Glucose detection method and application
By using the derivatization reaction of glucose and guanidine in the presence of sodium borate to generate an imidazole ring, and then using fluorescence intensity to detect glucose concentration, the problem of unpleasant reaction conditions and insufficient sensitivity in existing technologies is solved, thus achieving efficient and accurate glucose detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU HUASHANG UNIV
- Filing Date
- 2026-02-27
- Publication Date
- 2026-05-12
AI Technical Summary
Existing glucose detection methods suffer from harsh reaction conditions and insufficient sensitivity, making it difficult to achieve efficient and accurate glucose detection.
The derivatization reaction of glucose and guanidine in the presence of sodium borate produces an imidazole ring. The glucose concentration is detected by fluorescence intensity using a fluorescence spectrophotometer, and a working curve is established to achieve high sensitivity and accuracy.
A good linear relationship and high sensitivity for glucose detection were achieved within a certain concentration range. The relative standard deviation met the requirements, and the method is simple, safe, and reliable.
Smart Images
Figure CN122016748A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of glucose detection technology, and in particular to a glucose detection method and its application. Background Technology
[0002] Glucose is a substance that is widely found in nature and plays a very important role in both plants and animals. Its stable content is an important factor in human health. If the content is too low, it is hypoglycemia, which causes dizziness and weakness. If the content is too high, it is diabetes. Therefore, studying the properties of glucose is of great significance.
[0003] However, the detection of sugars is relatively difficult because sugars have neither strong UV absorption nor fluorescent groups, and are normally electroneutrally neutral. Therefore, the detection of sugars usually requires derivatization to give them UV absorption, fluorescence, or other measurable physicochemical properties. One important type of derivatization is the reaction between glucose and amines. The reaction with amine compounds is one of the earliest derivatization methods used for sugar determination. Its principle is that -NH₂ reacts with the functional group of sugar—the carbonyl group (aldehyde or ketone group)—to form -C=N. This type of method can be further divided into the following categories depending on the derivatization reagent.
[0004] (1) Reaction with alkylamines and aromatic amines: Honda et al. first discovered that reducing sugars react with ethylenediamine in a slightly alkaline phosphate buffer solution upon co-heating to produce strong fluorescence. This discovery could be used to determine reducing sugars. The detection limit of this derivatization method is less than 1 nmol for most sugars. Later, Kato and Kinoshita derivatized sugars with ethanolamine and 2-aminopropionitrile, and then determined them by fluorescence.
[0005] (2. Reductive Amination Derivatization: The principle of the reductive amination derivatization reaction is that the carbonyl group (aldehyde, ketone) at the reducing end of the sugar reacts with an aromatic compound containing a primary amino group to generate a Shiff base, which is then reduced to a secondary amine by NaBH3CN, and then detected by ultraviolet absorption or fluorescence.)
[0006] (3) Reaction with amide compounds: Amides are a type of sugar derivatization reagent that has been used for a long time and is widely applied. A representative example is 2-cyanoacetamide, which reacts with aldoses in a weakly alkaline solution to produce a strong fluorescent substance. This reaction is suitable for post-column derivatization, and the product can also be detected by spectrophotometry. The detection limit for aldoses can reach 0.1 nmol. This method can be used to distinguish isomers at low temperatures.
[0007] (4) Derivatization of acid sugars: Mechref and EI Rassi proposed a derivatization method for acid sugars. The principle is that the amino group of p-aminobenzenesulfonic acid (SA) or 7-aminonaphthalene-1,3-disulfonic acid (ANDSA) and the carbonyl group of the acid sugar are condensed into a peptide chain in the presence of water-soluble carbodiimide (HN:C:NH), and the reaction is detected by UV.
[0008] (5) Reaction with guanidine and its derivatives: Guanidine has two -NH2 groups, which react with sugars in the presence of borate to form an imidazole ring (as shown in Formula 1). Sugars can be detected by the fluorescence of the imidazole ring. The detection limits are: 5 pmol for pentoses and 6 pmol for hexoses. This reaction is easier, more sensitive, and more reproducible than the reaction of ethylene amino derivatives with sugars.
[0009] (Equation 1).
[0010] Two compounds with structures similar to guanidine—arginine and benzamide—are good fluorescent probes for carbohydrates. Arginine derivatization requires mild conditions, similar to the reaction of 2-cyanoacetamide and fatty amines in boric acid medium. Glucose and fructose have similar fluorescence intensities, with a detection limit of 25 pmol for glucose. Sucrose and raffinose only provide fluorescence intensity equivalent to 4% of glucose, while 2-deoxyglucoses show no fluorescence. Benzoamide and its p-methoxy derivatives exhibit high sensitivity as derivatizing agents, but the reaction requires a strongly alkaline medium.
