Colorimetric array sensor for qualitatively and quantitatively detecting phosphate

Abstract

The invention discloses a colorimetric array sensor for qualitatively and quantitatively detecting phosphate, each sensing unit is prepared by the following steps: preparing a total mixed solution from N-acetyl-L-cysteine polydopamine nanoparticles N-PDA NPs, a saline solution of Fe < 3 + >, Ag < + > or Hg < 2 + > and a mixed solution of TMB and H2O2, diluting the total mixed solution with redistilled water, and incubating the diluted solution at 25 DEG C for 50 minutes. The method can realize distinguishing detection of seven phosphates with the same concentration, fingerprint identification of phosphate mixed samples with different concentrations and different proportions coexisting in a physiological environment, quantitative analysis of the seven phosphates and blind sample identification. Besides, the array sensor can be applied to activity monitoring of protein kinase related to phosphate conversion, 100% distinguishing of different phosphates in a complex serum environment is achieved, and establishment of a method for visually judging whether the phosphate content in human serum meets the normal standard or not is achieved.

Description

Technical Field

[0001] The invention relates to a colorimetric array sensor, in particular to a colorimetric array sensor for qualitatively and quantitatively detecting phosphate. Background Art

[0002] Physiological phosphates, such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), hydrogen phosphate (HPO4 2- ), dihydrogen phosphate (H2PO4 - ), pyrophosphate (PPi) and orthophosphate (Pi), etc., which participate in important biological processes such as energy metabolism, cell signal transduction, enzyme activity regulation and DNA synthesis. Different phosphates can be converted by appropriate enzymes, and the balance of physiological phosphates maintains the normal operation of enzyme reactions, thereby ensuring the smooth progress of various biochemical processes in cells. Disorders in the enzymatic reactions between ATP, ADP and AMP may cause diseases such as circulatory shock, local ischemia and hypoglycemia. Abnormal levels of inorganic phosphate in human serum can lead to diseases such as thyroid dysfunction, renal insufficiency, uremia, and Fanconi syndrome. In addition, abnormal kinase activity is often accompanied by changes in phosphates such as ATP and ADP, and is closely related to various physiological and even pathological changes such as inflammation, hypoxia and cancer. Therefore, the development of high-throughput qualitative and quantitative analysis methods for different physiological phosphates and their mixtures is of great significance for deciphering their biological functions.

[0003] Array sensors break through the "lock-key" design concept of specific detection methods. The multiple sensing units they contain can cross-react with different analytes. Each analyte will produce a unique response signal. Combined with the multivariate statistical analysis method based on pattern recognition for signal data processing, it can achieve simultaneous identification of analytes with similar properties and their complex mixtures. It has the advantages of diverse detection objects, high detection throughput, and fast analysis speed. Among them, colorimetric array sensors have the advantages of simple detection methods, intuitive detection results, and stable detection signals. In the construction process of colorimetric array sensors, colorimetric array sensors usually need to be added. Compared with traditional colorimetric reagents (such as benzidine, o-benzidine, o-phenylenediamine, etc.), 3,3',5,5'-tetramethylbenzidine (TMB) has the advantages of high detection sensitivity and outstanding stability. Using the blue oxidation product (oxTMB) as the color signal, the visual detection of the analyte can be achieved. However, the existing colorimetric array sensors still have problems such as complex design, high construction cost, poor stability of sensing materials, limited target differentiation, and limited application range.

[0004] Polydopamine nanoparticles (PDA NPs) are an emerging nanomaterial with the advantages of simple preparation, diverse functionalization, good biocompatibility and thermal stability, and excellent physicochemical properties. Connecting biomolecules such as DNA, biothiol, peptide, antibody, etc. on the surface of PDA NPs can achieve diversified surface modification of PDA nanomaterials, making PDANPs an excellent candidate material for constructing sensing units for diverse array sensors. At present, there are no reports on the application of PDANPs in colorimetric array sensors for distinguishing and identifying phosphates. As a result, existing colorimetric array sensors for distinguishing and identifying phosphates have problems such as few types of phosphates that can be distinguished or quantified, poor stability, high detection cost, and difficulty in construction, which limits their further application. Summary of the invention

[0005] The present invention aims to solve the above technical problems existing in the prior art and provides a colorimetric array sensor for qualitative and quantitative detection of phosphate.

