Dual-mode sensor for detecting putrescine in food as well as preparation method and application of dual-mode sensor

Abstract

The invention discloses a dual-mode sensor for detecting putrescine in food as well as a preparation method and application of the dual-mode sensor. The dual-mode sensor (CDs / AuNCs) is composed of carbon dots (CDs) and gold nanoclusters (AuNCs), and putrescine can be rapidly and sensitively detected within 20 seconds. The interaction of putrescine and the sensor results in an increase in the fluorescence intensity of CDs at 430 nm and 525 nm, which is caused by the aggregation-induced emission (AIE) effect of CDs and the internal filter effect (IFE) of AuNCs. The detection limit of the CDs / AuNCs to putrescine in a ratio fluorescence mode is as low as 0.44 mu M, and the CDs / AuNCs has excellent selectivity and anti-interference performance. CDs / AuNCs also show good colorimetric linear response to putrescine, and the detection limit is 0.72 mu M. Finally, the CDs / AuNCs realizes rapid and accurate detection of putrescine in prawn meat by means of a smart phone, and shows the potential of food spoilage detection.

Description

Technical Field

[0001] This invention belongs to the field of food freshness analysis and detection, specifically relating to a dual-mode fluorescence colorimetric sensor for detecting putrescine in food, its preparation method, and its application. Background Technology

[0002] Food freshness, especially fresh seafood and meat, has received increasing attention due to growing public awareness of food safety. However, food spoilage caused by improper handling during food processing, transportation, and storage poses a significant threat to human health. The formation of biogenic amines, including histamine, putrescine, cadaverine, spermine, spermidine, tyramine, and tryptamine, are food hazard factors produced by the microbial decarboxylation of amino acids. Among these, putrescine is of particular concern due to its harmful effects, including unpleasant odors, bitter tastes, gastrointestinal discomfort, allergic reactions, and other health problems. Therefore, rapid and accurate detection of putrescine in food is crucial for monitoring food freshness.

[0003] Currently, putrescine detection based on chromatography, electrochemical techniques, and capillary electrophoresis typically relies on complex instruments, complicated procedures, and time-consuming synthesis processes, which are not conducive to real-time on-site detection. Therefore, it is necessary to develop a simple, rapid, and accurate method for putrescine detection. Compared with fluorescent molecules, sensor-based fluorescent nanomaterials have gradually attracted attention in recent years due to their advantages such as high sensitivity, low background interference, good stability, and fast detection speed. These fluorescent nanomaterials typically include carbon nanotubes, carbon dots, quantum dots, lanthanide materials, and metal nanoclusters. Among them, carbon dots (CDs) are luminescent carbon nanomaterials mainly composed of carbon elements, with a size of less than 10 nm, and possess excellent fluorescence properties, good biocompatibility, and tunable surface chemistry. They have wide applications in fluorescence imaging, bioanalysis, biomedicine, and energy. Gold nanoclusters (AuNCs) are innovative inorganic nanomaterials, molecular-level aggregates composed of several to hundreds of gold atoms formed under the protection of specific ligand molecules (such as thiols, polymers, and proteins). AuNCs, typically with a particle size of less than 2 nm, exhibit significant optical and catalytic properties, as well as relatively low reactivity, good biocompatibility and bioactivity, and a large Stokes shift.

[0004] Fluorescent sensors utilizing the combination of CDs and AuNCs have been partially reported. However, most studies use AuNCs as the reaction signal, with few focusing on using CDs as an enhancement signal. Furthermore, most reported fluorescent sensors rely on a single signal with a relatively long response time, which is sensitive to application environments, leading to inefficient and sometimes inaccurate detection results. Ratiometric fluorescence sensors offer high robustness against interference and provide more accurate and reliable quantification than conventional single-fluorescent probes. Moreover, compared to single-fluorescence detection modes, colorimetric fluorescence dual-mode detection enables cross-validation of detection results, further improving accuracy. Therefore, exploring novel multimode probes capable of generating ratiometric responses to biogenic amines (such as Put) is both interesting and valuable.

[0005] Current optical sensors initially characterized substances by detecting color changes. However, visual surveillance relies heavily on the human eye, making it difficult to capture subtle color variations. Furthermore, different detectors exhibit varying sensitivities to color. Smartphone-assisted analysis has become a popular method for the rapid detection of harmful and toxic substances. Compared to visual recognition analysis, smartphone-based colorimetric analysis (RGB / HSV) achieves higher sensitivity and accuracy, reducing errors associated with human observation. Moreover, given the rapid extraction and analysis capabilities of smartphones, portable intelligent detection devices can be developed for rapid on-site analysis and detection, thereby enhancing their practical application value.

