CN113655045A - Uric acid detection method and application thereof - Google Patents
Uric acid detection method and application thereof Download PDFInfo
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- CN113655045A CN113655045A CN202111137988.2A CN202111137988A CN113655045A CN 113655045 A CN113655045 A CN 113655045A CN 202111137988 A CN202111137988 A CN 202111137988A CN 113655045 A CN113655045 A CN 113655045A
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- uric acid
- urine
- solution
- detecting
- concentration
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- 229940116269 uric acid Drugs 0.000 title claims abstract description 218
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 title claims abstract description 214
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention belongs to the field of biochemical technology detection, and particularly relates to a uric acid detection method and application thereof. According to the method, the guanidine-based calix [5] arene and the fluorescein sodium are prepared into a mixed solution, a sample to be detected is added into the mixed solution, and the concentration of uric acid in the sample to be detected is detected through the change of a fluorescence signal. The detection method is hardly influenced by other interference components in urine, and can be used for detecting uric acid in urine; and the detection of uric acid can be realized through the fluorescence change of the sample under ultraviolet rays (366nm), so that the method is applied to the development of a portable and visual noninvasive uric acid detection method.
Description
Technical Field
The invention belongs to the field of biochemical technology detection, and particularly relates to a uric acid detection method and application thereof.
Background
Hyperuricemia is a disease caused by an excess of serum uric acid concentration due to insufficient uric acid excretion or/and kidney overload. Hyperuricemia in blood is a key risk factor for causing urate deposition and inducing gout. In addition, hyperuricemia is closely related to kidney diseases, cardiovascular diseases, diabetes and other diseases. Uric acid is a product of purine metabolism in the human body, and two thirds of uric acid is discharged out of the body through urine. In the body fluid sample detected, urine has the advantages of high biological information integration level, noninvasive sample collection, low biological risk and the like, and is the first choice biological sample for early diagnosis of various metabolic diseases.
At present, methods for detecting uric acid in urine include an electrochemical method, a fluorescence spectrometry method, a high performance liquid chromatography, a liquid chromatography-mass spectrometry combined method and the like. The methods have higher selectivity and sensitivity, but have the defects of complex sample pretreatment, complex operation, long detection time, high cost and the like. Therefore, the development of a rapid, simple, low-cost and high-sensitivity method for detecting uric acid in urine is urgently needed, and the method has important significance for early warning of hyperuricemia.
Disclosure of Invention
Aiming at the technical problems, the invention provides a method for detecting uric acid and application thereof, which realize the detection of uric acid in urine based on an Indicator Displacement (IDA) strategy.
In order to solve the above technical problems, a first aspect of the present invention provides a method for detecting uric acid, comprising: preparing mixed solution of guanidine-based calix [5] arene and fluorescein sodium, adding a sample to be detected into the mixed solution, and detecting the concentration of uric acid in the sample to be detected through the change of a fluorescence signal.
Compared with the prior art, the invention takes guanidino calix [5] arene (GC5A) and fluorescein sodium (Fl) as a host-guest pair, the added uric acid and the Fl compete to bind with a cavity of GC5A, the Fl is displaced from a GC5A Fl host-guest complex, the Fl recovers the luminescence property in the solution, and the fluorescence signal is sensitized (Switch-on). The simple, convenient and efficient detection of the uric acid can be realized through the linear relation between the fluorescence intensity and the uric acid concentration. Wherein the guanidino calix [5] arene is 5,11,17,23, 29-pentaguanidino-31, 32,33,34, 35-penta (4-methylpentanyloxy) calix [5] arene and is calix [5] arene with guanidino modified upper edge.
In combination with the first aspect, the final concentrations of guanidinylcalixarene and fluorescein sodium are 0.8 μmol/L and 1 μmol/L, respectively, when uric acid concentration is detected in HEPES buffer.
The second aspect of the invention provides the application of the detection method in detecting the concentration of uric acid in urine. The detection method is hardly influenced by other interference components in urine, and can be used for detecting uric acid in urine.
In combination with the second aspect, the following procedure can be used for detecting uric acid in urine by the above-mentioned detection method: taking artificial urine, centrifuging, collecting supernatant, diluting with HEPES buffer solution, mixing with a solution containing guanidino calix [5] arene and fluorescein sodium, gradually dropping uric acid solution with the artificial urine as a solvent, measuring the fluorescence intensity of the fluorescein sodium, establishing a linear relation of uric acid detection by using the uric acid concentration and the fluorescence intensity value of the fluorescein sodium, and detecting the uric acid concentration in the urine by using the linear relation.
Preferably, the concentration range of uric acid in the uric acid solution with the artificial urine as the solvent is 0-50 mu mol/L. Within this concentration range, the uric acid concentration and the fluorescence intensity have a good linear relationship.
