A novel bifunctional tyramide red light carbon dot and a preparation method and application thereof

Abstract

This invention discloses a novel bifunctional tyramine red-light carbon dot, its preparation method, and its applications. The novel bifunctional tyramine red-light carbon dot is a red-light carbon dot (Tyr-CDs) with tyramine structural residues on its surface. It is prepared using tyramine and o-phenylenediamine as precursors via a one-step hydrothermal method. The novel bifunctional tyramine red-light carbon dot possesses signal amplification and detection functions as well as cell fluorescence labeling functions. This carbon dot exhibits excellent luminescence performance, good stability, low preparation cost, a simple preparation method, and low biotoxicity. It can be combined with ELISA and TSA technologies to establish highly sensitive and low-cost immunofluorescence quantitative detection methods, and is expected to serve as a novel fluorescent probe to replace traditional fluorescent dyes. Furthermore, based on its excellent signal amplification and labeling functions, it can achieve ultra-high sensitivity fluorescence measurement of single yeast cells, providing new signal amplification reagents for biological techniques such as ELISA, cell fluorescence labeling, and immunohistochemistry, and has high application value in the field of biosensing.

Description

Technical Field

[0001] This invention relates to the field of fluorescent materials technology, and in particular to a novel bifunctional tyramine red carbon dot, its preparation method, and its application. Background Technology

[0002] The core technology system for biomolecular detection and imaging, with target identification, signal modulation, and result output as its core logic, aims to achieve accurate identification, quantitative analysis, and dynamic tracking of biomolecules (such as proteins, nucleic acids, and small molecule biomarkers). Signal modulation is the key link connecting target identification and result output, directly determining the sensitivity, accuracy, and applicability of the technology. Enzyme-linked immunosorbent assay (ELISA), fluorescent labeling imaging technology, and tyramine signal amplification (TSA) are all core supporting technologies in this process, collectively forming a closed-loop technology system of quantitative detection, dynamic imaging, and signal enhancement.

[0003] Enzyme-linked immunosorbent assay (ELISA) is a biochemical detection technique based on the combination of antigen-antibody specific reaction and enzyme catalysis, widely used in clinical diagnosis, food safety, and other fields. However, its application has significant limitations: First, its signal amplification capability is limited, and its adaptability to high-sensitivity detection scenarios is insufficient. Traditional ELISA relies on the signal amplification mechanism of enzyme-catalyzed substrate color development. For low-abundance targets such as early disease biomarkers, the signal amplification effect is weak, easily leading to missed diagnoses due to the inability to capture trace signals. Second, there are shortcomings in operating costs and detection accuracy. Special labeling strategies used to improve sensitivity require multiple steps and have high reagent costs, which increases the risk of experimental errors and limits large-scale screening applications. Third, when dealing with complex samples such as serum, non-specific binding is prone to occur, leading to false positive or false negative results.

[0004] Fluorescent labeling imaging technology is a core tool in life science research, enabling the visualization and tracking of biomolecules and cell imaging by binding fluorescent labels to target molecules. However, it suffers from significant technical limitations: fluorescent labels have poor selectivity and low signal-to-noise ratios; commonly used organic fluorescent dyes and other labels experience significant background interference and are prone to photobleaching under illumination, failing to meet the requirements for long-term dynamic observation of intracellular biomolecules; some labels are biotoxic, interfering with cellular physiological activities or even causing cell death, making them unsuitable for research with high biosafety requirements.

[0005] Tyramine signal amplification (TSA) is an enzyme-mediated signal amplification technique. TSA utilizes horseradish peroxidase (HRP) to catalyze the signal-carrying tyramine, demonstrating significant advantages in the highly sensitive detection of low-abundance targets and has become an important tool in enzyme-linked immunosorbent assays (ELISA). Fluorescence-based TSA technology is the most widely used. Currently, most fluorescent TSA techniques pre-label tyramine molecules with organic fluorescent dyes or fluorescent proteins; however, the labeling process is cumbersome, reagent costs are high, and the stability of the fluorescent substances used is limited. Therefore, it is necessary to develop a TSA technique that is simple to label, inexpensive, and uses stable fluorescent substances.

[0006] Carbon dots (CDs) are a novel type of carbon nanomaterial with excellent biocompatibility, outstanding luminescent properties, and the ability to retain some of the precursor structure. These superior properties have led to their widespread application in fields such as bioimaging, biosensing, and medical diagnosis. CDs can be divided into long-wavelength emitting carbon CDs and short-wavelength emitting CDs. In addition to the general luminescent properties of CDs, long-wavelength emitting CDs, compared to short-wavelength emitting CDs, also possess tunable luminescent properties, good photostability, and biocompatibility. In particular, they effectively avoid biological autofluorescence interference and exhibit higher fluorescence imaging contrast. Therefore, many researchers have sought to obtain long-wavelength emitting carbon dots by altering the precursor, changing reaction conditions, or performing post-modification.

[0007] Therefore, it is necessary to provide a novel carbon dot with excellent photostability and high selectivity, and simultaneously overcome the technical bottlenecks of insufficient ELISA signal amplification, large background interference in fluorescent labeling imaging technology, and cumbersome TSA labeling, so as to provide core material support for the field of biomolecular detection and imaging. Summary of the Invention

[0008] This invention provides a tyramine-derived carbon dot, its preparation method, and its application to solve the above-mentioned problems.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] This invention provides a novel bifunctional tyramine red light carbon dot, which is a red light carbon dot Tyr-CDs with tyramine structural residues on its surface. The novel bifunctional tyramine red light carbon dot is prepared by a one-step hydrothermal method using tyramine and o-phenylenediamine as precursors. The novel bifunctional tyramine red light carbon dot has signal amplification and detection functions as well as cell fluorescence labeling functions.

[0011] Another aspect of the present invention provides a method for preparing the novel bifunctional tyramine red carbon dots, comprising the following steps:

[0012] Tyramine and o-phenylenediamine were weighed and mixed, and a mixture of H2O and DMF solvent and H2SO4 were added. After tyramine and o-phenylenediamine were completely dissolved, they were transferred to a reaction vessel for reaction. After the reaction was completed, the product was taken out, cooled, centrifuged, dialyzed, filtered, concentrated and freeze-dried to obtain a novel bifunctional tyramine red carbon dots Tyr-CDs.

[0013] The prepared Tyr-CDs were dispersed in a solvent and set aside for later use.

