A carbon dot / quantum dot ratio fluorescent probe-based biological amine visual detection method and application

Abstract

This invention discloses a visual detection method and application of biogenic amines based on a carbon dot / quantum dot ratio fluorescent probe, belonging to the field of food safety detection technology. Addressing the problems of existing biogenic amine detection methods, such as complex operation, difficulty in rapid on-site screening, and susceptibility to interference from single fluorescent signals, this invention prepares a ratio fluorescent probe based on graphene carbon dots (G-CDs) and near-infrared quantum dots (QD700). This probe requires no chemical modification and achieves a synergistic response of G-CDs fluorescence enhancement and QD700 fluorescence quenching through the Spe-induced aggregation effect, achieving a detection limit of 0.0097 μM and a response time faster than 2 seconds. Combined with the DEIM intelligent model, it can accurately distinguish spermine, putrescine, cadaverine, histamine, and tyramine. Integrated into a paper-based smartphone platform, it enables visual identification of shrimp freshness (red for fresh, green for spoiled), showing a high correlation with the TVB-N standard method (R²=0.95), suitable for rapid on-site monitoring.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a dual-emission nanoprobe based on green carbon dots and near-infrared quantum dots, its construction method, its application in the detection and identification of biogenic amines, and a portable food freshness assessment system integrating a smartphone and a deep learning model. Background Technology

[0002] Biogenic amines (BAs) are nitrogen-containing organic compounds produced during food spoilage due to the decarboxylation of amino acids. Their content is closely related to the freshness of food, especially seafood, meat, and fermented foods. Among them, histamine (His), cadaverine (Cad), putrescine (Put), tyramine (Tyr), and spermine (Spe) are widely used as spoilage indicators. Traditional detection methods such as high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), while accurate, are expensive, complex to operate, and time-consuming, making them difficult to meet the needs of rapid on-site screening.

[0003] In recent years, fluorescence sensing technology has attracted attention due to its high sensitivity, fast response, and strong visualization potential. However, single fluorescent probes often lack the ability to distinguish between various structurally similar biogenic amines. Ratio fluorescence strategies can effectively eliminate environmental interference and improve detection reliability by monitoring changes in the intensity ratio of two emission peaks. However, existing ratio probes mostly rely on ultraviolet or visible light excitation, are easily affected by sample background fluorescence interference, and lack portable solutions for integration with smart terminals.

[0004] Therefore, there is an urgent need to develop a novel ratio fluorescence sensing system that combines high selectivity, anti-interference, visual output, and intelligent recognition capabilities for rapid on-site identification of biogenic amines and assessment of food freshness. Summary of the Invention

[0005] To address the problems of existing biogenic amine detection methods being complex to operate, difficult to conduct rapid on-site screening, and susceptible to interference from single fluorescence signals, this invention provides a ratiometric fluorescence sensor and sensor array based on G-CDs / QD700, achieving high selectivity and visual differentiation of five typical biogenic amines; and further constructs an integrated platform of "probe-smartphone-deep learning model" for portable, non-destructive, quantitative on-site detection of food freshness.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A ratiometric fluorescent nanoprobe for the detection of biogenic amines, the probe comprising green carbon dots G-CDs and near-infrared quantum dots QD700; the G-CDs emit fluorescence at 500 nm under 360 nm excitation, and the QD700 emits fluorescence at 700 nm under the same excitation; the probe exhibits a synergistic response of fluorescence enhancement at 500 nm and fluorescence quenching at 700 nm when interacting with spermine.

[0008] Furthermore, G-CDs and QD700 were mixed at a volume ratio of 1:1 (3 μL each), for a total volume of 1 mL, to form a nanocomposite system with dual emission peaks (~500 nm and ~700 nm), with an excitation wavelength of 360 nm.

[0009] Furthermore, the G-CDs are prepared from 3-hydroxybenzaldehyde by a solvothermal method, and the QD700 is a CdTe quantum dot.

[0010] Furthermore, G-CDs were prepared by solvothermal reaction of 3-hydroxybenzaldehyde in ethanol at 200°C for 4 hours, and purified by dialysis.