[0006] Several sensors for detecting food freshness have been developed. For example, patent CN117343054B, "A fluorescent probe and fluorescent tag for real-time non-destructive visual monitoring of meat freshness, its preparation method and application," designs a probe HBT-Bz, which has a simple structure and is easy to synthesize. It uses the HBT fluorophore with ESIPT properties and AIE effect as the parent compound, and the benzoxazole ester group as the amine compound recognition group to design and synthesize a fluorescence-activated probe that recognizes amines. The fluorescent probe HBT-Bz responds rapidly and sensitively to amine compounds, with a detection limit as low as 5.1 ppm. However, this probe provides a single fluorescence enhancement signal, which, compared to ratiometric fluorescence signals, has weaker resistance to environmental interference and is prone to inaccurate detection in complex environments. Patent CN119823063A, "A Fluorescent Probe for Ratio-Based Detection of Biogenic Amines and Its Preparation Method and Application," develops an ADA-type organic fluorescent small molecule material for ratio-based detection of biogenic amines. It utilizes a conjugated structure containing hydrogen-bonding sites as a donor. After introducing electron-withdrawing groups, it can form an intramolecular charge-transfer (ICT) state. The change in ICT properties before and after hydrogen bonding achieves the fluorescent detection of biogenic amines. This type of material exhibits advantages such as high sensitivity, rapid response, significant color change, and reusability for the fluorescent detection of biogenic amines. Furthermore, this invention constructs a portable detection platform based on a smartphone, enabling the visualization and quantitative detection of food freshness through digital analysis of fluorescence images. However, it is worth noting that this sensor only has a single fluorescence channel and cannot self-verify the detection results. Sensors with multiple detection modes can achieve mutual verification of detection results, further improving detection accuracy. Patent CN118032751A, "A Colorimetric / Fluorescence Dual-Response Sensor Tag and Its Preparation Method and Application in Visual Monitoring of Food Freshness," discloses a colorimetric / fluorescence dual-response sensor tag. It obtains pH-responsive carbon quantum dots through a hydrothermal reaction of 1,2,4-triaminobenzene hydrochloride and sodium hydroxide solution. Further, a suspension of carbon quantum dots and cellulose nanofibers is vacuum-filtered and freeze-dried to obtain the colorimetric / fluorescence dual-response smart sensor tag. The resulting tag exhibits flexibility, sensitivity, and excellent dual pH response (colorimetric and fluorescence), enabling real-time monitoring of the freshness of various food types, including aquatic products, meat, and fruits and vegetables, under visible and ultraviolet light. However, this tag only demonstrates qualitative analysis capabilities and cannot perform quantitative analysis. Sensors with qualitative plus semi-quantitative or qualitative plus quantitative analysis capabilities can help the public or businesses accurately control food spoilage. Therefore, the development of a sensor with both ratiometric, fluorescence, and colorimetric modes is of great significance for rapid, accurate, and sensitive on-site detection of food freshness. Summary of the Invention

[0007] To address the shortcomings and deficiencies of existing technologies, this invention aims to provide a dual-mode sensor for detecting putrescine, its preparation method, and its application. Specifically, it provides a ratiometric fluorescence and colorimetric dual-mode sensor for the rapid detection of putrescine in food. This sensor is based on carbon dot-gold nanoclusters and is a fluorescence-colorimetric dual-mode putrescine sensor. Notably, unlike existing sensors, this sensor uses carbon dots (CDs) as the response signal and AuNCs as an internal reference signal. This invention also provides a method for preparing this sensor and its application in the visual detection of putrescine in food using a smartphone. The dual-mode sensor of this invention has a very short detection time (within 20 seconds), with a detection range and detection limit of 1-80 μM and 0.44 μM for the ratiometric fluorescence channel, respectively. The sensor also exhibits good colorimetric linearity for putrescine, with a detection range and detection limit of 1-70 μM and 0.72 μM for the colorimetric channel, respectively. Furthermore, the sensor demonstrates good selectivity and anti-interference capabilities. Based on the above, an integrated platform for smartphone-assisted colorimetric and fluorescence detection is constructed, enabling the visualization and rapid detection of putrescine in food.