Preferably, in the solution containing the guanidine-based calix [5] arene and the sodium fluorescein, the concentrations of the guanidine-based calix [5] arene and the sodium fluorescein are 8 mu mol/L and 4 mu mol/L respectively.
The third aspect of the invention provides the application of the detection method in uric acid concentration detection by using a shooting tool. When the detection method is used for detecting the uric acid concentration by means of a shooting tool, the G value of the uric acid concentration can be extracted through the change of fluorescence generated by ultraviolet (366nm) irradiation through color processing, and the linear relation between the G value and the uric acid concentration is established, so that the portable and visual detection of the uric acid concentration in the urine is realized.
With reference to the third aspect, the detection method for uric acid using the shooting tool may include the following steps:
preparing a GC 5A-F1 host-guest pair mixed solution by using a HEPES buffer solution, and adding different amounts of uric acid to obtain a series of uric acid solutions with concentration; irradiating the uric acid solution under 366nm ultraviolet rays, photographing by using a photographing tool, extracting a green intensity value (G value) of the obtained picture, and establishing a linear relation of uric acid detection by using the uric acid concentration and the G value; and detecting the uric acid concentration in the sample to be detected by utilizing the linear relation.
When the detection method is used for detecting the uric acid concentration of urine by using a shooting tool, the following operations can be adopted:
taking real urine, centrifuging and collecting supernatant, adding HEPES buffer solution for dilution to obtain diluent, adding mixed solution of guanidine-based calix [5] arene and fluorescein sodium and different amounts of uric acid into the diluent, and uniformly mixing to obtain a series of uric acid solutions with concentration; irradiating the uric acid solution under 366nm ultraviolet rays, taking a picture by using a shooting tool under the same condition, extracting the G value of the obtained picture, and establishing a linear relation of uric acid detection by using the uric acid concentration and the G value; and detecting the uric acid concentration in the urine by using the linear relation.
The photographing function can be realized by a digital camera and a smart phone, the G value of the picture can be acquired by using image processing software after photographing, and the portable and visual detection of uric acid can be realized so as to be suitable for the analysis of urine samples of different people.
Preferably, the concentration of uric acid in the uric acid solution is 0-0.8 mmol/L, and within the concentration range, the uric acid concentration and the G value have a good linear relationship.
Preferably, the concentrations of the guanidine-based calix [5] arene and the fluorescein sodium in the uric acid solution are 10 mu mol/L and 5 mu mol/L respectively.
The uric acid detection method provided by the invention has the characteristics of high sensitivity, high selectivity, rapidness and simplicity, is hardly influenced by other interference components in urine, and can simply, conveniently and efficiently detect the concentration of uric acid in urine. By using the detection method, the detection of the uric acid can be realized through the fluorescence change of the sample under ultraviolet rays (366nm), the G value of the sample is extracted through color processing, and the linear relation between the uric acid concentration and the G value is established, so that the method is applied to the development of a portable and visual noninvasive uric acid detection method.
Drawings
FIG. 1 is a fluorescence spectrum of Fl (1. mu. mol/L) in example 1 showing that the fluorescence intensity increases with the uric acid concentrationGraph (lambda)ex＝500nm，λem＝505–650nm)；
FIG. 2 shows the trend of GC 5A. Fl (0.80. mu. mol/L/1.00. mu. mol/L) host-guest in example 2 in terms of the change of fluorescence intensity with the increase of uric acid concentration (. lamda.)ex＝500nm，λem505 and 650 nm); a is the change of a Fl fluorescence emission spectrum curve along with the increase of uric acid concentration (0-119.88 mu mol/L); b is a non-linear fitting curve (lambda) of Fl fluorescence intensity and uric acid concentrationem＝513nm)；
FIG. 3 is a graph showing the ratio of fluorescence intensity (I/I) in HEPES buffer in example 30) Linear fitting to uric acid concentration;
FIG. 4 is a view of the selective examination in example 4; a is Fl fluorescence intensity change when uric acid and an interferent are added into HEPES buffer solution; b is the ratio of fluorescence intensity (I/I) in artificial urine0) Linear fitting to uric acid concentration;
FIG. 5 is a graph showing the ratio of fluorescence intensity (I/I) in real urine of the volunteers in example 40) Linear fitting to uric acid concentration;
FIG. 6 shows the results of the detection of uric acid in real urine samples of example 4;
FIG. 7 is the visual detection procedure and the linear fit of the G values in HEPES buffer and real urine to the uric acid concentration in example 5;
in the above figures, I0And I as the fluorescence intensity without and with the added analyte, respectively.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Uric acid is a product of purine metabolism in the human body, and two thirds of uric acid is discharged out of the body through urine. The uric acid rise is closely related to gout, kidney diseases, cardiovascular diseases, diabetes and other diseases. At present, methods for detecting uric acid in urine comprise electrochemistry, fluorescence spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry and the like, and the detection methods have high selectivity and sensitivity, but have the defects of complex sample pretreatment, complex operation, long detection time, high cost and the like. Therefore, the development of a rapid, simple, low-cost and high-sensitivity method for detecting uric acid in urine is urgently needed, and the method has important significance for early warning of hyperuricemia.
