A method for rapid visual detection of phytic acid based on water-soluble perylene imide derivatives, application and kit

Abstract

The application relates to the field of phytic acid detection, and discloses a rapid detection method, application and kit based on a perylene imide derivative and a trivalent iron ion, and specifically discloses a method for detecting phytic acid, wherein a perylene imide derivative with a structure shown in formula (I) and a trivalent iron ion are used as probes; the perylene imide derivative has good water solubility and can realize high-sensitivity and specific detection of phytic acid; the detection method provided by the application has high detection efficiency and sensitivity, has the characteristics of visualization, and is convenient to apply.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection technology, and particularly relates to a rapid visual detection method for phytic acid based on water-soluble perylene imide derivatives, application and kit. BACKGROUND

[0002] Phytic acid (PA) is also known as myo-inositol hexakisphosphate, which is widely distributed in cereals, soybeans, fruits and vegetables and is the main form of phosphorus. It has strong reducing power and is a natural antioxidant, which has various applications in food production. Phytic acid can form coordination complexes with various metal cations, thereby affecting the normal physiological functions of metal ions in the body, and it also participates in many cellular functions such as RNA export, DNA repair, insulin secretion and innate immunity. Studies have shown that phytic acid plays a role in reducing the risk of diseases such as diabetes, cancer, heart disease and kidney stones.

[0003] Due to the application of phytic acid in food industry, biology, medicine and other fields, in order to accurately understand the role of phytic acid and provide accurate and effective information, it is necessary to qualitatively and quantitatively detect phytic acid. At present, there are many methods for detecting phytic acid, such as high performance liquid chromatography, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, titration, electrophoresis and Raman spectroscopy. However, these methods still have the shortcomings of complex detection methods, tedious processing process and the need for expensive equipment. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a rapid detection method for phytic acid based on water-soluble perylene imide derivatives, application and kit, which overcomes the defects of complex sample preparation, expensive equipment and poor sensitivity in the existing methods for detecting phytic acid.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] Firstly, the present application provides the application of the perylene imide derivative represented by formula (I) and the ferric ion probe in detecting phytic acid:

[0007]

[0008] The perylene imide derivative represented by formula (I) has good water solubility and can realize high sensitivity and specific detection of phytic acid, and has obvious specificity and anti-interference ability in detection.

[0009] The perylene imide derivative represented by the structure of formula (I) of the present application exhibits different states to external stimuli in the mixed solution with ferric ion, thereby causing changes in optical properties and fluorescence quenching phenomenon. Meanwhile, the perylene imide derivative and ferric ion are used for detection of phytic acid, and the fluorescence response effect can be achieved, i.e., the fluorescence of the perylene imide derivative is re-opened, and the fluorescence opening effect under ultraviolet light irradiation is visible to the naked eye.

[0010] In addition, the detection limit of the perylene imide derivative and ferric ion probe for phytic acid is as low as 0.185 μmol / L, and the visualization effect in the solution is obvious.

[0011] The perylene imide derivative and ferric ion probe provided by the present application has excellent phytic acid detection performance, which may be based on the following principle: the perylene imide derivative and ferric ion of the present application can form a complex through multiple non-covalent interactions in the solution, and phytic acid can displace ferric ion from the perylene imide derivative, resulting in changes in the fluorescence signal of the probe.

[0012] The source of the perylene imide derivative represented by the structure of formula (I) is not particularly limited, and can be generally commercially available or prepared according to methods well known to those skilled in the art.

[0013] Secondly, the present application provides a reagent, test paper or kit for detecting phytic acid, comprising the above-mentioned perylene imide derivative and ferric ion.

[0014] Thirdly, the present application provides a rapid visual detection method based on water-soluble perylene imide derivative. Specifically, the present application provides a method for detecting phytic acid, using the above-mentioned perylene imide derivative and ferric ion as a probe.

[0015] Preferably, the concentration of the probe is 0.2-3 μmol / L of the perylene imide derivative and 30-150 μmol / L of ferric ion. The concentration of the perylene imide derivative is further preferably 1 μmol / L, and the concentration of the ferric ion is further preferably 50 μmol / L.