[0006] The technical solution of the present invention is: a colorimetric array sensor for qualitative and quantitative detection of phosphate, which is composed of three sensing units and is prepared according to the following steps: Step 1. Under alkaline conditions, N-acetyl-L-cysteine ​​polydopamine nanoparticles N-PDA NPs were prepared by Michael addition reaction of dopamine hydrochloride and N-acetyl-L-cysteine; Step 2. In a citric acid-sodium citrate buffer solution at pH = 4, N-PDA NPs solution, trivalent iron salt solution, and a mixture of TMB and H2O2 were sequentially added to obtain a total mixed solution, and the total mixed solution was diluted with double distilled water to obtain a dilution solution, wherein the final concentration of N-PDA NPs in the dilution solution was 0.5 mg / mL, and the final concentration of Fe 3+ The final concentration of the mixture of TMB and H2O2 was 200 μM, and the dilution was incubated at 25°C for 50 min to obtain the first sensor unit; Step 3. Add N-PDA NPs solution, silver salt solution, TMB and H2O2 mixture to pH 4 citric acid-sodium citrate buffer solution in sequence to obtain a total mixture, dilute the total mixture with double distilled water to obtain a dilution, wherein the final concentration of N-PDA NPs in the dilution is 0.5 mg / mL, Ag is 0.5 mg / mL, and H2O is 0.5 mg / mL. + The final concentration of was 600 μM, the final concentration of the mixture of TMB and H2O2 was 200 μM, and the dilution was incubated at 25°C for 50 min to obtain the second sensor unit; Step 4. Add N-PDA NPs solution, mercury salt solution, TMB and H2O2 mixture to pH 4 citric acid-sodium citrate buffer solution in sequence to obtain a total mixture, dilute the total mixture with double distilled water to obtain a dilution, wherein the final concentration of N-PDA NPs in the dilution is 0.5 mg / mL, and the final concentration of Hg 2+ The final concentration of was 250 μM, the final concentration of the mixture of TMB and H2O2 was 200 μM, and the dilution was incubated at 25°C for 50 min to obtain the third sensor unit.

[0007] Compared with the prior art, the beneficial effects of the present invention are as follows: when detecting phosphate, the addition of phosphate will react with PDA NPs with different metal ions complexed on the surface, and finally the complexation between metal ions and PDA NPs will be weakened to varying degrees; at the same time, -SH on the surface of PDA NPs will be exposed, and because -SH has reducing properties, oxTMB will be reduced to colorless TMB, which is manifested as a decrease in the difference in absorbance at 650 nm in the UV-visible absorption spectrum after the addition of phosphate, providing three colorimetric signal channels for distinguishing and identifying different phosphates. After experimental testing, the colorimetric array sensor of the present invention can achieve the detection of 7 phosphates (ATP, ADP, AMP, HPO4 2- 、H2PO4 - , PPi and Pi), fingerprint recognition of mixed phosphate samples of different concentrations and different ratios coexisting in physiological environments, quantitative analysis and blind sample identification of 7 phosphates. In addition, the array sensor can be applied to the monitoring of protein kinase activity involving phosphate conversion; to achieve 100% discrimination of different phosphates in complex serum environments; and to establish an intuitive judgment method for whether the phosphate content in human serum meets normal standards. The experimental results show that the colorimetric array sensor constructed by the present invention has the advantages of excellent anti-interference performance, accurate detection results, simple operation, low cost, diverse analysis targets, and a wide range of applications. It has potential application value in the fields of biochemistry and biomedical research related to enzyme catalysis involving phosphates. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 It is a transmission electron microscope image and a particle size statistical distribution diagram of N-PDA NPs in an embodiment of the present invention.

[0009] Figure 2 It is the XPS graph of N-PDA NPs of the embodiment of the present invention.

[0010] Figure 3 It is the fluorescence spectrum of N-PDA NPs of the embodiment of the present invention.

[0011] Figure 4It is the FT-IR graph of N-PDA NPs of the embodiment of the present invention.

[0012] Figure 5 The embodiment of the present invention adds metal ions and HPO4 2- FT-IR images before and after.

[0013] Figure 6 The embodiment of the present invention adds metal ions and HPO4 2- Fluorescence spectra before and after.

[0014] Figure 7 The embodiment of the present invention adds HPO4 2- UV-visible absorption spectra before and after.

[0015] Figure 8 This is a comparative diagram of the detection effects of 7 phosphates with the same concentration.

