Method for constructing visual sensing array and analyzing sensing performance of visual sensing array

Abstract

The invention provides a construction and sensing performance analysis method of a visual sensor array, and belongs to the technical field of sensor array construction, the method comprises the following steps: step 1, fluorescent probes BDP, BTH, RFCC and CN-NBD and characterization; 2, carrying out a spectrum test; step 3, preparation of a sensing array: uniformly soaking non-fluorescence test paper in the probe solution for 20 minutes, then taking out the non-fluorescence test paper, exposing the non-fluorescence test paper in air for natural air drying to obtain a sensing material loaded with a probe, and storing the prepared sensing material in a low-temperature light-proof vacuum environment; step 4, detecting analyte steam by the sensing array; step 5, detecting the freshness of the seafood by the sensing array; step 6, testing volatile basic nitrogen; and 7, collecting array data. The invention develops a simple developing and fluorescent sensing array which is used for quickly identifying three kinds of steam, namely Cad, NH3 and H2S generated by food spoilage and detecting different concentrations.

Description

Technical Field

[0001] The present invention relates to the technical field of sensor array construction, and in particular to the construction of a visual sensor array and a sensor performance analysis method thereof. Background Art

[0002] Traditional colorimetric sensor arrays typically rely on weak hydrogen bonding interactions, resulting in less pronounced color changes and low sensitivity. In contrast, chemical sensor arrays have been widely used in the medical, environmental, and food sectors as a rapid and low-cost analytical tool. Their design mimics the olfactory or gustatory systems of animals for detection and analysis. Compared to individual sensors, each unit in a sensor array can interact non-specifically with odor molecules, generating a unique fingerprint pattern through differences in overall response. Sensor arrays are composed of chemical dyes and sensing materials. To improve sensitivity, specific chemical recognition sites need to be constructed to increase the speed of chemical reactions. Furthermore, the choice of sensing material in the sensor array is crucial. Recent studies have reported on sol-gel polyvinylidene membranes, porous titanium dioxide membranes, and carbon nanotube / porphyrin materials. These materials are complex to manufacture and expensive, making them difficult to visualize and transport. Therefore, choosing a low-cost and portable substrate is crucial. Flexible cellulose paper-based materials are considered due to their low cost, portability, and ease of operation. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for constructing a visual sensor array and analyzing its sensing performance, so as to solve the technical problems that the existing visual sensor array manufacturing process is relatively complex, the cost is high, and it is not conducive to visual portability.

[0004] Five fluorescent probes with different recognition sites were synthesized based on BODIPY, benzothiazole, resorufin, a near-infrared dye, and rhodamine B. These probes were then loaded onto test paper to successfully fabricate a multi-color and fluorescence cross-responsive sensor array for visual monitoring of seafood freshness. This sensor array can intuitively identify the main gases produced by shrimp spoilage—cadaverine (Cad), ammonia (NH3), and hydrogen sulfide (H2S)—and quantitatively detect the vapor concentrations of Cad, NH3, and H2S in the range of 0–120 ppm. Due to their distinct recognition sites and mechanisms of action, the probes exhibit distinct color and fluorescence changes. Furthermore, RGB patterns were generated by digital subtraction of array images before and after the reaction. Based on the high-dimensional difference vectors of the resulting data, combined with principal component analysis (PCA), dendrograms, and heatmaps, Cad, NH3, and H2S vapors and shrimp freshness at different storage times were successfully identified. Comparison of the sensor array results with the standard TVBN values demonstrated that the array can distinguish shrimp freshness at different time periods.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for constructing a visual sensor array and analyzing its sensing performance comprises the following steps:

[0007] Step 1: Synthesis and characterization of fluorescent probes BDP, BTH, RFCC, and CN-NBD;

[0008] Step 2: Perform spectrum test;

[0009] Step 3: Preparation of the sensor array: soak the non-fluorescent test paper evenly in the probe solution for 20 minutes, then take it out and expose it to air to dry naturally to obtain the probe-loaded sensing material. The prepared sensing material is stored in a low-temperature, light-proof, vacuum environment;

[0010] Step 4: Detection of analyte vapor by the sensing array;

[0011] Step 5: The sensor array detects the freshness of seafood;

[0012] Step 6: Volatile basic nitrogen test;

[0013] Step 7: Array data acquisition: perform three parallel experiments on each sample, use a smartphone to capture images of the visual array before and after the reaction, and use the software app to extract the average values of red (R), green (G), and blue (B) at different positions on the sensing material. Perform three parallel experiments, and the absolute value of the difference between the average values of R, G, and B before and after the reaction is used as the data set.

[0014] Furthermore, in step 1: the synthesis process of the fluorescent probe BDP is:

[0015] Under ice-bath conditions, pyrrole and N,N-diisopropylethylamine were dissolved in 1,2-dichloroethane at a molar ratio of 1:3 in a nitrogen atmosphere and reacted for 30 minutes. A certain amount of triphosgene was dissolved in 1,2-dichloroethane and added dropwise to the reaction system. The temperature was raised to room temperature and the reaction was continued for 2 hours. Finally, pyrrole was slowly added dropwise and refluxed at 65°C for 4 hours. After the reaction, the mixture was extracted with dichloroethane and water. The crude product was purified by silica gel column chromatography to obtain product 1.

[0016] The product 1, phosphorus oxychloride and 1,2-dichloroethane were reacted at a molar ratio of 1:8 at 80°C for 3.5 hours, then cooled to room temperature. An appropriate amount of triethylamine was added and stirred for 5 minutes in an ice bath, followed by slow dropwise addition of boron trifluoride ether. Finally, the temperature was raised to room temperature and stirred for 2 hours to terminate the reaction. The reaction mixture was extracted with dichloromethane and water, and the crude product was purified by silica gel column to obtain a reddish-yellow product BDP.

[0017] Furthermore, in step 1: the synthesis process of the fluorescent probe BTH is:

[0018] 2-Aminothiophenol and salicylaldehyde were dissolved in 5 mL of methanol at a molar ratio of 1:1. 37% concentrated hydrochloric acid and 30% hydrogen peroxide were added to the solution and reacted at room temperature for 3 h. At the end of the reaction, the mixture was filtered and washed with ethanol to obtain a white product HBT.

[0019] The product HBT and hexamethylenetetramine compound were dissolved in trifluoroacetic acid at a molar ratio of 1:2, refluxed at 90°C for 2 h, the solvent was evaporated, and the residue was purified by silica gel column chromatography to obtain a green solid product BH;

[0020] The product BH and 1,3-indandione compound were dissolved in 20 mL of anhydrous dichloromethane at a molar ratio of 1.2:1, and 2 drops of 4-methylpiperidine were added. The mixture was reacted at room temperature for 2 h. After the reaction, the solvent was evaporated and the crude product was purified by silica gel column chromatography to obtain a red solid BTH.

[0021] Furthermore, in step 1: the synthesis process of the fluorescent probe RFCC is:

[0022] Take resorufin (0.47 mol), K2CO3 (4.69 mol) and dissolve them in anhydrous dichloromethane. Under ice bath conditions, take hexanoyl chloride (0.94 mol) and dissolve it in CH2Cl2. Slowly add it dropwise to the reaction system and react at room temperature for 12 hours. The reaction process is monitored by TCL plate. At the end of the reaction, the solvent is removed by vacuum rotary evaporator. The crude product is purified by silica gel column chromatography to obtain yellow powder RFCC.

[0023] Furthermore, in step 1: the synthesis process of the fluorescent probe CN-NBD is:

[0024] Synthesis of Product 1: PBr3 was added dropwise to a mixture of DMF and chloroform at 0°C. The mixture was stirred for 45 minutes, followed by the addition of cyclohexanone at a molar ratio of 2.7:1. The reaction was allowed to proceed for 16 hours before termination. The crude product was extracted with dichloromethane and saturated brine, and the solvent in the lower layer was removed by rotary evaporation to yield Product 1.

[0025] Synthesis of Product 2: Compound 1 (0.08 mol), 2-hydroxy-4-methoxybenzaldehyde (0.9 mol), and Cs2CO3 (2.7 mol) were dissolved in DMF at room temperature and reacted for 16 hours. The product was extracted with dichloromethane and saturated brine. The organic solvent layer was removed by rotary evaporation, and the residue was purified by silica gel column chromatography to yield Product 2 as a yellow solid.

[0026] Synthesis of Product 3: Product 2, K2CO3, and dicyanoisophorone were dissolved in acetic anhydride at a molar ratio of 1:3:1.3 and reacted at 80°C under a nitrogen atmosphere for 16 hours to obtain a dark blue liquid. After removing the solvent from the reaction mixture, the product 3 was purified by silica gel column chromatography to obtain a blue solid.

[0027] Synthesis of Product 4: Product 3 (1 mol) was added to anhydrous dichloromethane, followed by BBr3 (20 mol) at 0°C. The reaction was allowed to warm to room temperature for 20 h. The pH was neutralized with NaHCO3 and the end point was determined when the pH reached approximately 7. The product was extracted with dichloromethane and water, and the crude product was purified by silica gel column chromatography to yield Product 4 as a bluish-black solid.