[0014] Another aspect of this invention provides the application of the novel bifunctional tyramine red carbon dots in signal amplification and detection. Using BSA as a model protein, scanning electron microscopy, fluorescence spectroscopy, and non-tyramine-derived carbon dot controls demonstrated that Tyr-CDs possess signal amplification capabilities similar to tyramine. Under optimal reaction conditions, the linear detection range of Tyr-CDs for BSA is 1.0–1.0 × 10⁻⁶. 6 The detection limit was 0.57 ng / mL.

[0015] Another aspect of the present invention provides the application of the novel bifunctional tyramine red carbon dots in enzyme-linked immunosorbent assay (ELISA).

[0016] Furthermore, quantitative immunofluorescence detection was performed using a combination of TSA and ELISA technologies. The specific detection method included the following steps:

[0017] S1: Mix horseradish peroxidase (HRP) with the antibody to be detected to obtain an HRP-labeled detection antibody;

[0018] S2: Co-incubate the standard solution of the antibody to be detected with the HRP-labeled detection antibody. After washing, add Tyr-CDs and H2O2 for further incubation. After incubation, discard the washing liquid, add DMF solvent, and then perform immunofluorescence quantitative detection. This protocol, based on ELISA and TSA technologies, establishes a highly sensitive and accurate immunofluorescence detection method for the accurate quantification of human serum hIgG, with a detection linear range of 1 ~ 1 × 10⁻⁶. 5 The detection limit is as low as 0.43 ng / mL. This method has good accuracy; compared with the traditional TMB colorimetric method, the relative error is within ±10%, and the relative standard deviation is less than 2.0%.

[0019] Another aspect of this invention provides the application of the novel bifunctional tyramine red carbon dots described above in cell fluorescence labeling. Using TSA technology, Tyr-CDs are catalytically deposited on yeast cells. Fluorescence microscopy reveals that the yeast cells appear bright red under 480 nm excitation light and retain their intact morphology. Based on the excellent signal amplification and labeling function of Tyr-CDs, ultra-high sensitivity fluorescence measurement of single yeast cells is achieved, with a dynamic detection range of 1 ~ 1 × 10⁻⁶. 7 cells / mL. Experimental results show that the novel bifunctional Tyr-CDs have certain application potential in ELISA and single-cell detection.

[0020] The beneficial effects of this invention are:

[0021] The novel bifunctional tyramine red carbon dots (Tyr-CDs) disclosed in this invention are prepared by a one-step solvothermal method using tyramine and o-phenylenediamine as precursors. They possess both tyramine signal amplification and detection functions and cell fluorescence labeling dual core functions, and have multiple performance advantages and significant application value.

[0022] (1) Significant advantages in preparation and performance: The preparation method is simple and low in cost, and it is easy to scale up and promote. At the same time, Tyr-CDs have excellent luminescence performance, strong stability and low biotoxicity, which solves the problems of complex preparation, high toxicity and poor stability of traditional fluorescent materials, and lays the material foundation for subsequent applications.

[0023] (2) High efficiency in detection and labeling: On the one hand, Tyr-CDs can be combined with ELISA and TSA technologies to construct a highly sensitive and low-cost immunofluorescence quantitative detection method, effectively improving the detection sensitivity of low-abundance targets; on the other hand, based on its excellent signal amplification and labeling functions, it can achieve ultra-sensitive fluorescence measurement of single yeast cells, breaking through the limitations of traditional fluorescence labeling technology in micro-detection and single-cell imaging.

[0024] (3) Wide application value and field adaptability: As a novel fluorescent probe, Tyr-CDs can replace traditional fluorescent dyes and provide novel signal amplification reagents for biological technologies such as ELISA, cell fluorescence labeling, and immunohistochemistry, showing high application potential and promotion value in the field of biosensing. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 These are quantum yield diagrams of Tyr-CDs prepared under different conditions in Examples 1-12;

[0027] Figure 2 These are transmission electron microscopy (TEM) images and particle size distribution diagrams of Tyr-CDs; among them, Figure 2 A is a TEM image of Tyr-CDs. Figure 2 B is the particle size distribution diagram of Tyr-CDs;

[0028] Figure 3 This is the infrared spectrum of Tyr-CDs;

[0029] Figure 4 These are the UV-Vis spectra of four Tyr-CDs with high quantum yields in the wavelength range of 200–800 nm. c (Tyr-RCDs) = 1 mg / mL, solvent: DMF);

[0030] Figure 5 This is the fluorescence spectrum of Tyr-CDs, where, Figure 5 A is the 3D fluorescence spectrum of Tyr-CDs. Figure 5 B represents the two-dimensional fluorescence spectrum of Tyr-CDs ( c (Tyr-RCDs) = 1 mg / mL, solvent: DMF);

[0031] Figure 6 This is a graph showing the effect of different excitation light intensities and illumination times on the fluorescence intensity of Tyr-CDs. c (Tyr-RCDs) = 1 mg / mL, solvent: DMF);

[0032] Figure 7 This is a graph showing the effect of temperature on the fluorescence intensity of Tyr-CDs. c (Tyr-RCDs) = 1 mg / mL, solvent: DMF);

[0033] Figure 8 This is a graph showing the effect of acidity on the fluorescence intensity of Tyr-CDs. c (Tyr-RCDs) = 1 mg / mL, solvent: DMF);

[0034] Figure 9 This is a graph showing the effect of NaCl concentration on the fluorescence intensity of Tyr-CDs. c (Tyr-RCDs) = 1 mg / mL, solvent: DMF);

[0035] Figure 10 This is a graph showing the effect of inorganic ions on the fluorescence intensity of Tyr-CDs. c(Tyr-RCDs) = 1 mg / mL c (metalions) = 5 × 10 -4 M, c (anion) = 5 × 10 -4 M);

[0036] Figure 11 This is a graph showing the effect of different biomolecules on the fluorescence intensity of Tyr-CDs. c (Tyr-RCDs) = 1 mg / mL c (metal ions) = 1 × 10 -3 M, c (base) = 1 × 10 -3 mg / mL);

[0037] Figure 12 These are the fluorescence spectra and fluorescence decay curves of Tyr-CDs-BSA with H2O2 added at different time intervals. Figure 12 A is the fluorescence spectrum of Tyr-CDs-BSA, where... Figure 12 B is the fluorescence decay curve of Tyr-CDs-BSA;

[0038] Figure 13 The images show the fluorescence excitation and emission spectra of Tyr-CDs-BSA with different concentrations of H2O2 added (the curve on the left is the excitation spectrum, and the curve on the right is the emission spectrum).