In order to detect uric acid quickly, simply and at low cost, the embodiment of the invention provides a method for detecting uric acid, which comprises the following steps: preparing GC5A and Fl into a mixed solution, adding a sample to be detected into the mixed solution, and detecting the concentration of uric acid in the sample to be detected through the change of a fluorescence signal. GC5A belongs to a macrocyclic molecule with a cavity that can be used as a receptor to selectively bind guest molecules (F1, uric acid) to form a complex. GC5A as a host reversibly binds to guest Fl, resulting in a decrease in Fl fluorescence intensity (Switch-off) based on the principle of Indicator Displacement Analysis (IDA); when uric acid serving as an analyte is introduced into the complex system as a competitive object, uric acid can compete Fl from the macrocyclic host to show fluorescence signal sensitization (Switch-on), and the uric acid can be sensed and detected according to the correlation between the fluorescence intensity change and the uric acid concentration. Experiments prove that the recognition of the uric acid by the GC5A is hardly influenced by other interference components in urine, and the method can be used for detecting the uric acid in the urine. Generally, some healthy people also have high uric acid due to abnormal metabolism, some gout patients have normal uric acid level due to acceleration of uric acid metabolism through self-regulation, and water intake and medicines also influence the uric acid level, so that the uric acid detection result in urine cannot be used for directly diagnosing diseases. However, uric acid detection can provide reference data for health risk early warning, diet guidance and the like, so that the uric acid detection in urine still has positive significance.
In order to facilitate the realization of uric acid visual detection in daily life, the embodiment of the invention provides a method for detecting uric acid by using a shooting tool, and provides a specific method for detecting uric acid by using the shooting tool, which comprises the following steps:
when the shooting tool is used for detecting the uric acid concentration of a sample to be detected with the HEPES buffer solution as a solvent, the following operations can be adopted: mixed solution containing GC5A & F1 host-guest pairs is prepared by HEPES buffer solution, and uric acid with different dosages is added into the mixed solution to obtain a series of uric acid solutions with concentrations (100 mu mol/L, 200 mu mol/L, 300 mu mol/L, 400 mu mol/L, 500 mu mol/L, 600 mu mol/L and 800 mu mol/L). Irradiating the uric acid solution under ultraviolet rays (366nm), taking a picture by using a shooting tool (such as a digital camera, a smart phone and the like), and extracting a green intensity value (G value) of the obtained picture by using color processing software, wherein the G value is in a linear relation with the uric acid concentration.
When the shooting tool is used for detecting the uric acid concentration of urine, the following operations can be adopted: centrifuging real urine (12700rpm, 15min), collecting supernatant, adding HEPES buffer solution for dilution to obtain a diluent, adding GC 5A. F1 host-guest pairs and different amounts of uric acid into the diluent, and uniformly mixing to obtain a series of uric acid solutions with concentrations (100 mu mol/L, 200 mu mol/L, 300 mu mol/L, 400 mu mol/L, 500 mu mol/L, 600 mu mol/L and 800 mu mol/L). Irradiating the uric acid solution under ultraviolet rays (366nm), taking pictures by using a shooting tool (such as a digital camera, a smart phone and the like) under the same condition, extracting a G value from the obtained pictures through color processing software, and establishing a linear relation of uric acid detection by using the concentration of uric acid and the G value. The portable and visual noninvasive uric acid detection method provides a feasible strategy for realizing household uric acid detection.
The following examples are intended to illustrate the invention in more detail.