[0028] CN-NBD synthesis process: Product 4, triethylamine and NBD-Cl compound were mixed in a molar ratio of 1:4:1.5, and the reaction mixture was reacted at room temperature for 3 h. The solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography. The product CN-NBD was a black solid.

[0029] Furthermore, the specific process of step 2 is:

[0030] BDP spectrum test: Use chromatographic grade acetonitrile to prepare BDP stock solution, use DMSO to prepare Cad and NH3 stock solutions, use distilled water to prepare H2S stock solution, fluorescence parameter setting: λ ex =392nm, slit: 2 / 2nm.

[0031] BTH spectral test: Use chromatographically pure dimethyl sulfoxide (DMSO) / distilled water to prepare the probe stock solution and analyte solution, and store in a refrigerator at 4°C. For spectral testing, dilute the probe to 10μM with DMSO, and then take 2mL into a quartz cuvette for testing. In the experiment, DMSO is used to prepare Cad and NH3, and distilled water is used to prepare H2S. The fluorescence parameters are set to: λ ex =400nm,λ em =462nm, slit: 1.5 / 1.5nm; λ ex =550nm,λ em =630nm, slit: 3.0 / 3.0nm;

[0032] RFCC spectrum test: Use chromatographically pure DMSO / distilled water to prepare the probe stock solution and analyte solution, and store in a refrigerator at 4°C. For spectrum testing, dilute the probe to 10μM using DMSO / PBS, and then take 2mL into a quartz cuvette for testing. The fluorescence parameters are set to: λ ex =550nm,λ em =592nm, slit: 2 / 2nm;

[0033] CN-NBD spectral test: Use chromatographically pure DMSO / distilled water to prepare CN-NBD probe stock solution and analyte solution, and store in a refrigerator at 4°C. For spectral testing, dilute the probe to 10 μM using DMSO / PBS, and then take 2 mL into a quartz cuvette for testing. Fluorescence parameter setting: λ ex =750nm,λ em =840nm, slit: 8 / 8nm;

[0034] RDE-B spectral test: CN-NBD probe stock solution and analyte solution were prepared using chromatographically pure DMSO / distilled water and stored in a refrigerator at 4°C. The probe was diluted to 10 μM using DMSO, and 2 mL was placed in a quartz cuvette for testing. Fluorescence parameter settings: λ ex =540nm,λ em =595nm, slit: 1 / 1nm.

[0035] Furthermore, the specific process for step 4 is as follows: A 96-well plate is used as a gas container. First, double-sided tape is applied to the edges of each 96-well plate for easy sealing. The prepared probe test strips are then secured to the 96-well lid. The analyte is then added, and the lid is closed and sealed. The plate is then placed in a 45°C incubator for 30 minutes. Finally, the test strips are removed and photographed both in daylight and at 365nm.

[0036] Furthermore, the specific process of step 5 is as follows: live shrimps are purchased and placed in a culture dish. Then, BDP, BTH, RFCC, CN-NBD and RDE-B labels are respectively attached to the upper cover of the culture dish with tape, without the labels contacting the shrimps. These samples are placed in a room temperature environment of 30°C for 0, 1, 3, 5, 7, 9, 12 and 24 hours. During the experiment, a ZF-7 UV darkbox is used to perform test paper fluorescence color analysis, an Apple 15 Pro Max mobile phone records the fluorescence image of the RFCC label, and then the mobile phone software RGBTest 1.6 is used to read the RGB value.

[0037] Furthermore, the specific process of step 6 is as follows: remove the shrimp heads, then blend the shrimp meat into a minced consistency. Place 10g of shrimp meat sample and 60mL of distilled water into a 150mL Erlenmeyer flask, shake to disperse the sample, and let the mixture settle for 30 minutes. Then, filter the filtrate through gauze. Add 50mL of the filtrate and 1g of magnesium oxide to a digestion tube for testing. The digestion tube is then placed in a semi-automatic Kjeldahl nitrogen analyzer (TVBN) to extract total volatile nitrogen. The instrument operation steps are as follows: set the boric acid receiving volume to 20mL and the distillation time to 3 minutes. Add 300μL of bromocresol green-methyl red indicator solution to the received liquid, then titrate with 0.1M hydrochloric acid solution. The titration endpoint is when the solution turns purple-red. The TVBN content is calculated according to the national standard GB 5009.228-2016.

[0038] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0039] (1) This paper develops a simple colorimetric and fluorescent sensor array for the rapid identification of three vapors, Cad, NH3, and H2S, produced by food spoilage, as well as for the detection of different concentrations. PCA and dendrograms were obtained by identifying RGB differences. The results showed that the array can identify the concentrations of Cad, NH3, and H2S at (0–120 ppm), and the sensing mechanism is consistent with the original intention of our design. In addition, we used the prepared sensor array to analyze the freshness of shrimp at different temperatures and verified it using the standard method TVBN, further confirming the reliability of the sensor. The sensor array is expected to be used in seafood transportation, supermarkets, and other occasions, and can also provide food practitioners with a non-destructive testing method.

[0040] (2) Five fluorescent probes with different recognition sites were synthesized based on BODIPY, benzothiazole, resorufin, near-infrared dye and rhodamine B. They were further loaded on test paper to successfully prepare a cross-response sensor array with multiple colors and fluorescence, and used for visual monitoring of the freshness of seafood. The sensor array can intuitively identify the main gases produced by shrimp spoilage (cadaverine (Cad), ammonia (NH3) and hydrogen sulfide (H2S)), and quantitatively detect the vapor concentrations of Cad, NH3 and H2S in the range of 0–120 ppm. Since the probes have different recognition sites and action mechanisms, they show different color and fluorescence changes. Furthermore, RGB pattern images were obtained by performing RGB digital subtraction on the array images before and after the reaction. Based on the high-dimensional difference vector of the obtained data, combined with principal component analysis (PCA), dendrogram and heat map analysis, Cad, NH3 and H2S vapors and the freshness of shrimp at different storage times were successfully identified. The TVBN value of the standard method was compared with the sensor array results, proving that the array can distinguish the freshness of shrimp at different time periods. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 (a) and (c) UV images, (b) and (d) fluorescence spectra of BDP and BTH before and after the reaction with Cad, NH3 and H2S;

[0042] Figure 2 (a) and (c) UV images of RFCC and CN-NBD of the present invention before and after the reaction of Cad, NH3 and H2S; (b) and (d) fluorescence spectra;

[0043] Figure 3 (a) UV and (b) fluorescence spectra of RDE-B of the present invention before and after the reaction of Cad, NH3 and H2S;

[0044] Figure 4 These are the daylight and fluorescence images of the sensor array of the present invention after being exposed to air and heated for different periods of time at 45°C;

[0045] Figure 5 These are the sunlight and fluorescence images of the sensor array of the present invention after being exposed to 80% air humidity and heated for different periods of time at 45°C;

[0046] Figure 6 are the daylight and fluorescence images of the sensor array of the present invention at different temperatures and storage times;

[0047] Figure 7 are the daylight and fluorescence images of the sensor array of the present invention exposed to different pH and heating environments at 45°C;

[0048] Figure 8 These are photographs of the array sensor of the present invention exposed to various interfering vapors under sunlight (1. blank, 2. triethylamine, 3. 4-methylpiperidine, 4. tyramine, 5. histamine, 6. spermine, 7. spermidine, 8. H2S, 9. NH3, 10. Cad);

[0049] Figure 9 These are photographs of the array sensor of the present invention exposed to various interfering vapors under ultraviolet light (1. blank, 2. triethylamine, 3. 4-methylpiperidine, 4. tyramine, 5. histamine, 6. spermine, 7. spermidine, 8. H2S, 9. NH3, 10. Cad);

[0050] Figure 10 This is the visual fingerprint of Cad, NH3 and H2S at a concentration of 120 ppm obtained by the present invention based on the visible light sensor array;

[0051] Figure 11 (a) 2D principal component score graph and (b) dendrogram of Cad, NH3, and H2S under the visible light sensing array of the present invention;

[0052] Figure 12 This is the visual fingerprint of Cad, NH3 and H2S at a concentration of 120 ppm obtained by the sensor array under ultraviolet light;

[0053] Figure 13 (a) principal component and (b) dendrogram of the sensor array Cad, NH3 and H2S under UV light according to the present invention;

[0054] Figure 14 is the visible light response image of the array of the present invention to different concentrations of Cad;

[0055] Figure 15 (a) principal component and (b) dendrogram of the sensor array of the present invention under sunlight for different concentrations of Cad;

[0056] Figure 16 is the fluorescence response image of the array of the present invention to different concentrations of Cad;

[0057] Figure 17 is the linear relationship between the RGB value of the sensor array under ultraviolet light of the present invention and Cad vapor (0–120 ppm);

