mol / L ipratropium bromide standard solution was added with 3 mL of C7@Q[8] supramolecular fluorescent probe to prepare ipratropium bromide-C7@Q[8] mixed sample solution 1 with an ipratropium bromide concentration of 20 μM; Take 1×10 -2 mol / L ipratropium bromide standard solution was added with 300 μL of C7@Q[8] supramolecular fluorescent probe to prepare ipratropium bromide-C7@Q[8] mixed sample solution 2 with an ipratropium bromide concentration of 20 μM; (4) Fluorescence emission spectrum detection: Respectively detecting the fluorescence emission spectra of the atropine sulfate-TPE-P@Q[7] mixed sample solution 1, the racemic hyoscine-TPE-P@Q[7] mixed sample solution 1, the scopolamine hydrobromide-TPE-P@Q[7] mixed sample solution 1, the ipratropium bromide-TPE-P@Q[7] mixed sample solution 1, the atropine sulfate-C7@Q[8] mixed sample solution 1, the racemic hyoscine-C7@Q[8] mixed sample solution 1, the scopolamine hydrobromide-C7@Q[8] mixed sample solution 1 and the ipratropium bromide-C7@Q[8] mixed sample solution 1 in step (3) to obtain the fluorescence wavelengths corresponding to the four drug standard solutions; (5) Preparation of supramolecular fluorescence sensing array: According to the fluorescence wavelengths corresponding to the four obtained drug standard solutions, the fluorescence intensities of the atropine sulfate-TPE-P@Q[7] mixed sample solution 2, the racemic hyoscine-TPE-P@Q[7] mixed sample solution 2, the scopolamine hydrobromide-TPE-P@Q[7] mixed sample solution 2, the ipratropium bromide-TPE-P@Q[7] mixed sample solution 2, the atropine sulfate-C7@Q[8] mixed sample solution 2, the racemic hyoscine-C7@Q[8] mixed sample solution 2, the scopolamine hydrobromide-C7@Q[8] mixed sample solution 2 and the ipratropium bromide-C7@Q[8] mixed sample solution 2 in step (3) are respectively detected; Five parallel sets of data were collected for each mixed sample solution. The fluorescence intensity of the samples at the detection wavelength was detected sequentially on a full-function microplate reader to obtain a data matrix consisting of 2 sensor units × 4 scopolamine drugs × 5 parallel experiments. SPSS 22.0 software was used to perform linear discriminant analysis on the above data matrix.

3. The method for preparing a supramolecular fluorescence sensor array according to claim 2, wherein: In the step (1), the preparation of TPE-P@Q[7] supramolecular fluorescent probe is as follows: S1: 0.33 mmol of TPE and 2.67 mmol of 1-bromopropane were dissolved in 10 mL of DMF solution and refluxed at 100°C for 48 h. After the reaction was completed, the mixture was cooled and acetone was gradually added dropwise to extract the precipitate. The precipitate was washed repeatedly with acetone 3-5 times and dried at 70°C for 6 h to obtain a white solid, namely TPE-P. S2: Dissolve 116.30 mg of the seven-membered cucurbit ring in ultrapure water, sonicate for 10 min, transfer to a volumetric flask and adjust the volume to a concentration of 1 × 10 -3 mol / L solution to obtain solution A, which is ready for use; S3: Take 76.87 mg of TPE-P, dissolve it in ultrapure water, transfer it to a volumetric flask and make up to a concentration of 1×10 - 3 mol / L solution to obtain solution B, which is ready for use; S4: Take the solution A and solution B in the above step and mix them in a volumetric flask. The molar ratio of TPE-P and Q[7] is 1:

10. Use ultrapure water to make up the volume, and the concentration is 1×10 -5 mol / L TPE-P@Q[7] supramolecular fluorescent probe.

4. The method for preparing a supramolecular fluorescence sensor array according to claim 2, wherein: In the step (2), the preparation of C7@Q[8] supramolecular fluorescent probe: SS1: Take 13.28 mg of eight-membered cucurbitacin ring, dissolve it in ultrapure water, sonicate for 10 min, transfer it to a volumetric flask and adjust the volume to a concentration of 1×10 -4 mol / L solution to obtain solution C, which is ready for use; SS2: Dissolve 33.34 mg of coumarin 7 in ultrapure water, transfer to a volumetric flask and dilute to a concentration of 1 × 10 - 3 mol / L solution to obtain solution D, which is ready for use; SS3: Take the above solution C and solution D and mix them in a volumetric flask. The molar ratio of Q[8] and C7 is 3:

2. Use ultrapure water to make up the volume to obtain a concentration of 1×10 -5 mol / L C7@Q[8] supramolecular fluorescent probe.

5. The method for preparing a supramolecular fluorescence sensor array according to claim 2, wherein: In step (4), the fluorescence emission spectrum detection conditions of the probe TPE-P@Q[7] are an excitation wavelength of 255 nm, a voltage of 490 V, and a slit of 10 nm; the fluorescence emission spectrum detection conditions of the probe C7@Q[8] are an excitation wavelength of 467 nm, a voltage of 500 V, and a slit of 10 nm.

6. Use of the supramolecular fluorescence sensor array according to any one of claims 1 to 5 in detecting tropane anticholinergic drugs, characterized in that: The application is to detect and distinguish four tropane anticholinergic drugs, namely atropine sulfate, racemic anisodamine, scopolamine hydrobromide and ipratropium bromide, by a supramolecular fluorescence sensor array. The detection method is to take a standard solution of the tropane anticholinergic drug to be detected, add it to two supramolecular fluorescent probes, TPE-P@Q[7] and C7@Q[8], respectively, detect the fluorescence intensity, input the detection value into SPSS22.0 software, and perform LDA analysis.

7. Use of the supramolecular fluorescence sensor array according to claim 6 in detecting tropane anticholinergic drugs, characterized in that: The supramolecular fluorescence sensing array can be unaffected by 17 interfering substances, including K + 、Na + , Ca 2+ Mg 2+ 、Cu 2+ 、HCO3 - 、Cl - , alanine, glycine, leucine, methionine, valine, serine, proline, urea, glucose, glutathione.

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