Uric acid Fl, eosin Y disodium salt (EY), trans-4- [4- (dimethylamino) styryl]-1-methylpyridine iodide (DSMI), 8-hydroxypyrene-1, 3, 6-trisulfonic acid trisodium salt (HPTS), Lucigenin (LCG), Acridine Orange (AO), rhodamine b (rhb), α -cyclodextrin (α -CD), β -cyclodextrin (β -CD), methyl- β -cyclodextrin (Me- β -CD), hydroxypropyl- β -cyclodextrin (HP- β -CD), sulfobutyl- β -cyclodextrin (SBE- β -CD), γ -cyclodextrin (γ -CD), cucurbituril [6] urea](CB[6]) Cucurbituril [7]](CB[7]) Cucurbituril [8]](CB[8]) Guanine, xanthine, hypoxanthine, allopurinol, and creatinine are all purchased from Sigma-Aldrich, usa. N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid (HEPES) and Neutral Red (NR) are commercially availableFrom Tianjin Xiansi Biotechnology Ltd. Methylene Blue (MB) was purchased from Merck, Inc., Germany. Bovine serum albumin was purchased from Beijing Sun Biotechnology, Inc. Glutamic acid and aspartic acid were purchased from Shanghai-derived leaf Biotech, Inc. Urea, sodium chloride, potassium chloride and sodium phosphate (monobasic) were purchased from Tianjin Fengchuan chemical reagents science and technology, Inc. N, N' -dimethyl-2, 7-diazapyrene (Me)2DAP) from the institute of nanotechnology of the college of carlshure physics, germany. Guanidine-based cup [5]]Arene (GC5A), 5,11,17,23, 29-pentakis (trimethylammonium) -31,32,33,34, 35-pentakis (4-methylpentyloxy) calix [5]]Aromatic hydrocarbon (QAC5A) and sulfonated azo cup [4 ]]Aromatic hydrocarbons (SAC4A), 5,11,17, 23-tetraguanidine-25, 26,27, 28-tetrabutoxy calix [4 ]]Arene (GC4A-4C), oligo-polyethylene glycol modified guanidyl calixarene (GC4AOEG), and sulfonated calix [4 ]]Aromatic hydrocarbon (SC4A) and sulfonated calix [5]]Aromatic hydrocarbons (SC5A) and sulfonated calix [6]]Aromatic hydrocarbons (SC6A) were all from the chemical college of southern opening university.
Example 1
The embodiment of the invention inspects the interference condition of uric acid on the fluorescent signal of F1.
1. Preparation of the solution
2.38g of HEPES was weighed out, dissolved in about 0.9L of ultrapure water, the pH of the solution was adjusted with a sodium hydroxide solution (pH 13), and the solution was made up to 1L with ultrapure water to obtain a HEPES buffer (10mmol/L, pH 7.4).
Preparing a dye stock solution: precisely weighing F1, preparing into 1mmol/L with HEPES buffer solution to obtain dye guest stock solution (F1 stock solution), and storing at 4 deg.C.
Preparing a uric acid stock solution: uric acid is precisely weighed, dissolved by sodium hydroxide solution (pH 13), diluted by HEPES buffer solution to prepare 10mmol/L, namely uric acid stock solution, and stored at 4 ℃ for later use.
2. Fluorescence spectrum experimental conditions
A Varian Cary Eclipse fluorescence spectrometer (Agilent technologies, USA) is used, and a temperature control device of Cary Single-cuvette Peltier type is provided, and a quartz cell (10X 45 mm) is used3) Has an optical length of 10 mm. Titration experiments were performed at room temperature (25 ℃).
3. Examination of interference condition of uric acid on fluorescence signal of F1
Taking F1 stock solution and uric acid stock solution, and diluting a titration solution containing 2mmol/L uric acid and 1 mu mol/L Fl by using HEPES buffer solution so as to ensure that the Fl concentration in the fluorescence pool is unchanged during titration. F1 stock solution was taken, diluted to 1. mu. mol/L F1 with HEPES buffer solution, and placed in a fluorescence cell. Dropwise adding the titration solution into 1 mu mol/LFl, and adding uric acid (0-447.08 mu mol/L) to obtain a Fl fluorescence signal (lambda)ex＝500nm，λem505 to 650nm), as shown in fig. 1, indicating that uric acid (447.08 μmol/L) does not interfere with Fl fluorescence signal.
Example 2
The bonding constant of GC5A and uric acid was examined in the examples of the present invention.
1. Preparation of the solution
HEPES buffer, Fl stock solution and uric acid stock solution were the same as in example 1.
Preparing a large ring main body stock solution: GC5A was taken, precisely weighed, and prepared into 50. mu. mol/L, i.e., macrocyclic main body stock solution, with HEPES buffer solution, and stored at 4 ℃ for further use.
2. Fluorescence spectrum experimental conditions
The same as in example 1.
3. Determination of bonding constant of GC5A to uric acid
Binding constant (Association constant, K)a) The complex is a physical constant between a supermolecule chemical host-object pair, and the larger the bonding constant is, the stronger the bond and the strong ability between the macrocyclic host and a fluorescent molecule or an analyte are, and the more stable the complex formed by the host-object inclusion is.
