Method for detecting and identifying physiological phosphate by using multi-channel array sensor based on carbon dot-metal ion pair

Abstract

The invention discloses a method for detecting and identifying physiological phosphate by using a multichannel array sensor based on a carbon dot-metal ion pair, which comprises the following steps: (1) synthesis of carbon dots, including synthesis of blue light carbon dots, synthesis of green light carbon dots and synthesis of red light carbon dots; the invention relates to the technical field of physiological phosphate detection and identification. The multi-channel array sensor based on the carbon dot-metal ion pair is used for a detection and identification method of physiological phosphate, the detection method based on the array sensor comprises a plurality of cross reaction sensing units, signals of various analytes can be read out at the same time according to physicochemical property differences among the analytes with similar structures, and the detection accuracy is improved. Classification and quantification of analytes can be realized by means of pattern recognition, and the method can be used for detection and identification of various physiological phosphates in complex samples; the constructed array sensor not only can accurately detect and distinguish different physiological phosphates, but also shows excellent detection performance in the aspect of quantitative detection.

Description

Technical Field

[0001] The present invention relates to the technical field of physiological phosphate detection and identification, and in particular to a method for detecting and identifying physiological phosphate using a multi-channel array sensor based on carbon dot-metal ion pairs. Background Art

[0002] Adenosine triphosphate (ATP) is an energy molecule in cells. It participates in the metabolism of fat, protein, nucleic acid and sugar in the body and plays an important role in various life activities. ATP is composed of one adenosine molecule and three phosphate groups. When the body's metabolic process needs to consume energy, the high-energy phosphate bonds far away from adenosine are easily hydrolyzed under the catalysis of related enzymes and are hydrolyzed into adenosine diphosphate (ADP) and phosphate. ADP can be further hydrolyzed into adenosine monophosphate (AMP) and phosphate under the catalysis of enzymes. The hydrolysis process releases energy to provide cells with normal physiological activities. Various forms of physiological phosphates related to the ATP hydrolysis process play a key role in human life and health activities. Through the hydrolysis and synthesis of ATP, cells can regulate the storage and release of energy according to demand, thereby maintaining normal life activities; however, when ATP hydrolysis is unbalanced, it will lead to abnormal fluctuations in the body's physiological phosphate levels, disrupting the body's normal metabolic process, thereby inducing a series of diseases, such as renal failure, hypoglycemia, Parkinson's disease, rickets, etc. Therefore, monitoring of ATP-related physiological phosphate levels in the body is very important for preventing the occurrence and development of diseases.

[0003] The ATP hydrolysis process involves the presence of multiple physiological phosphates. Traditional methods for detecting physiological phosphates, such as high-performance liquid chromatography, electrochemical method, fluorescence method and colorimetry, can usually only detect one specific physiological phosphate, which is not conducive to detection and identification in a mixture of multiple physiological phosphates with similar structures. Summary of the invention

[0004] 1. Technical issues to be resolved In view of the deficiencies of the prior art, the present invention provides a multi-channel array sensor based on carbon dot-metal ion pairs for the detection and identification of physiological phosphate, which solves the problems mentioned in the above background technology.

[0005] (II) Technical solution To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for detecting and identifying physiological phosphate based on a multi-channel array sensor of carbon dots-metal ion pairs, specifically comprising the following steps: (1) Synthesis of carbon dots, including synthesis of blue light carbon dots, synthesis of green light carbon dots and synthesis of red light carbon dots; (2) Construction of array sensor: Use NaAc-HAc buffer to dilute the three carbon dot solutions in (1), then add metal ions of corresponding concentrations and mix well to form a multi-channel fluorescence array sensor; (3) Add physiological phosphate and observe the fluorescence changes of carbon dots.

[0006] Preferably, the synthesis method of the blue light carbon dots is as follows: first, 40 mL of ultrapure water is transferred into 40 mg of folic acid and completely dissolved, and the solution is placed in a 50 mL high pressure reactor and reacted at 180 ° C for 6 hours. After the reaction is cooled to room temperature, a yellow-brown carbon dot solution is obtained, and then the carbon dot solution is filtered with a 0.22 μm filter membrane, and dialyzed to obtain a yellow-brown aqueous solution, which is then dried to obtain a yellow solid powder.