[0058] Figure 18 (a) principal component and (b) dendrogram of the sensor array of the present invention under ultraviolet light for different concentrations of Cad;

[0059] Figure 19 This is the visible light response image of the array of the present invention to different concentrations of NH3;

[0060] Figure 20 (a) Principal component and (b) dendrogram of the sensor array of the present invention for different NH3 concentrations under sunlight;

[0061] Figure 21 It is the UV response image of the array of the present invention to different concentrations of NH3;

[0062] Figure 22 is the linear relationship between the RGB value of the sensor array under ultraviolet light and NH3 vapor (0–120 ppm);

[0063] Figure 23 (a) Principal component and (b) dendrogram of the sensor array of the present invention under UV light for different concentrations of NH3;

[0064] Figure 24 is the visible light response image of the array of the present invention to different concentrations of H2S;

[0065] Figure 25 (a) Principal component and (b) dendrogram analysis of the sensor array of the present invention under sunlight for different H2S concentrations;

[0066] Figure 26 It is the UV response image of the array of the present invention to different concentrations of H2S;

[0067] Figure 27 is the linear relationship between the RGB value of the sensor array under ultraviolet light of the present invention and H2S vapor (0–120 ppm);

[0068] Figure 28 (a) Principal component and (b) dendrogram analysis of the sensor array of the present invention under UV light for different H2S concentrations;

[0069] Figure 29 It is the response of the sensor array to shrimp under different temperatures and time intervals under sunlight of the present invention;

[0070] Figure 30 This is the PCA graph of shrimp under sunlight, different temperatures and time intervals of the present invention;

[0071] Figure 31 This is the thermal map of shrimp under sunlight at different temperatures and time intervals according to the present invention;

[0072] Figure 32 It is the response of the sensor array to shrimp under the ultraviolet light of the present invention at different temperatures and time intervals;

[0073] Figure 33 This is the PCA graph of shrimp under the UV lamp of the present invention at different temperatures and time intervals;

[0074] Figure 34 This is a thermal image of shrimp under the ultraviolet light of the present invention at different temperatures and time intervals;

[0075] Figure 35 is the TVBN value of shrimp at different temperatures and time intervals of the present invention;

[0076] Figure 36 This is the synthetic route of the fluorescent probe BDP of the present invention;

[0077] Figure 37 This is the synthesis route of the fluorescent probe BTH of the present invention;

[0078] Figure 38 This is the synthesis route of the fluorescent probe RFCC of the present invention;

[0079] Figure 39 This is the synthesis route of the fluorescent probe CN-NBD of the present invention. DETAILED DESCRIPTION

[0080] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and by way of preferred embodiments. However, it should be noted that many of the details listed in this specification are merely provided to help the reader gain a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be practiced even without these specific details.

[0081] A method for constructing a visual sensor array and analyzing its sensing performance comprises the following steps:

[0082] Step 1: Synthesis and characterization of fluorescent probes BDP, BTH, RFCC, and CN-NBD, such as Figures 36-39 shown.

[0083] The synthesis process of the fluorescent probe BDP is as follows:

[0084] Pyrrole (0.7 mL, 1 mmol) and N,N-diisopropylethylamine (1.8 mL, 3 mmol) were dissolved in 1,2-dichloroethane (10 mL) under an N₂ atmosphere and reacted for 30 min. Subsequently, 1 g of triphosgene was dissolved in 1,2-dichloroethane (8 mL) and added dropwise to the reaction system. The temperature was raised to room temperature and the reaction was continued for 2 h. Finally, pyrrole (0.7 mL) was slowly added dropwise, and the mixture was refluxed at 65°C for 4 h. After the reaction, the mixture was extracted with dichloroethane and water, and the crude product was purified by silica gel column chromatography (CH₂Cl₂) to obtain product 1 (200 mg, 40% yield).

[0085] Product 1 (100 mg, 1 mmol), phosphorus oxychloride (0.11 mL, 8 mmol), and 1,2-dichloroethane (8 mL) were reacted at 80°C for 3.5 h and then cooled to room temperature. In an ice bath, 1.4 mL of triethylamine was added and stirred for 5 min, followed by the slow dropwise addition of boron trifluoride etherate (1.5 mL). Finally, the mixture was warmed to room temperature and stirred for 2 h to terminate the reaction. The reaction mixture was extracted with dichloromethane and water, and the crude product was purified on a silica gel column (petroleum ether / ethyl acetate = 10:1) to obtain a reddish-yellow product, BDP (80 mg, yield: 35%).

[0086] Characterization: 1 H NMR (500MHz, CDCl3): 7.90 (s, 2H), 7.42 (d, 2H, J = 4.00Hz), 6.59 (s, 2H, J = 4.00Hz); 13 C NMR (126MHz, CDCl3) δ (ppm) 145.1, 141.2, 134.0, 128.7, 119.1.HR-MS (ESI): [C9H6N2F2ClB+H] +Theoretical value: 227.0342; actual value: 227.0335.

[0087] In step 1: the synthesis process of the fluorescent probe BTH is:

[0088] The synthesis process of the fluorescent probe BTH is as follows:

[0089] 2-Aminothiophenol (250 mg, 2 mmol) and salicylaldehyde (244 mg, 2 mmol) were dissolved in 5 mL of methanol. Concentrated hydrochloric acid (37%, 169 μL) and hydrogen peroxide (30%, 188 μL) were added to the solution and reacted at room temperature for 3 h. At the end of the reaction, the mixture was filtered and washed with ethanol to obtain a white product, HBT.

[0090] The product HBT (454 mg, 2 mmol) and hexamethylenetetramine (577 mg, 4 mmol) were dissolved in trifluoroacetic acid and refluxed at 90°C for 2 h. The solvent was evaporated and the residue was purified by silica gel column chromatography (dichloromethane) to obtain the green solid product BH.

[0091] Characterization: 1 HNMR(500MHz,Chloroform-d)δ / ppm 14.28(s,1H),10.64(s,1H),10.03(s,1H),8.60(s,1H),8.45(d,J=2.0Hz,1H),8.10(d,J =8.2Hz,1H),8.02(d,J=7.8Hz,1H),7.64–7.60(m,1H),7.57–7.52(m,1H).HR-MS(ESI): [C 15 H8NO3S-H] ﹣ Theoretical value: 282.0225; actual value: 282.0240.

[0092] The product BH (628 mg, 1.2 mmol) and 1,3-indandione (300 mg, 1 mmol) were dissolved in 20 mL of anhydrous dichloromethane, and 2 drops of 4-methylpiperidine were added. The mixture was allowed to react at room temperature for 2 hours. After completion of the reaction, the solvent was evaporated, and the crude product was purified by silica gel column chromatography (CH2Cl2 / ethanol = 100 / 1) to obtain BTH as a red solid.

[0093] Characterization: 1HNMR(500MHz,DMSO-d6)δ / ppm 9.89(s,1H),9.67(s,1H),8.80(s,1H),8.52(s,1H),8.18(d,J=7.8Hz,1H),8.09( d,J=8.2Hz,1H),7.96(d,J=17.9Hz,3H),7.57(s,1H),7.47(s,1H).HR-MS(ESI):[C 24 H 11 NO4S-H] ﹣ Theoretical value: 410.0487; actual value: 410.0464.

[0094] In step 1: the synthesis process of the fluorescent probe RFCC is:

[0095] Take resorufin (100mg, 0.47mmol), K2CO3 (648.3mg, 4.69mmol) and 10mL anhydrous dichloromethane and be placed in a 50mL round-bottomed two-necked flask. Under ice bath condition, take hexanoyl chloride (0.13mL, 0.94mmol) and be dissolved in 10mL CH2Cl2, slowly be added dropwise in the reaction system, and at room temperature react 12h. Monitor the reaction process with TCL plate, when reaction finishes, remove solvent with vacuum rotary evaporator, crude product obtains yellow powder RFCC (65mg, productive rate: 53.4%) through silica gel column chromatography purification (petroleum ether / ethyl acetate=3 / 1). 1 H NMR, 13 Its chemical structure was characterized by C NMR and HR-MS.

[0096] Characterization: 1 H NMR(500MHz,CDCl3-d)δ / ppm 7.82(d,J=8.6Hz,1H),7.46(d,J=9.8Hz,1H),7.17(s,1H),7.14(d,J=8.8Hz,1H),6.89(d,J=9.8Hz,1H),6 .36(s,1H),2.63(t,J=7.5Hz,2H),2.37(t,J=7.6Hz,2H),1.80(m,2H),1.35(m,2H),0.97(t,J=6.8Hz,3H). 13 CNMR(125MHz,CDCl3-d)δ / ppm 186.4,171.4,153.7,149.4,148.2,144.4,135.14,134.8,131.2,125.0,119.3,109.7,107.2,34.3,31.2,29.7,22.3,13.9.HR-MS(ESI):[C 18 H17 NO4+H] + Theoretical value 312.1236, actual value 312.1238.