The bonding constant of GC5A and uric acid was determined by fluorescence competition titration. Taking F1 stock solution, macrocyclic host stock solution and uric acid stock solution, diluting with HEPES buffer solution to obtain titration solution containing 2mmol/L uric acid and GC 5A. Fl (0.80. mu. mol/L/1.00. mu. mol/L) host-object pair, so as to ensure that the Fl concentration in the fluorescence pool is unchanged during titration. As shown in FIG. 2A, the titration solution was added dropwise to a solution of GC 5A. Fl (0.80. mu. mol/L/1.00. mu. mol/L) host-guest pair. Uric acid (0-120.30 mu mol/L) competes Fl from a GC5A cavity in a GC 5A. Fl host-guest complex, and the Fl recovers the luminescent property in the solution and the fluorescence signal sensitization(Switch-on). As shown in fig. 2B, the Fl fluorescence intensity value and the uric acid concentration value are subjected to nonlinear fitting by using a subject-guest 1:1 competitive inclusion model fitting formula (oneHost _ oneGuest _ onemodulator), and a bonding constant (K) between GC5A and uric acid is obtained by fittinga) Is (2.87 +/-0.23) multiplied by 105M-1. The determination of the bonding constants was carried out in HEPES buffer (25 ℃ C.).
Bonding constant K of GC 5A. Fl by fluorescence titration method and ultraviolet-visible spectroscopyaVerification is performed. Fluorescence titration was performed using a Varian Cary Eclipse fluorescence spectrometer (Agilent technologies, USA) with a quartz cell (10X 45 mm)3) Has an optical length of 10 mm. To eliminate the influence of temperature changes on the measurement results, a temperature control device of Cary Single-cuvette Peltier type was used, and the fluorescence titration experiments were carried out at room temperature (25 ℃). UV-visible Spectroscopy UV-2600 UV spectrophotometer (Shimadzu science and technology, Inc., Japan) was used. The verification result determines the bonding constant K of GC 5A. FlaIs (5.00 +/-1.00) multiplied by 106M-1。
Comparative example
This comparative example provides the use of other macrocyclic host molecules and dye guest in uric acid assays.
1. Preparation of the solution
HEPES buffer and uric acid stock solution were the same as in example 1.
Preparing a large ring main body stock solution: taking macrocyclic main molecule cyclodextrin (alpha-, beta-, Me-beta-, SBE-beta-, HP-beta-, gamma-CD), calixarene (SC4A, SC5A, SC6A, SAC4A, QAC5A, GC4A-4℃, GC4AOEG) and cucurbituril (CB [6], CB [7] and CB [8]), accurately weighing, preparing stock solutions with proper concentration by using HEPES buffer solution, and storing at 4 ℃ for later use.
Preparing a dye stock solution: taking dyes MB, RhB, HPTS, LCG, DSMI, AO, Me2DAP, NR and EY are precisely weighed, and prepared into stock solution with proper concentration by using HEPES buffer solution, and the stock solution is stored at 4 ℃ for later use.
2. Fluorescence spectrum condition
The same as in example 1.
3. Isothermal calorimetric titration conditions
The measurement was carried out at a standard atmospheric pressure using a PEAQ-ITC isothermal calorimeter (Microcal, USA).
3. Bonding constant of alpha-CD to uric acid
The bonding constant of α -CD to uric acid was determined by isothermal titration calorimetry. The uric acid solution is automatically injected into a reaction tank filled with the alpha-CD solution and is automatically stirred and uniformly mixed. Because the reaction of alpha-CD and uric acid is low in heat and unstable, the bonding constant of alpha-CD and uric acid cannot be measured, namely the alpha-CD cannot be used for detecting uric acid.