[0007] Preferably, the synthesis method of the green light carbon dots is: first, 2 g of citric acid and 4 g of urea are mixed with 20 mL of DMF, then transferred to a 25 mL high-pressure reactor, and the reactor is placed in an oven at 160°C for 4 hours to obtain a dark brown carbon dot solution, which is then dialyzed and dried to obtain a black solid powder.

[0008] Preferably, the synthesis method of the red light carbon dots is: first, 2 g of citric acid and 4 g of urea are mixed with 20 mL of formic acid, reacted at 160° C. for 4 h in a 25 mL high pressure reactor to obtain a dark reddish brown solution, washed three times with ethanol, and then dried to obtain a black solid powder.

[0009] Preferably, the metal ions in (2) include Nd 3+ 、Ag + and Fe 3+ .

[0010] Preferably, in (3), the specific operation method is: in a 96-well plate, 150 μL of carbon dot-metal ion solution is added to each well, and then 10 μL of physiological phosphate solution and 40 μL of NaAc-HAc buffer are added thereto to make the total volume of each well 200 μL, and finally, the fluorescence intensity of different emission channels is collected using an ELISA instrument.

[0011] Preferably, the molar concentration of the NaAc-HAc buffer solution is 10 mmol / L and the pH is 5.

[0012] (III) Beneficial effects The present invention provides a method for detecting and identifying physiological phosphates using a multi-channel array sensor based on carbon dot-metal ion pairs. Compared with the prior art, the method has the following beneficial effects: the method for detecting and identifying physiological phosphates using a multi-channel array sensor based on carbon dot-metal ion pairs, the detection method based on the array sensor has multiple cross-reaction sensing units, and the signals of multiple analytes can be read out simultaneously according to the differences in the physical and chemical properties between structurally similar analytes. The classification and quantification of analytes can be achieved with the help of pattern recognition, and the method can be used for the detection and identification of multiple physiological phosphates in complex samples; the constructed array sensor can not only accurately detect and distinguish different physiological phosphates, but also show excellent detection performance in quantitative detection. In addition, the array sensor has strong anti-interference ability, high detection sensitivity, and outstanding sensing performance in serum samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The design diagram of the three-channel fluorescence array sensor for physiological phosphate detection and identification of the present invention; Figure 2 TEM images of BCDs of the present invention and corresponding particle size distribution diagram; Figure 3 TEM images of GCDs of the present invention and corresponding particle size distribution diagram; Figure 4 TEM images of RCDs of the present invention and corresponding particle size distribution diagram; Figure 5 The UV-visible absorption spectra of three carbon dots of the present invention are shown; Figure 6 The fluorescence effects of different metal ions on three carbon dots of the present invention; Figure 7 This is the effect of different physiological phosphates on the fluorescence of the three carbon dots of the present invention.

[0014] Figure 8 This is a fluorescence titration experiment result diagram of the present invention; Fig. 9 BCDs-Nd 3+ Fluorescence response spectra of the system in the presence of different concentrations of physiological phosphate; Fig.10 GCDs-Ag + Fluorescence response spectra of the system in the presence of different concentrations of physiological phosphate; Fig.11 RCDs-Fe 3+ Fluorescence response spectra of the system in the presence of different concentrations of physiological phosphate; Fig.12 The sensing performance of the array sensor of the present invention to five physiological phosphates (1 μM) was studied; Fig.13 The fluorescence response bar graph and LDA graph of the array sensor of the present invention to different concentrations of physiological phosphate; Fig.14 The sensing performance of the array sensor of the present invention to different concentrations of ATP is studied; Fig.15 The fluorescence response bar graph and LDA graph of the array sensor of the present invention to different ratios of ATP, ADP and AMP binary physiological phosphate mixture; Fig.16 The fluorescence response bar graph and LDA graph of the array sensor of the present invention to ternary physiological phosphate mixtures of ATP, ADP and AMP in different proportions; Fig.17 The fluorescence response graph and LDA graph of the sensor of the present invention to 10 μM and 50 μM of five physiological phosphates in the presence of 0.1% FBS; Fig.18 This paper studies the anti-interference ability of the array sensor of the present invention. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] In this technical scheme, ultrapure water (18.25 MΩ·cm) produced by the Millipore system was used to prepare all solutions in the experiment.