[0008] The objective of this invention is achieved through the following technical solution: A dual-mode fluorescent colorimetric sensor for detecting putrescine in food is disclosed. The sensor is a dual-mode fluorescent colorimetric putrescine sensor based on carbon dot-gold nanoclusters, expressed as CDs / AuNCs. It is synthesized via a hydrothermal method using 2,4-dihydroxybenzaldehyde as a precursor; AuNCs are synthesized via a template etching method under heating, and the mixture yields the CDs / AuNCs sensor. The specific preparation method includes the following steps: (1) Adjust the pH of the aqueous solution of 2,4-dihydroxybenzaldehyde with NaOH, perform hydrothermal reaction, filter and dialyze to obtain CDs; (2) Casein was completely dissolved in NaOH solution, HAuCl4 solution was added to it, and the mixture was heated and incubated in the dark. Then, it was centrifuged, filtered and dialyzed to obtain AuNCs. (3) The prepared CDs and AuNCs are directly physically mixed in proportion to obtain the CDs / AuNCs sensor.

[0009] Further, in step (1), the pH of the 2,4-dihydroxybenzaldehyde aqueous solution is adjusted to 5-9 using NaOH.

[0010] Furthermore, in step (1), the concentration of the 2,4-dihydroxybenzaldehyde aqueous solution is 10-20 mM.

[0011] Furthermore, in step (1), the hydrothermal reaction temperature is 150-180℃ and the time is 5-10h.

[0012] Furthermore, in step (1), the dialysis bag required for filtering CDs is 1000 Da in size, and the dialysis time is 12-24 h.

[0013] Furthermore, in step (2), the casein concentration is 30-50 g / L, and the chloroauric acid HAuCl4 solution concentration is 50-100 mM.

[0014] Furthermore, in step (2), the heating reaction temperature is 40-60℃ and the time is 2-6h.

[0015] Furthermore, in step (2), the dialysis bag required for filtering AuNCs is 10000 Da in size, and the dialysis time is 12-36h.

[0016] Further, in step (3), CDs are diluted to 0.04-0.36 v / v of the original solution, and the diluted CDs are mixed with AuNCs at a volume ratio of 1 / 3-3 / 1 (v / v).

[0017] The present invention provides a dual-mode putrescine sensor based on carbon dot-gold nanoclusters prepared by the method described above. The dual-mode sensor (CDs / AuNCs) is composed of carbon dots (CDs) and gold nanoclusters (AuNCs).

[0018] The present invention provides a dual-mode putrescine sensor based on carbon dot-gold nanoclusters prepared by the method described above, wherein the sensor has both ratio fluorescence and colorimetric dual detection modes for putrescine.

[0019] This invention provides a dual-mode putrescine sensor based on carbon dot-gold nanoclusters prepared by the method described above. The sensor is used with the aid of a smartphone to detect the putrescine content in food (such as shrimp, fruit, vegetables, etc.).

[0020] Furthermore, the stock solution of the dual-mode putrescine sensor based on carbon dot-gold nanoclusters was mixed with an aqueous putrescine solution (fluorescent mode putrescine concentration of 0-250 μM and colorimetric mode putrescine concentration of 0-140 μM) at a volume ratio of 1:1, stirred evenly, and allowed to stand for 0-180 seconds. Then, the detection results were observed under excitation light of 300-400 nm using a fluorescence spectrophotometer and a smartphone.

[0021] The dual-mode sensor (CDs / AuNCs) of this invention consists of carbon dots (CDs) and gold nanoclusters (AuNCs), enabling rapid and sensitive detection of putrescine within 20 seconds. The interaction between putrescine and the sensor leads to increased fluorescence intensity of CDs at 430 nm and 525 nm, resulting from the aggregation-induced emission (AIE) effect of CDs and the internal filtering effect (IFE) with AuNCs. The CDs / AuNCs exhibit a detection range of 1-80 μM for putrescine in ratiometric fluorescence mode, with a detection limit as low as 0.44 μM, accompanied by a fluorescence color ranging from dark green to green to cyan. Furthermore, the sensor demonstrates excellent selectivity and anti-interference performance. The CDs / AuNCs also exhibit good colorimetric linearity for putrescine, with a detection range of 1-80 μM and a detection limit of 0.72 μM, accompanied by a color ranging from light brown to dark brown. Finally, CDs / AuNCs, with the help of smartphones to extract RGB colors, enabled rapid and qualitative and quantitative detection of putrescine in shrimp meat, demonstrating the potential of food spoilage detection.

