Method for quantitatively detecting bilirubin based on assembly of dual-emission gold nanocluster and polyallylamine hydrochloride

Abstract

The invention relates to the field of bilirubin detection, in particular to a method for quantitatively detecting bilirubin through a double-emission gold nanocluster fluorescent probe. According to the method, cyclodextrin is taken as a ligand, novel double-emission gold nano-clusters with two unique emission are synthesized, and then the novel double-emission gold nano-clusters and PAH are synthesized into a DE-CD-AuNCs (at) PAH assembly through electrostatic interaction. Based on the principle that after bilirubin is combined with an assembly, the fluorescence of an emission peak at 530 nm of the assembly is enhanced, and the fluorescence of an emission peak at 730 nm of the assembly is reduced, a ratio detection method for detecting bilirubin is established according to the ratio of two self-contained emission fluorescence intensities. When the method is used for detecting bilirubin, a good linear relation can be achieved within the range of 0.01-8.0 [mu] mol.L <-1 >, and the detection limit is 5.36 + / -0.23 nmol.L <-1 > (S / N = 3). In the detection process, the method is not detected by common coexisting components in serum, and has relatively high sensitivity and relatively high reaction speed.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly relates to a method for quantitatively detecting bilirubin based on an assembly of dual-emission gold nanoclusters and polyacrylamine hydrochloride. Background Art

[0002] Bilirubin formed by the decomposition of red blood cells is a key biomarker for jaundice. The quantitative detection of bilirubin is of great significance for clinical diagnosis and health management. Currently, various methods for bilirubin detection have been established, including colorimetry, electrochemistry, Raman spectroscopy, high performance liquid chromatography, fluorescence analysis, etc. Among these methods, fluorescence analysis has become a more promising method for bilirubin detection due to its advantages such as high sensitivity, convenient operation, and easy reading.

[0003] Gold nanoclusters (AuNCs) are a new type of inorganic nanomaterial composed of several to dozens of gold atoms and a ligand shell with multiple charges. They are prone to corresponding optical transitions and emissions, providing unique optical properties for gold nanoclusters. Moreover, their synthesis methods have the advantages of simplicity, rapidity, and environmental friendliness. Most reported gold nanocluster fluorescence probes have a single emission wavelength. Although ratio fluorescence probes have been developed, most reported ratio sensors are constructed by assembling or conjugating two independent light sources. The ratio sensors constructed in this way may have problems such as being easily interfered by external factors, cumbersome design, and complex synthesis, which have certain limitations for the detection and practical application of substances. The dual-emission gold nanocluster fluorescence probe not only retains the advantages of the single-emission gold nanocluster fluorescence probe, but also can detect different substances simultaneously, improving the detection efficiency. According to research, there is currently no practical application of a self-contained dual-emission gold nanocluster fluorescence probe for bilirubin detection. Summary of the Invention

[0004] Based on the problems existing in the current bilirubin detection methods and the fluorescence method for bilirubin detection, the present invention designs a method for quantitatively detecting bilirubin based on an assembly of dual-emission gold nanoclusters and polyacrylamine hydrochloride.

[0005] To achieve the above detection method, the present invention adopts the following detection scheme:

[0006] (1) Dissolve and prepare HAuCl4 (1 M) and SH-β-CD solution (0.06 mM) with 0.1 M NaHCO3 solution respectively. At room temperature, add 40 μL of HAuCl4 and 1.04 mL of NaHCO3 solution into a round-bottom flask, and gradually add 1.96 mL of SH-μ-CD solution dropwise under vigorous stirring. At this time, the solution changes from yellow to colorless. After stirring for 5 min, add another 22 mL of NaHCO3 solution to the mixture, and then react at 80 °C for 48 h. During this process, the reaction solution will slowly change from colorless to pink. After stopping the reaction, a clear light brownish-red solution is obtained. Filter the obtained solution through a microporous filter membrane to remove large particles, and then remove the excess reactants by ultrafiltration centrifugation (10 KDa, 7500 rpm) to obtain cyclodextrin-gold nanoclusters with dual emission (ED-CD-AuNCs).