[0039] Figure 14 This is a graph showing the effect of HRP concentration on the fluorescence spectrum of Tyr-CDs-BSA in the TSA group;

[0040] Figure 15 These are fluorescence spectra of Tyr-CDs-BSA at different concentrations of Tyr-CDs and a comparison of fluorescence signals between the TSA group and the co-incubation group. Figure 15 A represents the fluorescence spectra of Tyr-CDs-BSA obtained under different concentrations of Tyr-CDs. Figure 15 B is a comparison of the fluorescence signals of Tyr-CDs-BSA in the co-incubation groups under different concentrations of Tyr-CDs;

[0041] Figure 16 This is a graph showing the fluorescence spectra of Tyr-CDs-BSA at different concentrations of BSA and the relationship between fluorescence intensity and BSA concentration. Figure 16 A represents the fluorescence spectra of Tyr-CDs-BSA at different concentrations of BSA. Figure 16 B is a graph showing the relationship between the fluorescence intensity of Tyr-CDs-BSA and the concentration of BSA.

[0042] Figure 17 This is a graph showing the trend of Tyr-CDs-hIgG fluorescence intensity changes at different Tyr-CDs concentrations;

[0043] Figure 18 These are the fluorescence spectra and magnification diagrams of Tyr-CDs-hIgG under different concentrations of H2O2, among which... Figure 18 A represents the fluorescence spectra of Tyr-CDs-hIgG under different concentrations of H2O2. Figure 18 B is a magnification diagram of the fluorescence signal of Tyr-CDs-hIgG under different concentrations of H2O2;

[0044] Figure 19 These are the fluorescence spectra and magnification images of Tyr-CDs-hIgG obtained at different TSA reaction times. Figure 19 A represents the fluorescence spectra of Tyr-CDs-hIgG at different time points. Figure 19 B shows the magnification of the fluorescence signal of Tyr-CDs-hIgG at different time points;

[0045] Figure 20 The graph shows the fluorescence spectra of Tyr-CDs-hIgG with different concentrations of hIgG added, and the linear relationship between the fluorescence intensity of Tyr-CDs-hIgG and the concentration of hIgG.

[0046] Figure 21 These are fluorescence micrographs (bright field and dark field) of yeast cells in the control group and the TSA group. Figure 21 A is the bright-field fluorescence microscopy image of the blank group. Figure 21 B is the fluorescence microscopy dark-field image of the blank group. Figure 21 C is the bright-field fluorescence microscopy image of the TSA group. Figure 21 D is the fluorescence microscopy dark-field image of the TSA group;

[0047] Figure 22 These are fluorescence spectra of yeast cells incubated for different times. Figure 22 Group A is the co-incubation group. Figure 22 B is the co-incubation group (washed out). Figure 22 C is the TSA group. Figure 22 D represents the TSA group (elution). Figure 22 E is the magnification factor;

[0048] Figure 23 These are fluorescence spectra of Tyr-CDs-labeled yeast at different concentrations and curves showing the relationship between fluorescence intensity and yeast concentration. Figure 23 A is the fluorescence spectrum. Figure 23 B is a graph showing the relationship between fluorescence intensity and yeast concentration;

[0049] Figure 24 These are cell counting chamber images of yeast cells at different concentrations observed under a microscope. Figure 24 A is a cell counting plate image with 1 cell / mL. Figure 24 B is 1 × 10 2 Cell counting plate image (cells / mL) Figure 24 C is 1 × 10 4 Cell counting plate image (cells / mL) Figure 24 D is 1 × 10 6 Cell counting plate image (cells / mL). Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] All reagents used in this embodiment were commercially available.

[0052] Example 1:

[0053] A novel bifunctional tyramine red light carbon dot was prepared using the following method:

[0054] Accurately weigh 0.1370 g of tyramine and 0.1080 g of o-phenylenediamine into a 25 mL beaker (molar ratio 1:1), add 10 mL of a mixed solvent of H2O and DMF (volume ratio of H2O to DMF 3:7) and 1 mL of H2SO4 (0.1 M), and after complete dissolution, transfer to a polytetrafluoroethylene reactor and react at 200 °C for 12 h. After the reaction is complete, cool to room temperature and centrifuge at 8000 rpm for 20 min. Dialyze the supernatant through a dialysis bag with a molecular weight cutoff of 1000 Da for 12 h (changing the water every 2 h) until no fluorescence is observed in the external solution of the dialysis bag. Filter the dialysate through a 0.22 μm microporous membrane, concentrate it in an oven at 50 °C, and freeze-dry to obtain a novel bifunctional tyramine red-light carbon, designated Tyr-CDs-3. Disperse the CDs in a solvent and store in a sealed refrigerator.

[0055] Example 2:

[0056] The only difference between this embodiment and Embodiment 1 is that in this embodiment, the molar ratio of tyramine to o-phenylenediamine is 2:1, and everything else is the same as in Embodiment 1.

[0057] Example 3:

[0058] The only difference between this embodiment and Embodiment 1 is that in this embodiment, the molar ratio of tyramine to o-phenylenediamine is 3:2, and everything else is the same as in Embodiment 1.

[0059] Example 4:

[0060] The only difference between this embodiment and Embodiment 1 is that in this embodiment, the molar ratio of tyramine to o-phenylenediamine is 2:3, and everything else is the same as in Embodiment 1.

[0061] Example 5:

[0062] The only difference between this embodiment and Embodiment 1 is that in this embodiment, the molar ratio of tyramine to o-phenylenediamine is 1:2, and everything else is the same as in Embodiment 1.

[0063] Example 6:

[0064] The only difference between this embodiment and Embodiment 1 is that the concentration of H2SO4 in the reaction system is 0.001M in this embodiment, while the rest is the same as in Embodiment 1.

[0065] Example 7:

[0066] The only difference between this embodiment and Embodiment 1 is that the concentration of H2SO4 in the reaction system is 0.01M in this embodiment, otherwise it is the same as Embodiment 1.

[0067] Example 8:

[0068] The only difference between this embodiment and Example 1 is that the concentration of H2SO4 in the reaction system is 0.18M in this embodiment, otherwise it is the same as Example 1.

[0069] Example 9:

[0070] The only difference between this embodiment and Embodiment 1 is that the reaction time in this embodiment is 4 hours; otherwise, it is the same as in Embodiment 1.

[0071] Example 10:

[0072] The only difference between this embodiment and Embodiment 1 is that the reaction time in this embodiment is 8 hours; otherwise, they are the same as in Embodiment 1.

[0073] Example 11:

[0074] The only difference between this embodiment and Embodiment 1 is that the reaction time in this embodiment is 24 hours; otherwise, they are the same as in Embodiment 1.