[0011] Currently, there is a need for a glucose detection method with mild reaction conditions and high sensitivity. Summary of the Invention
[0012] The purpose of this application is to provide a glucose detection method and its application to solve the following technical problem: how to detect glucose efficiently and accurately. The detection method of this invention utilizes the excellent fluorescence emission resulting from the derivatization reaction between glucose and guanidine. The concentration of glucose is detected by the fluorescence intensity. In the same experiment, the relative standard deviation meets the requirements, and it exhibits high sensitivity and a good linear relationship within a certain concentration range, thus achieving accurate and efficient detection of glucose.
[0013] The first aspect of this application provides a glucose detection method, the detection method comprising the following steps: S1. Prepare sodium borate solution and guanidine solution; S2. Weigh glucose and prepare glucose working solutions of different concentrations. Add the sodium borate solution and guanidine solution from step S1 to the glucose working solution, heat in a water bath, cool, and then bring the volume to 10 mL. Perform fluorescence detection. Based on the detection results, take the logarithm of the concentration and fluorescence intensity to perform a linear regression equation to obtain the working curve. S3. Take the sodium borate solution and guanidine solution from step S1 and add them to the test solution. Heat in a water bath and measure the fluorescence intensity. Then, substitute the detection results into the working curve in step S2 to calculate the glucose concentration in the test solution.
[0014] Optionally, in step S1, the concentration of the sodium borate solution is 0.04-0.2 mol / L.
[0015] Alternatively, in step S1, the concentration of the sodium borate solution is 0.1004 mol / L or 0.16 mol / L.
[0016] Optionally, in step S1, the concentration of the guanidine solution is 0.002-0.04 mol / L.
[0017] Alternatively, in step S1, the concentration of the guanidine solution is 0.0295 mol / L or 0.0311 mol / L.
[0018] Optionally, in steps S2 and S3, the water bath heating time is 30-180 minutes.
[0019] Optionally, in steps S2 and S3, the water bath heating time is 50-150 minutes.
[0020] Alternatively, the water bath heating time can be 30 min, 60 min, 90 min, 120 min, 150 min, or 180 min.
[0021] Alternatively, the water bath heating time may be 100 min, 120 min, or 150 min.
[0022] Optionally, in step S2, the working curve is: InI f =-0.4877InC+9.3259; Among them, InI f ν represents fluorescence intensity, and InC represents glucose concentration.
[0023] Optionally, in step S3, the volume ratio of the test solution to the sodium borate solution and the guanidine solution is (1-2):1:1.
[0024] Further optionally, in step S3, the volume ratio of the test solution to the sodium borate solution and the guanidine solution can be 1:1:1 or 2:1:1.
[0025] Optionally, in step S3, the volume ratio of the test solution to the sodium borate solution and the guanidine solution is 1:1:1. Optionally, the amount of sodium borate solution added is 1 mL, the amount of guanidine solution added is 1 mL, and the amount of test solution added is 1 mL.
[0026] The second aspect of this application provides the application of the glucose detection method as described in the first aspect in food testing.
[0027] Optionally, the food may be vegetables or fruits.
[0028] The third aspect of this application provides a method for detecting glucose content in food, comprising the following detection steps: The food to be tested is processed to obtain a test solution, which is then tested using the detection method described in the first aspect to obtain the glucose concentration in the test solution, and the glucose content of the food to be tested is then calculated.
[0029] The technical solutions provided in this application have the following advantages compared with the prior art: 1. The detection method of the present invention utilizes the excellent fluorescence absorption of glucose after derivatization with guanidine, and achieves high-sensitivity detection of glucose concentration by measuring the fluorescence intensity; moreover, the relative standard deviation meets the requirements in the same experiment, and shows a good linear relationship within a certain concentration range, thus realizing accurate and efficient detection of glucose.
[0030] 2. Traditional glucose determination using the Dublis method (i.e., the sulfuric acid-phenol method) involves heating with concentrated sulfuric acid, which is unsafe and complex. Compared with traditional methods, this invention has the following significant advantages: the method is simple, sensitive, safe, and reliable. Attached Figure Description
[0031] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.