[0007] (2) Add 50 μL of polyallylamine hydrochloride aqueous solution (PAH: MW ~ 15000, 1 mg / mL) to 1 mL of ED-CD-AuNCs solution (0.06 mg / mL), and stir at 50 °C for 90 min to prepare a polyallylamine hydrochloride-gold nanocluster assembly (ED-CD-AuNCs@PAH). Centrifuge the obtained solution at 6000 rpm for 5 min, and take the supernatant to obtain ED-CD-AuNCs@PAH.

[0008] (3) Take 1 mL of ED-CD-AuNCs@PAH solution, add 10.0 μL of serum sample, and let it react for 2 min. Under the excitation light of 450 nm, set the slit width as EX = 10 nm and EM = 5 nm, and measure the fluorescence intensity ratio (F 530 nm / F 730 nm) of ED-CD-AuNCs@PAH before and after adding bilirubin, and measure the bilirubin concentration of the serum sample at this time according to the fitting equation of F 530 / F 730 and bilirubin concentration C.

[0009] Further, the ED-CD-AuNCs prepared in step (1) are prepared after improvement based on the reference document "One-pot synthesis of β-cyclodextrin modified Au nanoclusters with near-infrared emission".

[0010] Further, based on the ED-CD-AuNCs prepared in step (1), an ED-CD-AuNCs@PAH assembly is prepared and used as a fluorescence probe for bilirubin detection.

[0011] Furthermore, for the preparation method of the ED-CD-AuNCs@PAH assembly in step (2), the optimal preparation conditions are pH = 10, reaction time of 3 h, and the optimal reaction temperature of 50 °C.

[0012] Furthermore, for the bilirubin detection process in the serum sample in step (3), the optimal detection conditions are a PAH concentration of 3.5 uM, the reaction environment of ED-CD-AuNCs@PAH with bilirubin is an alkaline environment (pH = 10 is optimal), the reaction temperature is 37 °C, and the reaction time is 2 minutes.

[0013] Furthermore, for the bilirubin detection process in the serum sample in step (3), F 530 / F 730 The fitting equation with bilirubin concentration C is F 530 / F 730 = 0.1676C (μmol·L-1) + 0.4956. The square of the correlation coefficient R 2 is 0.99. The detection limit of bilirubin is calculated to be 5.36 ± 0.23 nmol·L-1 (S / N = 3).

[0014] Furthermore, for the bilirubin detection process in the serum sample in step (3), the bilirubin concentration for detection should be in the range of 0.01 - 8.0 μmoL-1

[0015] The ED-CD-AuNCs and ED-CD-AuNCs@PAH were respectively characterized by HRTEM morphology, FTIR infrared spectroscopy, and XRD photoelectron spectroscopy. The results clearly showed that ED-CD-AuNCs and PAH were combined through electrostatic interaction to form the ED-CD-AuNCs@PAH nanocomposite, presenting a spherical morphology with an average particle size of 5.65 ± 0.02 nm.

[0016] The assembly based on the gold nanoclusters with dual emissions and the cationic polymer PAH developed in the present invention achieved fluorescence enhancement of the dual emission peaks through the induced emission effect. The cyclodextrin on the assembly was used as a bilirubin-selective recognition unit, and bilirubin was recognized by the host-guest interaction, making the assembly structure rigid and causing the fluorescence intensity of the emission peak at 530 nm to increase, and the fluorescence intensity of the emission peak at 730 nm to decrease through energy transfer. Based on the different changes of these dual emission peaks, low-level bilirubin ratio quantification was achieved. This method does not require additional auxiliary fluorophores, is easy to operate, has a rapid reaction, high sensitivity. Moreover, the common coexisting components in human serum do not interfere with the determination of bilirubin. Description of the Drawings

[0017] Figure 1Schematic diagram of a method for quantitatively detecting bilirubin based on an assembly of dual-emission gold nanoclusters and polyallylamine hydrochloride;

[0018] Figure 2 is an analysis diagram of the optimal conditions for the preparation of the ED-CD-AuNCs@PAH assembly, where Figure 2a is the analysis of PAH concentration, Figure 2b is the pH analysis, Figure 2c is the reaction time analysis, Figure 2d is the reaction temperature analysis;