[0075] Example 12:

[0076] The only difference between this embodiment and Embodiment 1 is that the reaction time in this embodiment is 36 hours; otherwise, they are the same as in Embodiment 1.

[0077] For the above embodiments 1-12, as follows Figure 1 As shown, the molar ratio of tyramine to o-phenylenediamine ( n Tyramine: n When the reaction ratio of OPD is 1:1, the H2SO4 concentration is 0.1 M, the reaction temperature is 200 °C, and the reaction time is 12 h (Example 1), the Tyr-CDs obtained have the longest emission wavelength of 580 nm and the highest quantum yield of 41.78%.

[0078] Comparative Example 1:

[0079] The only difference between this embodiment and Embodiment 1 is that, in this comparative example, the volume ratio of H2O to DMF is 0:1, that is, no H2O is added. Otherwise, it is the same as Embodiment 1, and the carbon dots obtained are denoted as Tyr-CDs-DMF.

[0080] Comparative Example 2:

[0081] The only difference between this embodiment and Example 1 is that, in this comparative example, the volume ratio of H2O to DMF is 1:9, and everything else is the same as in Example 1. The carbon dots obtained are denoted as Tyr-CDs-1.

[0082] Comparative Example 3:

[0083] The only difference between this embodiment and Example 1 is that, in this comparative example, the volume ratio of H2O to DMF is 2:8, while the rest is the same as in Example 1. The carbon dots obtained are denoted as Tyr-CDs-2.

[0084] Comparative Example 4:

[0085] The only difference between this embodiment and Example 1 is that, in this comparative example, the volume ratio of H2O to DMF is 4:6, and everything else is the same as in Example 1. The carbon dots obtained are denoted as Tyr-CDs-4.

[0086] Comparative Example 5:

[0087] The only difference between this embodiment and Example 1 is that, in this comparative example, the volume ratio of H2O to DMF is 5:5, while the rest is the same as in Example 1. The carbon dots obtained are denoted as Tyr-CDs-5.

[0088] Comparative Example 6:

[0089] The only difference between this embodiment and Example 1 is that, in this comparative example, the volume ratio of H2O to DMF is 6:4, while the rest is the same as in Example 1. The carbon dots obtained are denoted as Tyr-CDs-6.

[0090] Comparative Example 7:

[0091] The only difference between this embodiment and Example 1 is that, in this comparative example, the volume ratio of H2O to DMF is 7:3, while the rest is the same as in Example 1. The carbon dots obtained are denoted as Tyr-CDs-7.

[0092] Comparative Example 8:

[0093] The only difference between this embodiment and Example 1 is that, in this comparative example, the volume ratio of H2O to DMF is 8:2, while the rest is the same as in Example 1. The carbon dots obtained are denoted as Tyr-CDs-8.

[0094] Comparative Example 9:

[0095] The only difference between this embodiment and Example 1 is that, in this comparative example, the volume ratio of H2O to DMF is 9:1, while the rest is the same as in Example 1. The carbon dots obtained are denoted as Tyr-CDs-9.

[0096] Comparative Example 10:

[0097] The only difference between this embodiment and Embodiment 1 is that, in this comparative example, the volume ratio of H2O to DMF is 1:0, while the rest is the same as in Embodiment 1, i.e., no DMF is added, and the resulting carbon dots are denoted as Tyr-CDs-H2O.

[0098] The luminescence properties and quantum yield results of Tyr-CDs prepared by Example 1 and Comparative Examples 1-10 with different H2O and DMF solvent ratios are listed in Table 2.

[0099] Table 1. Luminescent properties and quantum yield of CDs prepared with different H2O and DMF solvent ratios

[0100]

[0101] As shown in Table 2, in a single reaction solvent, the excitation and emission wavelengths of Tyr-CDs are 480 nm / 580 nm and 370 nm / 470 nm, respectively, with relative quantum yields of 40.33% and 17.53%. In a mixed reaction solvent, Tyr-CDs-1 has the highest relative quantum yield of 64.21%; Tyr-CDs-2 and Tyr-CDs-3 have the largest emission wavelengths of 605 nm; Tyr-CDs-3 has the largest excitation wavelength of 525 nm. As the proportion of H2O in the solvent increases, the emission wavelength of CDs blue-shifts to 590 nm, and the relative quantum yield decreases to 22.28%. Among these, the optimal ratio (Example 1) has the largest excitation and emission wavelengths when the volume ratio of H2O to DMF is 3:7.

[0102] I. Characterization studies of Tyr-CDs-3 prepared in Example 1:

[0103] 1. Transmission electron microscopy (TEM)

[0104] Carbon dots were tested using a JEM-2000EX transmission electron microscope. The test results are as follows: Figure 2 As shown, Figure 2 A is a TEM image of Tyr-CDs, which shows that Tyr-CDs have good dispersion and relatively uniform size distribution; Figure 2 B is the particle size distribution diagram of Tyr-CDs. The particle size data of 200 CDs were statistically analyzed. The particle size is mainly distributed in the range of 2.4 nm to 7.6 nm, and the average particle size is calculated to be 4.98 nm.

[0105] 2. Fourier Transform Infrared Spectroscopy (FT-IR)

[0106] Tensor II Fourier transform infrared spectrometer was used in the 400-4000 cm⁻¹ range. -1 Tyr-CDs were analyzed within the range, and their infrared spectra are shown below. Figure 3 As shown: at 3350 cm -1 (NH / OH stretching vibration), 2910 cm -1 (Methyl / methylene CH stretching vibration), 1600 cm⁻¹ -1 (Aromatic ring C=C vibration), 1260 cm -1 (phenolic hydroxyl CO stretching vibration) and 795 cm -1 A characteristic absorption peak exists near the (out-of-plane bending vibration of the aromatic ring CH group), with an intensity lower than the corresponding peak of the control tyramine, indicating that the surface of Tyr-CDs may contain tyramine-like structures such as phenolic hydroxyl groups, amino groups, and aromatic ring skeletons. Furthermore, a characteristic peak distinct from tyramine, at 1660 cm⁻¹, was observed in Tyr-CDs. -1 Corresponding to C=O stretching vibration, 1130 cm -1 Corresponding to S=O stretching vibration and 620 cm -1 The corresponding CS stretching vibration confirms that sulfur (S) was successfully incorporated into Tyr-CDs after the introduction of H2SO4, a result consistent with XPS analysis. In summary, Tyr-CDs exhibit structural features derived from tyramine and have been successfully doped with sulfur.