4. Determination of the linkage constant to uric acid for other macrocyclic hosts
The binding constant of the dye guest to the macrocyclic host was determined by fluorescent direct titration in HEPES buffer. Taking a dye stock solution, diluting the dye stock solution with a HEPES buffer solution, and placing the dye stock solution into a fluorescence pool; taking the macrocyclic host stock solution and the dye stock solution, diluting the macrocyclic host stock solution and the dye stock solution by using a HEPES (high efficiency particulate ES) buffer solution to be used as a macrocyclic host titration solution, wherein the concentration of a dye object in the macrocyclic host titration solution is the same as that of the dye object in a fluorescence pool, so that the concentration of the dye object in the fluorescence pool is ensured to be unchanged in the titration process. With the addition of the macrocyclic host titrant, the fluorescence signal is enhanced or weakened due to the complexation of the host and the guest. The maximum emission wavelength of the dye (Fl: 513nm, MB: 688nm, EY: 537nm, HPTS: 435nm, LCG: 505nm, DSMI: 582nm, AO: 510nm, RhB: 576nm, NR: 573nm, Me2DAP: 449nm) and the concentration value of the macrocyclic host are fitted by adopting a host-guest 1:1 inclusion model fitting formula (oneHost _ oneGuest), so as to obtain the bonding constant of the host and the guest. All bond constants were the average of the fitting data of triplicate titration experiments
And (3) determining the bonding constant of other macrocyclic main bodies and uric acid by a fluorescence competition titration mode. Selecting proper host-object pairs, beta-CD.MB (1.00mmol/L/10.00 mu mol/L), HP-beta-CD.MB (1.00 mmol/L/10.0)0μmol/L)、SBE-β-CD·NR(1.00mmol/L/10.00μmol/L)、Me-β-CD·NR(1.00mmol/L/10.00μmol/L)、γ-CD·HPTS(10.00mmol/L/10.00μmol/L)、CB[6]·DSMI(8.00μmol/L/1.00μmol/L)、CB[7]·AO(15.00μmol/L/0.50μmol/L)、CB[8]·Me2DAP (2.00. mu. mol/L/1.00. mu. mol/L), SC 4A. LCG (0.50. mu. mol/L/0.50. mu. mol/L), SC 5A. LCG (1.00. mu. mol/L/1.00. mu. mol/L), SC 6A. LCG (0.25. mu. mol/L/1.00. mu. mol/L), GC4A-4℃ Fl (8.00. mu. mol/L/1.00. mu. mol/L), GC4 EG. EY (4.00. mu. mol/L/0.50. mu. mol/L), QAC 5A. EY (0.40. mu. mol/L/0.50. mu. mol/L), SAC 4A. RhB (1.00. mu. mol/L/0.80. mu. mol/L). Dropwise adding the titration solution into the solutions of the subject-object pairs, wherein the final concentration range of uric acid is 0-185.12 mu mol/L, the fluorescence intensity is basically unchanged, and the uric acid cannot displace the dye from the macrocyclic cavity, which indicates that the bonding capacity of the macrocyclic host molecules and uric acid is weak and is not enough to realize IDA analysis, namely, the sensing detection of uric acid cannot be realized.
Example 3
The embodiment of the invention provides a method for detecting uric acid in a HEPES buffer solution.
1. Preparation of the solution
HEPES buffer, Fl stock solution and uric acid stock solution were the same as in example 1. The GC5A macrocyclic bulk stock solution was prepared as in example 2.
2. Fluorescence spectrum condition
The same as in example 1.
3. Establishment of uric acid standard curve in HEPES buffer solution
Taking F1 stock solution, macrocyclic host stock solution and uric acid stock solution, diluting with HEPES buffer solution to obtain titration solution containing 2mmol/L uric acid and GC 5A. Fl (0.80. mu. mol/L/1.00. mu. mol/L) host-object pair, so as to ensure that the Fl concentration in the fluorescence pool is unchanged during titration. The titration solution is added dropwise into a GC 5A. Fl host-guest pair (0.80 mu mol/L/1.00 mu mol/L) solution, dropwise addition of uric acid can displace Fl from a GC5A macrocyclic cavity, and Fl fluorescence signals are enhanced. The Fl fluorescence intensity ratio (I/I) is in the range of 0-20 mu mol/L0) In linear relation to uric acid concentration (y-25943.68 x +1.0285, R)20.988), as shown in fig. 3, wherein I0The GC 5A. Fl host-guest pair is not added with uric acidThe fluorescence intensity of (1) is the fluorescence intensity after the system is stabilized after adding uric acid. The uric acid detection Limit (LOD) was determined according to the 3. sigma./slope method, where "σ" is I0The "slope" is the slope of a linear equation obtained by linearly fitting the uric acid concentration to the Fl fluorescence intensity value, and is calculated according to the formula (LOD 3 σ/slope), and the detection limit of uric acid in HEPES buffer is 1.53 μmol/L. In the HEPES buffer solution, a standard curve for detecting uric acid is established by taking GC 5A. Fl (0.80 mu mol/L/1.00 mu mol/L) as a host-object pair, so that the uric acid in the HEPES buffer solution can be detected.
Example 4
The embodiment of the invention provides a method for detecting uric acid in artificial urine and real urine.
1. Preparation of the solution
HEPES buffer, Fl stock solution, GC5A stock solution, and uric acid stock solution were the same as in example 3.
Preparing artificial urine: urea (36.40g), sodium chloride (15.00g), sodium phosphate (monobasic solution, 9.60g), potassium chloride (9.00g), creatinine (4.00g) and bovine serum albumin (100mg) were dissolved in 2L of ultrapure water, the pH of the artificial urine was adjusted to 6.0 with sodium hydroxide, and the solution was stored at 4 ℃ for future use.