[0011] Furthermore, the preparation of G-CDs was as follows: 0.2 g of 3-hydroxybenzaldehyde was dissolved in 40 mL of anhydrous ethanol and transferred to a 100 mL polytetrafluoroethylene-lined stainless steel high-pressure reactor. The reactor was then subjected to a solvothermal reaction at 200°C for 4 hours. After natural cooling, the mixture was filtered sequentially through medium-speed qualitative filter paper (pore size ~7 μm), centrifuged at 8000 rpm for 15 minutes, filtered through a 0.22 μm syringe, and dialyzed against deionized water for 5 hours using a dialysis bag with a molecular weight cutoff of 500 Da to remove unreacted substances and small molecule byproducts. Finally, the solid G-CDs were freeze-dried and stored at 4°C in the dark.

[0012] Furthermore, the QD700 is dispersed in water and stored after being settled with anhydrous ethanol, washed three times by centrifugation at 8000 rpm.

[0013] Furthermore, the preparation of QD700 involved an aqueous solvothermal method using cadmium chloride (CdCl2·2.5H2O) as the cadmium source, sodium telluride (NaHTe) as the tellurium source, and 3-mercaptopropionic acid (MPA) as the stable ligand. After nucleation at pH ≈ 10, the mixture was reacted in a sealed environment at 160°C for 20 minutes to obtain near-infrared CdTe quantum dots (QD700) emitting a wavelength of approximately 700 nm.

[0014] A method for detecting biogenic amines, employing the aforementioned ratiometric fluorescent nanoprobe, includes the following steps:

[0015] Step 1: Mix the ratiometric fluorescent nanoprobe with the sample to be tested to form a detection system;

[0016] Step 2: Excite the detection system with 360 nm light and measure the fluorescence intensity F at 500 nm and 700 nm. 500 and F 700 ;

[0017] Step 3, based on the F 500 and F 700 Calculate the ratio signal, and determine the presence or concentration of biogenic amines in the sample based on the ratio signal.

[0018] Furthermore, in step S3, the dual ratio parameter F is specifically calculated. 500 / F 700 and / or F (0)500 / F 500 , where F (0)500 The initial fluorescence intensity at 500 nm before sample addition; a response vector is constructed based on the aforementioned dual ratio parameters, and multivariate statistical analysis is used to distinguish at least one of spermine, histamine, cadaverine, putrescine, and tyramine.

[0019] Furthermore, the multivariate statistical analysis includes linear discriminant analysis (LDA), radar chart or heatmap analysis.

[0020] A bioamine intelligent recognition system, comprising:

[0021] The aforementioned ratiometric fluorescent nanoprobes;

[0022] An ultraviolet excitation source is used to excite the probe with 365 nm light;

[0023] An image acquisition module is used to acquire fluorescence images after the probe reacts with the sample;

[0024] The processing module is equipped with a trained deep learning model for extracting color channel ratios from the fluorescence image and outputting the identification results of the types and concentrations of biogenic amines.

[0025] Furthermore, the image acquisition module is a smartphone camera, the processing module is a smartphone with an integrated recognition app, and the deep learning model is a DEIM model based on DETR.

[0026] Visual identification of biogenic amines: Response tests were conducted by setting gradient concentrations for five biogenic amines (Spe, His, Cad, Put, Tyr); two ratio parameters were used: F... 500 / F 700 and F (0)500 / F 500 (where F) (0)500The fluorescence intensity at 500 nm before the addition of biogenic amines was used as the sensing signal; multivariate statistical analysis of the response patterns was performed by linear discriminant analysis (LDA), radar charts and heatmaps to achieve clear differentiation of the five biogenic amines.

[0027] The specific steps are as follows:

[0028] ① Probe solution preparation: Take G-CDs aqueous solution and QD700 aqueous solution (both with a concentration of 1 mM, calculated as Cd), mix them at a volume ratio of 1:1 (e.g., take 3 μL of each) to prepare the probe working solution. This probe exhibits dual emission peaks under 360 nm excitation, located at ~500 nm (green, from G-CDs) and ~700 nm (near-infrared red, from QD700).