[0107] 3. Ultraviolet-Vis spectrum (UV-vis)

[0108] The carbon dots of Example 1 and Comparative Examples 2-4 were analyzed using a U-3900 UV-Vis spectrophotometer. Figure 4The UV spectra of four high-quantum-yield Tyr-CDs in the 200-800 nm range are shown: the 290 nm peak is attributed to the π-π* transition of the conjugated double bond, the 370 nm peak to the n-π* transition of C=O / C=N, the 480 nm peak to the n-electron n-π* transition, and the shoulder peak at 520 nm coincides with its maximum emission wavelength. The consistent UV absorption peak positions of Tyr-CDs prepared with different solvent ratios indicate that changing the reaction solvent ratio of H2O to DMF does not affect the properties of Tyr-CDs other than the excitation and emission wavelengths.

[0109] II. Performance testing of Tyr-CDs-3 prepared in Example 1:

[0110] 1. Study on the luminescent properties of Tyr-CDs-3 prepared in Example 1

[0111] The photoluminescence properties of Tyr-CDs-1 (1 mg / mL) were analyzed using an F-7000 fluorescence spectrometer, and the results are as follows: Figure 5 As shown. Figure 5 As shown in Figure A, the emission peak redshifts from 570 nm to 605 nm when excited at 400–480 nm, exhibiting a small excitation wavelength dependence. When excited at 480–560 nm, the emission peak stabilizes at 605 nm, exhibiting excitation wavelength-independent photoluminescence behavior. Figure 5 B determined its maximum excitation / emission wavelengths to be 525 nm / 605 nm. The inset shows that the carbon dots exhibit red fluorescence in solution under 520 nm illumination.

[0112] 2. Study on the photobleaching resistance of Tyr-CDs-3 prepared in Example 1

[0113] Tyr-CDs were continuously irradiated with 525 nm excitation light for 60 min. The excitation light intensity (excitation slit width Slit) was changed by adjusting the size of the incident light slit. ex The fluorescence intensity of Tyr-CDs changed over time at 1 nm, 2.5 nm, 5 nm, and 10 nm (respectively). Figure 6 The fluorescence intensity changes of Tyr-CDs under four different excitation intensities were -1.36%, -2.17%, 1.31%, and -4.20%, respectively, all within the allowable error range of ±5%. Due to the low excitation energy of 525 nm, Tyr-CDs exhibit excellent photostability and are suitable for long-term fluorescence sensing and imaging.

[0114] 3. Investigation of the temperature, acidity, and salt tolerance stability of Tyr-CDs-3 prepared in Example 1.

[0115] The effect of temperature on Tyr-CDs is as follows Figure 7 As shown, the fluorescence intensity of Tyr-CDs remained essentially unchanged within the temperature range of 5 to 55℃, indicating good thermal stability. The effect of acidity on Tyr-CDs is shown in the following results. Figure 8 As shown, Tyr-CDs exhibited good stability with fluorescence intensity variations within ±5% in the pH range of 4 to 10. The fluorescence intensity was highest at pH 6 to 7. Considering the optimal pH environment in vivo, pH 7.4 was chosen for subsequent detection. The effect of NaCl on Tyr-CDs is shown below. Figure 9 As shown, the fluorescence intensity of Tyr-CDs remained essentially unchanged within a concentration range of 0 to 200 mM, indicating that the Tyr-CDs-3 prepared in Example 1 exhibited good salt resistance.

[0116] 4. Study on the effects of inorganic ions and biomolecules on Tyr-CDs-3 prepared in Example 1

[0117] This invention examines 5×10 -4 The effects of 12 metal ions and 12 anions of M on the fluorescence intensity of Tyr-CDs-3. The results are as follows: Figure 10 As shown, except for Cu 2+ and Fe 3+ Apart from the quenching of Tyr-CDs fluorescence intensity by 14% and 16% by the two metal ions, the effects of other inorganic ions on the fluorescence intensity of CDs were all within the allowable error range of ±10%, indicating that the carbon dots prepared by this method have good selectivity for most inorganic ions.

[0118] In addition, the effects of 12 amino acids, 4 bases, and 5 proteins on the fluorescence intensity of Tyr-CDs were investigated, and the results are as follows: Figure 11 As shown. From Figure 11 As can be seen, the fluorescence intensity of Tyr-CDs decreased by 18% and 22% respectively after the addition of bovine hemoglobin (BHb) and human hemoglobin (Hb). This phenomenon is due to the presence of Fe in BHb and Hb, which reduces the fluorescence intensity of Tyr-CDs. The effects of all other biomolecules investigated on the fluorescence intensity of Tyr-CDs were within the allowable error range of ±10%, and had no significant effect.

[0119] III. Application Performance Verification and Condition Optimization of Tyr-CDs Prepared in Example 1

[0120] 1. Validation of the signal amplification function of Tyr-CDs-3 prepared in Example 1 and its application in the detection of protein-based target analytes using ELISA-TSA (enzyme-linked immunosorbent assay combined with tyramine signal amplification technology).

[0121] (1) Experimental methods

[0122] (A) TSA method for Tyr-CD

[0123] Preparation of Tyr-CDs-BSA / hIgG in the TSA group: Take 6 mL of PBS buffer (pH = 7.4) into a 25 mL beaker, and add 1 mL of 0.01 mg / mL BSA or hIgG, 1 mL of 1 × 10⁻⁶ PBS buffer, and so on. -4 mg / mL HRP and 1 mL 0.05 mg / mL Tyr-CDs (Tyr-CDs-3 prepared in Example 1) were stirred at room temperature in the dark for 1 h, during which 200 μL H2O2 (1 × 10⁻⁶ mg / mL) was added dropwise every 12 min. -4 (M), for a total of 5 times. After the reaction, the solution was transferred to a 7000 Da dialysis bag and dialyzed for 24 h (changing the water every 2 h) until the outside of the dialysis bag showed no fluorescence, in order to remove unbound Tyr-CDs. The dialysate was freeze-dried to obtain Tyr-CDs-BSA, which was then sealed and stored at -20℃. Before testing, it was ultrasonically dispersed in 2 mL of DMF.

[0124] Co-incubation control group: Take 8 mL of PBS buffer (pH = 7.4) in a 25 mL beaker, add 1 mL of 10 ng / mL BSA or hIgG and 1 mL of 1 mg / mL Tyr-CDs, and stir at room temperature in the dark for 1 h. After the reaction, the purification and storage methods are the same as those for the TSA group.