Treatment of real urine: collecting urine of morning urine of volunteers (age, 22 +/-1 year) and hyperuricemia patients (age, 55 +/-18 years), centrifuging (12700rpm, 15min), collecting supernatant, and diluting with HEPES buffer solution by 50 times to obtain real urine to be detected.
2. Fluorescence spectrum condition
The same as in example 1.
3. Sensing system selectivity investigation
To evaluate the selectivity of the GC 5A. Fl host-guest for the detection of uric acid by the sensing system, we examined the effect of interfering components in urine on the GC 5A. Fl host-guest pair. Urine contains many interfering components, such as creatinine, urea, ions, proteins, purines, etc., which may bind to GC5A, thereby interfering with uric acid detection. In order to verify the selectivity of the sensing system, uric acid and each interferent (final concentration, uric acid 10 mmol/L; bovine serum albumin 0.40 mg/L; urea, chloride, glutamic acid, phenylalanine 0.30 mmol/L; xanthine, hypoxanthine, allopurinol, adenine, guanine, creatinine 10mmol/L) were added to a GC 5A. Fl (0.80. mu. mol/L/1.00. mu. mol/L) host-guest pair, respectively, and the change in fluorescence intensity was measured. As shown in FIG. 4A, the interferents in urine were all weakly complexed with GC5A, Fl was difficult to compete out, and there was no significant change in fluorescence intensity. Therefore, GC5A preferentially binds uric acid in artificial urine, real urine, with high selectivity for uric acid.
4. Establishment of standard curve of uric acid in artificial urine
The standard curve and detection limit of uric acid in HEPES buffer solution in example 3 were determined. As shown in FIG. 4B, in the artificial urine, the ratio of the change in Fl fluorescence intensity is linear with the uric acid concentration (0-50. mu. mol/L) (y: 8543.61x +1.0013, R)20.987) and a uric acid detection limit of 1.65 ± 0.17 μmol/L. Therefore, in the artificial urine, a standard curve for detecting uric acid is established by taking GC 5A. Fl (8.00. mu. mol/L/4.00. mu. mol/L) as a host-object pair, so that the uric acid can be sensed and detected.
5. Establishment of standard curve of uric acid in real urine
The standard curve and detection limit of uric acid in HEPES buffer solution in example 3 were determined. As shown in FIG. 5, in the real urine to be tested from six volunteers, the Fl fluorescence intensity change ratio and the uric acid concentration (0-250. mu. mol/L) are in a linear relationship. Therefore, in the above real urine of the volunteer, GC 5A. Fl (10.00. mu. mol/L/5.00. mu. mol/L) is used as a subject-object pair to establish a standard curve for detecting uric acid, which shows that the interference component in the real urine does not substantially influence the sensory detection of uric acid.
6. Sensory detection of uric acid in urine to be detected of volunteers and hyperuricemia patients
GC 5A. Fl (10.00. mu. mol/L/5.00. mu. mol/L) subject-object pair solution is added into urine to be tested (diluted) of volunteers and hyperuricemia patients, and the fluorescence intensity value range is measured to be 201.99-437.65 (figure 6A). Meanwhile, the content of uric acid in urine is determined by adopting a high performance liquid chromatography. As shown in fig. 6B, the results of the fluorescence method and the hplc method showed a positive correlation (pearson correlation coefficient, r ═ 0.798, p <0.01), confirming the reliability of the results of the fluorescence method for detecting uric acid. As shown in FIG. 6C, there are distinct divisions of fluorescence intensity in the urine to be tested for volunteers and hyperuricemia patients. As shown in fig. 6D, there was a significant difference in fluorescence intensity between the urine to be tested in the hyperuricemia patients (413.06 ± 18.18) and the volunteers (330.89 ± 44.71) (. p <0.001), the urine to be tested in the hyperuricemia patients (410.20 ± 19.37) and the hyperuricemia patients with renal insufficiency (307.11 ± 31.96) (. p <0.001), and there was no statistical difference in fluorescence intensity between the hyperuricemia patients with renal insufficiency and the volunteers (p > 0.05). The supermolecule subject-object pair sensing system established by the invention can realize the sensing detection of uric acid in urine, has the advantages of high sensitivity and high accuracy, realizes the distinction of volunteers and hyperuricemia patients (except hyperuricemia patients with renal insufficiency) according to the fluorescence intensity value of the detected urine to be detected, and has important significance for early warning of hyperuricemia.
Example 5
The embodiment of the invention provides a method for detecting uric acid by using a smart phone, so as to realize the normalization and the convenience of uric acid detection.