[0016] The specific detection method of the present application is preferably as follows: the above probe concentration is 1 μmol / L of perylene imide derivative and 50 μmol / L of ferric ion in 10 mmol / L Tris-HCl buffer solution with pH=7.0 for the spectral test of phytic acid, the excitation wavelength is 485 nm during detection, and the slit width of excitation light and emission light is 5.0 nm. The fluorescence intensity at the emission wavelength of 546 nm is selected to calculate the fluorescence change ratio of the probe, and the standard curve for detection is established by taking the phytic acid concentration as the abscissa and the fluorescence ratio of the probe as the ordinate. The above probe concentration is 1 μmol / L of perylene imide derivative and 50 μmol / L of ferric ion in 10 mmol / L Tris-HCl buffer solution with pH=7.0 for the visual detection of phytic acid, and the probe generates fluorescence that can be recognized by naked eyes under the irradiation of ultraviolet lamp with 365 nm.

[0017] Compared with the prior art, the present application provides a rapid detection method based on water-soluble perylene imide derivative, taking perylene imide derivative with the structure shown in formula (I) and ferric ion as the probe. The above perylene imide derivative has good water solubility and can realize the specific detection of phytic acid with high sensitivity. The detection method provided by the present application has high detection efficiency and sensitivity, and has the characteristics of visualization, and is convenient to apply. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0019] Figure 1 The fluorescence spectrum (λ ex =485 nm, λ em =505-750 nm) of the probe (perylene imide derivative 1 μmol / L, ferric ion 50 μmol / L) after adding different concentrations of phytic acid in Tris-HCl buffer solution (10 mmol / L pH=7.0);

[0020] Figure 2 The fluorescence recovery degree (λ ex =485 nm, λ em =546 nm) of the probe (perylene imide derivative 1 μmol / L, ferric ion 50 μmol / L) after adding different concentrations of phytic acid in Tris-HCl buffer solution (10 mmol / L pH=7.0), Figure 2 The inset is the photo under the irradiation of ultraviolet lamp with 365 nm before and after adding phytic acid;

[0021] Figure 3 The fluorescence recovery degree (λ ex = 485 nm, λ em = 546 nm) of the probe (perimide derivative 1 μmol / L, ferric ion 50 μmol / L) in Tris-HCl buffer (10 mmol / L pH = 7.0) in the presence of phytic acid (5 μmol / L) and other interfering substances (50 μmol / L) (Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , Cl - , SO4 2- , methionine (Met), lysine (Lys), alanine (Ala), glutamic acid (Glu), glucose (Glucose));

[0022] Figure 4 The photo of the probe solution (perimide derivative 5 μmol / L, ferric ion 250 μmol / L) under the irradiation of 365 nm ultraviolet light after the addition of phytic acid in Tris-HCl buffer (10 mmol / L pH = 7.0) at different concentrations;

[0023] Figure 5 The photo of the probe (perimide derivative 5 μmol / L, ferric ion 250 μmol / L) in Tris-HCl buffer (10 mmol / L pH = 7.0) in the presence of phytic acid (50 μmol / L) and other interfering substances (500 μmol / L) (Na + , K + , Ca 2+ , Mg 2 + , Zn 2+ , Cl - , SO4 2- , methionine (Met), lysine (Lys), alanine (Ala), glutamic acid (Glu), glucose (Glucose)) under the irradiation of 365 nm ultraviolet light;

[0024] Figure 6 The relationship between the R value of the probe solution (perimide derivative 5 μmol / L, ferric ion 250 μmol / L) under the irradiation of 365 nm ultraviolet light and the concentration of phytic acid after the addition of phytic acid in Tris-HCl buffer (10 mmol / L pH = 7.0) at different concentrations;

[0025] Figure 7The relationship between the G value of the probe solution (perylene imide derivative 5 μmol / L, trivalent iron ion 250 μmol / L) under the irradiation of 365 nm ultraviolet lamp and the concentration of phytic acid was investigated when different concentrations of phytic acid were added to Tris-HCl buffer solution (10 mmol / L, pH=7.0). DETAILED DESCRIPTION

[0026] In order to further illustrate the present application, the rapid visual detection method of phytic acid based on water-soluble perylene imide derivative provided by the present application is described in detail below in combination with examples.