[0125] (B) ELISA-Tyr-CDs fluorescence method for the determination of hIgG

[0126] Dilute 480 μg / mL hIgG standard solution to the desired concentration serially with PBS (pH = 7.4) and store sealed at 4°C for later use. Take a microplate strip embedded with hIgG antibody, place it in an aluminum foil bag, and equilibrate to room temperature for 20 min. Add 50 μL of hIgG standard solution of different concentrations and 100 μL of HRP-labeled detection antibody to each well. Seal the reaction wells with sealing film and incubate at 37°C for 60 min. Discard the liquid, pat dry on absorbent paper, fill each well with washing buffer, let stand for 1 min, discard the washing buffer, pat dry on absorbent paper, and repeat this washing process 5 times. Add 2.5 mg / mL Tyr-CDs and 5 × 10⁻⁶ ppm of HCl to each well. -3 Incubate 100 μL each of M and H2O2 at 37°C in the dark for 60 min. After incubation, discard the liquid and wash the plate as before. Finally, add 200 μL of DMF to each well, repeatedly pipet at least 5 times, transfer the solution to a 200 μL sample cell, and measure its fluorescence spectrum using an F-7000 fluorescence spectrometer. Quantify the hIgG in the sample based on the fluorescence intensity at 580 nm.

[0127] (C) ELISA-TMB colorimetric method for detecting hIgG

[0128] Following the kit instructions, a standard curve was plotted using the OD values ​​of the six sample wells against the hIgG concentration of the standard test sample. A linear regression equation was obtained (OD = 0.0023 chIgG + 0.0901, R² = 0.9927). Human serum samples were used as the actual test samples, and their OD values ​​at 450 nm were measured using a Syunergy H1 / H1MD multi-mode microplate reader. The calculated hIgG concentration in these samples was the actual value.

[0129] (D) Serum sample processing methods

[0130] Collect blood samples and place them in a test tube without anticoagulant. Centrifuge at 3000 rpm for 10 min. The supernatant is the serum, which should be frozen and stored. Dilute 1000 times with 0.1 M PBS (pH = 7.4) before testing.

[0131] (2) Experimental results:

[0132] (2.1) Detection of BSA based on Tyr-CD signal amplification (TSA method for Tyr-CD in Experimental Method (A)):

[0133] (A) Optimization of H2O2 addition method and concentration

[0134] Figure 12 The fluorescence spectra of Tyr-CDs-BSA prepared by adding H2O2 at different time intervals (no interval, and intervals of 12 min, 15 min, and 30 min) within 1 hour are as follows: Figure 12 As can be seen from the fluorescence spectrum of Tyr-CDs-BSA, the fluorescence intensity of the Tyr-CDs-BSA complex is significantly higher than that of BSA; and when the time interval of H2O2 addition is shortened, the fluorescence intensity increases and the maximum emission peak blue shifts. Figure 12 Results B showed that the shortening of fluorescence lifetime decreased synchronously with the time interval. Therefore, in subsequent BSA labeling experiments, H2O2 was added in a manner that was added 5 times every 12 minutes within 1 hour.

[0135] Figure 13 Different concentrations of H2O2 (1 × 10⁻⁶) are added to the TSA reaction. -2 ~1 × 10 -6The fluorescence excitation and emission spectra of Tyr-CDs-BSA (M) showed that as the H2O2 concentration decreased, the excitation wavelength (350-550 nm) exhibited a blue shift, while the emission wavelength (500-650 nm) remained essentially unchanged; when the concentration was 1 × 10⁻⁶, the excitation wavelength remained relatively constant. -4 At time M, Tyr-CDs-BSA exhibited high fluorescence intensity, therefore, subsequent experiments selected an H2O2 concentration of 1 × 10⁻⁶. -4 M.

[0136] (B) HRP concentration optimization

[0137] Figure 14 Different concentrations of HRP (1 × 10⁻⁶) were added to the TSA reaction. -2 ~1 × 10 -6 The fluorescence spectrum of Tyr-CDs-BSA obtained under HRP catalysis (mg / mL) shows that the emission wavelength remains unchanged within the investigated HRP concentration range, while the luminescence intensity slightly increases (1 × 10⁻⁶). -2 At a concentration of mg / mL, Tyr-CDs-BSA showed relatively high fluorescence intensity. However, HRP was also labeled by Tyr-CDs to improve the detection sensitivity of the target analyte; therefore, subsequent experiments selected an HRP concentration of 1 × 10⁻⁶ mg / mL. -4 mg / mL.

[0138] (C) Optimization of Tyr-CDs Concentration

[0139] Optimization results Figure 15 As shown, Figure 15 A shows the fluorescence spectra of Tyr-CDs-BSA obtained under different concentrations of Tyr-CDs (solid line represents the TSA group, dashed line represents the co-incubation group). As the concentration of Tyr-CDs increases, the fluorescence intensity of Tyr-CDs-BSA also increases. At the same concentration of Tyr-CDs, the fluorescence intensity of the TSA group is significantly higher than that of the co-incubation group. Figure 15 As shown in B, the amplification factor is the largest at a Tyr-CDs concentration of 0.05 mg / mL, which is 298.4%. Therefore, this concentration was selected as the optimal labeling concentration for Tyr-CDs.

[0140] (D) BSA detection

[0141] Under optimal conditions, the fluorescence spectra of Tyr-CDs-BSA after TSA operation with different concentrations of BSA were measured, and the results are as follows: Figure 16 As shown, from Figure 16 As shown in Figure A, the fluorescence peak position of Tyr-CDs-BSA remains unchanged, while the intensity increases with BSA concentration. A working curve was plotted using fluorescence intensity versus BSA concentration. Figure 16 B), in the range of 1.0 ~ 1.0 × 10 6Within the range of ng / mL, the fluorescence intensity of Tyr-CDs-BSA shows a good linear relationship with the concentration of BSA, and the regression equation is F = 33.40 log( c BSA ) + 77.03 (R 2 = 0.9707). According to the IUPAC recommendation, the detection limit of this method for BSA is calculated to be 0.57 ng / mL, demonstrating high sensitivity for BSA detection.

[0142] (2.2) Detection of hIgG by ELISA-TSA method based on Tyr-CDs

[0143] (A) Optimization of Tyr-CDs concentration (optimization of Tyr-CDs concentration in experimental method (B))

[0144] By changing the concentration of Tyr-CDs or hIgG, the influence on the fluorescence intensity of Tyr-CDs-hIgG was measured, and the results are as Figure 17 shown. It can be seen from the figure that when the concentration of Tyr-CDs is 2.5 mg / mL, the fluorescence intensity is the highest, and the change rate of its fluorescence intensity increases with the increase of hIgG concentration. Therefore, Tyr-CDs with a concentration of 2.5 mg / mL and hIgG with a concentration of 50 μg / mL were selected for subsequent experiments.