[0029] ② Preparation of biogenic amine standard solutions: Prepare a series of standard aqueous solutions for the five target biogenic amines (spermine (Spe), histamine (His), cadaverine (Cad), putrescine (Put), and tyramine (Tyr), with concentrations set according to their physiological relevance:

[0030] Spe: 0, 0.01, 0.02, 0.03, 0.05, 0.07, 0.1, 0.3, 1, 15, 30, 50, 100μM;

[0031] His, Cad, Put, Tyr: 0, 0.03, 0.3, 1, 15, 30, 50, 100μM.

[0032] ③ Fluorescence response test: Take 1 mL of the above G-CDs / QD700 probe working solution into a quartz cuvette and record its initial fluorescence spectrum (excitation wavelength λex = 360 nm). Record the fluorescence intensity at 500 nm as F. (0)500 / F 500 Add 15 μL of the target biogenic amine standard solution to the solution (to achieve the set concentration, such as 15 μM), and record the fluorescence spectrum again. Read the fluorescence intensity at 500 nm and 700 nm, and record them as F1. 500 and F 700 Repeat the above procedure for each biogenic amine at its entire concentration gradient.

[0033] ④ Ratio signal calculation: Calculate two dimensionless ratio parameters for each sample: F500 / F700 and F(0)500 / F500.

[0034] ⑤ Compile the data of the five biogenic amines at the same concentration (e.g., 15 μM) into a 5×2 response matrix, import it into OriginPro 2021 software, and perform linear discriminant analysis (LDA). Project the high-dimensional response into the low-dimensional space to achieve category separation. At the same time, draw radar charts and heatmaps (with the response intensity at all concentrations as the matrix) to intuitively show the unique "fingerprint" response patterns induced by different bio-glues. Verify whether the five biogenic amines are clearly distinguished through clustering results.

[0035] A method for assessing food freshness employs the aforementioned detection method or intelligent recognition system to non-destructively detect biogenic amines on the surface of the food to be tested, wherein a change in fluorescence color from red to green or a decrease in the G / R ratio indicates increased food spoilage.

[0036] Furthermore, the food product is an aquatic product, preferably shrimp; the detection is performed in a portable darkroom, which integrates an ultraviolet light source, a sample stage, and filters.

[0037] A paper-based visual detection device for rapid on-site screening of biogenic amines includes a test paper carrier loaded with the aforementioned ratiometric fluorescent nanoprobes. After the test paper comes into contact with a sample containing biogenic amines and is excited by ultraviolet light, the change in its fluorescence color is used to visually determine the freshness of the food, wherein red indicates freshness and green indicates spoilage.

[0038] A ratiometric fluorescence sensor and sensing array of G-CDs / QD700 obtained by the above construction method.

[0039] The above-mentioned ratio fluorescence sensor and sensor array are used in the detection of biogenic amines in food. Among them, the linear detection range of spermine fluorescence detection is 0~100µM, and the detection limit is 0.0097µM.

[0040] Compared with the prior art, the present invention has the following advantages:

[0041] This invention prepares a ratiometric fluorescent probe based on graphene carbon dots (G-CDs) and near-infrared quantum dots (QD700). This probe exhibits differentiated responses to five typical biogenic amines (spermine, histamine, cadaverine, putrescine, and tyramine). By monitoring the changes in fluorescence intensity at 500 nm and 700 nm, two independent ratio signals can be obtained. The ratio signals show characteristic differences with changes in the type and concentration of different biogenic amines, thereby achieving highly selective identification and semi-quantitative analysis of the target analytes.

[0042] This invention constructs a visual ratiometric fluorescence sensor and sensor array based on G-CDs / QD700, and combines it with smartphone imaging and a portable ultraviolet detection device for rapid on-site screening of biogenic amines in aquatic products. It effectively addresses the problems of existing chromatographic or mass spectrometric methods, such as reliance on expensive instruments, complex operation, and difficulty in on-site deployment. It also overcomes the shortcomings of single fluorescence signals, which are susceptible to environmental interference and prone to misjudgment. This dual-ratio signal strategy significantly improves the reliability and anti-interference capability of detection, providing a highly sensitive, highly specific, and practical new method for intelligent assessment of food freshness. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 The detection response of the G-CDs / QD700 probe to spermine (Spe) is shown in the following diagrams: (a) Fluorescence emission spectra at different Spe concentrations (0~100 μM), with the corresponding UV-excited fluorescence photograph in the lower right inset; (b) Dotted line graph of fluorescence intensity as a function of Spe concentration; (c) Corresponding CIE chromaticity coordinates; (d)~(f) Fluorescence spectra, calibration curves (fluorescence photograph in the upper left inset), and CIE plots for the low concentration range (0~100 nM); (g)~(i) Fluorescence spectra, calibration curves (upper left inset), and CIE plots for the range of 0.1~100 μM.

