CN102053085A - Method for detecting glucose by ferroferric oxide nano particle catalytic chemiluminescence - Google Patents
Method for detecting glucose by ferroferric oxide nano particle catalytic chemiluminescence Download PDFInfo
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- CN102053085A CN102053085A CN 200910236714 CN200910236714A CN102053085A CN 102053085 A CN102053085 A CN 102053085A CN 200910236714 CN200910236714 CN 200910236714 CN 200910236714 A CN200910236714 A CN 200910236714A CN 102053085 A CN102053085 A CN 102053085A
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Abstract
The invention discloses a method for detecting glucose by ferroferric oxide nano particle catalytic chemiluminescence, which comprises the following steps of: at the temperature of between 10 DEG C and 60 DEG C, adding a glucose solution into a ferroferric oxide nano granule-luminol-H2O2 solution system, the pH of which is 4.0 to 9.0; and detecting the concentration of the glucose by using a SpectraMaxM2 microplate reader, wherein the concentration of the ferroferric oxide nano particles is more than or equal to 10ppm. The average granule diameter of the ferroferric oxide nano particles is 25 nanometers. By using the characteristics of high adsorption, biocompatibility, zymoid catalytic property and reuse of the ferroferric oxide nano particles, the glucose chemiluminescence detection method with simple operation, low cost, wide linear range and low detection limit is established to realize simple, convenient and high-sensitivity detection of the glucose content in the fields of clinic and food.
Description
Technical field
The present invention relates to a kind of detection method of glucose content, the chemoluminescence method that relates to a kind of ferroferric oxide nano granules catalysis particularly detects the method for glucose content.
Background technology
Glucose is the main energy sources of human body cell, the ANOMALOUS VARIATIONS of its level can directly reflect the situation of organism metabolic disorder, various diseases such as the content by measuring glucose in blood of human body, urine sample, the cerebrospinal fluid in the clinical examination can diagnosing diabetes, cell tumour, pulmonary tuberculosis, meningitis.In addition, whether up to standard the detection of glucose content in various food such as fruit juice, honey and the grape wine etc. be to weigh various food a important indicator.Therefore the fast quantification of glucose detects significant in biological chemistry, clinical chemistry and field of food industry.
At present, the detection method of glucose mainly comprises [Wu ZS such as spectrophotometric method, electrochemical process, Surface enhanced raman spectroscopy spectroscopic methodology and chemoluminescence method, Zhou GZ, Jiang JH, et al.Gold colloid-bienzyme conjugates for glucose detection utilizing surface-enhanced Raman scattering.Talanta, 2006,70,533-539.].Yet the spectrophotometric method accuracy is low, poor selectivity, and electrochemical process and Surface enhanced raman spectroscopy law part harshness, instrument complexity.Chemoluminescence method is the main analysis means of present clinical medicine and food industry glucose detection owing to have advantages such as instrument is simple, detection limit is low, the range of linearity is wide, highly sensitive and easy and simple to handle.It is to utilize glucose to generate gluconic acid and hydrogen peroxide under the effect of glucose oxidase that chemoluminescence method detects glucose, hydrogen peroxide can produce chemiluminescence by the oxidation luminol in the presence of catalyzer, its luminous intensity directly is directly proportional with the concentration of hydrogen peroxide, because Luminol-H in the present chemoluminescence method
2O
2The luminous intensity of system is more weak, therefore generally adopts the Chemiluminescence Apparatus with light amplification effect to carry out input.Therefore with Chemiluminescence Apparatus measure luminous intensity can be indirectly the quantitative content of glucose in the sample.Up to now, in order to improve the detection limit of chemoluminescence method, the detection (enzymatic reaction and flow injection technology are technology as well known to those skilled in the art) that chemoluminescence method combines and carries out biological sample with enzymatic reaction and flow injection technology usually.