[0022] The working mechanism of the CDs / AuNCs sensor of this invention is as follows: Upon addition of putrescine, CDs aggregate due to hydrogen bonding interactions and imine bond formation, resulting in aggregation-induced emission (AIE) and enhanced fluorescence of CDs. Furthermore, the enhanced internal filtering effect (IFE) from AuNCs to CDs further increases the fluorescence intensity of CDs, thereby enabling fluorescence detection of putrescine. Additionally, based on the above mechanism, the addition of putrescine causes a shift and enhancement of the ultraviolet peak position (n-π* transition peak of C=O) in CDs / AuNCs (from 460 nm to 494 nm), thus achieving colorimetric detection of putrescine.

[0023] Compared with existing technologies, the outstanding advantages of the fluorescent colorimetric dual-mode putrescine sensor based on carbon dot-gold nanoclusters provided by this invention include: (1) The sensor of the present invention has a dual detection mode of ratio fluorescence and colorimetry for putrescine. Compared with a single fluorescence signal or a single detection mode, it can resist background interference well and realize mutual verification of detection results.

[0024] (2) The sensor of the present invention has a fast detection speed and can achieve detection within 20s; in addition, the sensor has high sensitivity and a detection limit as low as 0.44μM.

[0025] (3) In this invention, the CDs / AuNCs sensor undergoes aggregation-induced emission (AIE) after the addition of putrescine, resulting in enhanced fluorescence. In addition, the internal filtering effect (IFE) from AuNCs to CDs is enhanced, further enhancing the fluorescence intensity of CDs. With the help of AIE and IFE, the CDs / AuNCs can detect putrescine fluorescence. The addition of putrescine causes a new ultraviolet absorption peak to appear in CDs / AuNCs, thereby realizing the colorimetric detection of putrescine.

[0026] (4) The sensor of this invention has strong selectivity and anti-interference ability. It does not respond to common interference factors such as potassium ions, sodium ions, calcium ions, magnesium ions, zinc ions, nitrate ions, nitrite ions, sulfate ions, glycine (Gly), alanine (Ala), phenylalanine (Phe), proline (Pro), leucine (Leu), valine (Val), serine (Ser), threonine (Thr), tyrosine (Tyr), tryptophan (Trp), glutamine (Gln), histidine (His), and cysteine ​​(Cys), but has a good response to putrescine. When this sensor is applied to the detection of putrescine in shrimp meat, it is simple to operate, fast, accurate, and highly reliable.

[0027] (5) The sensor of the present invention uses a smartphone for digital analysis, which can achieve higher sensitivity and accuracy compared with visual recognition analysis, thereby reducing errors related to human observation. Attached Figure Description

[0028] Figure 1 The images show the transmission electron microscope (TEM) image of CDs (a), the particle size distribution of 100 CDs in TEM (b), the X-ray diffraction pattern (c), the ultraviolet absorption spectrum (d), the infrared spectrum (e), and the Raman spectrum (f).

[0029] Figure 2 The fluorescence spectrum (a) and ultraviolet spectrum (b) of CDs / AuNCs are shown.

[0030] Figure 3 This describes the optimization of reaction conditions for detecting putrescine using CDs / AuNCs in Example 1. (a) shows the effect of the concentrations of CDs and AuNCs before doping on the results; (b) shows the effect of the doping volume ratio of CDs and AuNCs on the results; and (c) shows the effect of the reaction time of CDs / AuNCs with putrescine on the results.

[0031] Figure 4 The images show the fluorescence spectrum (a), linear correlation graph (b), and CIE colorimetry (c) of putrescine detected by CDs / AuNCs in Example 2, as well as the fluorescence spectrum (d) and ultraviolet spectrum (e) of putrescine detected by AuNCs.

[0032] Figure 5 The UV spectrum (a) and linear correlation diagram (b) of putrescine detected by CDs / AuNCs in Example 3 are shown.

[0033] Figure 6 This refers to the selectivity and anti-interference capability of the CDs / AuNCs fluorescence mode in Application Example 4.

[0034] Figure 7 This refers to the selectivity and anti-interference capability of the CDs / AuNCs colorimetric mode in Application Example 5.

[0035] Figure 8 Infrared spectra of CDs with and without putrescine. Detailed Implementation

[0036] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0037] Example 1 A fluorescent colorimetric dual-mode putrescine sensor based on carbon dot-gold nanoclusters includes the following preparation steps: (1) Synthesis of CDs: 0.2 mmol of 2,4-dihydroxybenzaldehyde was added to 10 mL of ultrapure water and the solution was adjusted to pH 9 with 1 mol / L NaOH. The solution was transferred to a hydrothermal reactor and reacted in an oven at 180 °C for 10 hours, then cooled to room temperature. Larger particles were filtered out using a 0.22 μm filter, and the resulting solution was dialyzed through a 1000 Da dialysis bag for 24 hours. The dialysate was collected and stored at 4 °C with a mass concentration of 1.54 mg / mL.