[0016] Fig. 9 It is a diagram showing the detection effect of seven phosphates with the same concentration according to an embodiment of the present invention.

[0017] Fig.10 It is a diagram showing the detection effect of different concentrations of phosphate according to an embodiment of the present invention.

[0018] Fig.11 It is a diagram showing the detection effect of a phosphate mixture according to an embodiment of the present invention.

[0019] Fig.12 This is a diagram showing the quantitative analysis results of ATP, ADP, AMP and ppi at different concentrations according to an embodiment of the present invention.

[0020] Fig.13 The present invention is an example of HPO4 at different concentrations 2- 、H2PO4 - Quantitative analysis results of Pi.

[0021] Fig.14 This is a diagram showing the test results of kinase activity in an embodiment of the present invention.

[0022] Fig.15 2 is a diagram showing the anti-interference performance analysis results of an embodiment of the present invention.

[0023] Fig.16 This is a graph showing the evaluation results of serum phosphate content of 4 healthy volunteers according to an embodiment of the present invention. DETAILED DESCRIPTION Example

[0024] A colorimetric array sensor for qualitative and quantitative detection of phosphate of the present invention is composed of three sensing units and is prepared in the following steps: Step 1. Under alkaline conditions, N-acetyl-L-cysteine ​​polydopamine nanoparticles N-PDA NPs were prepared by Michael addition reaction of dopamine hydrochloride and N-acetyl-L-cysteine; Specific method: 4 mg dopamine hydrochloride was dissolved in 2 mL Tris-HCl (pH=8.5) buffer solution at room temperature. Under magnetic stirring, 16 mmol N-acetyl-L-cysteine ​​was added after the solution color became darker. After continuous stirring for 2 h, 200 μL H2O2 (30 wt%) was injected dropwise into the above solution, and the solution was hydrothermally reacted at 50°C for 7 h. The prepared PDA NPs solution was dialyzed in double distilled water using a 500-1000Da dialysis bag for 48 h. The purified product was freeze-dried in vacuum and stored in a refrigerator (-20°C) for a long time. Step 2. Add 5 g / L N-PDA NPs solution, ferric chloride solution, and a mixture of TMB and H2O2 to a pH 4 citric acid-sodium citrate buffer solution to obtain a total mixed solution, and dilute the total mixed solution with double distilled water to obtain a dilution solution. 3+ The final concentration of the mixture of TMB and H2O2 was 200 μM, and the dilution was incubated at 25°C for 50 min to obtain the first sensor unit; Step 3. Add 5 g / L N-PDA NPs solution, silver nitrate solution, and a mixture of TMB and H2O2 to a pH=4 citric acid-sodium citrate buffer solution to obtain a total mixed solution, and dilute the total mixed solution with double distilled water to obtain a dilute solution. + The final concentration of was 600 μM, the final concentration of the mixture of TMB and H2O2 was 200 μM, and the dilution was incubated at 25 °C for 50 min to obtain the second sensor unit; Step 4. Add 5 g / L N-PDA NPs solution, mercuric nitrate solution, and a mixture of TMB and H2O2 to a citric acid-sodium citrate buffer solution at pH = 4 to obtain a total mixed solution, and dilute the total mixed solution with double distilled water to obtain a diluted solution. 2+ The final concentration of was 250 μM, the final concentration of the mixture of TMB and H2O2 was 200 μM, and the dilution was incubated at 25°C for 50 min to obtain the third sensor unit.

[0025] experiment: 1. Experimental Reagents The reagents required for the experiment are shown in Table 1.

[0026] Table 1

[0027] A 25 mL polytetrafluoroethylene-lined hydrothermal reactor was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; a dialysis bag (MW: 500-1000 Da) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and the experimental water was double distilled water.

[0028] 2. Experimental Instruments The instruments required for the experiment are shown in Table 2.

[0029] Table 2

[0030] 3. Experimental Methods When the embodiment of the present invention detects phosphate, 450 μL of each sensor unit solution is taken, and 50 μL of the phosphate solution to be tested is added respectively, and the mixture is incubated at room temperature for 50 min, and the UV-visible absorption spectrum signal is collected. Each sample is measured 4 times in parallel, and the absorbance of the system after adding each phosphate is recorded as A; Blank control: Take 450 μL of each sensor unit solution in the embodiment of the present invention, add 50 μL of buffer solution to form a system as a blank control, and record the absorbance at 650 nm as A0; The value A / A0 is used to represent the UV-visible absorption response of the system; linear discriminant analysis (LDA) is performed on the processed data.