At present, chemiluminescence reaction adopts metal complex and peroxidase to come catalysis Luminol-H as catalyzer usually
2O
2System realizes detection [the Kricka LJ of glucose, Voyta JC, Bronstein I.Chemiluminescent methods for detecting and quantitating enzyme activity.Methods Enzymol, 2000,305,370-390.], yet, disturbs metal complex because producing chemiluminescence signal easily, and peroxidase costs an arm and a leg, less stable and can not reclaim [topaz literary composition, Feng Manliang, Zhang Zhujun.Flow Injection Chemiluminescence Method injected chemical luminescence method is measured glucose.Analytical chemistry 1997,25,34-36], therefore need to seek new chemiluminescence and strengthen system, realize simple, the convenient and high-sensitivity detection of glucose.Detection method commonly used at present has:
1) metal complex is made catalyzer
For the activity that improves glucose oxidase and the stability of enzyme, the researchist adopts enzyme immobilization technology, is about to glucose oxidase and is adsorbed on certain matrix, to improve the life-span and the activity of enzyme.Yang Minli etc. are fixed on glucose oxidase on the glass microballoon of porous and make long-life and highly active immobilized enzyme, by enzymatic glucose oxidase reaction of solid phase and portable injection chemiluminescence system (luminol-H
2O
2-K
3Fe (CN)
4) in conjunction with detecting glucose, by optimizing temperature of reaction, the concentration of luminol and the flow velocity of glucose solution, the range of linearity of this method can reach 0.4～100 μ g/mL, it is beautiful that detection limit reaches 0.08 μ g/mL[Yang Min, Xiong Chuming.Immobilised enzymes portable injection chemiluminescence method is measured glucose.Ningxia University's journal natural science edition, 1995,16,28-31].
2) peroxidase is made catalyzer
Upward fixedly gold nano grain and shitosan are made immobilized enzyme as the fixedly matrix of glucose oxidase with glucose liquid phase enzyme to Lin etc. at glass microballoon (through silane pretreatment).Luminol luminescence system (luminol-H in conjunction with horseradish peroxidase enzyme catalytic
2O
2-HRP), by optimizing the concentration of reaction pH value, reaction time and luminol, on flow injection-Chemiluminescence Apparatus, carry out chemiluminescence detection, the range of linearity of the method can reach 0.01～6.0mmol/L, detect and be limited to 5.0 μ mol/L[Lin JH, Zhang H, Zhang SS.New bienzymatic strategy for glucose determination by immobilized-gold nanoparticle-enhanced chemiluminescence.Sci China Ser B-Chem, 2009,52,196-202].
Lan etc. adsorb glucose oxidase and horseradish peroxidase with gold nano grain, the gold nano grain of adsorptive enzyme is fixed on the inside surface in chemiluminescence pond by sol-gel process, can strengthen the activity of glucose oxidase and horseradish peroxidase simultaneously by this method, thereby the signal of enhanced chemiluminescence reaction, in conjunction with flow injection technical Analysis glucose, the range of linearity of this method can reach 1.0 * 10
-5Mol/L～1.0 * 10
-3Mol/L, detection limit reaches 5 * 10
-6Mol/L[Lan D, Li BX, Zhang ZJ.Chemiluminescence flow biosensor for glucose based on gold nanoparticle-enhanced activities of glucose oxidase and horseradish peroxidase.Biosensors and Bioelectronics, 2008,24,934-938].
Summary of the invention
The object of the present invention is to provide a kind of method of chemiluminescence detection glucose content of ferroferric oxide nano granules catalysis, the present invention is simple to operate, cost is low, the range of linearity is wide, detection limit is low.
For achieving the above object, the present invention has introduced the magnetic ferroferric oxide nanometer particle in chemical luminous system, makes catalyzer with the magnetic ferroferric oxide nanometer particle, has substituted normally used peroxidase, metallic ion or metal complex.
In detail, method provided by the invention is in 10-60 ℃, and glucose solution is added to ferroferric oxide nano granules-luminol that pH is 4.0-9.0-H
2O
2In the solution system, detect concentration of glucose with the SpectraMaxM2 microplate reader; Wherein, the concentration of ferroferric oxide nano granules is for being greater than or equal to 10ppm (preferred 10-30ppm), and luminol concentration is 1-2mmol/L.The mean grain size of ferroferric oxide nano granules is 25nm.
The present invention has utilized high adsorption, biocompatibility, the catalysis characteristics of fermentoid and the characteristics that can reuse of magnetic ferroferric oxide nanometer particle, set up glucose chemistry luminous detection method simple to operate, that cost is low, the range of linearity is wide, detection limit is low, to realize clinical simple, convenient and highly sensitive detection with the field of food glucose content.
The difference of the present invention and existing chemiluminescence detecting method:
(1) the present invention has added the magnetic ferroferric oxide nanometer particle with strong catalytic activity in chemical luminous system, has replaced catalyzer such as existing metallic ion, metal complex and peroxidase.