[0038] CDs were characterized by transmission electron microscopy, X-ray diffraction patterns, XPS spectroscopy, Raman spectroscopy, ultraviolet spectroscopy, and infrared spectroscopy. Figure 1 As shown. Figure 1 In (a), the carbon dots are in the ellipse. Figure 1 In (b), the particle size distribution obtained by transmission electron microscopy shows that the CDs have a particle size of approximately 10.48 nm; the X-ray diffraction pattern ( Figure 1 (c) shows two peaks at 23° and 43°, confirming the crystalline nature of CDs. Compared to the CDs precursor 2,4-dihydroxybenzaldehyde, the UV absorption spectrum of CDs ( Figure 1(d) A broad characteristic absorption band centered at 464 nm was observed, attributed to the n-π* transition, further confirming the synthesis of CDs. Infrared spectrum ( Figure 1 In (e)), CDs are at 3400 cm -1 The broad absorption band in the vicinity is attributed to the stretching vibration of the OH group on the benzene ring. (3000-3150 cm⁻¹) -1 The absorption band in the region corresponds to the CH stretching vibration of the benzene ring, while the 2500-3000 cm⁻¹ region... -1 The vibrations between them are attributed to the CH stretching vibrations of the aldehyde functional groups. (1600 cm⁻¹) -1 The absorption peak at 1100-1340 cm⁻¹ is attributed to the C=O stretching vibration. -1 The region between these is related to CO stretching vibrations. Furthermore, the Raman spectra of CDs (…) Figure 1 (f) at 1366cm -1 1563 cm -1 It shows distinct D-bands and G-bands, which are respectively associated with sp. 2 Hybrid and disordered graphite are related, I D / I G The value is 0.933, indicating a good degree of graphitization.

[0039] (2) Synthesis of AuNCs: 0.5 g of casein was completely dissolved in 10 mL of NaOH solution (0.2 M). Then, 0.5 mL of HAuCl4 solution (100 mM) was added to the previous solution, and the mixture was incubated at 40 °C with vigorous stirring (400 rpm) in the dark for 6 h. Subsequently, the reaction mixture was centrifuged (10000 rpm, 10 min) and filtered through a 0.45 μm membrane filter to remove larger products. Dialysis was performed using a 10000 Da membrane in the dark for 36 h. Finally, the sample was stored at 4 °C for subsequent use.

[0040] (3) Synthesis of CDs / AuNCs: CDs with a concentration of 0.2 v / v (v / v is the volume ratio before and after dilution) and AuNCs with a concentration of 0.05 v / v can be directly and physically mixed to obtain CDs / AuNCs.

[0041] Fluorescence and UV spectra of CDs / AuNCs are as follows: Figure 2As shown in the fluorescence spectrum, CDs / AuNCs exhibit three distinct emission peaks at 430 nm, 525 nm, and 640 nm. The emission peaks at 430 nm and 525 nm are primarily attributed to CDs, while the emission peak at 640 nm is attributable to AuNCs. When excited with 365 nm UV light, CDs / AuNCs show a pale green fluorescence. In the UV spectrum, CDs / AuNCs exhibit strong absorption bands corresponding to the characteristic absorptions of CDs at 464 nm and AuNCs at 274 nm. Importantly, no new absorption peaks appear, indicating that no chemical reaction occurs between CDs and AuNCs. Figure 2 As can be seen in the inner illustration of (a), CDs appear dark green and light brown under ultraviolet and visible light, respectively; AuNCs appear purplish-red and transparent under ultraviolet and visible light, respectively; CDs / AuNCs appear light green and brown under ultraviolet and visible light, respectively.

[0042] Application Example 1 Optimization of reaction conditions for CDs / AuNCs detection of putrescine, such as Figure 3 As shown: Fluorescence detection procedure: Take the CDs / AuNCs aqueous solution from Example 1 and mix it with 0-250μM putrescine aqueous solution at a volume ratio of 1:1. Record the fluorescence spectrum at an excitation wavelength of 370 nm.