[0145] (B) Optimization of H2O2 concentration

[0146] The fluorescence spectrum results and magnification results of Tyr-CDs-hIgG with different concentrations of H2O2 added are as Figure 18 shown ( Figure 18 A is the fluorescence spectrum diagram under different concentrations of H2O2, Figure 18 B is the magnification result). As the concentration of H2O2 increases, the fluorescence intensity of Tyr-CDs-hIgG shows a trend of first increasing and then decreasing. The fluorescence intensity is the largest when the concentration of H2O2 is 5 × 10 -4 M, and the fluorescence method magnification is 265%. In subsequent experiments, 5 × 10 -4 M was selected as the H2O2 concentration used in the ELISA-TSA system.

[0147] (C) Optimization of TSA reaction time (incubation time)

[0148] The optimization results of the TSA reaction time in the ELISA-TSA system based on Tyr-CDs are as Figure 19 shown. Figure 19 A shows that when the TSA reaction time is extended to 60 min, the fluorescence intensity of Tyr-CDs-hIgG reaches the maximum value; as Figure 19As shown in Figure B, the amplification factor first increases and then decreases as the response time increases, with the signal amplification factor being the largest at 60 min. Therefore, the optimal response time for TSA is 60 min.

[0149] (D) Performance Analysis:

[0150] Under optimal conditions, the analytical performance of the Tyr-CDs-based ELISA-TSA method for detecting hIgG was evaluated, and the results are as follows: Figure 20 As shown. Figure 20 A shows the fluorescence spectra of Tyr-CDs-hIgG with different concentrations of hIgG added. It can be seen that the fluorescence intensity of Tyr-CDs-hIgG increases significantly with the increase of hIgG concentration. Figure 20 B shows that when the hIgG concentration is between 1 and 1 × 10⁻⁶, 5 Within the ng / mL range, the fluorescence intensity of Tyr-CDs-hIgG showed a good linear relationship with the hIgG concentration, and the regression equation was F = 31.89 log[hIgG] + 214.8 (R²). 2 = 0.9867). Eleven parallel experiments were performed on the fluorescence intensity of hIgG at a concentration of 1 ng / mL. The relative standard deviation (RSD) was calculated to be 2.2%, and the limit of detection was 0.43 ng / mL, indicating high sensitivity for the detection of hIgG.

[0151] 2. Actual sample determination

[0152] To evaluate the reliability of the established ELISA-TSA assay in real-world sample testing, the levels of hIgG in the serum of four healthy adult volunteers were measured. Prior to the assay, the serum samples from the four healthy adults were diluted 1000-fold to ensure that the hIgG concentration was within the linear range.

[0153] The experimental results are shown in Table 4. The Tyr-CDs fluorescence method detected serum hIgG concentrations ranging from 7.6 mg / mL to 11.0 mg / mL, all within the normal range for human serum hIgG. The TMB colorimetric kit (Jiangsu Enzyme Labeling Biotechnology Co., Ltd.: MB-0157B) detected serum hIgG concentrations ranging from 8.6 mg / mL to 11.9 mg / mL. Compared with the TMB method, the relative error of this method was within ±10%, indicating high accuracy. Recovery experiments showed recoveries between 91.0% and 106.0%, with relative standard deviations all less than 2.0%, demonstrating that the established new hIgG detection method has good accuracy and precision, and great potential for practical application.

[0154] Table 4. Detection results of hIgG in human serum samples (n=3)*

[0155]

[0156] *The normal human serum hIgG level is 6 mg / mL ~ 16 mg / mL.

[0157] 3. Investigation of the cellular fluorescent labeling function of Tyr-CDs-3 prepared in Example 1

[0158] (1) Experimental methods

[0159] (A) Yeast culture method

[0160] Prepare a 3% glucose solution using PBS (pH 7.4), sterilize at high temperature, and then filter through a 0.22 μm filter membrane for sterilization. Mix 0.5 g of yeast with 5 mL of the above glucose solution in a 10 mL centrifuge tube, and incubate at 37°C for 1 h. Open the centrifuge tube every 20 min to release air and prevent excessive pressure from the CO2 produced by the yeast breaking down glucose, which could cause the centrifuge tube cap to open. After incubation, disperse the suspension evenly in the tube, and take 1 mL of each suspension and place it in several identical centrifuge tubes. Centrifuge at 8000 rpm for 5 min, remove the supernatant, wash with PBS buffer, and repeat this step three times. Then disperse the yeast suspension in PBS and store it under cold.

[0161] (B) Fluorescent labeling method for yeast

[0162] Co-incubation group (control group): 500 μL of suspension, 200 μL of 1 mg / mL Tyr-CDs and 1.3 mL of PBS were added to a 5 mL centrifuge tube and reacted in a 37℃ water bath with a constant temperature shaker for 1 h. After incubation, the supernatant was removed by centrifugation at 8000 rpm for 5 min. The supernatant was washed with PBS at pH = 7.4 and centrifuged again. This step was repeated three times. The mixture was then eluted by sonication in a 50℃ water bath for 30 min. The yeast was dispersed in 2 mL of DMF and its fluorescence spectrum was measured.

[0163] TSA group: 500 μL suspension, 200 μL 1 mg / mL Tyr-CDs, and 200 μL 1×10⁻⁶ ppm were added to a 5 mL centrifuge tube. -4 M HRP, 100 μL 1×10 -4 M H2O2 and 1 mL pH = 7.4 PBS were reacted in a 37°C water bath with a constant temperature shaker for 1 h. The washing and elution methods were the same as those for the co-incubation group.

[0164] (C) Yeast fluorescence imaging method

[0165] Yeast cells labeled with Tyr-CDs were observed using a ZEISS Axio vert5 inverted fluorescence microscope. All images were taken at 40x magnification.

[0166] (D) Yeast counting method

[0167] After evenly dispersing the cultured yeast suspension, use a pipette to add 10 μL of the yeast suspension to the edge of the counting cell of the cell counting chamber. The droplet will enter the counting cell below the coverslip under the siphon effect (try to ensure that the volume of filling the cell is consistent each time, generally around 10 μL / cell). Add the sample to the counting cell on the other side in the same way. Let the counting chamber stand for a few minutes to allow the yeast to diffuse and settle. Observe under an optical microscope, record the number of yeast cells in 8 medium-sized squares on the counting chamber, and take the average value. According to the parameters of the cell counting chamber, the area of ​​each medium square is 1 / 400 mm². 2 After covering with a coverslip, the height above the counting area is 0.1 mm, and the volume of each square counting area is 1 / 4000 mm. 2 = 2.5 × 10 -7 mL, yeast concentration = average number of yeast cells / volume of counting zone.