(2) this method has adopted simpler widely used microplate reader (having the luminous test function of additional chemical), need not special chemiluminescent analyzer, for the detection of glucose provides convenience.
Description of drawings
Fig. 1 has shown ferroferric oxide nano granules-luminol of the present invention-H
2O
2The luminosity curve of solution system when different pH value.
Fig. 2 has shown ferroferric oxide nano granules-luminol of the present invention-H
2O
2The luminosity curve of solution system when different temperatures.
Fig. 3 has shown ferroferric oxide nano granules-luminol of the present invention-H
2O
2In the solution system, the ferroferric oxide nano granules luminosity curve of variable concentrations.
Fig. 4 is ferroferric oxide nano granules-luminol of the present invention-H
2O
2Solution system detects the range of linearity of glucose content.
Embodiment
Ferroferric oxide nano granules is owing to have the characteristic of appraising at the current rate, be proved to be biologically active with class peroxidase, and particle size is more little, the biologically active of such peroxidase high more [Gao L, Zhuang J, Nie L, et al.Intrinsic peroxidase-like activity of ferromagnetic nanoparticles.Nature Nanotech, 2007,2,577-58].
The present invention utilizes this character catalysis Luminol-H of magnetic ferroferric oxide nanometer particle in conjunction with the enzymatic reaction of glucose
2O
2Chemical luminous system adopts the SpectraMaxM2 microplate reader (U.S. molecule instrument company) that has the chemiluminescent assay function to measure the concentration of glucose.And the present invention optimizes the condition (pH value, temperature and ferroferric oxide nano granules concentration) of making the chemiluminescence reaction of catalyzer with ferroferric oxide nano granules.Studied the influence of luminol concentration to chemiluminescence intensity in the reference literature 5, increased though consider the increase luminous signal with luminol concentration, background values also increases thereupon, and the luminol concentration that the present invention selects for use is 2mmol/L.The mean grain size of the ferroferric oxide nano granules that the present invention adopts is 25nm.
Embodiment:
1) the pH value is optimized
Preparation ferroferric oxide nano granules-luminol-H
2O
2Solution system is at constant ferroferric oxide nano granules, luminol and H
2O
2(concentration of ferroferric oxide nano granules is 10ppm for concentration and temperature of reaction; Luminol concentration is 2mmol/L; H
2O
2Concentration is 50 μ mol/L; Temperature of reaction is 25 ℃) condition under, by in solution, adding the pH value (pH value scope is 4～10) of the method regulation system of HCl and NaOH, after measuring the pH value with pH meter, measure the luminous intensity of each system with the SpectraMaxM2 microplate reader, the result as shown in Figure 1.As can be seen: in pH value scope is 4～9 o'clock, and luminous intensity is bigger, and when pH=7.0, the luminous intensity maximum.
2) temperature optimization
Preparation ferroferric oxide nano granules-luminol-H
2O
2Solution system is at constant ferroferric oxide nano granules, luminol and H
2O
2(concentration of ferroferric oxide nano granules is 10ppm for concentration and pH value; Luminol concentration is 2mmol/L; H
2O
2Concentration is 50 μ mol/L; The pH value is 7.0) condition under, with the constant-temperature shaking culture case system is carried out different temperatures control, temperature controlling range is 4～70 ℃, and measures the luminous intensity of each system with the SpectraMaxM2 microplate reader, the result is as shown in Figure 2.As can be seen: when temperature of reaction was 10～60 ℃, luminous intensity was bigger, and when temperature of reaction is 40 ℃, the luminous intensity maximum.
3) ferroferric oxide nano granules concentration is optimized
Preparation ferroferric oxide nano granules-luminol-H
2O
2Solution system is at constant luminol and H
2O
2(concentration range of ferroferric oxide nano granules is 3～60ppm for concentration and pH value and temperature; Luminol concentration is 2mmol/L; H
2O
2Concentration is 50 μ mol/L; The pH value is 7.0; Temperature of reaction is 40 ℃) condition under.Measure the luminous intensity of each system with the SpectraMaxM2 microplate reader, the result as shown in Figure 3.As can be seen: when the concentration of ferroferric oxide nano granules is 3～10ppm, increase along with ferroferric oxide nano granules concentration, luminous intensity strengthens rapidly, when the concentration of ferroferric oxide nano granules during greater than 10ppm, luminous intensity is bigger, and along with the increase of ferroferric oxide nano granules concentration also strengthens gradually; And when the concentration of ferroferric oxide nano granules during greater than 25ppm, along with the increase of ferroferric oxide nano granules concentration, luminous intensity remains unchanged substantially, so the optimization concentration of ferroferric oxide nano granules is 25ppm.