[0043] The procedure for colorimetric detection is as follows: Take the CDs / AuNCs aqueous solution from Example 1 and mix it with 0-140μM putrescine aqueous solution at a volume ratio of 1:1. Finally, measure the ultraviolet absorption value at 494 nm.

[0044] Test results as follows Figure 3 As shown, F0 represents the state without the addition of 1 mM putrescine (F 430 +F 525 ), F 430 Represents the fluorescence intensity at a wavelength of 430 nm; I = (F 430 +F 525 ) / F 640 I0 represents the state without the addition of 1 mM putrescine (F). 430 +F 525 ) / F 640Dilution factors of 0.04, 0.12, 0.2, 0.28, and 0.36 were set for CDs. The fluorescence intensity ratio before and after adding putrescine reached its maximum when CDs were diluted to 0.2. Doping volume ratios of 3 / 1, 2 / 1, 1 / 1, 1 / 2, and 1 / 3 were set for CDs and AuNCs. The fluorescence intensity ratio before and after adding putrescine reached its maximum when the doping volume ratio was 1:1. A time gradient of 0-180 seconds was set for the CDs / AuNCs putrescine reaction time. It was found that the fluorescence intensity ratio before and after adding putrescine stabilized at 20 seconds. When detecting actual samples, putrescine solutions of different concentrations of shrimp sample matrix were added, while other operations remained unchanged. The reaction conditions for CDs / AuNCs to detect putrescine were optimized. The optimal conditions were: CDs dilution concentration of 5%; CDs to AuNCs volume ratio of 1:1; and CDs / AuNCs reaction time to putrescine of 20 seconds.

[0045] Application Example 2 Fluorescence spectrum, linear correlation plot, and CIE chromaticity diagram of putrescine detected by CDs / AuNCs, such as Figure 4 As shown in (a)-(c): In Example 1, an aqueous solution of CDs / AuNCs was used in the experiment. In the fluorescence spectrum, the fluorescence intensity of CDs at 430 nm and 525 nm gradually increased with increasing putrescine concentration. However, as an effective internal reference against environmental interference, the fluorescence intensity of AuNCs at 640 nm remained relatively constant. In lg[(F 430 +F 525 ) / F 640 A linear relationship was observed between the concentration range of putrescine and 1-80 μM. The limit of detection (LOD) was calculated to be 0.44 μM according to the formula LOD = 3σ / k (where σ is the standard deviation of 10 blank measurements and k is the slope of the calibration curve). Notably, a distinct fluorescence color change from light green to green and then to cyan was observed to the naked eye, further confirmed by the CIE chromaticity diagram. Mechanistically, the addition of putrescine leads to CD aggregation due to the formation of hydrogen bonds (non-covalent interactions) and imine bonds (covalent interactions, where the aldehyde groups on the surface of CDs react with the amino groups of putrescine in a Schiff base reaction to form imine bonds), resulting in aggregation-induced emission (AIE) and enhanced fluorescence of CDs. Furthermore, the enhanced internal filtration effect (IFE) from AuNCs to CDs further increases the fluorescence intensity of CDs, thus enabling the fluorescence detection of putrescine.

[0046] For comparison, the fluorescence and UV spectra of putrescine detected by AuNCs, such as... Figure 4 As shown in (d)-(e): In Example 1, an aqueous solution of AuNCs was used in the experiment. The fluorescence intensity at 430 nm, 525 nm, and 640 nm remained stable with the addition of putrescine. Furthermore, the addition of putrescine had no effect on the UV spectrum of AuNCs. This indicates that CDs are the core unit for detecting putrescine.

[0047] Application Example 3 UV spectra and linear correlation diagrams for the detection of putrescine by CDs / AuNCs, as follows: Figure 5 As shown: In Example 1, the CDs / AuNCs aqueous solution was used in the experiment. Under visible light, the color of the CDs / AuNCs solution changed significantly from light brown to dark brown with increasing putrescine concentration. In the ultraviolet spectrum, after the addition of putrescine, a distinct absorption peak appeared at 494 nm, and its intensity gradually increased with increasing putrescine level. lg(A 494 The CDs / AuNCs exhibit a good linear relationship with putrescine in the concentration range of 1-90 μM, with a detection limit of 0.72 μM. These test results demonstrate that CDs / AuNCs have a good colorimetric response to putrescine and can characterize changes in putrescine concentration through color changes, making them suitable for detecting food freshness. From a mechanistic perspective, the addition of putrescine leads to the aggregation of CDs, causing a shift and enhancement of the UV peak position (the n-π* transition peak of C=O) in CDs / AuNCs (from 460 nm to 494 nm), thus enabling colorimetric detection of putrescine.