[0032] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0033] Figure 1 The excitation and emission spectra of the glucose standard solution of Example 1 of this application are shown. Figure 2 This is a graph showing the effect of guanidine solution concentration on fluorescence intensity in Example 2 of this application; Figure 3This is a graph showing the effect of sodium borate solution concentration on fluorescence intensity in Example 2 of this application; Figure 4 This is a graph showing the effect of reaction time on fluorescence intensity in Example 2 of this application; Figure 5 This is a working curve diagram for this application. Detailed Implementation
[0034] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0035] The range descriptions used in this application, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values within that range. For example, the range descriptions of "1 to 6" or "1~6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "comprising" and others used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships involved in this document, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained by purchasing from the market or by existing methods.
[0036] This application provides a glucose detection method and its application, the detection method comprising the following steps: S1. Prepare sodium borate solution and guanidine solution; S2. Weigh glucose and prepare glucose working solutions of different concentrations. Add the sodium borate solution and guanidine solution from step S1 to the glucose working solution, heat in a water bath, cool, and then bring the volume to 10 mL. Perform fluorescence detection. Based on the detection results, take the logarithm of the concentration and fluorescence intensity to perform a linear regression equation to obtain the working curve. S3. Take the sodium borate solution and guanidine solution from step S1 and add them to the test solution. Heat in a water bath and measure the fluorescence intensity. Then, substitute the detection results into the working curve in step S2 to calculate the glucose concentration in the test solution.
[0037] In step S2, the working curve is: InI f =-0.4877InC+9.3259
[0038] Where InC is the glucose concentration, and InI is the glucose concentration. f The fluorescence intensity is [value], with a correlation coefficient of 0.9976 and a linear range of 1×10⁻⁶. -5 ~2×10 -4 mol / L.
[0039] It should be noted that a fluorescence spectrophotometer can be used for fluorescence detection.
[0040] The specific mechanism is as follows: Guanidine has two -NH2 groups. In the presence of borate, it reacts with glucose to form an imidazole ring. Glucose can be detected by the fluorescence of the imidazole ring.
[0041] In some optional embodiments, in step S1, the concentration of the sodium borate solution is 0.04-0.2 mol / L.
[0042] Alternatively, in step S1, the concentration of the sodium borate solution can be 0.1004 mol / L or 0.16 mol / L.
[0043] In some optional embodiments, in step S1, the concentration of the guanidine solution is 0.002-0.04 mol / L.
[0044] Alternatively, in step S1, the concentration of the guanidine solution can be 0.0295 mol / L or 0.0311 mol / L.
[0045] In some optional implementations, the water bath heating time in steps S2 and S3 is 30-180 min.
[0046] Alternatively, the water bath heating time can be 30 min, 60 min, 90 min, 120 min, 150 min, or 180 min.
[0047] In some optional implementations, the water bath heating time in steps S2 and S3 is 50-150 minutes.
[0048] Alternatively, the water bath heating time can be 100 min, 120 min, or 150 min.
[0049] In some optional embodiments, in step S3, the volume ratio of the test solution to the sodium borate solution and the guanidine solution is (1-2):1:1.
[0050] Further optionally, in step S3, the volume ratio of the test solution to the sodium borate solution and the guanidine solution can be 1:1:1 or 2:1:1.
[0051] Optionally, in step S3, the volume ratio of the test solution to the sodium borate solution and the guanidine solution is 1:1:1. Optionally, the amount of sodium borate solution added is 1 mL, the amount of guanidine solution added is 1 mL, and the amount of test solution added is 1 mL.
[0052] The second aspect of this application provides the application of the glucose detection method as described in the first aspect in food testing.
[0053] Optionally, the food may be vegetables, fruits, or other foods.
[0054] The third aspect of this application provides a method for detecting glucose content in food, comprising the following detection steps: The food to be tested is processed to obtain a test solution, which is then tested using the detection method described in the first aspect to obtain the glucose concentration in the test solution, and the glucose content of the food to be tested is then calculated.
[0055] It should be noted that when the food to be tested is a fruit or vegetable, weigh 100g of the edible portion of the fruit or vegetable to be tested, add an equal mass of double-distilled water, crush and stir to make a homogenate, weigh 10-20g of the homogenate, filter, and dilute the filtrate to a 100ml volumetric flask to obtain the test solution.
[0056] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national / industry standards; if there is no corresponding national / industry standard, they are performed according to general international standards, conventional conditions, or conditions recommended by the manufacturer.
[0057] The instruments and reagents involved in the embodiments of this application are shown in Table 1 below: Table 1. Reagents and Instruments Covered in This Patent
[0058] The 4500 fluorescence spectrometer (Japan) used in this application embodiment has the following fluorescence parameters set as follows: excitation / emission slit 5nm / 5nm, scan speed 1200nm / min.