[0019] Figure 3 is an analysis diagram of the optimal conditions for the reaction of the ED-CD-AuNCs@PAH assembly with bilirubin, where Figure 3a is the analysis of PAH concentration, Figure 3b is the pH analysis, Figure 3c is the reaction time analysis, Figure 3d is the reaction temperature analysis;

[0020] Figure 4a is the linear relationship diagram between the fluorescence of ED-CD-AuNCs@PAH and the bilirubin concentration, Figure 4b is the selectivity analysis diagram of the reaction of this assembly with bilirubin; Specific implementation method

[0021] The principle of the present invention will be further elaborated below in conjunction with the drawings and examples, but it is not a limitation of the present invention. The instruments and reagents required for the present invention are as follows:

[0022] All reagents used in the preparation are analytical pure reagents, and the reagents are configured based on this standard, including mono(6-mercapto-6-deoxy)β-cyclodextrin, bilirubin, chloroauric acid trihydrate (H[AuCl4]·3H2O), NaHCO3 solution, polyallylamine hydrochloride aqueous solution, NaOH solution, ultrapure water;

[0023] The main instruments for reagent preparation include a thermostatic magnetic stirrer (Shanghai Lichen), an ultra-thin carbon film copper mesh (acceleration voltage of 200 kV), a tabletop high-speed refrigerated centrifuge, a WD-9403A ultraviolet instrument, and a freeze dryer. The main analytical instruments include an F-4600 fluorescence spectrometer (Hitachi High-Technologies), a Hitachi UH5300 ultraviolet-visible spectrophotometer, a Zetasizer Nano ZS90 particle size analyzer, a JEOL 2100 high-resolution transmission electron microscope (HRTEM), a Nicolet iS10 FTIR spectrometer (FTIR), a Thermoescalab 250Xi, a Bruker D8 Advance x-ray diffractometer, and an Edinburgh FLS1000 photoluminescence spectrometer.

[0024] The specific process is as follows:

[0025] 1) Prepare PAH at concentrations of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 μmol / L respectively, and detect the fluorescence intensity of ED-CD-AuNCs@PAH ( Figure 2a ). Select the optimal assembly concentration of PAH as 3.5 μM; set 2, 4, 6, 8, 10, and 12 respectively, and detect the fluorescence intensity of the assembly at 6 pH values ( Figure 2b ). Select an environment with pH = 10 to prepare the assembly to ensure that the fluorescence intensity of ED-CD-AuNCs@PAH is the maximum; detect the fluorescence intensity of ED-CD-AuNCs@PAH at 5 time points during the reaction time from 0.5 to 4 h ( Figure 2c ), and select the optimal reaction time as 3 h; detect the fluorescence intensity of ED-CD-AuNCs@PAH at 5 temperature nodes from 25 to 60 °C ( Figure 2d ), and select the optimal reaction temperature as 50 °C;

[0026] 2) Under the optimal conditions for preparing ED-CD-AuNCs@PAH, conduct a feasibility test for detecting bilirubin. Use ED-CD-AuNCs@PAH as a fluorescent probe to quantitatively detect bilirubin. The gold core serves as the fluorescent reporting unit, and the fluorescence changes of bilirubin and β-CD are measured. Dissolve 1.2 mg of bilirubin in 1 mL of NaOH (1 M), add ultrapure water to 10 mL to prepare a bilirubin stock solution of 2×10 - 4 M, and dilute it to different concentrations. Take 1 mL of the ED-CD-AuNCs@PAH solution, add 10.0 μL of the bilirubin stock solution, allow it to react for 2 min, under an excitation light of 450 nm, set the slit width as EX = 10 nm and EM = 5 nm, and measure the fluorescence intensity ratio (F530nm / F730 nm) of ED-CD-AuNCs@PAH before and after adding bilirubin to achieve the determination of bilirubin. The experimental results show that the fluorescence of ED-CD-AuNCs@PAH is quenched after binding to bilirubin, the fluorescence peak at 530 nm increases, and the fluorescence peak at 730 nm decreases, verifying the feasibility of this scheme;