[0097] In step 1: the synthesis process of the fluorescent probe CN-NBD is:

[0098] Synthesis of Product 1: To a mixture of DMF (5.6 mL) and chloroform (25 mL) at 0°C was added PBr (6.2 mL, 130.5 mmol) dropwise. The mixture was stirred for 45 min, followed by the addition of cyclohexanone (2.5 mL, 48.4 mmol). The reaction was allowed to proceed for 16 h before termination. The crude product was extracted with dichloromethane and saturated brine, and the lower layer was freed from the solvent using a rotary evaporator to afford Product 1 (200 mg, 84% yield).

[0099] Synthesis of Product 2: Compound 1 (200 mg, 1.08 mmol), 2-hydroxy-4-methoxybenzaldehyde (134 mg, 0.90 mmol), and Cs2CO3 (880 mg, 2.7 mmol) were dissolved in 6 mL of DMF at room temperature and reacted for 16 hours. The product was extracted with dichloromethane and saturated brine. The organic solvent layer was removed by rotary evaporation, and the residue was purified by silica gel column chromatography (dichloromethane) to obtain Product 2 as a yellow solid (300 mg, 60% yield).

[0100] Synthesis of Product 3: Product 2 (300 mg, 1 mmol), K2CO3 (513 mg, 3 mol), and dicyanoisophorone (299 mg, 1.3 mmol) were dissolved in 10 mL of acetic anhydride. The reaction was allowed to proceed at 80°C under an N2 atmosphere for 16 h to yield a dark blue liquid. After removing the solvent from the reaction mixture, the product was purified by silica gel column chromatography (dichloromethane / petroleum ether = 3:2) to obtain Product 3 as a blue solid (254 mg, 50% yield).

[0101] Synthesis of Product 4: Product 3 (150 mg, 1 mmol) was added to 20 mL of anhydrous dichloromethane. BBr (1.4 mL, 20 mmol) was added at 0°C and the mixture was allowed to react at room temperature for 20 h. The pH was neutralized with NaHCO, and the endpoint was determined when the pH reached approximately 7. Extraction was performed with dichloromethane and water, and the crude product was purified by silica gel column chromatography (dichloromethane). Product 4 was obtained as a bluish-black solid (100 mg, 69% yield).

[0102] Characterization: 1HNMR(500MHz, CDCl3)δ7.32(d,J=2.4Hz,1H),7.10(dd,J=8.2,2.4Hz,1H),7.02–6.95(m, 2H),6.68–6.58(m,3H),2.64–2.56(m,4H),1.85–1.79(m,4H),1.45(s,2H),1.10(s,6H). 13 C NMR (126MHz, CDCl3) δ156.9,151.8,142.9,135.2,124.1,123.5,115.9,43.0,34.7,34.3,33.8,30.3,29.4,28.1.

[0103] Synthesis process of CN-NBD: Product 4 (100 mg, 1 mmol), triethylamine (0.1 mL, 4 mmol) and NBD-Cl (75 mg, 1.5 mmol), the reaction mixture was reacted at room temperature for 3 h, the solvent was removed by rotary evaporator, and the crude product was purified by silica gel column chromatography (dichloromethane), and the product 4 was a black solid (60 mg, yield: 43%).

[0104] Characterization: 1 HNMR(500MHz,)δ8.48(dd,J=8.3Hz,1H),7.52–7.48(m,1H),7.20(dd,J=8.4Hz,1H),7.00(d,J=2.1Hz,1H),6.92–6.89(m,1H),6.77(s,1H),6. 69(dd,J=8.4Hz,1H),6.44(d,J=16.4Hz,2H),2.62–2.57(m,4H),2.53–2.50(m,2H),2.44(s,2H),1.13(d,J=6.8Hz,2H),1.06(d,J=1.6Hz,6H). 13 CNMR (126MHz, CDCl3) δ154.8,154.2,152.9,145.1,133.1,131.7,131.2,127.5,127.2,12 2.6,121.5,115.5,114.1,113.3,108.1,108.1,42.9,39.2,32.0,31.6,28.0,24.6,20.6.

[0105] Step 2: Perform spectral testing. For BDP, prepare a BDP stock solution (1 mM) using chromatographic grade acetonitrile. For the analytes, prepare Cad and NH3 stock solutions (10 mM) using DMSO, and prepare a H2S stock solution (1 mM) using distilled water. Store in a refrigerator at 4°C. For spectral testing, dilute the probe to 10 μM using acetonitrile, then place 2 mL in a quartz cuvette for testing. Fluorescence parameters are set to: λ ex =392nm, slit: 2 / 2nm

[0106] BTH: Use chromatographically pure dimethyl sulfoxide (DMSO) to prepare the probe stock solution (1mM) and the analyte (cadaverine (Cad), ammonia (NH3)) solution (10mM). Use distilled water to prepare the hydrogen sulfide (H2S) solution (1mM) stock solution and store it in a refrigerator at 4°C. For spectral testing, dilute the probe to 10μM in DMSO, and then place 2mL in a quartz cuvette for testing. The fluorescence parameters are set as follows: λ ex =400nm,λ em =462nm, slit: 1.5 / 1.5nm; λ ex =550nm,λ em =630nm, slit: 3.0 / 3.0nm.

[0107] RFCC: Use chromatographically pure dimethyl sulfoxide (DMSO) to prepare the probe stock solution (1 mM) and the analyte (cadaverine (Cad), ammonia (NH3)) solution (10 mM). Use distilled water to prepare the hydrogen sulfide (H2S) solution (1 mM) stock solution and store it in a refrigerator at 4°C. For spectral testing, dilute the probe to 10 μM using DMSO / PBS (v / v = 3 / 7, pH 7.4), and then take 2 mL into a quartz cuvette for testing. The fluorescence parameters are set as follows: λ ex =550nm,λ em =592nm, slit: 2 / 2nm.

[0108] CN-NBD: Use chromatographically pure dimethyl sulfoxide (DMSO) to prepare the probe stock solution (1 mM) and the analyte (cadaverine (Cad), ammonia (NH3)) solution (10 mM). Use distilled water to prepare the hydrogen sulfide (H2S) solution (1 mM) stock solution and store in a refrigerator at 4°C. For spectral testing, dilute the probe to 10 μM using DMSO / PBS (v / v = 1 / 9), and then take 2 mL and place it in a quartz cuvette for testing. The fluorescence parameters are set as follows: λ ex =750nm,λ em =840nm, slit: 8 / 8nm.

[0109] RDE-B: Use chromatographically pure dimethyl sulfoxide (DMSO) to prepare the probe stock solution (1 mM) and the analyte (cadaverine (Cad), ammonia (NH3)) solution (10 mM). Use distilled water to prepare the hydrogen sulfide (H2S) solution (1 mM) and store in a refrigerator at 4°C. For spectral testing, dilute the probe to 10 μM in DMSO, then take 2 mL and place it in a quartz cuvette for testing. The fluorescence parameters are set as follows: λ ex =540nm,λ em =595nm, slit: 1 / 1nm.

[0110] Step 3: Prepare the sensor array. Soak non-fluorescent test paper (size: 1.0 cm × 3.0 cm) evenly in the probe solution (1 mM) for 20 minutes. Then, remove and air-dry to obtain the probe-loaded sensing material. Store the prepared sensing material in a cool, dark, and vacuum environment.

[0111] Step 4: The sensor array detects analyte vapors, using a 96-well plate as a gas container. First, double-sided tape is applied to the edges of each 96-well plate for easy sealing. The prepared probe strips are then secured to the top cover of the 96-well plate. Analytes are then added, and the plate is sealed on all sides. The plate is then placed in a 45°C incubator for 30 minutes. Finally, the strips are removed and photographed in both daylight and at 365nm.

[0112] Step 5: The sensor array detects the freshness of seafood. Live shrimp were purchased from a supermarket in Nanning, Guangxi. Fresh shrimp were placed in a culture dish, and then BDP, BTH, RFCC, CN-NBD, and RDE-B labels (size: 1.0 cm × 3.0 cm) were attached to the top of the culture dish with tape. The labels did not come into contact with the shrimp and fish. These samples were placed in a room temperature environment of 30°C for 0, 1, 3, 5, 7, 9, 12, and 24 hours. During the experiment, a ZF-7 UV darkroom was used to analyze the fluorescence color of the test paper. The Apple 15 Pro Max mobile phone recorded the fluorescence image of the RFCC label, and then the mobile phone software RGB Test 1.6 was used to read the RGB value.