[0059] Example 1
[0060] Weigh out glucose and prepare a solution with a concentration of 1×10⁻⁶. -3Prepare a 0.0100 mol / L glucose standard solution; weigh out guanidine and prepare a 0.1000 mol / L guanidine solution; weigh out sodium borate and prepare a 0.1000 mol / L sodium borate solution. Add 1 mL of sodium borate solution and 1 mL of guanidine solution to 1 mL of glucose standard solution, heat in a water bath for 120 min, and perform excitation and emission spectra detection. The results are as follows: Figure 1 As shown.
[0061] Figure 1 1×10 of Example 1 -3 Excitation and emission spectra of mol / L glucose standard solution, from Figure 1 As can be seen, after glucose is added to guanidine and sodium borate, excitation and emission spectra can be clearly detected. The excitation spectrum has two high-intensity excitation peaks at 259 nm and 301 nm, respectively, while the emission spectrum has only one peak at 420 nm.
[0062] Example 2
[0063] 1. Effect of guanidine concentration on fluorescence intensity
[0064] The reaction detection system of Example 1 was used, with the only difference from Example 1 being the change in the concentration of the guanidine solution. The detection results are as follows: Figure 2 As shown.
[0065] Figure 2 This is a graph showing the effect of guanidine solution concentration on fluorescence intensity in Example 2. Figure 2 As can be seen, when the concentrations of glucose and sodium borate solution remain constant, the fluorescence intensity gradually increases as the concentration of guanidine is gradually increased.
[0066] 2. Effect of sodium borate concentration on fluorescence intensity
[0067] Using the reaction detection system of Example 1, the concentration of sodium borate solution was changed. Specifically, the concentrations of glucose and guanidine remained constant. 0.2 ml, 0.4 ml, 0.8 ml, 1.0 ml, 2.0 ml, 3.0 ml, and 4.0 ml of 0.4 mol / L sodium borate solution were added to seven colorimetric tubes numbered 1, 2, 3, 4, 5, 6, and 7, respectively. The fluorescence detection results are as follows: Figure 3 As shown.
[0068] Figure 3 This is a graph showing the effect of sodium borate solution concentration on fluorescence intensity in Example 2. Figure 3 As can be seen, the fluorescence intensity no longer increases when the sodium borate solution concentration reaches 0.16 mol / L.
[0069] 3. Effect of reaction time on fluorescence intensity
[0070] Weigh out glucose and prepare a solution with a concentration of 1×10⁻⁶. -3 Prepare a 0.0100 mol / L glucose standard solution; weigh out guanidine and prepare a 0.16000 mol / L guanidine solution; weigh out sodium borate and prepare a 0.16000 mol / L sodium borate solution. Take six identical colorimetric tubes, add 1 ml of glucose solution, 1 ml of guanidine solution, and 1 ml of sodium borate solution to each tube, and then heat for 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min respectively. The fluorescence detection results are as follows. Figure 4 As shown.
[0071] Figure 4 This is a graph showing the effect of reaction time on fluorescence intensity in Example 2. Figure 4 As can be seen, the fluorescence intensity reaches its maximum when heated in a water bath for 120 minutes.
[0072] Example 3
[0073] Precision testing: Calculation of relative standard deviation
[0074] The same components were added to seven identical colorimetric tubes: 1 ml of 0.0100 mol / L glucose, 1 ml of 0.0311 mol / L guanidine solution, and 1 ml of 0.16 mol / L sodium borate solution. The tubes were heated in a water bath for 120 min, and then the fluorescence intensity was measured. The results are shown in Table 2 below. Table 2. Fluorescence intensity values under the same conditions
[0075] The relative standard deviation can be calculated to be 3.06% based on the data in Table 2. The relative standard deviation of the detection method of the present invention meets the requirements.
[0076] Example 4
[0077] 1. Drawing working curves
[0078] Prepare a sodium borate solution with a concentration of 0.16 mol / L and a guanidine solution with a concentration of 0.0311 mol / L; Weigh out glucose and prepare glucose working solutions of different concentrations. Add the above sodium borate solution and guanidine solution to the glucose working solution, heat in a water bath for 120 min, cool and bring the volume to 10 mL, and perform fluorescence detection. Based on the detection results, take the logarithm of concentration and fluorescence intensity to perform a linear regression equation to obtain the working curve.