[0027] Preparation of standby substances:

[0028] Preparation of buffer solution: weigh the solid trimethylaminomethane, and prepare 500 mL of Tris-HCl buffer solution with a concentration of 10 mmol / L using distilled water, and adjust the pH value to 7.0 using 1 mol / L hydrochloric acid standard solution. Store it in a 4°C refrigerator for standby.

[0029] Preparation of perylene imide derivative mother liquor: weigh the solid shown in formula (I), and prepare a mother liquor with a concentration of 1 mmol / L using distilled water, and divide it into solutions with the same volume in vials for standby. Dilute to a certain concentration using the prepared Tris-HCl buffer solution (10 mmol / L, pH=7.0) for testing.

[0030] Preparation of trivalent iron ion mother liquor: weigh the solid anhydrous ferric chloride, and prepare a mother liquor with a concentration of 10 mmol / L using distilled water, and divide it into solutions with the same volume in vials for standby. Dilute to a certain concentration using the prepared Tris-HCl buffer solution (10 mmol / L, pH=7.0) for testing.

[0031] Preparation of test substances and interferents: prepare a phytic acid stock solution with a concentration of 1 mmol / L using distilled water for standby. Prepare other interferent stock solutions with a concentration of 10 mmol / L using distilled water. Store these solutions in a 4°C refrigerator for standby.

[0032] Preparation of actual samples: fresh oranges are purchased at a local fruit and vegetable market, and after peeling, juice is extracted using a juicer, and the filtrate is obtained by filtering through gauze. Dilute 10 times with distilled water, and filter through a 0.22 μm microporous filter membrane. Dilute 50 times with Tris-HCl buffer solution, and then perform the experiment.

[0033] Example 1

[0034] Test of fluorescence spectra :

[0035] 1 μL of the perylene imide derivative mother liquor, 5 μL of the anhydrous ferric chloride mother liquor and 994 μL of 10 mmol / L Tris-HCl buffer were mixed and added into 1 mL of a sample cell, after mixing, the fluorescence spectrum of the probe buffer was measured, then a certain concentration gradient of phytic acid was gradually added into the sample cell, after mixing, the corresponding fluorescence spectrum was measured.

[0036] The results are shown in Table 1. Figure 1 Figure 1 The fluorescence spectrum (λ ex =485 nm, λ em =505-750 nm) of the probe (perylene imide derivative 1 μmol / L, trivalent iron ion 50 μmol / L) after adding different concentrations of phytic acid in Tris-HCl buffer (10 mmol / L pH=7.0) is shown in Table 1.

[0037] Example 2

[0038] Qualitative detection method establishment

[0039] The fluorescence intensity I0 and I at 546 nm before and after adding different concentrations of phytic acid were tested by the fluorescence spectrum of Example 1, and the fluorescence opening degree was represented by I / I0.

[0040] The results are shown in Table 2. Figure 2 Figure 2 The fluorescence opening degree (λ ex =485 nm, λ em =546 nm) of the probe (perylene imide derivative 1 μmol / L, trivalent iron ion 50 μmol / L) after adding different concentrations of phytic acid in Tris-HCl buffer (10 mmol / L pH=7.0) is shown in Table 2.

[0041] The experimental results show that the aforementioned perylene imide derivative and trivalent iron ion probe are excited at an excitation wavelength of 485 nm, emitting an emission peak at 546 nm, with the addition of phytic acid, the fluorescence emission intensity of the aforementioned perylene imide derivative gradually increases, when the phytic acid concentration is added to 1 μmol / L, the fluorescence ratio I / I0 of the aforementioned perylene imide derivative and trivalent iron ion probe is more than 7. And the fluorescence ratio I / I0 has a good linear correlation with the concentration of phytic acid, the correlation coefficient R 2 =0.990, according to the detection limit calculation method, the detection limit of the probe for phytic acid is 0.185 μmol / L.

[0042] Figure 2 ​​​The middle figure is the photo under 365 nm ultraviolet light before and after adding phytic acid. It can be seen that after adding phytic acid, the fluorescence of the corresponding solution is turned on.

[0043] Example 3

[0044] Selective study:

[0045] Common interferents for detecting phytic acid were selected, Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , Cl - , SO4 2- , methionine (Met), lysine (Lys), alanine (Ala), glutamic acid (Glu), glucose (Glucose). In the test, the concentration of the aforementioned perylene imide derivative was 1 μmol / L, the concentration of ferric ion was 50 μmol / L, the concentration of phytic acid was 5 μmol / L, and the concentration of all interferents was 50 μmol / L. The fluorescence spectrum was tested under the same test conditions. The fluorescence quenching degree of the probe at 546 nm before and after adding the interferents was taken as the parameter for measuring the influence degree of the aforementioned probe on the measured substance.