[0113] Step 6: Volatile Basic Nitrogen (TVBN) Testing: Remove the shrimp heads and blend the shrimp meat into a minced consistency. Place 10g of shrimp meat sample and 60mL of distilled water into a 150mL Erlenmeyer flask and shake to disperse the sample. Allow the mixture to settle for 30 minutes, then filter through gauze to obtain the filtrate. Add 50mL of the filtrate and 1g of magnesium oxide to a digestion tube for testing. The digestion tube is placed in a semi-automatic Kjeldahl nitrogen analyzer (K9840) to extract total volatile nitrogen. The instrument operation steps are as follows: Set the boric acid receiving volume to 20mL and the distillation time to 3 minutes. Add 300μL of bromocresol green-methyl red indicator solution (1g / L) to the receiving liquid and titrate with 0.1M hydrochloric acid solution. The titration endpoint is when the solution turns purple-red. TVBN content is calculated according to the national standard GB5009.228-2016.

[0114] Step 7: Array data acquisition: Perform three parallel experiments on each sample. Use a smartphone to capture images of the array before and after the reaction. Use the software app to extract the average values of red (R), green (G), and blue (B) at different locations on the sensing material. Perform three parallel experiments. The absolute value of the difference between the average values of R, G, and B before and after the reaction is used as the data set. The specific calculation method is as follows:

[0115] ΔR=|R a -R b ∣

[0116] ΔG=|G a -G b ∣

[0117] ΔB=|B a -B b ∣

[0118] Among them, ΔR, ΔG and ΔB are the absolute differences of the mean values of R, G and B before and after the sensor response respectively; R a , G a and B a are the average values of R, G, and B before the sensor responds; R b , G b and B b They are the average values of R, G, and B after the sensor response.

[0119] Optical response of array probes:

[0120] First, we studied the UV and fluorescence spectra of BDP and BTH to Cad, NH3 and H2S. Figure 1As shown in (a), the initial maximum UV absorption peak of BDP appears at 450nm. After BDP reacts with Cad, the maximum absorption peak blue-shifts to 390nm, and the color changes from yellow to white. Furthermore, the maximum emission peak of BDP is at 512nm. After the addition of Cad, the maximum emission peak completely blue-shifts to 452nm, and the fluorescence color changes from green fluorescence to blue fluorescence. The BDP probe has a slight reaction to NH3, but no reaction to H2S ( Figure 1 (a) and (b)). Afterwards, we studied the UV and fluorescence spectra of BTH to Cad, NH3 and H2S. Figure 1 As shown in (c), the absorbance of BTH at 560 nm shifts blue to 441 nm with the addition of Cad, and the color changes from pink to yellow. Simultaneously, a new fluorescence emission peak appears at 462 nm, and the fluorescence color changes from red to green. Furthermore, BTH shows no reaction to NH₃ and H₂S.

[0121] Next, we studied the spectral response of RFCC to Cad, NH3 and H2S. Figure 2 As shown in (a), RFCC has a weak absorption band in the wavelength range of 450–620 nm and is colorless. When RFCC reacts with Cad and NH3, a clear absorption band appears at 578 nm. The absorbance of Cad is slightly higher than that of NH3, mainly because the pK a Greater than NH3, the color changes from light yellow to pink. At the same time, the maximum emission peak appears at 592nm, and the fluorescence color changes from colorless to red fluorescence. In addition, H2S has no reaction with RFCC ( Figure 2 (b)). Afterwards, we selected the near-infrared fluorescent probe CN-NBD to detect Cad, NH3 and H2S. The results are shown in Figure 2 .

[0122] like Figure 2 As shown in (c) and (d), the UV absorption peak of CN-NBD appears at 550 nm. After the addition of H₂S, the UV absorption peak shifts to 800 nm, and the color changes from purple to blue. Furthermore, we observed that CN-NBD exhibits almost no fluorescence. However, upon addition of H₂S, a distinct fluorescence emission peak emerges at 840 nm in the near-infrared region. CN-NBD shows no reaction to Cad or NH₃.

[0123] Finally, the spectral responses of the RDE-B probe to Cad, NH3, and H2S were studied. Figure 3As shown, the RDE-B probe exhibits both color and fluorescence quenching responses to Cad, NH3, and H2S. RDE-B initially displays a pink color. Upon reaction with Cad, NH3, and H2S, the absorption peak at 590 nm decreases, while the maximum emission peak at 600 nm disappears, shifting the fluorescence color from red to colorless. In summary, BDP, BTH, RFCC, CN-NBD, and RDE-B exhibit distinct, visually recognizable colors in response to Cad, NH3, and H2S. These three analytes are produced during shrimp spoilage, suggesting that these five probes have potential for monitoring seafood freshness.

[0124] Stability of array fluorescent test paper:

[0125] Effects of air and humidity on the stability of fluorescent test paper

[0126] In order to apply the sensor array to actual places, the stability of the fluorescent test paper in the air was investigated. First, it was attached to the top of a culture dish and then placed in a 45°C oven for 0h, 1h, and 3h, while recording the color and fluorescence color changes of the array. Figure 4 As shown in the figure, the color and fluorescence of the array test paper changed very little with the extension of heating time. The color and fluorescence after 3 hours of heating were basically the same as those at 0 hours. The results show that the array has good stability in air.

[0127] Furthermore, the color and fluorescence changes of the sensor array after being placed in 80% air humidity for different periods of time were simulated. Figure 5 It was found that the color and fluorescence of the test paper after heating for 3 hours were basically the same as those at 0 hours, and it was difficult to observe the changes with the naked eye, indicating that the fluorescent test paper still maintained good light stability at 80% air humidity.

[0128] Effects of temperature and pH value on the stability of fluorescent test paper

[0129] Due to different storage temperatures, the performance of fluorescent test paper may change. Based on this, the color and fluorescence color changes of fluorescent test paper at different temperatures and time were tested. The results are as follows Figure 6 As shown. After being placed at 25℃ for 3 days, it was observed that the color and fluorescence of the fluorescent test paper had basically not changed. In addition, since the storage temperature at 4℃ and -20℃ is relatively low, the color and fluorescence color did not change after being placed for 3 days, indicating that the fluorescent test paper itself has good stability during storage and can be used for the detection of actual samples. The photostability of the fluorescent test paper at different pH values was also tested. First, three pH environments (acidic, neutral, and alkaline) were selected, and the results are shown in Figure 2. Figure 7As shown. 200 μL of pH buffer was added to the bottom of the culture dish and sealed, then placed at a constant temperature of 45°C to allow it to completely evaporate. The results showed that BTH changed from red to yellow fluorescence at pH = 1, while in neutral and alkaline environments, the array color and fluorescence decreased slightly, but it was basically difficult to identify with the naked eye. In addition, during the actual spoilage process of meat, the pH value gradually changes from a weakly acidic state to an alkaline condition, so the fluorescent test paper can be used for visual detection of actual samples.

[0130] Array selectivity:

[0131] The presence of various interfering analytes in the food environment can affect the accuracy of the probe. Therefore, we need to perform selective analysis of BDP, BTH, RFCC, and RDE-B. First, we tested the selectivity of the array test paper for various interfering vapors commonly found in shrimp (1. blank, 2. triethylamine, 3. 4-methylpiperidine, 4. tyramine, 5. histamine, 6. spermine, 7. spermidine, 8. H2S, 9. NH3, 10. Cad). Figure 8 and 9 As shown, when the test strips were exposed to interfering gases 1–8, almost no color or fluorescence changes were observed for the BDP, BTH, RFCC, and RDE-B array test strips. This is primarily due to the low volatility of most bio-volatile substances, and also partly due to the inability of the probes themselves to react with the analytes. When exposed to vapors of 9 and 10, the BDP test strips changed color from yellow to white, and their fluorescence changed from green to blue and light blue, respectively. This indicates that BDP can undergo nucleophilic substitution reactions with both NH₃ and Cad. Cad is more basic than NH₃, resulting in a more pronounced fluorescence with Cad. For the BTH test strips, upon reaction with vapors of 9 and 10, their colors changed from red to yellow and white, respectively, and their fluorescence changed from red to green and light red, respectively, indicating that BTH only reacts with Cad. NH₃ caused some fading of the BTH test strips, but they still exhibited red fluorescence. This is primarily due to the dual-reactive sites of BTH, which can only undergo cyclization reactions with long-chain primary amines.

[0132] For the RFCC test paper, both Cad and NH3 vapors undergo esterification reactions with ester bonds, causing the RFCC test paper to change color from yellow to pink and its fluorescence from colorless to red. However, no reaction occurred with other biogenic amines due to their low volatility. For the CN-NBD test paper, except for 8, which caused the test paper to change color from purple to blue and its fluorescence from dark blue to light blue, no other vapors showed any color or fluorescence changes. The fluorescence color change of 8 with CN-NBD was minimal, primarily because CN-NBD is a near-infrared fluorophore, making a significant fluorescence change difficult to detect under UV light. In summary, these five array probes exhibited varying degrees of color and fluorescence changes in response to only three gases: Cad, NH3, and H2S. Research has shown that Cad, NH3, and H2S are produced during seafood spoilage. Therefore, a sensor array constructed with these five probes has potential for monitoring seafood freshness. Next, the sensor array's recognition performance for Cad, NH3, and H2S, as well as its ability to identify actual shrimp freshness, will be tested.