[0027] 3) Conduct a sensitivity test under the conditions of detecting feasibility. Optimize a series of conditions such as the concentration of PAH, the pH of the medium, the response temperature, and time, and use the fluorescence enhancement amount Δ(F 530 / F 730 ) of the double emission peak ratio to reflect the fluorescence response of ED-CD-AuNCs@PAH to bilirubin. By using ED-CD-AuNCs solutions assembled with different concentrations of PAH, the effect of the concentration of PAH on the fluorescence response of bilirubin was studied ( Figure 3a)。The PAH concentration of ED-CD-AuNCs@PAH was selected as 3.5 uM, with the highest fluorescence intensity and the best binding effect with bilirubin; the pH value of the medium plays a decisive role in the reaction between ED-CD-AuNCs@PAH and bilirubin( Figure 3b )。The reaction between ED-CD-AuNCs@PAH and bilirubin was carried out under alkaline conditions; the effect of temperature on the reaction between ED-CD-AuNCs@PAH and bilirubin was investigated in the range of 20 - 50 °C( Figure 3c ),and at the same time, the effect of reaction time on the fluorescence response was carried out( Figure 3d )。The reaction temperature was selected as 37 °C and the reaction time was 2 minutes as the best;

[0028] 4) Selective detection was carried out under the optimal conditions, and the influence of some coexisting substances in serum on the determination of bilirubin was studied through coexistence experiments. Urea, heme, cholesterol, fructose, cysteine, galactose, dopamine, human serum albumin (HSA), uric acid, glutathione, ascorbic acid, hemoglobin, and (2 μM) and other typical exogenous biomolecules with a concentration of 65 μM (2.5 μg / mL) coexisted with bilirubin, and the analysis was carried out according to the protocol in step 3) Figure 4b )。The results showed that these coexisting components did not interfere with the detection of bilirubin;

[0029] 5) Using SH-β-CD as a reducing agent and stabilizer, gold nanoclusters with dual emission were prepared by a one-pot hydrothermal reduction method under the optimal conditions. ED-CD-AuNCs and the cationic polymer PAH were further assembled into a stable ED-CD-AuNCs@PAH assembly through electrostatic attraction;

[0030] 6) Establish a quantitative relationship between the fluorescence of ED-CD-AuNCs@PAH and the concentration of bilirubin. Under the optimal experimental conditions, ED-CD-AuNCs@PAH interacted with bilirubin at different concentrations, and the fluorescence spectra and fluorescence intensities were recorded respectively. In the range of bilirubin concentration from 0.01 to 8.0 μmol·L-1, the (F 530 / F 730 ) of ED-CD-AuNCs@PAH showed a linear relationship with the bilirubin concentration. The fitting equation of F 530 / F 730 and the bilirubin concentration C was F 530 / F 730 = 0.1676C (μmol·L-1) + 0.4956, and the square of the correlation coefficient R2 was 0.99( Figure 4a )。The detection limit of bilirubin was calculated to be 5.36 ± 0.23 nmol·L-1 (S / N = 3).

[0031] 7) Add 10 μL of serum to 1 mL of the ED-CD-AuNCs@PAH test solution, react for 2 min, and measure the fluorescence intensity. Detect the bilirubin concentration in the serum sample at this time according to the quantitative relationship fitting equation between the fluorescence of ED-CD-AuNCs@PAH and the bilirubin concentration established in step 6);

[0032] Furthermore, add a bilirubin standard solution with a known concentration according to step 7), record the fluorescence intensity, and calculate the spiked concentration and recovery rate results of bilirubin, as shown in Table 1. The spiked recovery rate is in the range of 97.8 - 103.47%, indicating that the proposed method has good applicability.