[0031] Comparative Example: Using only three metal ions (Fe 3+ 、Ag + and Hg 2+ ) with TMB and H2O2 as the three sensing units of the colorimetric array sensor.

[0032] 3.1 Distinguishing between phosphates of the same concentration, different concentrations and different ratios Differentiation of phosphates at the same concentration: 7 phosphates (ATP, ADP, AMP, HPO4 2- 、H2PO4 - , PPi and Pi) were distinguished, and each sample was tested 4 times in parallel to obtain a data matrix of 3 sensor units × 7 analytes × 4 repetitions. The obtained data were then subjected to LDA analysis.

[0033] Differentiation of phosphates at different concentrations: Two phosphates (ATP and ADP) at 3 μM, 6 μM, and 18 μM, and three phosphates (ATP, ADP, and AMP) at 3 μM and 9 μM were differentiated. Each sample was tested four times in parallel, and the LDA method was used to differentiate and identify phosphates at different concentrations.

[0034] Differentiation of phosphates with different ratios: ATP and ADP were mixed, and the total final concentration of the two in the control system was 6 μM. The molar ratios were 0:10, 2:8, 3:7, 7:3, 8:2 and 10:0, respectively. Each sample was measured four times in parallel, and the mixed sample data were subjected to LDA analysis.

[0035] 3.2 Quantitative analysis Taking ATP as an example, spectral measurements were performed on sample systems with final ATP concentrations of 12 μM, 15 μM, 18 μM and 21 μM, respectively. Each sample was tested four times in parallel to obtain a data matrix of 3 sensor units × 4 concentrations × 4 repetitions. The processed data were subjected to LDA analysis, and a linear analysis of phosphate concentration was performed based on the value of Factor (1) obtained from the LDA analysis.

[0036] 3.3 Blind sample identification Using 6 μM samples of seven phosphates (ATP, ADP, AMP, HPO4 2- 、H2PO4 - A known training set was constructed based on the differentiation results of phosphate (PPi, PPi and Pi), and 35 unknown phosphate samples with a final concentration of 6 μM were identified. The recognition accuracy was verified by LDA analysis.

[0037] 3.4 Protein kinase activity assay Add 10 μL of 0-2.5 KU / L creatine kinase, 50 μL of 1 mM ATP, 50 μL of 1 mM creatine, and 250 μL of 100 mM HEPES buffer (pH 7.4) to a 1.5 mL centrifuge tube and dilute to 500 μL with water. Oscillate the reaction at 37°C and 120 rpm for 50 min. Take 50 μL of the reaction solution and add it to the three sensor units mentioned above. Continue the reaction for 50 min, collect the colorimetric response signal, and measure each sample 4 times in parallel. Plot the protein kinase concentration using Factor (1) of the LDA analysis results.

[0038] 3.5 Anti-interference ability test In order to evaluate the anti-interference ability of the embodiment of the present invention, its discrimination performance of 7 phosphates in a complex matrix environment was first investigated. The collected healthy human serum was diluted 100 times as a complex system, and 40 μL of the diluted serum and 7 phosphates (final concentration of 6 μM) were added to the three sensor units respectively, and the reaction continued for 50 min. The colorimetric response signal was collected using a UV-visible spectrophotometer, and each sample was measured in parallel 4 times.

[0039] Next, the effects of proteins, biothiols, and inorganic salts on the recognition of phosphate by the constructed array sensor were further investigated. Interfering substances with a final concentration of 30 μM and seven phosphates with a final concentration of 6 μM were added to the three sensor units, and the reaction was continued for 50 min. The colorimetric response signals were collected using a UV-visible spectrophotometer. Each sample was measured in parallel 4 times.

[0040] 3.6 Differentiation and identification of phosphates in real samples First, the accuracy of the present invention in detecting serum phosphate was investigated by the continuous addition method. 50 μL of serum sample diluted 100 times was added to each of the three sensor units, and 6 μM, 12 μM, and 18 μM HPO4 were added successively. 2- The solution was reacted for 50 min. The UV-visible absorption spectrum was collected by UV-visible spectrophotometer, and each sample was measured four times in parallel.