[0133] Sensing and identification of Cad, NH3, and H2S:

[0134] By using five dyes loaded on test paper to construct a sensor array, we first identified the fingerprints of three volatile vapors (Cad, NH3, H2S) produced by meat spoilage under visible light. The C, N, and S in the figure are the abbreviations of Cad, NH3, and H2S, respectively. The same is true below and will not be repeated. Figure 10 As shown, the array shows different color changes for the three volatile vapors, but it is difficult to distinguish them with the naked eye. First, we performed principal component analysis (PCA) on the data. By converting a set of correlated variables into linearly uncorrelated variables, we obtained a two-dimensional PCA graph, as shown in Figure 11 As shown in (a), the first two principal components of PCA cover 96.8% of the total variance (PC1 = 80.5%, PC2 = 16.3%). In addition, it is clearly observed that these three substances have a good classification trend. Further, we analyzed the fingerprint spectrum under UV light. Figure 12 As shown in Figure 2, the array shows different fluorescence color changes for the three vapors. The naked eye can see that the colors of Cad and H2S are more obvious, while NH3 can only be seen to be slightly weaker than Cad. We then performed PCA analysis, as shown in Figure 2. Figure 13 As shown, the first two principal components of PCA cover 99.0% of the total variance (PC1=80.9%, PC2=18.1%). In addition, it is clearly observed that the three vapors have a good classification trend.

[0135] Quantitative detection of three vapors at different concentrations:

[0136] Quantitative detection of Cadmium vapor: To evaluate the quantitative detection capability of the sensor array, the visualization pattern of Cadmium vapor in the range of 0–120 ppm was tested under sunlight. Figure 14 As shown in the figure, as the Cad vapor concentration gradually increases from 0ppm to 120ppm, BDP gradually changes from yellow to white; BTH gradually changes from red to yellow; RFCC gradually changes from light yellow to pink; CN-NBD has no reaction and its color is purple; RDE-B gradually fades from pink to light pink. Further, the PCA diagram is used to analyze its response pattern. Figure 15 As shown in (a), PC1 and PC2 account for 66.2% and 14.2% of the total variance, respectively. The figure shows that samples with the same concentration cluster together, forming nine groups overall. Among them, 0 and 1 ppm are relatively close together, with some overlap visible. Figure 15 (b) shows a dendrogram of 27 samples. It shows that the two concentrations with the smallest difference first cluster together, such as 0 and 1 ppm, and 80 and 120 ppm, before clustering with other adjacent concentrations. This indicates that the two concentrations with the smallest difference are similar. Overall, two major clusters are formed: low concentrations (0–4 ppm) and high concentrations (8–120 ppm).

[0137] Next, we tested the fluorescence patterns of the arrays in the range of 0–120 ppm. Figure 16 As shown in the figure, with the increase of Cad vapor concentration, BDP gradually changes from green fluorescence to blue fluorescence; BTH gradually changes from red fluorescence to green fluorescence; RFCC gradually changes from no fluorescence to red fluorescence; CN-NBD has no fluorescence change; and RDE-B red fluorescence gradually weakens.

[0138] According to the color change ( Figure 17 ), Cad at concentrations of 0–120 ppm was fitted with BDP (B / G), BTH (G / R), and RFCC (R) values, and the fitting coefficients were 0.990, 0.991, and 0.995, respectively, showing good linear correlation. Among them, RDE-B (R) had no linear relationship with Cad vapor concentration, and only a decrease in R value was observed. PCA and dendrograms were further used for data analysis. Figure 18 As shown in (a), PC1 and PC2 account for 63.1% and 19.4% of the total variance, respectively. It is clear from the figure that the same concentration is grouped into 9 groups. Figure 18(b) shows the dendrogram of 27 samples. It can be seen that low concentrations of 0 and 1 ppm first form a cluster and then cluster with the surrounding ones. The overall categories are low concentrations (0–8 ppm) and high concentrations (20–120 ppm). It is obvious that the classification effect of the fluorescence photos is more obvious. This is mainly because the fluorescence color changes of the sensor array are more obvious, resulting in more obvious differences in the data sets.

[0139] Quantitative detection of NH3 vapor,In order to evaluate the quantitative detection capability of the sensor array for NH3 vapor, the visualization pattern of NH3 vapor in the range of 0–120 ppm under sunlight was tested. Figure 19 As shown in the figure, as the concentration of NH3 vapor gradually increases from 0ppm to 120ppm, it is observed that the overall array color change pattern is similar to that of Cad, but it is more sensitive to Cad vapor. This is mainly because the alkalinity of Cad is greater than that of NH3, resulting in a more obvious color change. Among them, RDE-B is more sensitive to NH3. It can be seen that when the concentration increases from 2ppm to 120ppm, the pink color of RDE-B gradually begins to fade. This may be because the NH3 molecular structure is smaller and it is easier to attack RDE-B, causing RDE-B to produce spirolactam to remain closed, and the color gradually disappears. Further, PCA and dendrograms were used to analyze its response pattern. As shown in the figure, Figure 20 As shown, PC1 and PC2 account for 42.2% and 26.3% of the total variance, respectively. The 0 ppm and 1 ppm data sets overlap, resulting in overlapping clusters in the dendrogram. This is primarily due to the nearly identical colors of the 0 and 1 ppm sensors on the sensor array, leading to data overlap. There is no overlap at any other concentration, and the dendrogram also shows distinct clusters with no overlap.

[0140] Next, we tested the fluorescence patterns of the arrays in the range of 0–120 ppm. Figure 21 As shown in the figure, as the NH3 vapor concentration increases from 0ppm to 120ppm, the fluorescence color of the BDP test paper changes from green to blue; the red fluorescence of the BTH test paper does not change, mainly because BTH can only react with long-chain diprimary amines to form a ring, so it has almost no reaction with NH3; RFCC gradually changes from no fluorescence to red fluorescence; CN-NBD has no fluorescence change; and the red fluorescence of RDE-B gradually weakens. Through the overall fluorescence color change, we found that the array's reaction activity to NH3 is lower than that of Cad, which is mainly due to the strong alkalinity of Cad. According to the fluorescence color change of the array ( Figure 22 ), we fitted 0–120 ppm NH3 vapor with BDP(B / G) and RFCC(R) values, and the fitting coefficients were 0.991 and 0.971, respectively. We also found that BTH(G / R) and RDE-B(R) values could not be linearly related to NH3 vapor.

[0141] Furthermore, three-dimensional PCA and dendrogram were used for data analysis. Figure 23 (a), PC1, PC2, and PC3 account for 64.3%, 15.8%, and 10.2% of the total variance, respectively. It is found that the same concentration is a group, and it is also divided into 9 groups. There is no overlap between the data. By analyzing the dendrogram, such as Figure 23 As shown in (b), 2 ppm clustered first with 8 ppm and then with 4 ppm. This may be due to the similar fluorescence colors of the 4 and 8 ppm arrays, resulting in a subtle difference. However, no discrepancies were observed between the two datasets. Overall, the main clusters were divided into low concentrations (0–20 ppm) and high concentrations (40–120 ppm). Again, the fluorescence images of the NH3 array showed a more pronounced classification effect.

[0142] Quantitative detection of H2S vapor: To evaluate the quantitative detection capability of the sensing array for H2S vapor, we tested the visualization pattern of H2S vapor in the range of 0–120 ppm under sunlight. Figure 24 As shown in the figure, as the H2S vapor concentration (0–120 ppm) continued to increase, the color of the BDP, BTH, and RFCC test papers did not change. The color of CN-NBD changed from purple to blue; the pink color of RDE-B gradually faded. We then used PCA and dendrograms to analyze their response patterns. Figure 25 As shown in (a), PC1 and PC2 account for 78.3% and 7.7% of the total variance respectively, and there is no overlap between samples. At the same time, the tree diagram also shows clear classification without overlap. Figure 25 (b) The concentrations with the smallest difference in concentration clustered first, then formed clusters with the surrounding concentrations. The concentrations of 1 and 2 ppm, 4 and 8 ppm, 20 and 40 ppm, and 80 and 120 ppm each formed their own cluster. Subsequently, two major clusters formed: low concentration (0–2 ppm) and high concentration (4–120 ppm).

[0143] Next, we tested the fluorescence patterns of the H2S array in the range of 0–120 ppm. Figure 26 As shown in the figure, with the increase of H2S vapor concentration, the fluorescence colors of BDP, BTH and RFCC all maintain their original colors without fluorescence color change. In contrast, the fluorescence color of CN-NBD changes from dark blue to bright blue; the red fluorescence of RDE-NBD still shows a trend of gradual quenching. According to the fluorescence color change of the array, as shown in the figure, Figure 27 As shown in the figure, we fitted the CN-NBD(B) values with H2S vapor concentrations from 0–120 ppm, and the fitting coefficients were 0.990. Since the colors of the other probes barely changed, the RGB values could not be linearly fitted.