[0033] Table 1 Determination of bilirubin in human serum samples (n = 3)

[0034]

[0035] The above steps have detailed the principle of the experimental method of the present invention, the best environment for the preparation of the required fluorescent probe, and the experimental process of the best environment for reacting with bilirubin. It is summarized from the above steps that the present method synthesizes cyclodextrin-capped gold nanoclusters with dual emission, and assembles them with PAH through electrostatic interaction. The assembly realizes the fluorescence enhancement of the dual emission peaks through the aggregation-induced emission effect. The cyclodextrin on the assembly is used as a bilirubin-selective recognition unit to recognize bilirubin by host-guest interaction. Based on the different changes of this dual emission peak, low-level bilirubin ratio quantitative determination has been achieved, with the advantages of simple operation, rapid reaction, high sensitivity, strong stability, and strong selectivity.

[0036] It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A method for quantitatively detecting bilirubin based on an assembly of dual-emission gold nanoclusters and polyacrylamine hydrochloride, characterized in that: The method comprises the following steps: (1) Dissolve HAuCl4 (1M) and SH-β-CD solution (0.06mM) in 0.1M NaHCO3 solution. Add 40μL HAuCl4 and 1.04mL NaHCO3 solution into a round-bottom flask at room temperature, and add 1.96mL SH-μ-CD solution dropwise under vigorous stirring. At this time, the solution changes from yellow to colorless. After stirring for 5 minutes, add 22mL of NaHCO3 solution to the mixture, and then react at 80℃ for 48h. During this process, the reaction solution will slowly change from colorless to pink, and a clear light brown-red solution will be obtained after stopping the reaction. The resulting solution is filtered with a microporous filter membrane to remove large particles, and then ultrafiltration centrifugation (10KDa, 7500rpm) is used to remove excess reactants to obtain self-contained dual-emission cyclodextrin-gold nanoclusters (ED-CD-AuNCs). (2) Add 50 μL of polyallylamine hydrochloride aqueous solution (PAH: MW ~ 15000, 1 mg / mL) to 1 mL of ED-CD-AuNCs solution (0.06 mg / mL), heat and stir at 50 ° C for 90 min to prepare polyallylamine hydrochloride-gold nanocluster assembly (ED-CD-AuNCs @ PAH). The resulting solution was centrifuged at 6000 rpm for 5 min, and the supernatant was taken to obtain ED-CD-AuNCs @ PAH. (3) Take 1 mL of ED-CD-AuNCs@PAH solution, add 10.0 μL of serum sample, and let it react for 2 min. Under the excitation light of 450 nm, set the slit width to EX = 10 nm, EM = 5 nm, and measure the fluorescence intensity ratio (F) of ED-CD-AuNCs@PAH before and after adding bilirubin. 530 nm / F 730 nm), and according to F 530 / F 730 The bilirubin concentration of the serum sample at this time was measured by the fitting equation with the bilirubin concentration C.

2. The method for quantitatively detecting bilirubin based on the assembly of dual-emission gold nanoclusters and polyacrylamine hydrochloride according to claim 1, characterized in that: The preparation method of ED-CD-AuNCs was improved.

3. The method for quantitatively detecting bilirubin based on the assembly of dual-emission gold nanoclusters and polyacrylamine hydrochloride according to claim 1, characterized in that: ED-CD-AuNCs@PAH was prepared and used as a fluorescent probe for bilirubin detection.

4. According to the preparation method of ED-CD-AuNCs@PAH assembly as described in claim 3, the optimal preparation conditions are pH=10, reaction time is 3h, and the optimal reaction temperature is 50°C.

5. According to the bilirubin detection process in step (3) of claim 1, the optimal detection conditions are that the concentration of PAH is 3.5uM, the reaction environment of ED-CD-AuNCs@PAH and bilirubin is an alkaline environment (pH=10 is the best), the reaction temperature is 37°C, and the reaction time is 2 minutes.

6. The bilirubin detection process in the serum sample in step (3) according to claim 1, 530 / F 730 The fitting equation for bilirubin concentration C is F 530 / F 730 =0.1676C(μmol·L-1)+0.4956. Correlation coefficient R 2 The square of is 0.

99. The detection limit of bilirubin is calculated to be 5.36±0.23nmol·L-1 (S / N=3).

7. According to the bilirubin detection process in step (3) of claim 1, the bilirubin concentration used for detection should be in the range of 0.01 to 8.0 μmoL-1.

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