[0041] Subsequently, the serum phosphate content of different volunteers was evaluated to examine the semi-quantitative ability of the array sensor and its potential for disease diagnosis. The evaluation model was established based on the normal range of blood phosphorus content in healthy humans (lower limit: 7 μM, upper limit: 14 μM), and HPO4 2- For example, the array sensor constructed by the present invention was used to detect HPO4 with a final concentration of 7 μM and 14 μM. 2- The phosphate content in the serum samples of the volunteers was determined by LDA to determine whether it was within the normal range.

[0042] 4. Experimental Results 4.1 Characterization of polydopamine particles In order to obtain the particle size information of the prepared N-PDA NPs, TEM characterization was performed. The results are as follows: Figure 1 As shown. The particle size of N-PDA NPs is in the range of 1.6-4.4 nm, with an average particle size of 3.2 nm. N-PDA NPs have good dispersion and a relatively uniform size distribution (the particle size results are obtained by counting the particle size data of 100 N-PDA NPs using ImageJ).

[0043] The surface element composition and molecular structure of N-PDA NPs were studied by XPS characterization. Figure 2 As shown. From the full spectrum of X-ray photoelectron spectroscopy ( Figure 2A) It can be seen that there are four obvious peaks in N-PDA NPs, which correspond to C, N, O and S elements. There are three different bonding energy peaks in the high-resolution spectrum of C 1s of N-PDA NPs, among which the signal peaks at 288 eV, 285 eV and 284 eV are attributed to C=O, CO / CN and CC / CH2 / C-NH2 components, respectively ( Figure 2 B). The peak at 399 eV in the N 1s spectrum is attributed to the NH2 / CN / R2NH component ( Figure 2 C). The peaks at 532.6 eV and 531.5 eV in the high-resolution spectrum of O 1s correspond to CO and C=O / -OH groups, respectively ( Figure 2 D). The peak at 168 eV in the S 2p spectrum corresponds to the SC bond ( Figure 2 E). The above results indicate the presence of S element in N-PDA NPs, proving that N-acetyl-L-cysteine ​​has been successfully modified on the surface of polydopamine nanoparticles.

[0044] Fluorescence spectroscopy of N-PDA NPs was scanned and the results were as follows: Figure 3 According to the 3D fluorescence image ( Figure 3 A) and 2D fluorescence spectra ( Figure 3 B) It can be seen that the maximum excitation wavelength of N-PDA NPs is 330 nm, and the maximum emission wavelength is 410 nm.

[0045] In order to obtain the surface functional group information of N-PDA NPs, FT-IR characterization was performed. The results are shown in Figure 4 As shown. 3500cm -1 The peak at 2900 cm is the stretching vibration of OH in the hydroxyl group or NH in the amino group. -1 The peak at 1600 cm is the stretching vibration of the C-H bond. -1 The peak at 1300 cm is attributed to the stretching vibration of C=O and C—C bonds. -1 The peak at is the stretching vibration of the CN bond. The above results indicate that the surface of N-PDA NPs contains abundant hydroxyl and amino functional groups.

[0046] The Zeta potential of N-PDA NPs was measured by dynamic light scattering. The results showed that the charge was negative (-12.8 mV) and the absolute value was relatively high, indicating that the electrostatic repulsion between the nanoparticles was large, resulting in high stability of the prepared N-PDA NPs. At the same time, based on the potential value, it can be preliminarily inferred that electrostatic interactions may occur between metal ions and N-PDA NPs.

[0047] 4.2 Sensing mechanism research In the embodiment of the present invention and HPO4 2- Taking the system of interaction as an example, the mechanism of array sensor sensing phosphate was explored. The addition of HPO4 2- The FT-IR results of the last three sensors are as follows Figure 5 As shown in the figure, after the addition of metal ions, the 3500 cm-1 wavelength corresponding to the stretching vibration of the phenolic OH group and the amino NH group in the three sensors -1 The peak at 1620 cm -1 The corresponding peak values ​​of the in-plane bending vibration of the amino group all decreased to varying degrees, indicating that complexation occurred between the metal ions and the amino or hydroxyl groups of N-PDANPs. 2- , 3500 cm -1 and 1620 cm -1 The peak values ​​at the 2- After HPO4 2- The stronger interaction with metal ions will bring the metal ions away from the surface of N-PDA NPs, destroying the complexation between metal ions and N-PDA NPs. -1 A small peak appeared at the , which indicates that part of the N-acetyl-L-cysteine ​​in the N-PDA NPs left the surface of the N-PDA NPs along with the metal ions, resulting in the appearance of reducing -SH in the system.