[0144] Furthermore, PCA and dendrogram were used for statistical data analysis. Figure 28 As shown in (a), PC1 and PC2 account for 78.0% and 11.9% of the total variance respectively. The data are divided into 9 groups, and there is no overlap between them. In addition, the dendrogram results clearly show that the data do not overlap and are similar to the PCA results ( Figure 28 (b) These results demonstrate that the sensor array can quantitatively detect Cad, NH3, and H2S vapors, which are commonly generated during the storage and transportation of seafood. Therefore, the sensor array is expected to be used for seafood freshness assessment.

[0145] Seafood freshness monitoring:

[0146] Inspired by the superior performance of the sensor array, the sensor array was used to non-destructively evaluate the freshness of shrimp at different storage temperatures (25, 4, -20°C) and storage times. The sensor array was fixed on the top of the culture dish and

[0147] They are the first (BDP), second (BTH), third (RFCC), fourth (CN-NBD) and fifth (RDE-B) sensor strips. The order of all the test strips below is the same and will not be explained one by one. Figure 29 As shown, after 0 h of storage at 25°C, the color of the test strips for fresh shrimp changed from yellow, red, white, purple, and pink, respectively. After 6 h of storage, the BD changed from yellow to light yellow; the BTH from red to light red; the RFCC from white to light pink; the CN-NBD from purple to light blue; and the RDE-B from pink. These slight color changes indicate that the shrimp have begun to produce Cad, NH3, and H2S. After 12 h of storage, the BDP changed from yellow to white; the BTH from red to yellow; the RFCC from white to pink; the CN-NBD from purple to blue; and the RDE-B pink faded, indicating that the shrimp have released significant amounts of Cad, NH3, and H2S. Similarly, after 10 days of storage at 4°C, the color changes of the array test strips were similar to those observed at 12 h, but the color was less pronounced. This may be because microbial growth is inhibited at 4°C, resulting in a less pronounced color change than at 25°C. Furthermore, at -20°C, the color remained essentially unchanged, indicating that little Cad, NH3, and H2S vapor was produced.

[0148] Furthermore, the RGB values of the sensor array were obtained, and PCA and heat maps were drawn to evaluate the freshness of the shrimp. It should be noted that the suffix "-1" refers to shrimp at -20℃. Figure 30, it is clearly observed that PC1 and PC2 account for 47.7% and 17.8% of the total variance respectively. The data overlap, indicating that the data are similar. By analyzing the data, it is found that the data are divided into four categories: fresh (0h, 0d, 0d-1; 1d-1, 5d-1; 3d-1; 1d; 7d-1, 10d-1), slightly corrupt (3h; 3d), corrupt (5d, 7d; 6h); completely corrupt (48h; 24h; 12h; 10d). Afterwards, we conducted a heat map analysis ( Figure 31 ), heat map analysis revealed that data points with the same concentration clustered together, with no overlap between different data points. The study found that the same color first clustered together, such as 3h, 3d; 7d-1, 10d-1; 5d, 7d, and then clustered with the surrounding data. This result is consistent with the PCA results.

[0149] Next, we tested the UV sensor array for shrimp freshness at different storage temperatures. Figure 32 As shown, after storage at 25°C for 0h, the fluorescent colors of the test paper of fresh shrimp showed green, red, colorless, colorless and red. After storage for 24h, it was clearly observed that BDP changed from green to blue; BTH changed from red to green; RFCC changed from colorless to red; CN-NBD showed dark blue, and the fluorescence color change was not obvious. This may be because CN-NBD is a near-infrared probe with a weaker fluorescence color. In addition, the culture dish may have a light absorption effect, resulting in its fluorescence color change not being obvious enough; the red fluorescence of RDE-B weakened, indicating that the shrimp has released a large amount of Cad, NH3, and H2S. Similarly, when the shrimp was stored at 4°C for 10d, the fluorescence color change of the array test paper was similar to the color at 24h, but the fluorescence color intensity was significantly weaker. In addition, the fluorescence color basically did not change at -20°C, indicating that almost no Cad, NH3, and H2S vapor was produced. Further, PCA and heat maps were used to evaluate the freshness of the shrimp. From Figure 33 , the first two principal components scored 84.9% (>80%), indicating that the freshness values were clearly classified. The data was divided into four freshness levels: fresh (0d-1, 1d-1; 0h, 0d; 1d; 7d-1, 5d-1; 3d-1; 10d-1), slightly corrupted (3h, 3d), corrupted (6h; 5d; 7d), and severely corrupted (24h; 12h; 48h; 10d). In addition, heat map analysis found that similar data first clustered into one cluster, and then formed another cluster with the surrounding data ( Figure 34 ), and the overall grouping is as follows: 10d, 7d; 12h, 24h, 48h; 0d-1, 1d-1; 1d, 0d, 0h; 3d-1; 10d-1, 5d-1, 7d-1; 3h; 6h; 5d, 3d. In summary, the data collected by the sensor array can be used to evaluate the freshness of shrimp.

[0150] In order to verify the effectiveness of the sensor array method, the standard method TVBN value was used as a control method to monitor the freshness of shrimp. TVBN value was divided into: TVBN ≤ 12mg / 100g, fresh; 20mg / 100g ≥ TVBN ≤ 12mg / 100g, slightly corrupt but still edible; TVBN ≥ 25mg / 100g, corrupt and inedible. Figure 35 As shown,

[0151] The TVBN value of fresh shrimp is 1.40mg / 100g. After being stored at 25℃ for 3 hours, the TVBN value increases to 13.99mg / 100g, indicating slight spoilage and edible. The array results also show slight spoilage and edible. After being stored for 6 hours,

[0152] When the TVBN value rises to 30.77mg / 100g (TVBN>25mg / 100g), the shrimp has begun to rot and cannot be eaten. The array results show that the shrimp has begun to rot and cannot be eaten. At 4℃, after 1 day of storage, the TVBN value is 7.12mg / 100g.

[0153] This indicates that the shrimp are fresh. After 3 days of storage, the TVBN value reaches 16.45 mg / 100g (12 mg / 100g ≥ TVBN ≤ 20 mg / 100g), indicating slight decomposition and edible status. Subsequently, after 5 days of storage, the TVBN value reaches 26.76 mg / 100g (TVBN > 25 mg / 100g), indicating that the shrimp have begun to decompose and are inedible. These results are also consistent with the freshness classification of the array.

[0154] Furthermore, TVBN levels were <15 mg / 100 g within 10 days of storage at -20°C, indicating that the shrimp remained fresh while frozen. Overall, TVBN levels were graded as follows: fresh (0 hours, 0 days, 1 day, 0-day-1, 1-day-1, 3-day-1, 5-day-1, 7-day-1, 10-day-1), slightly spoiled (3 hours, 3 days), spoiled (6 hours, 5 days, 7 days), and severely spoiled (24 hours, 12 hours, 48 hours, 10 days). This result is consistent with that obtained with the array. However, the array does not require sample destruction or pretreatment, and it also requires less expensive, large-scale equipment, making it more suitable for non-destructive monitoring of shrimp freshness in the field.

[0155] A simple colorimetric and fluorescent sensor array was developed for the rapid identification of Cad, NH3, and H2S vapors produced by food spoilage, as well as for the detection of varying concentrations. PCA and dendrograms, generated by identifying RGB differences, demonstrated that the array could identify Cad, NH3, and H2S concentrations within a range of 0–120 ppm, and that the sensing mechanism was consistent with our design. Furthermore, the prepared sensor array was used to analyze shrimp freshness at different temperatures and validated using the standard TVBN method, further confirming the reliability of the sensor. This sensor array is expected to find use in seafood transportation, supermarkets, and other environments, and could also provide a nondestructive testing method for food professionals.

[0156] Matters not covered by the present invention are known technologies.

[0157] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Construction of a visual sensor array and a method for analyzing its sensor performance, characterized by: The method comprises the following steps: Step 1: Synthesis and characterization of fluorescent probes BDP, BTH, RFCC, and CN-NBD; Step 2: Perform spectrum test; Step 3: Preparation of the sensor array: soak the non-fluorescent test paper evenly in the probe solution for 20 minutes, then take it out and expose it to air to dry naturally to obtain the probe-loaded sensing material. The prepared sensing material is stored in a low-temperature, light-proof, vacuum environment; Step 4: Detection of analyte vapor by the sensing array; Step 5: The sensor array detects the freshness of seafood; Step 6: Volatile basic nitrogen test; Step 7: Array data acquisition: perform three parallel experiments on each sample, use a smartphone to capture images of the visual array before and after the reaction, and use the software APP to extract the average values of red, green, and blue at different positions on the sensing material. Perform three parallel experiments, and the absolute value of the difference between the average values of R, G, and B before and after the reaction is used as the data set.