[0017] Citric acid, formic acid, N,N-dimethylformamide (DMF), ferric chloride hexahydrate, silver nitrate: Model-AR, Sinopharm Chemical Reagent Co., Ltd.; Neodymium nitrate, folic acid: Model-AR, Aladdin Shanghai Reagent Co., Ltd.; Urea, adenosine triphosphate (ATP), adenosine-5´-diphosphate sodium salt (ADP), adenosine-5´-monophosphate sodium salt (AMP), trisodium phosphate dodecahydrate (Pi), sodium pyrophosphate (PPi): Model-AR, Shanghai MacLean Biochemical Technology Co., Ltd.; UV-visible absorption spectrometer: Model-UV-2600i, Shimadzu, Japan; Fluorescence spectrometer: Model-QM40, PTI, Canada; X-ray photoelectron spectrometer (XPS): Model-K-Alpha 1063, Thermo Scientific, USA; Nanopotential system: Model-ZS90, Malvern, UK; Transmission electron microscope (TEM): model-JEOL-2100, JEOL, Japan; Fourier transform infrared spectrometer (FTIR): Model-NEXUS-870, NEXUS, USA; Microplate reader: Model-Synergy Mx, Bio Tek, USA.

[0018] The embodiment of the present invention provides the following technical solutions: Example 1 Synthesis of three types of carbon dots (CDs): Synthesis of blue light carbon dots (BCDs): First, 40 mL of ultrapure water was added to 40 mg of folic acid and completely dissolved. The solution was placed in a 50 mL autoclave and reacted at 180 ° C for 6 h. After the reaction was cooled to room temperature, a yellow-brown carbon dot solution was obtained. The carbon dot solution was then filtered with a 0.22 μm filter membrane and dialyzed to obtain a yellow-brown aqueous solution, which was then dried to obtain a yellow solid powder. Synthesis of green carbon dots (GCDs): First, 2 g of citric acid and 4 g of urea were mixed with 20 mL of DMF, then transferred to a 25 mL autoclave, and the autoclave was placed in an oven at 160 °C for 4 h to obtain a dark brown carbon dot solution, which was then dialyzed and dried to obtain a black solid powder. The synthesis of red carbon dots (RCDs) is as follows: first, 2 g of citric acid and 4 g of urea were mixed with 20 mL of formic acid, and reacted at 160 ° C for 4 h in a 25 mL high-pressure reactor to obtain a dark red-brown solution, which was washed three times with ethanol and then dried to obtain a black solid powder. The morphology and particle size of the three CDs were characterized by transmission electron microscopy (TEM), which showed that the three CDs were spherical and evenly distributed. The particle size distribution statistics showed that the particle sizes of BCDs, GCDs and RCDs were 2.7±0.4nm, 3.3±0.4nm and 4.0±0.5nm, respectively (Figure 2). Figure 2-Figure 4 as shown).

[0019] The spectral properties of the three CDs were characterized by UV-visible absorption spectra and fluorescence spectra. The UV-visible absorption spectra showed that the three CDs all had π-π* and n-π* transition absorption peaks, such as Figure 5 shown.

[0020] Example 2 Construction of array sensor: Using NaAc-HAc buffer (10mM, pH=5.0), the three carbon dot solutions were diluted, including 6μg / mL of BCDs, 20μg / mL of GCDs, and 5μg / mL of RCDs. Then, metal ions of corresponding concentrations (Nd3+: 40μM; Ag+: 100μM; Fe3+: 30μM) were added. After the three carbon dot-metal ion solutions were evenly mixed, a multi-channel fluorescence array sensor was formed; Figure 1 shown.

[0021] Example 3 Effects of different metal ions on carbon dots fluorescence: Nd 3+ 、Ag + and Fe 3+ They were mixed with three CDs solutions respectively, with the final concentration of metal ions being 100 μM. Then, QM40 was used to record the changes in CDs fluorescence intensity before and after incubation with metal ions and CDs solutions. Each group of experiments was repeated three times. Figure 6 shown.

[0022] Effects of different physiological phosphates on carbon dot fluorescence: Five physiological phosphates (ATP, ADP, AMP, Pi, PPi) were mixed with three CDs solutions, respectively, with a final concentration of 100 μM. Then, QM40 was used to record the changes in CDs fluorescence intensity before and after incubation with physiological phosphates. Each experiment was repeated three times. Figure 7 shown.

[0023] In summary, by examining the effects of different metal ions on the fluorescence of the three CDs, when different metal ions were added to the CDs solution, only Nd 3+ can significantly quench its fluorescence, while Ag + and Fe 3+ There is almost no effect on its fluorescence; similarly, for GCDs and RCDs, only Ag + and Fe 3+ It can significantly quench its fluorescence, and the other two metal ions have almost no effect on its fluorescence intensity. In the presence of physiological phosphate, the five physiological phosphates (ATPADP, AMP, Pi, PPi) have no obvious effect on the fluorescence of CDs. These experimental results lay the foundation for the construction of physiological phosphate array sensors.