[0048] From Figure 6 The fluorescence spectrum shown shows that in HPO4 2- After addition, the fluorescence intensity of N-PDA NPs, which was originally reduced due to the addition of metal ions, increased, which further proved that HPO4 2- The addition of destroys the complexation between metal ions and N-PDA NPs. Figure 7 In the UV-visible absorption spectrum shown, HPO4 2- After that, the characteristic peak absorbance at 650 nm corresponding to oxTMB decreased. Since -SH has reducing properties, oxTMB can be reduced to colorless TMB, which further proves the existence of -SH in the system.

[0049] Only three metal ions (Fe 3+ 、Ag + and Hg 2+ ) were used as the three sensing units of the colorimetric array sensor (comparative example). The recognition ability of 7 phosphates (6 μM) was as follows Figure 8As shown in the figure, the reproducibility of the results of distinguishing the same type of phosphates is poor, and there is a serious overlap between different types of phosphates. In addition, the LDA analysis results also show that the array sensor has a classification accuracy of 92.2% for the seven phosphates. Therefore, the array sensor with only metal ions as sensing units cannot completely distinguish the seven phosphates, which further shows that the N-PDA NPs-based material, Fe 3+ 、Ag + and Hg 2+ Necessity for constructing colorimetric array sensors for regulators to discriminate between target phosphates.

[0050] 4.3 Detection of phosphates at the same concentration, different concentrations and different ratios In order to test the recognition sensitivity of the colorimetric array sensor constructed in the present invention for different types of phosphates, it was used to distinguish 7 phosphates of the same concentration (3 μM, 6 μM and 18 μM) under the same experimental conditions. Each sample was measured 4 times, and the obtained data were subjected to linear discriminant analysis (LDA). The results are shown in Figure 2. Fig. 9 As shown. Fig. 9 As can be seen from B, when the final concentration of phosphate is 6 μM, the same type of phosphate is concentrated in one area, and different types of phosphate are distributed in different areas, indicating that the array sensor constructed by the present invention can completely distinguish and identify phosphate. Fig. 9 As can be seen in A, when the phosphate concentration is 3 μM, the classification accuracy still reaches 100%, but the data points overlap in the two-dimensional LDA graph, indicating that if the phosphate concentration continues to decrease, the constructed array sensor cannot achieve complete distinction of the seven phosphates. Therefore, the detection limit of the colorimetric array sensor constructed in this experiment for the recognition of the seven phosphates is 3 μM. Fig. 9 C shows that when the phosphate concentration is 18 μM, the classification accuracy is 100%, and overlap also occurs in the LDA graph. Therefore, the concentration range of phosphate recognition by the array sensor constructed in this experiment is 3~18 μM.

[0051] ATP, ADP and AMP play a vital role in cells. The three phosphates usually coexist in the cell environment and can be converted into each other under the catalysis of enzymes. Their concentration changes are closely related to various functional reactions of cells, such as energy metabolism, signal transduction, and synthesis of DNA and RNA. Therefore, the evaluation of the concentration and ratio of the three is of great significance. The colorimetric array sensor constructed in the embodiment of the present invention can distinguish different concentrations of ATP and ADP (3 μM, 6 μM and 9 μM) as shown in the figure. Fig.10 As shown. Fig.10As can be seen in A, the test points of the same phosphate with the same concentration are clustered into one area. The three concentrations of ATP and ADP are clearly distinguished. The results of distinguishing different concentrations of ATP, ADP and AMP (3 μM and 9 μM) are shown in Figure 1. Fig.10 As shown in B, two concentrations of ATP, ADP, and AMP can also achieve 100% discrimination. Therefore, the constructed array sensor has good discrimination performance for phosphate samples of different concentrations and has potential application value in the fields of biochemistry and biomedical research related to enzyme catalysis involving phosphate.

[0052] ATP and ADP were selected as coexisting components, and the final total concentration of 6 μM ATP and ADP were mixed in the ratios of 0:10, 2:8, 3:7, 7:3, 8:2 and 10:0 to obtain the detection solution. The array sensor was used to distinguish the ATP and ADP mixtures of the six ratios, and the scores of the two most significant factors were used to plot the results, as shown in Figure 2. Fig.11 As shown. The results show that the colorimetric array sensor of the present invention has obvious discrimination ability for ATP / ADP binary mixtures of different ratios. The data points of the mixed ATP / ADP sample are located between the data points of the pure ATP and ADP samples. Relatively speaking, the mixtures with high ADP content (samples: 2:8 and 3:7) are close to the data points of ADP and are located in the right half of the LDA graph; the mixtures with high ATP content (samples: 7:3 and 8:2) are close to the data points of ADP and are located in the left half of the LDA graph. It shows that the colorimetric array sensor of the present invention has good performance in distinguishing mixed phosphate samples, and the possible ATP / ADP ratio can be predicted according to the position of the data points in the LDA graph.