[0048] Figure 5 (a) can also confirm the hydrogen bond interaction. After adding putrescine, the absorption peak at 460 nm red-shifted to 494 nm and the absorbance increased, which proves the existence of the hydrogen bond interaction.

[0049] Application Example 4 Selectivity and anti-interference capability in CDs / AuNCs fluorescence mode: Some of the analytes included putrescine, common ions (KCl, CaCl2, MgCl2, ZnCl2, NaCl, Na2SO4, NaNO2, KNO3, NaHCO3), and amino acids (Gly, Ala, Phe, Pro, Leu, Val, Ser, Thr, Tyr, Trp, Gln, His, Cys), all dissolved in deionized water to obtain solutions of the corresponding concentrations (100 μM). In Example 1, CDs / AuNCs were mixed with the analytes at a 1:1 volume ratio, and after 20 s, the fluorescence spectra were recorded at an excitation wavelength of 370 nm. The results are as follows: Figure 6 As shown. Figure 6As can be seen, the fluorescence intensity of the test groups with the addition of various other substances did not change significantly, and only showed a ratiometric fluorescence response in the presence of putrescine. The experimental results indicate that CDs / AuNCs exhibit good selective response to putrescine, and that the sensor has high chemical stability, showing inertness to a variety of chemical substances.

[0050] Application Example 5 Selectivity and interference resistance in CDs / AuNCs colorimetric modes: Some of the analytes included putrescine, common ions (KCl, CaCl2, MgCl2, ZnCl2, CuCl2, Na2SO4, NaNO2, Zn(NO3)2), and amino acids (Gly, Ala, Phe, Pro, Leu, Val, Ser, Thr, Tyr, Trp, Gln, His, Cys), all dissolved in deionized water to obtain solutions of the corresponding concentrations (100 μM). A mixed solution of the analytes and putrescine was prepared at a 1:1 volume ratio. The CDs / AuNCs from Example 1 were mixed with the mixed solution at a 1:1 volume ratio. After 20 seconds, the UV absorbance at 494 nm was measured. The results are as follows: Figure 7 As shown. Figure 7 As can be seen, adding common interfering substances in the food matrix to the CDs / AuNCs detection system for putrescine does not affect the colorimetric response of CDs / AuNCs to putrescine, indicating that CDs / AuNCs are suitable for use in food environments with complex compositions, and the aforementioned substances will not interfere with the detection signal.

[0051] Application Example 6 Putrescine spiking experiments of CDs / AuNCs and putrescine spiking experiments of CDs / AuNCs using a smartphone platform: To evaluate the sensor's practical applicability, fresh shrimp meat purchased from a local supermarket was selected as the real sample for freshness assessment. The shrimp meat sample (5.0 g) was transferred to a 50 mL centrifuge tube, and 20 mL of 3% (w / v) trichloroacetic acid aqueous solution was added. The mixture was shaken horizontally for 5 minutes, then centrifuged at 10,000 rpm for 10 minutes at 4°C. Subsequently, 20 mL of n-hexane was added to the supernatant, and the mixture was vigorously vortexed for 5 minutes to remove fat. The upper organic phase was discarded. The remaining aqueous phase was filtered through a 0.22 μm membrane filter, and the pH was adjusted to 6 with 1 mol / L sodium hydroxide. The final extract was stored at 4°C.

[0052] The putrescine solution was diluted with shrimp meat extract to prepare a putrescine gradient solution for spiking experiments. CDs / AuNCs from Example 1 were mixed with the mixed solution at a 1:1 volume ratio. After 20 seconds, the fluorescence spectrum was recorded at an excitation wavelength of 370 nm. The basic operation of colorimetric detection was the same as that of fluorescence detection, measuring the ultraviolet absorbance at 494 nm.

[0053] The results are shown in Table 1. The recoveries for ratiometric fluorescence detection ranged from 93.88% to 103.21%, with relative standard deviations (RSDs) between 0.81% and 1.43%. In colorimetric mode, the recoveries ranged from 93.45% to 107.45%, with RSDs between 1.97% and 6.78%. These results indicate that CDs / AuNCs sensors can accurately assess putrescine levels during food spoilage using both ratiometric fluorescence and colorimetric modes, thereby evaluating food freshness.