[0024] Fluorescence responses of carbon dots-metal ion systems to different physiological phosphates: Towards BCDs-Nd 3+ 、GCDs-Ag + and RCDs-Fe 3+Different concentrations of five physiological phosphates (ATP, ADP, AMP, Pi, PPi) were added to the three systems respectively. After mixing, QM40 was used to record the fluorescence changes of the three fluorescence channels in the array sensor after adding different concentrations of physiological phosphates; Figure 9-11 shown.

[0025] Example 4 Fluorescence titration experiment: Add various concentrations of metal ion solutions (BCDs with Nd 3+ 、GCDs added to Ag + 、RCDs added Fe 3+ ), the fluorescence spectra of the mixture of CDs and metal ions were scanned at room temperature using a QM40 fluorescence spectrophotometer (PTI, Canada), and each set of experiments was repeated three times; Figure 8 As shown; the excitation wavelengths of BCDs, GCDs, and RCDs are 370nm, 470nm, and 560nm, respectively.

[0026] The Stern-Volmer equation was used to calculate the binding constants between the three CDs and the three metal ions (the results are shown in Table 1). The calculation formula is as follows: , in, and F represent the fluorescence intensity of CDs before and after the addition of metal ions, respectively; represents the Stern-Volmer quenching constant; Indicates the concentration of metal ions.

[0027] Table 1

[0028] Example 5 The array sensor was used to identify and differentiate different physiological phosphates: 150 μL of carbon dot-metal ion solution was added to each well of a 96-well plate, followed by 10 μL of physiological phosphate solution and 40 μL of NaAc-HAc buffer (10 mmol / L, pH=5.0) to make the total volume of each well 200 μL. Finally, the fluorescence intensity of different emission channels was collected using an ELISA reader; each set of experiments was repeated five times.

[0029] For the detection of different concentrations of physiological phosphate, five physiological phosphate solutions with a series of concentrations (final concentrations were: 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) were prepared respectively, and then the constructed array sensor was used to detect the five physiological phosphates with different concentrations, and each group of experiments was repeated five times. Fig.12 and Fig.13 shown.

[0030] Example 6 Identification and differentiation of different concentrations of ATP by array sensors: For the detection of different concentrations of ATP, a series of ATP solutions were prepared (the final concentrations were: 0.1μM, 1μM, 3μM, 5μM, 8μM, 10μM, 15μM, 20μM, 50μM), and the group without ATP was used as the control group. The constructed array sensor was used to detect it, and the data were processed and analyzed by LDA. Each group of experiments was repeated five times. Fig.14 shown.

[0031] Example 7 The array sensor was used to detect and distinguish binary physiological phosphate mixtures: first, a series of binary physiological phosphate mixture samples with different ratios (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0) were prepared with ATP and ADP, ATP and AMP, and ADP and AMP, respectively. Then, the constructed array sensor was used to detect binary physiological phosphate mixtures with different ratios. The final concentration of the physiological phosphate mixture was 10 μM, and each group of experiments was repeated five times; Fig.15 shown.

[0032] Example 8 The array sensor was used to detect and distinguish ternary physiological phosphate mixtures: a series of ternary physiological phosphate mixture samples with different ratios were prepared with ATP, ADP and AMP (1:1:8, 2:2:6, 3:3:4, 4:4:2, 1:8:1, 2:6:2, 3:4:3, 4:2:4, 8:1:1, 6:2:2, 4:3:3, 2:4:4), and then the constructed array sensor was used to detect the ternary physiological phosphate mixture samples with different ratios, where the final concentration of the physiological phosphate mixture was 10 μM, and each group of experiments was repeated five times; Fig.16 shown.

[0033] Example 9 Array sensor detects physiological phosphate in serum samples: 1) Prepare five physiological phosphate solutions containing fetal bovine serum (FBS) and FBS solution without physiological phosphate; 2) In a 96-well plate, the blank group, FBS group and five physiological phosphate groups containing FBS were detected using an array sensor, where the final concentration of FBS was 0.1% (V:V), the final concentration of physiological phosphate was 10 μM, and each group of experiments was repeated five times; 3) Prepare a series of solutions with the same operation, in which the final concentration of FBS is 1% (V:V) and the final concentration of physiological phosphate is 50 μM. Use the constructed array sensor to detect the blank group, 1% FBS group and five physiological phosphate groups containing 1% FBS. Repeat each experiment five times. Fig.17 shown.