1. Preparation of the solution
HEPES buffer, GC5A stock solution, F1 stock solution, and uric acid stock solution were prepared in the same manner as in example 4.
Treatment of real urine: collecting the urine of the middle morning urine of the volunteers and hyperuricemia patients, centrifuging (12700rpm, 15min), collecting the supernatant, and diluting 200 times with HEPES buffer solution to obtain the real urine to be detected.
2. Visual detection
In HEPES buffer, uric acid solutions containing GC 5A. Fl (10.0. mu. mol/L/5.0. mu. mol/L) at a range of concentrations (100. mu. mol/L, 200. mu. mol/L, 300. mu. mol/L, 400. mu. mol/L, 500. mu. mol/L, 600. mu. mol/L, 800. mu. mol/L) of the host-guest pair were prepared. As shown in fig. 7, the uric acid solution has no obvious color difference under the daylight lamp; irradiating by ultraviolet rays (366nm), taking a picture by using an intelligent mobile phone (iPhone XR), processing the obtained picture by Color processing software (Color Name), extracting a green intensity value (G value), and establishing a linear relation of the G value and a uric acid concentration value for detecting uric acid. Similarly, a linear relation of uric acid detection is successfully established between the volunteer and the urine to be detected for hyperuricemia. The research shows that the simple and sensitive detection of uric acid in urine can be realized by combining a sensing system by the supermolecule host-object with a smart phone, and a feasible strategy is provided for developing a portable and visual household noninvasive uric acid detection method.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The method for detecting the uric acid is characterized in that guanidine-based calix [5] arene and fluorescein sodium are prepared into a mixed solution, a sample to be detected is added into the mixed solution, and the concentration of the uric acid in the sample to be detected is detected through the change of a fluorescence signal.
2. The method for detecting uric acid according to claim 1, wherein the final concentrations of guanidinylcalixarene and fluorescein sodium in HEPES buffer are 0.8 μmol/L and 1 μmol/L, respectively.
3. The use of the method for detecting uric acid according to claim 1 in detecting the concentration of uric acid in urine.
4. The use according to claim 3, wherein said detecting uric acid concentration in urine comprises the following operations:
taking artificial urine, centrifuging, collecting supernatant, diluting with HEPES buffer solution, mixing with a solution containing guanidino calix [5] arene and fluorescein sodium, gradually dropping uric acid solution with the artificial urine as a solvent, measuring the fluorescence intensity of the fluorescein sodium, establishing a linear relation of uric acid detection by using the uric acid concentration and the fluorescence intensity value of the fluorescein sodium, and detecting the uric acid concentration in the urine by using the linear relation.
5. The use according to claim 4, wherein the uric acid solution with artificial urine as a solvent has a uric acid concentration ranging from 0 to 50 μmol/L.
6. The use according to claim 4, wherein the concentrations of guanidinylcalixarene and sodium fluorescein in the solution containing guanidinylcalixarene and sodium fluorescein are 8 μmol/L and 4 μmol/L, respectively.
7. Use of the method for detecting uric acid according to claim 1 in uric acid concentration detection using a camera.
8. The use according to claim 7, wherein the using of the photographing tool to detect the uric acid concentration of the sample to be detected with HEPES buffer solution as a solvent comprises the following operations:
preparing mixed solution of guanidine-based calix [5] arene and fluorescein sodium by using HEPES buffer solution, and adding different amounts of uric acid to obtain a series of uric acid solutions with different concentrations; irradiating the uric acid solution under 366nm ultraviolet rays, photographing by using a photographing tool, extracting a green intensity value of an obtained picture, and establishing a linear relation of uric acid detection by using the uric acid concentration and the green intensity value; detecting the uric acid concentration in the sample to be detected by utilizing the linear relation;
the method for detecting the uric acid concentration of the urine by using the shooting tool comprises the following operations:
taking real urine, centrifuging and collecting supernatant, adding HEPES buffer solution for dilution to obtain diluent, adding mixed solution of guanidine-based calix [5] arene and fluorescein sodium and different amounts of uric acid into the diluent, uniformly mixing to obtain a series of uric acid solutions, irradiating the uric acid solutions under 366nm ultraviolet rays, taking pictures by using a shooting tool under the same condition, extracting a green intensity value of the obtained picture, and establishing a linear relation of uric acid detection by using the uric acid concentration and the green intensity value; and detecting the uric acid concentration in the urine by using the linear relation.
9. The use according to claim 8, wherein the uric acid solution has a uric acid concentration of 0 to 0.8 mmol/L.
10. The use according to claim 8, characterized in that the concentrations of guanidinylcalix [5] arene and fluorescein sodium in the uric acid solution are 10 μmol/L and 5 μmol/L, respectively.
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