[0054] Table 1

[0055] To facilitate on-site detection and reduce errors in visual judgment, smartphone-assisted colorimetric fluorescence detection was used. Smartphones captured colorimetric and fluorescence images before and after the addition of putrefaction at a fixed location, then uploaded to Photoshop for RGB value extraction to construct a standard curve and detect the putrefaction concentration in unknown samples. Shrimp meat samples were adulterated with a specified concentration of putrefaction, and subsequently analyzed. In smartphone-assisted colorimetric detection, recoveries ranged from 91.56% to 102.71%, with RSDs ranging from 1.59% to 8.78%. For smartphone-assisted fluorescence detection, recoveries ranged from 98.2% to 106.52%, with corresponding RSDs ranging from 3.48% to 11.8%. These results collectively confirm the feasibility of the proposed sensing strategy for detecting putrefaction in complex real-world samples.

[0056] like Figure 8 When putrescine is added to CDs, the stretching vibration peaks of C=O and OH shift, while the peaks corresponding to CH and CO weaken. This can be attributed to the strong hydrogen bonding interactions between the amino groups on putrescine and the hydroxyl, carboxyl, and aldehyde groups on the carbon dot surface. Furthermore, at 1630 cm⁻¹... -1 The presence of a C=N peak nearby further confirms the formation of an imine bond, indicating that the amino group on putrescine (Put) reacts with the aldehyde group on carbon dots (CDs) to form an imine bond.

Claims

1. A dual-mode fluorescence colorimetric sensor for detecting putrescine in food, characterized in that, The sensor is a dual-mode fluorescent and colorimetric putrescine sensor based on carbon dot-gold nanoclusters, expressed as CDs / AuNCs. It is synthesized via a hydrothermal method using 2,4-dihydroxybenzaldehyde as a precursor, and AuNCs are synthesized via a template etching method. The mixture yields the CDs / AuNCs sensor. The sensor uses carbon dot CDs as the response signal and AuNCs as the internal reference signal, and has both ratiometric fluorescence and colorimetric dual detection modes for putrescine.

2. The method for preparing a dual-mode sensor (CDs / AuNCs) for detecting putrescine in food as described in claim 1, characterized in that, Includes the following steps: (1) Adjust the pH of the aqueous solution of 2,4-dihydroxybenzaldehyde with NaOH, perform hydrothermal reaction, filter and dialyze to obtain CDs; (2) Casein was completely dissolved in NaOH solution, HAuCl4 solution was added to it, and the mixture was heated and incubated in the dark. Then, it was centrifuged, filtered and dialyzed to obtain AuNCs. (3) The prepared CDs and AuNCs are directly physically mixed in proportion to obtain the CDs / AuNCs sensor.

3. The preparation method according to claim 2, characterized in that, In step (1), the pH of the 2,4-dihydroxybenzaldehyde aqueous solution is adjusted to 5-9 with NaOH; the concentration of the 2,4-dihydroxybenzaldehyde aqueous solution is 10-20 mM.

4. The preparation method according to claim 2, characterized in that, In step (1), the hydrothermal reaction temperature is 150-180℃ and the time is 5-10h.

5. The preparation method according to claim 2, characterized in that, In step (2), the casein concentration is 30-50 g / L and the chloroauric acid HAuCl4 solution concentration is 50-100 mM.

6. The preparation method according to claim 2, characterized in that, In step (2), the heating reaction temperature is 40-60℃ and the time is 2-6h.

7. The preparation method according to claim 2, characterized in that, In step (1), the dialysis bag required for filtering CDs is 1000 Da in size, and the dialysis time is 12-24 h; in step (2), the dialysis bag required for filtering AuNCs is 10000 Da in size, and the dialysis time is 12-36 h.

8. The preparation method according to claim 2, characterized in that, In step (3), CDs are diluted to 0.04-0.36 v / v of the original solution, and the diluted CDs are mixed with AuNCs diluted 20 times, i.e., 0.05 v / v, at a volume ratio of 1 / 3-3 / 1 (v / v).

9. The application of the dual-mode fluorescence colorimetric sensor for detecting putrescine in food as described in claim 1, characterized in that, The sensor can detect the putrescine content in food using a smartphone.

10. The application according to claim 9, characterized in that, The aqueous solution of the dual-mode putrescine sensor based on carbon dot-gold nanoclusters is mixed with the aqueous solution of putrescine at a volume ratio of 1:1, stirred evenly, and allowed to stand for 0-180 seconds. The detection results are then observed using a smartphone. The concentration of putrescine in the fluorescence mode is 0-250 μM, and the concentration of putrescine in the colorimetric mode is 0-140 μM.

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