[0034] Example 10 Evaluation of the anti-interference ability of array sensors: Ca 2+ , K + 、Na + , Cl - 、SO4 2- ,Ac - , urea (Urea), glucose (Glu), ascorbic acid (AA), glycine (Gly), tryptophan (Try), proline (Pro), aspartic acid (Asp) solution as interfering substances; at the same time, five physiological phosphate solutions (ATP, ADP, AMP, Pi, PPi) were prepared respectively, and the constructed array sensor was used to detect the interfering substances and five physiological phosphates, and the data were processed and analyzed by LDA and HCA. 2+ , K + 、Na + , Cl - 、SO4 2- ,Ac - The final concentration was 50 μM, other interfering substances (Glu, Urea, AA, Gly, Try, Pro, Asp) were added at a final concentration of 10 μM, and the final concentration of the five physiological phosphates was 10 μM. Each group of experiments was repeated five times; Fig.18 shown.

[0035] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0036] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0037] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting and identifying physiological phosphate using a multi-channel array sensor based on carbon dots-metal ion pairs, characterized in that: The specific steps include: (1) Synthesis of carbon dots, including synthesis of blue light carbon dots, synthesis of green light carbon dots and synthesis of red light carbon dots; (2) Construction of array sensor: Use NaAc-HAc buffer to dilute the three carbon dot solutions in (1), then add metal ions of corresponding concentrations and mix well to form a multi-channel fluorescence array sensor; (3) Add physiological phosphate and observe the fluorescence changes of carbon dots.

2. The method for detecting and identifying physiological phosphate using a multi-channel array sensor based on carbon dots-metal ion pairs according to claim 1, characterized in that: The synthesis method of the blue light carbon dots is as follows: first, 40 mL of ultrapure water is added to 40 mg of folic acid and completely dissolved, and the solution is placed in a 50 mL high-pressure reactor and reacted at 180° C. for 6 hours. After the reaction is cooled to room temperature, a yellow-brown carbon dot solution is obtained. The carbon dot solution is then filtered with a 0.22 μm filter membrane and dialyzed to obtain a yellow-brown aqueous solution, which is then dried to obtain a yellow solid powder.

3. The method for detecting and identifying physiological phosphate using a multi-channel array sensor based on carbon dots-metal ion pairs according to claim 1, characterized in that: The synthesis method of the green light carbon dots is as follows: first, 2 g of citric acid and 4 g of urea are mixed with 20 mL of DMF, and then transferred to a 25 mL high-pressure reactor, and the reactor is placed in an oven at 160° C. for reaction for 4 hours to obtain a dark brown carbon dot solution, which is then dialyzed and dried to obtain a black solid powder.

4. The method for detecting and identifying physiological phosphate using a multi-channel array sensor based on carbon dots-metal ion pairs according to claim 1, characterized in that: The synthesis method of the red light carbon dots is as follows: first, 2 g of citric acid and 4 g of urea are mixed with 20 mL of formic acid, and reacted at 160° C. for 4 h in a 25 mL high pressure reactor to obtain a dark red-brown solution, which is washed three times with ethanol and then dried to obtain a black solid powder.

5. The method for detecting and identifying physiological phosphate using a multi-channel array sensor based on carbon dots-metal ion pairs according to claim 1, characterized in that: The metal ions in (2) include Nd 3+ 、Ag + and Fe 3+ .

6. The method for detecting and identifying physiological phosphate using a multi-channel array sensor based on carbon dots-metal ion pairs according to claim 1, characterized in that: In (3), the specific operation method is as follows: in a 96-well plate, 150 μL of carbon dot-metal ion solution is added to each well, and then 10 μL of physiological phosphate solution and 40 μL of NaAc-HAc buffer are added thereto to make the total volume of each well 200 μL. Finally, the fluorescence intensity of different emission channels is collected using an ELISA reader.

7. The method for detecting and identifying physiological phosphate using a multi-channel array sensor based on carbon dots-metal ion pairs according to claim 1, characterized in that: The molar concentration of the NaAc-HAc buffer solution is 10 mmol / L, and the pH is 5.

Images