[0168] (2) Experimental results:

[0169] (2.1) TSA-based fluorescent labeling function of Tyr-CDs cells

[0170] (A) Fluorescence microscopy image results

[0171] The effect of yeast labeling was observed using a ZEISS Axio vert5 microscope (×40). Figure 21 Bright-field images show that the blank group ( Figure 21 A) and TSA group ( Figure 21 C) All cells maintained a complete spherical structure, indicating that Tyr-CDs have good biocompatibility. Dark-field images showed no fluorescence in the blank group ( Figure 21 B), the TSA group exhibited bright red fluorescence under 480 nm excitation ( Figure 21 (D) This confirms that Tyr-CDs can be specifically deposited in cells using TSA technology while maintaining their red light emission properties.

[0172] (B) Optimization of experimental conditions

[0173] This invention optimizes experimental procedures and conditions, and investigates the fluorescence intensity and signal amplification effect of yeast fluorescence spectra before and after elution at 50℃ under different incubation times.

[0174] result Figure 22 As shown, Figure 22A, Figure 22 C represents the fluorescence spectra of yeast in the uneluted TSA group and the incubated group, respectively. Figure 22 B. Figure 22 D represents the fluorescence spectra of yeast in the TSA group and the incubation group after elution at 50℃. It can be seen that the fluorescence intensity of the labeled yeast increases with increasing yeast concentration. Both the incubation group and the TSA group showed a decrease in fluorescence intensity before and after ultrasonic elution at 50℃. The average fluorescence intensity of the incubation group decreased by 62.31% after elution, while that of the TSA group decreased by an average of 38.61%. Figure 22 As can be seen in E, the signal amplification factor of Tyr-CDs-labeled yeast increased after elution compared to before elution.

[0175] (C) Quantitative detection of yeast cells using TSA-based Tyr-CDs fluorescent labeling

[0176] To evaluate the cellular fluorescence labeling performance of Tyr-CDs at low concentration levels, different concentrations of yeast cells were labeled using the TSA reaction of Tyr-CDs. The resulting fluorescence spectra ( Figure 23 A) and its fluorescence intensity versus yeast concentration curve ( Figure 23 As shown in B). The results indicate that the fluorescence intensity of the labeled yeast cells is related to the logarithm of their concentration in the range of 1 to 1 × 10⁻⁶. 6 The study showed good linearity in the cell / mL range (F = 155.0 log[yeast] - 10.20, R0). 2 = 0.9893). This result indicates that Tyr-CDs possess the ability to perform fluorescent labeling on single cells, demonstrating high sensitivity and good application potential in cellular fluorescent labeling.

[0177] To verify the accuracy of the above results, four concentration samples of yeast cells (1 cells / mL, 1 × 10⁻⁶ ... 2 cells / mL, 1 × 10 4 cells / mL, 1 × 10 6 cells / mL, observed under a microscope using a cell counting chamber ( Figure 24 (A-24D), the calculated actual yeast concentration is 2 cells / mL, 2.5 × 10⁻⁶. 2 cells / mL, 1.6 × 10 4 cells / mL, 1.8 × 10 6 The cells / mL reading is on the same order of magnitude as the detection result, indicating that the quantitative results of Tyr-CDs cell fluorescent labeling are accurate.

[0178] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A novel bifunctional tyramine red light carbon dot, characterized in that, The novel bifunctional tyramine red-light carbon dots are red-light carbon dots (Tyr-CDs) with tyramine structural residues on their surface. These novel bifunctional tyramine red-light carbon dots are prepared by using tyramine and o-phenylenediamine in a molar ratio of 1~3:1~3 as precursors in a mixed solvent containing 0.001 M~0.18 M H2SO4 and a volume ratio of H2O to DMF of 3:7, followed by hydrothermal reaction at 200°C for 4~36 hours. The resulting carbon dots are then purified by sequential cooling, centrifugation at 8000 rpm for 20 min, dialyzing through a 1000 Da dialysis bag for 12 h, and filtration through a 0.22 μm microporous membrane. The novel bifunctional tyramine red-light carbon dots possess signal amplification and detection functions as well as cell fluorescence labeling functions.

2. A method for preparing the novel bifunctional tyramine red carbon dots as described in claim 1, characterized in that, Includes the following steps: Tyramine and o-phenylenediamine were weighed and mixed, and a mixture of H2O and DMF solvent and H2SO4 were added. After tyramine and o-phenylenediamine were completely dissolved, the mixture was transferred to a reaction vessel for reaction. After the reaction was completed, the product was taken out, cooled, centrifuged, dialyzed, filtered, concentrated, and freeze-dried to obtain a novel bifunctional tyramine red carbon dots Tyr-CDs. The obtained Tyr-CDs were dispersed in a solvent for later use. The molar ratio of tyramine to o-phenylenediamine is 1~3:1~3; the volume ratio of H2O to DMF in the mixed solvent is 3:7; the concentration of H2SO4 is 0.001 M~0.18 M; the reaction time is 4~36 h, and the reaction temperature is 200℃; the centrifugation method is centrifugation at 8000 rpm for 20 min; the dialysis method is dialysis for 12 h using a dialysis bag with a molecular weight cutoff of 1000 Da to obtain the dialysate; the filtration method is to filter the dialysate using a 0.22 μm microporous membrane.

3. The application of a novel bifunctional tyramine red carbon dot as described in claim 1 in signal amplification and detection.

4. The application of a novel bifunctional tyramine red carbon dot as described in claim 1 in enzyme-linked immunosorbent assay (ELISA).

5. The application according to claim 4, characterized in that, The application in enzyme-linked immunosorbent assay (ELISA) refers to quantitative immunofluorescence detection based on the combined use of TSA and ELISA technologies. The specific method for quantitative immunofluorescence detection includes the following steps: S1: Mix horseradish peroxidase (HRP) with the antibody to be detected to obtain an HRP-labeled detection antibody; S2: The standard solution of the antibody to be detected is incubated with the HRP-labeled detection antibody. After washing, the novel bifunctional tyramine red carbon dots Tyr-CDs and H2O2 are added and incubated. After incubation, the liquid is discarded and washed. DMF solvent is added, and then immunofluorescence quantitative detection is performed.

6. The application of a novel bifunctional tyramine red carbon dot as described in claim 1 in cell fluorescent labeling.

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