[0046] The results are shown in Table 1 (that is, Figure 3 Figure 3 , which represents the fluorescence recovery degree (λ ex = 485 nm, λ em = 546 nm) of the probe (perylene imide derivative 1 μmol / L, ferric ion 50 μmol / L) in Tris-HCl buffer (10 mmol / L pH = 7.0) in the presence of phytic acid (5 μmol / L) and other interfering substances (50 μmol / L).

[0047] As can be seen from the figure, the I / I0 of all other substances except phytic acid is approximately 1, and the I / I0 of phytic acid is > 40, which is much higher than that of other compounds. This result shows that the aforementioned probe has excellent selectivity for phytic acid.

[0048] Example 4

[0049] Visual detection: The concentration of the aforementioned probe perylene imide derivative was 5 μmol / L, the concentration of ferric ion was 250 μmol / L, and different concentrations of phytic acid were added.

[0050] Figure 4 The figure is the photo under 365 nm ultraviolet light with the addition of 0-60 μmol / L phytic acid. With the increase of the concentration of phytic acid, the fluorescence of the responding solution is gradually turned on, and increases to bright yellow. The visual detection limit is 30 μmol / L.​

[0051] Example 5

[0052] Visual specific detection: In order to more intuitively observe the effect of the aforementioned phthalimide derivative ferric ion probe after interacting with phytic acid and other interferents, the fluorescence change of the aforementioned phthalimide derivative in aqueous solution during the selective investigation of phytic acid was monitored under 365 nm ultraviolet light irradiation.

[0053] Phytic acid and the aforementioned interfering substances, such as Figure 5 As shown in the figure, when the aforementioned phthalimide derivative 5 μmol / L, the concentration of ferric ion in the probe was 250 μmol / L, and the concentration of the interfering substance was 500 μmol / L, the fluorescence of the solution did not change, but when 50 μmol / L of phytic acid was added, the fluorescence of the solution recovered obviously.

[0054] Example 6

[0055] Application of real samples :

[0056] In order to verify the feasibility of the detection method in actual samples, fresh orange juice was selected for the determination of the recovery rate, as shown in Table 1.

[0057] Table 1 Application of actual samples in fresh orange juice

[0058]

[0059] After adding 0.1 μmol / L, 0.2 μmol / L and 0.4 μmol / L of phytic acid, the recovery rates were 99.77%, 93.38% and 97.68% (RSD < 3%), respectively, as shown in the table below. The results show that the method has good accuracy and can be applied to the detection of phytic acid in orange juice.

[0060] Example 7

[0061] Application of visual quantitative detection :

[0062] In order to verify the visual quantitative detection effect of the detection method, the aforementioned probe phthalimide derivative concentration 5 μmol / L, ferric ion concentration 250 μmol / L, and different concentrations of phytic acid were added under 365 nm ultraviolet light irradiation. The color of the photo was taken, as shown in Figures 6-7 The R value and G value obtained have good linear relationship with the concentration of phytic acid, and the correlation coefficients R 2 are 0.993 and 0.989, respectively.

[0063] From the above results, the perylene imide derivative of the structure represented by formula (I) has good water solubility, and can realize high sensitivity and specific detection of phytic acid.

[0064] The above only describes the preferred embodiments of the present application, and it should be pointed out that for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Application of the perylene imide derivative and ferric ions shown in formula (I) in the detection of phytic acid; The detection limit for phytic acid is 0.185 μmol / L.

2. A method for detecting phytic acid, characterized in that, The perylene imide derivative and ferric ions shown in formula (I) were used as probes; The detection limit for phytic acid is 0.185 μmol / L.

3. The method according to claim 2, characterized in that, The concentration of the perylene imide derivative shown in formula (I) is 0.5-3.0 μmol / L, and the concentration of the ferric ions is 30-150 μmol / L.

4. A reagent, test strip, or kit for detecting phytic acid, characterized in that, Including the perylene imide derivative shown in formula (I) and ferric ions;

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