[0045] Figure 2 The following figures show the detection results of histamine (His) and cadaverine (Cad) by the G-CDs / QD700 probe: (a)~(c) are the fluorescence emission spectra, calibration curves (including insets of UV-excited fluorescence images) and CIE chromaticity diagrams for 0~100μM His, respectively; (d)~(f) are the fluorescence emission spectra, calibration curves (including insets of UV-excited fluorescence images) and CIE chromaticity diagrams for 0~100μM Cad, respectively.

[0046] Figure 3 The following figures show the detection results of putrescine (Put) and tyramine (Tyr) by the G-CDs / QD700 probe: (a)~(c) are the fluorescence emission spectra, calibration curves (including insets of UV-excited fluorescence images) and CIE chromaticity diagrams for 0~100μM Put, respectively; (d)`(f) are the fluorescence emission spectra, calibration curves (including insets of UV-excited fluorescence images) and CIE chromaticity diagrams for 0~100μM Tyr, respectively.

[0047] Figure 4 The following are the anti-interference performance evaluation graphs for the G-CDs / QD700 system: (a)~(b) show the response to 24 potential interfering substances without the addition of Spe, including radial bar graphs, 3D histograms (with fluorescent photographs in the top inset), and parallel coordinate graphs; (c)~(d) show the response results to the same interfering substances in the presence of 15 μM Spe.

[0048] Figure 5 The identification results for five biogenic amines (Spe, His, Cad, Put, Tyr) are shown in the following figures: (a) fluorescence emission spectrum; (b) fingerprint response map composed of dual ratio parameters; (c) linear discriminant analysis (LDA) score map; and (d) classification confusion matrix.

[0049] Figure 6 Portable detection device and shrimp freshness assessment diagram: (a) Overall appearance of the device; (b) Exploded view of the structure; (c) Schematic diagram of paper-based visualization detection of Spe; (d) Standard calibration curve of G / R ratio against Spe concentration (0~120 μM); (e) Photographs of fluorescence color changes of shrimp samples stored at 25℃, 4℃ and -20℃ for 0~24 h; (f) Total volatile basic nitrogen (TVBN) content at corresponding time points; (g) Linear relationship between G / R ratio and TVBN content; (h) Linear relationship between G / R ratio and storage time. Detailed Implementation

[0050] To gain a deeper understanding of this invention, we will provide a comprehensive and detailed description. However, this invention has various implementations and is not limited to the specific examples listed herein. These examples are presented to enhance a full understanding of the disclosure of this invention.

[0051] Example 1

[0052] Preparation of G-CDs:

[0053] Preparation of HOF-101 precursor solution:

[0054] 0.2 g of 3-hydroxybenzaldehyde was dissolved in 40 mL of anhydrous ethanol and transferred to a 100 mL high-pressure reactor lined with PTFE and stainless steel. The reactor was sealed and subjected to a solvothermal reaction at 200 °C for 4 h. After natural cooling to room temperature, the reaction solution was filtered through medium-speed qualitative filter paper (pore size ~7 μm) and then centrifuged at 8000 rpm for 15 min to remove insoluble matter. The supernatant was filtered through a 0.22 μm syringe filter membrane and placed in a dialysis bag with a molecular weight cutoff of 500 Da. Dialysis was performed with deionized water for 5 h to remove unreacted small molecules. The dialysate was freeze-dried to obtain a brownish-yellow solid, G-CDs, which was stored at 4 °C protected from light. A 1 mM stock solution was prepared with deionized water before use.