2. The construction of the visual sensor array and the method for analyzing its sensor performance according to claim 1 are characterized by: In step 1: the synthesis process of the fluorescent probe BDP is: The synthesis process of BDP: Under ice bath conditions, pyrrole and N,N-diisopropylethylamine were dissolved in 1,2-dichloroethane at a molar ratio of 1:3 in an N2 environment and reacted for 30 minutes. A certain amount of triphosgene was dissolved in 1,2-dichloroethane and added dropwise to the above reaction system. The temperature was raised to room temperature and the reaction was continued for 2 hours. Finally, pyrrole was slowly added dropwise and refluxed at 65°C for 4 hours. After the reaction, the mixture was extracted with dichloroethane and water. The crude product was purified by silica gel column chromatography to obtain product 1. The product 1, phosphorus oxychloride and 1,2-dichloroethane were reacted at a molar ratio of 1:8 at 80°C for 3.5 hours, then cooled to room temperature. An appropriate amount of triethylamine was added and stirred for 5 minutes in an ice bath, followed by slow dropwise addition of boron trifluoride ether. Finally, the temperature was raised to room temperature and stirred for 2 hours to terminate the reaction. The reaction mixture was extracted with dichloromethane and water, and the crude product was purified by silica gel column to obtain a reddish-yellow product BDP.

3. The construction of the visual sensor array and the method for analyzing its sensor performance according to claim 1, characterized in that: In step 1: the synthesis process of the fluorescent probe BTH is: 2-Aminothiophenol and salicylaldehyde were dissolved in 5 mL of methanol at a molar ratio of 1:

1. 37% concentrated hydrochloric acid and 30% hydrogen peroxide were added to the solution and reacted at room temperature for 3 h. At the end of the reaction, the mixture was filtered and washed with ethanol to obtain a white product HBT. The product HBT and hexamethylenetetramine compound were dissolved in trifluoroacetic acid at a molar ratio of 1:2, refluxed at 90°C for 2 h, the solvent was evaporated, and the residue was purified by silica gel column chromatography to obtain a green solid product BH; The product BH and 1,3-indandione compound were dissolved in 20 mL of anhydrous dichloromethane at a molar ratio of 1.2:1, and 2 drops of 4-methylpiperidine were added. The mixture was reacted at room temperature for 2 h. After the reaction, the solvent was evaporated and the crude product was purified by silica gel column chromatography to obtain a red solid BTH.

4. The construction of a visual sensor array and the method for analyzing its sensor performance according to claim 1, characterized in that: In step 1: the synthesis process of the fluorescent probe RFCC is: Take resorufin and K2CO3 and dissolve them in anhydrous dichloromethane. Under ice bath conditions, take hexanoyl chloride and dissolve it in CH2Cl2, slowly add it dropwise to the reaction system, and react at room temperature for 12 hours. Monitor the reaction process with a TCL plate. At the end of the reaction, remove the solvent with a vacuum rotary evaporator. Purify the crude product by silica gel column chromatography to obtain yellow powder RFCC.

5. The construction of the visual sensor array and the method for analyzing its sensor performance according to claim 1 are characterized by: In step 1: the synthesis process of the fluorescent probe CN-NBD is: Synthesis of Product 1: PBr3 was added dropwise to a mixture of DMF and chloroform at 0°C. The mixture was stirred for 45 minutes, followed by the addition of cyclohexanone at a molar ratio of 2.7:

1. The reaction was allowed to proceed for 16 hours before termination. The crude product was extracted with dichloromethane and saturated brine, and the solvent in the lower layer was removed by rotary evaporation to yield Product 1. Synthesis of Product 2: Compound 1, 2-hydroxy-4-methoxybenzaldehyde, and Cs2CO3 were dissolved in DMF at room temperature and reacted for 16 hours. The product was extracted with dichloromethane and saturated brine. The organic solvent layer was removed by rotary evaporation, and the residue was purified by silica gel column chromatography. Product 2 was obtained as a yellow solid. Synthesis of Product 3: Product 2, K2CO3, and dicyanoisophorone were dissolved in acetic anhydride at a molar ratio of 1:3:1.3 and reacted at 80°C under a nitrogen atmosphere for 16 hours to obtain a dark blue liquid. After removing the solvent from the reaction mixture, the product 3 was purified by silica gel column chromatography to obtain a blue solid. Synthesis of Product 4: Product 3 was added to anhydrous dichloromethane, followed by BBr3 at 0°C. The reaction was allowed to warm to room temperature for 20 h. The pH was neutralized with NaHCO3, and the end point was determined when the pH reached approximately 7. The reaction was then extracted with dichloromethane and water, and the crude product was purified by silica gel column chromatography. Product 4 was a blue-black solid. CN-NBD synthesis process: Product 4, triethylamine and NBD-Cl compound were mixed in a molar ratio of 1:4:1.5, and the reaction mixture was reacted at room temperature for 3 h. The solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography. The product CN-NBD was a black solid.

6. The construction of a visual sensor array and the method for analyzing its sensor performance according to claim 1, characterized in that: The specific process of step 2 is: BDP spectrum test: Use chromatographic grade acetonitrile to prepare BDP stock solution, use DMSO to prepare Cad and NH3 stock solutions, use distilled water to prepare H2S stock solution, fluorescence parameter setting: λ ex =392nm, slit: 2 / 2nm; BTH spectrum test: Use chromatographically pure dimethyl sulfoxide or distilled water to prepare the probe stock solution and analyte solution, and store them in a refrigerator at 4°C. During the spectrum test, use DMSO to dilute the probe to 10μM, then take 2mL and place it in a quartz cuvette for testing. In the experiment, use DMSO to prepare Cad and NH3, and use distilled water to prepare H2S. The fluorescence parameters are set to: λ ex =400nm,λ em =462nm, slit: 1.5 / 1.5nm; λ ex =550nm,λ em =630nm, slit: 3.0 / 3.0nm; RFCC spectrum test: Use chromatographically pure dimethyl sulfoxide or distilled water to prepare the probe stock solution and analyte solution, and store them in a refrigerator at 4°C. For spectrum testing, dilute the probe to 10μM using DMSO / PBS, then take 2mL and place it in a quartz cuvette for testing. The fluorescence parameters are set to: λ ex =550nm,λ em =592nm, slit: 2 / 2nm; CN-NBD spectral test: Use chromatographically pure dimethyl sulfoxide or distilled water to prepare CN-NBD probe stock solution and analyte solution, store in a refrigerator at 4°C. For spectral testing, dilute the probe to 10μM using DMSO / PBS, then take 2mL and place it in a quartz cuvette for testing. Fluorescence parameter setting: λ ex =750nm,λ em =840nm, slit: 8 / 8nm; Spectral test of RDE-B: Use chromatographically pure DMSO / distilled water to prepare CN-NBD probe stock solution and analyte solution, store in a refrigerator at 4°C, dilute the probe to 10 μM with DMSO, and then take 2 mL into a quartz cuvette for testing. The fluorescence parameter setting is: λ ex =540nm,λ em =595nm, slit: 1 / 1nm.

7. The construction of a visual sensor array and the method for analyzing its sensor performance according to claim 1, characterized in that: The specific process for step 4 is as follows: A 96-well plate is used as a gas container. First, double-sided tape is applied to the edges of each well for easy sealing. The prepared probe strips are then secured to the top cover of the 96-well plate. The analyte is then added, and the plate is sealed on all sides with the cover closed. Next, the plate is placed in a 45°C incubator for 30 minutes. Finally, the strips are removed and photographed both in daylight and at 365nm.

8. The construction of a visual sensor array and the method for analyzing its sensor performance according to claim 1 are characterized by: The specific process of step 5 is as follows: purchase live shrimp, place the fresh shrimp in a culture dish, and then stick the BDP, BTH, RFCC, CN-NBD and RDE-B labels to the top cover of the culture dish with tape, without the labels contacting the shrimp. Place the samples in a room temperature environment of 30°C for 0, 1, 3, 5, 7, 9, 12 and 24 hours. During the experiment, a ZF-7 UV darkbox was used to perform test paper fluorescence color analysis. The fluorescence image of the RFCC label was recorded by an Apple 15 Pro Max mobile phone, and the RGB value was then read using the mobile phone software RGB Test 1.

6.

9. The construction of a visual sensor array and the method for analyzing its sensor performance according to claim 1, characterized in that: Step 6 involves removing the shrimp heads and blending the meat until it becomes a fine paste. Place 10g of the meat sample and 60mL of distilled water into a 150mL Erlenmeyer flask, shake to disperse the sample, and allow the mixture to settle for 30 minutes. Filter the resulting filtrate through gauze. Add 50mL of the filtrate and 1g of magnesium oxide to a digestion tube for testing. The digestion tube is then placed in a semi-automatic Kjeldahl nitrogen analyzer to extract total volatile nitrogen. The instrument's operating procedures are as follows: Set the boric acid receiving volume to 20mL and the distillation time to 3 minutes. Add 300μL of bromocresol green-methyl red indicator solution to the received liquid, then titrate with 0.1M hydrochloric acid. The titration endpoint is determined when the solution turns purple-red. The TVB content is calculated according to GB 5009.228-2016.

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