[0053] 4.4 Quantitative analysis Taking ATP as an example, the same phosphate at different concentrations was analyzed for identification. Fig.12 As shown. Fig.12 A shows that after LDA analysis, when the ATP concentration is in the range of 12~21 μM, different concentrations of ATP can be accurately distinguished, and there is no overlap between different concentrations. At the same time, the two-dimensional LDA graph shows that Factor (1) is 90.5%, and the value is greater than 85%, indicating that Factor (1) is the main factor affecting the distinction of different concentrations of ATP and can be used to correlate ATP concentrations. Fig.12 B shows that as the ATP concentration increases, the value of Factor (1) shows a good linear correlation with the ATP concentration in the range of 12~21 μM (R 2 =0.9906). The ability of the colorimetric array sensor of the present invention to quantitatively analyze the other six phosphates was further investigated. The results are as follows Fig.12 CH and Fig.13 As shown in the figure, it can be seen that the linear range of ADP is 3~21 μM, the linear range of AMP is 1~21 μM, the linear range of PPi is 3~18 μM, and the linear range of HPO4 2- The linear range of H2PO4 is 2~12 μM. - The linear range of is 6~18 μM, and the linear range of Pi is 3~21 μM. The above results show that the colorimetric array sensor of the present invention has the potential to quantify 7 types of phosphates. As shown in Table 3, compared with previous literature, the colorimetric array sensor of the present invention explores more comprehensive types of phosphates when performing quantitative analysis. In addition, based on practical application considerations, after the actual samples such as serum are appropriately diluted, the constructed array sensor can meet the quantitative detection requirements of phosphates therein.

[0054] Table 3

[0055] 4.5 Blind sample identification The ability of the array sensor of the embodiment of the present invention to identify unknown samples was investigated. A known training set was established using the response signals of 28 known 6 μM phosphate samples, and 35 unknown 6 μM phosphate samples were identified. The unknown phosphate types were compared with the LDA analysis results to evaluate the recognition accuracy of the constructed array sensor for unknown phosphates. As shown in Table 4, the colorimetric array sensor of the embodiment of the present invention has a recognition accuracy of 100% for unknown samples, indicating that it has good robustness in the identification and recognition of phosphates.

[0056] Table 4

[0057] 4.6 Kinase activity assay Creatine kinase (CK), a biomarker of myocardial infarction, can catalyze creatine and ATP to generate phosphocreatine and ADP. Based on the significantly different colorimetric responses of the array sensor to ATP and ADP, we were inspired to further develop a creatine kinase activity monitoring platform. The effect of the reaction of catalyzing creatine and ATP in the presence of different concentrations of creatine kinase on the colorimetric response signal of the constructed array sensor was investigated, and the obtained data were subjected to LDA analysis. Factor (1) was used to plot the creatine kinase concentration. Fig.14 As shown in Figure 2, with the increase of the final concentration of creatine kinase, the value of Factor (1) showed a good linear relationship with the concentration of creatine kinase in the range of 0.4~10 U / L (R 2 =0.9809). This indicates that the array sensor has high sensitivity and reliability in monitoring kinase activity involving phosphate changes.

[0058] 4.7 Anti-interference performance analysis Serum is a complex biological fluid containing thousands of chemical substances, which poses a challenge to the identification of phosphate by array sensors. Therefore, the invention verifies the effect of the constructed colorimetric array sensor on the identification of phosphate in healthy human serum. The results are as follows: Fig.15 shown. Fig.15 A is to add 7 kinds of phosphates with a final concentration of 6 μM to the diluted serum sample. The LDA analysis results show that the colorimetric array sensor of the embodiment of the present invention can completely distinguish the 7 kinds of phosphates, and the recognition accuracy rate reaches 100%.