[0055] Example 2

[0056] Preparation of QD700:

[0057] Weigh 0.2284 g of CdCl2·2.5H2O (1.0 mmol) and 210 μL of 3-mercaptopropionic acid (MPA, ~2.0 mmol), dissolve in 100 mL of deionized water, and adjust the pH to 10.0 with 1.0 M NaOH while stirring at 60 °C. Separately, mix 0.3062 g of tellurium powder (2.4 mmol) with 24 mL of water, add 0.35 g of NaBH4 at 40 °C, and react until the solution turns clear purple to obtain NaHTe stock solution. Quickly inject 5 mL of fresh NaHTe solution into the above cadmium precursor (Cd:Te:MPA = 1:2.4:2), and the solution immediately turns orange. Transfer to an autoclave and react at 160 °C for 20 min to obtain CdTe quantum dots (QD700) with an emission peak at 700 nm. The product was precipitated in anhydrous ethanol for 12 h, centrifuged at 8000 rpm for 10 min, washed three times, and finally dispersed in water at a concentration of 1 mM (Cd). It was stored at room temperature in the dark.

[0058] Example 3

[0059] Construction and characterization of G-CDs / QD700 ratiometric fluorescent probes:

[0060] Take 3 μL each of G-CDs and QD700 stock solutions, add water to a final volume of 1 mL, and mix well to obtain the G-CDs / QD700 probe working solution. Figure 1 As shown, TEM revealed that G-CDs were uniform nanoparticles (~3 nm), QD700 had a size of about 5 nm, and there was no obvious aggregation after compositing; EEM spectroscopy confirmed the dual emission characteristics (500 nm / 700 nm); UV-Vis, XPS and FTIR showed that the two coexisted successfully and the surface functional groups were well preserved.

[0061] Example 4

[0062] Detection and identification of spermine and four biogenic amines:

[0063] A 1 mL detection system was prepared, containing 6 μL of G-CDs / QD700 probe solution and the target biogenic amine. Spermine (Spe), histamine (His), cadaverine (Cad), putrescine (Put), or tyramine (Tyr) were added to a final concentration of 15 μM, and their fluorescence emission spectra were measured. The results showed that only in the presence of spermine did fluorescence significantly increase at 500 nm, while fluorescence was significantly quenched at 700 nm, forming a unique dual-signal change pattern. The other four biogenic amines had weak effects on the emission intensity at 500 nm and 700 nm, and exhibited distinguishable cross-response characteristics. Based on the ratio parameter F... 500 / F 700 and F (0)500 / F 500 The constructed response vectors can effectively distinguish five biogenic amines, among which spermine is specifically identified due to a significant fluorescence enhancement-quenching synergistic effect, while the other four are classified through differences in cross-response. Five standard working curves were constructed, Spe1: Y = 0.12 + 1.54X (0–0.1 µM, R 2 =0.99), Spe2: Y=0.29+0.04X (0.1-100 µM, R 2 =0.99)His (0-100 µM) Y=0.11+0.0016X (R 2 =0.98), Cad (0-50µM) Y=0.14+0.0081X (R 2 =0.99), Put(0-100 µM) Y=0.12+0.0037X (R 2 =0.99), Tyr (0-100 µM) Y=0.12+0.0021X (R 2 =0.99)

[0064] Example 5

[0065] Distinguishing Anti-interference Performance from Multi-component Mixtures:

[0066] In the presence of 15 μM interfering substances (including 24 types such as metal ions and amino acids), the probe response to 15 μM Spe showed no significant change. Further detection of five biogenic amines revealed that each mixture exhibited a unique pattern in radar, LDA, and heatmaps, allowing for effective differentiation.

[0067] Example 6

[0068] Smartphone-assisted intelligent recognition system:

[0069] Fluorescence images of the probe reacting with different concentrations of Spe (0–100 μM) were captured using a smartphone under a 365 nm UV lamp. The G / R ratio was calculated by extracting RGB values ​​using Color Grab. A dataset of 980 images was constructed, the DEIM model was trained, and integrated into a mobile app. It can quantify and distinguish five biogenic amines with an accuracy exceeding 93%.