Therefore, the chemiluminescence condition of optimum of the present invention is: pH=7, and temperature of reaction is 40 ℃, the concentration of ferroferric oxide nano granules is 25ppm.
4) measurement of glucose content under the optimal conditions
With the glucose solution of variable concentrations respectively with the glucose oxidase effect, under the luminescent condition of optimizing, its chemiluminescence intensity is tested with the SpectraMaxM2 microplate reader, make the typical curve of chemiluminescence intensity with the sugared Portugal of grape concentration change.As shown in Figure 4.Optimize under the luminescent condition at this as can be seen, the range of linearity that grape sugar Portugal concentration detects can reach 2 * 10
-6Mol/L～1 * 10
-3Mol/L (is equivalent to that 0.36mg/L～180mg/L), related coefficient is 0.9990.By to (2mmol/L luminol, 25ppm Fe under the optimal conditions
3O
4, pH=7 and temperature of reaction are 40 ℃) blank background test, calculate detection limit with the concentration that 3 σ (standard deviations that blank replicate determination is 11 times) are corresponding, can obtain this method and detect detecting of glucose and be limited to 0.4 μ mol/L.
The beneficial effect of technical solution of the present invention
(1) range of linearity of detection glucose is 2 * 10 under the optimal conditions of the present invention
-6Mol/L～1 * 10
-3Mol/L (is equivalent to that 0.36mg/L～180mg/L), detection limit reaches 0.4 μ mol/L.As shown in table 1 below with the contrast of existing correlation technique.As can be seen: the present invention not only has the wide characteristics of the range of linearity that detect glucose, and detection limit reduced by 5 times than prior art, can realize that the glucose content of lower concentration detects.
Need to prove that (2) detecting instrument that the present invention uses is the SpectraMaxM2 microplate reader.Compare with the special detecting instrument-chemiluminescent analyzer that uses of chemical luminescence method, the SpectraMaxM2 microplate reader does not possess optical amplification function, therefore, if the ferroferric oxide nano granules-luminol-H that uses the chemiluminescent analyzer measurement the present invention relates to
2O
2Glucose content in the-glucose solution system, its detection limit will be lower.Simultaneously,, only need simpler widely used microplate reader (having the luminous test function of additional chemical), therefore provide convenience for the detection of glucose content because the present invention need not special chemiluminescent analyzer.
(3) because ferroferric oxide nano granules has magnetic, therefore utilize the action of a magnetic field, as magnet etc., can realize the separation of ferroferric oxide nano granules, thereby realize and to reuse, overcome the shortcoming that is difficult to reuse of making catalyzer with peroxidase, metallic ion and metal complex, more helped reducing the detection cost.
Table 1
Annotate: the testing result of mark * hurdle for using the SpectraMaxM2 microplate reader to obtain; Do not mark the hurdle and obtain testing result for using chemiluminescent analyzer.
Document
[5]For: Lin JH, Zhang H, Zhang S S.New bienzymatic strategy for glucose determination by immobilized-gold nanoparticle-enhanced chemiluminescence.Sci China Ser B-Chem, 2009,52,196-202.
Document
[6]For: Lan D, Li BX, Zhang ZJ.Chemiluminescence flow biosensor for glucose based on gold nanoparticle-enhanced activities of glucose oxidase and horseradish peroxidase.Biosensors and Bioelectronics, 2008,24,934-938.
Claims (5)
1. the method for a chemiluminescence detection glucose is characterized in that, adopts ferroferric oxide nano granules catalysis luminol-H
2O
2Chemical luminous system; In 10-60 ℃, glucose solution is added to ferroferric oxide nano granules-luminol that pH is 4.0-9.0-H
2O
2Detect concentration of glucose in the solution system;
Wherein, the concentration of ferroferric oxide nano granules is for being greater than or equal to 10ppm.
2. the method for claim 1, wherein the mean grain size of ferroferric oxide nano granules is 25rm.
3. the method for claim 1, wherein detecting is to adopt the SpectraMaxM2 microplate reader.
4. the method for claim 1, wherein luminol concentration is 1-2mmol/L.
5. the method for claim 1, wherein the concentration of ferroferric oxide nano granules is 10-30ppm.
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