[0079] The specific process for preparing glucose working solutions of different concentrations is as follows: Accurately weigh 0.1982g of glucose, dissolve it in deionized water, and dilute to a final volume of 250ml in a volumetric flask to prepare 10... -3Take another 10 ml of the stock solution and dilute it to a 100 ml volumetric flask to obtain 10 mol / L. -4 Take another 1 ml of the working solution (mol / L), and dilute to 100 ml with 1 ml of the stock solution to obtain 10 mol / L. -5 A working solution of mol / L was prepared. Then, different amounts of the working solution were diluted to prepare a series of glucose working solutions of different concentrations.
[0080] The working curve is: InI f =-0.4877InC+9.3259, correlation coefficient is 0.9976, linear range is 1×10 -5 ~2×10 -4 mol / L. The working curve is as follows: Figure 5 As shown.
[0081] 2. Actual sample testing
[0082] Actual samples: Onions, tomatoes, bell peppers, grapes, fennel, potatoes, and green peppers were all purchased from local supermarkets.
[0083] A glucose detection method, the detection method comprising the following steps: Wash fresh vegetables and fruits thoroughly. Weigh 100g of the edible portion of the sample and place it in a multi-functional juicer. Add an equal amount of double-distilled water, blend, and homogenize. Weigh 10-20g of the homogenate, filter, and dilute the filtrate to a 100ml volumetric flask. Take 1ml of the solution into a colorimetric tube, add 1ml of 0.16mol / L sodium borate solution and 1ml of 0.0311mol / L guanidine solution, heat in a water bath for 120min, and measure the fluorescence intensity. Substitute the values into a linear regression equation to calculate the corresponding solution concentration, thus obtaining the glucose content of the sample. The results are shown in Table 3. Taking green bell peppers as an example, calculate the glucose content in 100g of sample: ㏑I f =4.8032 Substituted into the linear regression equation InI f The concentration to be measured was obtained from -0.4877InC + 9.3259. C = 9.42 × 10 -5 mol / L; Multiply the concentration being measured by the molar mass of glucose: C = 1.6956 × 10 -8 g / mL; Then multiply by the dilution factor of 10. 4 The mass of glucose in the 100g actual sample can then be obtained: m = 0.16956g; Therefore, the glucose content of the fresh green pepper sample is 0.169%, and the glucose content of other vegetables is calculated in the same way.
[0084] Table 3. Glucose content in different vegetables or fruits
[0085] In summary, the glucose detection method and application provided in this application are simple in process, highly accurate, and highly sensitive.
[0086] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed in this application.
Claims
1. A method for detecting glucose, characterized in that, The detection method includes the following steps: S1. Prepare sodium borate solution and guanidine solution; S2. Weigh glucose and prepare glucose working solutions of different concentrations. Add the sodium borate solution and guanidine solution from step S1 to the glucose working solution, heat in a water bath, cool, and then bring the volume to 10 mL. Perform fluorescence detection. Based on the detection results, take the logarithm of the concentration and fluorescence intensity to perform a linear regression equation to obtain the working curve. S3. Take the sodium borate solution and guanidine solution from step S1 and add them to the test solution. Heat in a water bath and measure the fluorescence intensity. Then, substitute the detection results into the working curve in step S2 to calculate the glucose concentration in the test solution.
2. The glucose detection method according to claim 1, characterized in that, In step S1, the concentration of the sodium borate solution is 0.04-0.2 mol / L.
3. The glucose detection method according to claim 1, characterized in that, In step S1, the concentration of the guanidine solution is 0.002-0.04 mol / L.
4. The glucose detection method according to claim 1, characterized in that, In steps S2 and S3, the water bath heating time is 30-180 minutes.
5. The glucose detection method according to claim 4, characterized in that, In steps S2 and S3, the water bath heating time is 50-150 minutes.
6. The glucose detection method according to claim 1, characterized in that, In step S2, the working curve is: InI f =-0.4877InC+9.3259; Among them, InI f ν represents fluorescence intensity, and InC represents glucose concentration.
7. The glucose detection method according to claim 1, characterized in that, In step S3, the volume ratio of the test solution to the sodium borate solution and the guanidine solution is (1-2):1:
1.
8. The glucose detection method according to claim 7, characterized in that, In step S3, the volume ratio of the test solution to the sodium borate solution and the guanidine solution is 1:1:
1.
9. The application of the glucose detection method according to any one of claims 1-8 in food testing.
10. A method for detecting glucose content in food, characterized in that, The following testing steps are included: The food to be tested is processed to obtain a test solution, which is then tested using the detection method described in any one of claims 1-8 to obtain the glucose concentration in the test solution, and the glucose content of the food to be tested is then calculated.