[0070] Example 7

[0071] Portable devices for on-site testing of shrimp freshness:

[0072] A self-made portable UV imaging darkroom was constructed, incorporating a 365 / 302 nm dual-wavelength lamp, an adjustable sample stage, and filters. Fresh shrimp were placed in the apparatus, and fluorescence images were captured at 0, 4, 8, 12, and 24 hours. TVBN content was simultaneously measured. The results showed that the G / R ratio decreased linearly with storage time and was highly correlated with TVBN (RG). 2 = 0.95), enabling non-destructive quantitative assessment of freshness.

[0073] Contents not described in detail in this specification are prior art known to those skilled in the art. Although illustrative specific embodiments of the invention have been described above to facilitate understanding by those skilled in the art, it should be understood that the invention is not limited to the scope of the specific embodiments. Various modifications are readily apparent to those skilled in the art as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of this invention are protected.

Claims

1. A ratiometric fluorescent nanoprobe for the detection of biogenic amines, characterized in that, The probe comprises green carbon dots (G-CDs) and near-infrared quantum dots (QD700); the G-CDs emit fluorescence at 500 nm under 360 nm excitation, and the QD700 emits fluorescence at 700 nm under the same excitation; when the probe interacts with spermine, it exhibits a synergistic response of fluorescence enhancement at 500 nm and fluorescence quenching at 700 nm. ‌ 2. The ratiometric fluorescent nanoprobe according to claim 1, characterized in that, The G-CDs were prepared from 3-hydroxybenzaldehyde via a solvothermal method, and the QD700 was a CdTe quantum dot.

3. A method for detecting biogenic amines, characterized in that, The method of using the ratiometric fluorescent nanoprobe according to claim 1 or 2 includes the following steps: Step 1: Mix the ratiometric fluorescent nanoprobe with the sample to be tested to form a detection system; Step 2: Excite the detection system with 360 nm light and measure the fluorescence intensity F at 500 nm and 700 nm. 500 and F 700 ; Step 3, based on the F 500 and F 700 Calculate the ratio signal, and determine the presence or concentration of biogenic amines in the sample based on the ratio signal.

4. The detection method according to claim 3, characterized in that, In step S3, the dual ratio parameter F is specifically calculated. 500 / F 700 and / or F (0)500 / F 500 , where F (0)500 The initial fluorescence intensity at 500 nm before sample addition; A response vector is constructed based on the aforementioned dual ratio parameters, and at least one of spermine, histamine, cadaverine, putrescine, and tyramine is distinguished through multivariate statistical analysis.

5. The detection method according to claim 4, characterized in that, The multivariate statistical analysis includes linear discriminant analysis, radar chart or heatmap analysis.

6. A bioamine intelligent identification system, characterized in that, include: ‌ The ratiometric fluorescent nanoprobe according to any one of claims 1 or 2; ‌ An ultraviolet excitation source is used to excite the probe with 365 nm light; ‌ The image acquisition module is used to acquire fluorescence images after the probe reacts with the sample; ‌ The processing module is equipped with a trained deep learning model for extracting color channel ratios from the fluorescence image and outputting the identification results of the types and concentrations of biogenic amines. ‌ 7. The bioamine intelligent recognition system according to claim 6, characterized in that, The image acquisition module is a smartphone camera, and the processing module is a smartphone with an integrated recognition app; the deep learning model is a DEIM model based on DETR.

8. A method for assessing the freshness of food, characterized in that, Using the detection method of any one of claims 3 to 5 or the intelligent identification system of any one of claims 6 to 7, non-destructive detection of biogenic amines on the surface of the food to be tested is performed, wherein a change in fluorescence color from red to green or a decrease in the G / R ratio indicates an increase in the degree of food spoilage. ‌ 9. The food freshness assessment method according to claim 8, characterized in that, The food is aquatic product; the detection is carried out in a portable darkroom, which integrates an ultraviolet light source, a sample stage and a filter. ‌ 10. A paper-based visual detection device for rapid on-site screening of biogenic amines, characterized in that, A test paper carrier comprising a ratiometric fluorescent nanoprobe as described in claim 1 or 2, wherein the test paper, after contacting a sample containing biogenic amines and being excited by ultraviolet light, exhibits a change in fluorescence color that is used to visually determine the freshness of food, wherein red indicates freshness and green indicates spoilage.

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