[0059] Proteins, biothiols and inorganic salts are also common interfering substances in physiological analysis. In order to further evaluate the anti-interference ability of the colorimetric array sensor constructed by the present invention, the colorimetric array sensor was used to distinguish and identify 7 phosphates at 6 μM and 30 μM of interfering substances. The results are shown in 15 B. Interfering substances such as biothiols, proteins, inorganic salts (GSH, L-cys, Na2CO3, NaNO3, MgSO4, KBr, CaCl2, BSA, Hb and glucose) are distributed in different areas from the 7 phosphates, and it can be seen from the illustration that the 7 phosphates can be completely distinguished at this time. This shows that the constructed colorimetric array sensor has good specificity and discrimination ability for phosphates. Combining the above two results, it is proved that the prepared array sensor has good anti-interference ability and practical application feasibility.

[0060] 4.8 Differentiation and identification of phosphates in real samples Blood phosphorus refers to the inorganic phosphorus in the blood, mainly including H2PO4 - and HPO4 2- Its content can reflect the body's ability to metabolize and regulate phosphorus, help us understand bone metabolism and related diseases, and has important clinical guidance significance. The normal range of inorganic phosphorus in human serum is 0.74 mM-1.39 mM. If the test value is not within this range, there will be a risk of illness. The present invention uses HPO4 2- As an example, the accuracy of the array sensor of the present invention in detecting phosphate in serum was explored using the continuous standard addition method. The results are shown in Table 5. The recovery rates of the three additions were all between 87% and 99%, which shows that the array sensor has a high degree of reliability in detecting phosphate in complex body fluids.

[0061] Table 5

[0062] Subsequently, serum HPO4 2-Since the content of inorganic phosphorus in human serum is at the mM level, and the constructed array sensor detects phosphate at the μM level, the serum samples need to be diluted 1000 times in advance for semi-quantitative analysis. Four serum samples of healthy volunteers diluted 1000 times and HPO4 with a final concentration of 7 μM and 14 μM were tested. 2- Perform differentiation test, the result is as follows Fig.16 shown. Fig.16 Factor (1) was 90.3%, which was greater than 85%, indicating that Factor (1) was the main factor affecting the differentiation and could be used to correlate concentrations. 2- The contents were all within the concentration range of 7 μM-14 μM, indicating that the array sensor of the present invention has semi-quantitative ability for the phosphate content in complex body fluids, and is expected to be further applied to the model construction and intuitive diagnosis of phosphate-related diseases.

Claims

1. A colorimetric array sensor for qualitative and quantitative detection of phosphate, comprising three sensing units, characterized in that Prepare according to the following steps: Step 1. Under alkaline conditions, N-acetyl-L-cysteine ​​polydopamine nanoparticles N-PDA NPs were prepared by Michael addition reaction of dopamine hydrochloride and N-acetyl-L-cysteine; Step 2. In a citric acid-sodium citrate buffer solution at pH = 4, N-PDA NPs solution, trivalent iron salt solution, and a mixture of TMB and H2O2 were sequentially added to obtain a total mixed solution, and the total mixed solution was diluted with double distilled water to obtain a dilution solution, wherein the final concentration of N-PDA NPs in the dilution solution was 0.5 mg / mL, and the final concentration of Fe 3+ The final concentration of the mixture of TMB and H2O2 was 200 μM, and the dilution was incubated at 25°C for 50 min to obtain the first sensor unit; Step 3. Add N-PDA NPs solution, silver salt solution, TMB and H2O2 mixture to pH 4 citric acid-sodium citrate buffer solution in sequence to obtain a total mixture, dilute the total mixture with double distilled water to obtain a dilution, wherein the final concentration of N-PDA NPs in the dilution is 0.5 mg / mL, Ag is 0.5 mg / mL, and H2O is 0.5 mg / mL. + The final concentration of was 600 μM, the final concentration of the mixture of TMB and H2O2 was 200 μM, and the dilution was incubated at 25°C for 50 min to obtain the second sensor unit; Step 4. Add N-PDA NPs solution, mercury salt solution, TMB and H2O2 mixture to pH 4 citric acid-sodium citrate buffer solution in sequence to obtain a total mixture, dilute the total mixture with double distilled water to obtain a dilution, wherein the final concentration of N-PDA NPs in the dilution is 0.5 mg / mL, and the final concentration of Hg 2+ The final concentration of was 250 μM, the final concentration of the mixture of TMB and H2O2 was 200 μM, and the dilution was incubated at 25°C for 50 min to obtain the third sensor unit.

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