CN102053085A - Method for detecting glucose by ferroferric oxide nano particle catalytic chemiluminescence - Google Patents
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Abstract
一种四氧化三铁纳米颗粒催化的化学发光检测葡萄糖的方法，于10-60℃，将葡萄糖溶液加入至pH为4.0-9.0的四氧化三铁纳米颗粒-鲁米诺-H2O2溶液体系中，用SpectraMaxM2酶标仪检测葡萄糖浓度；其中，四氧化三铁纳米颗粒的浓度为大于或者等于10ppm。四氧化三铁纳米颗粒的平均粒径为25nm。本发明利用了磁性四氧化三铁纳米颗粒的高吸附性、生物相容性、类酶的催化特性及可重复利用的特点，建立了操作简单、成本低、线性范围宽、检出限低的葡萄糖化学发光检测法，以实现临床和食品领域葡萄糖含量简单、方便和高灵敏度的检测。A method for detecting glucose by chemiluminescence catalyzed by iron ferric oxide nanoparticles. At 10-60°C, glucose solution is added to iron ferric oxide nanoparticles-luminol-H 2 O 2 solution with a pH of 4.0-9.0 In the system, the glucose concentration is detected with a SpectraMaxM2 microplate reader; wherein, the concentration of ferric oxide nanoparticles is greater than or equal to 10ppm. The average particle diameter of ferric oxide nanoparticles is 25nm. The present invention utilizes the characteristics of high adsorption, biocompatibility, enzyme-like catalytic properties and reusability of magnetic ferroferric oxide nanoparticles, and establishes a method with simple operation, low cost, wide linear range and low detection limit. Glucose chemiluminescence detection method to realize simple, convenient and highly sensitive detection of glucose content in clinical and food fields.
Description
技术领域technical field
本发明涉及一种葡萄糖含量的检测方法，具体地涉及一种四氧化三铁纳米颗粒催化的化学发光法检测葡萄糖含量的方法。The invention relates to a method for detecting glucose content, in particular to a method for detecting glucose content by a chemiluminescence method catalyzed by iron ferric oxide nanoparticles.
背景技术Background technique
葡萄糖是人体细胞的主要能源，其水平的异常变化能够直接反映机体代谢紊乱的情况，临床检验上通过测定人体血液、尿样、脑脊液中葡萄糖的含量可以诊断糖尿病、细胞肿瘤、肺结核、脑膜炎等各种疾病。此外，各种食品如果汁、蜂蜜和葡萄酒等中葡萄糖含量的检测，是衡量各种食品是否达标的一个重要指标。因此葡萄糖的快速定量检测在生物化学、临床化学和食品工业领域具有重要意义。Glucose is the main energy source of human cells. Abnormal changes in its level can directly reflect the metabolic disorder of the body. In clinical tests, the glucose content in human blood, urine samples, and cerebrospinal fluid can be used to diagnose diabetes, cell tumors, tuberculosis, meningitis, etc. various diseases. In addition, the detection of glucose content in various foods such as juice, honey and wine is an important indicator to measure whether various foods meet the standards. Therefore, the rapid quantitative detection of glucose is of great significance in the fields of biochemistry, clinical chemistry and food industry.
目前，葡萄糖的检测方法主要包括分光光度法、电化学法、表面增强拉曼散射光谱法和化学发光法等[Wu ZS，Zhou GZ，Jiang JH，et al.Gold colloid-bienzyme conjugates for glucose detection utilizing surface-enhanced Raman scattering.Talanta，2006，70，533-539.]。然而，分光光度法准确度低，选择性差，而电化学法和表面增强拉曼光谱法条件苛刻，仪器复杂。化学发光法由于具有仪器简单、检出限低、线性范围宽、灵敏度高和操作简便等优点，是目前临床医学以及食品工业葡萄糖检测的主要分析手段。化学发光法检测葡萄糖是利用葡萄糖在葡萄糖氧化酶的作用下生成葡萄糖酸和过氧化氢，过氧化氢在催化剂存在下能够氧化鲁米诺产生化学发光，其发光强度直接与过氧化氢的浓度成正比，由于目前化学发光法中Luminol-H2O2体系的发光强度比较弱，因此普遍采用具有光放大作用的化学发光仪进行信号检测。因此用化学发光仪测量发光强度可间接定量样品中葡萄糖的含量。迄今为止，为了提高化学发光法的检出限，化学发光法通常与酶促反应和流动注射技术相结合进行生物样品的检测(酶促反应和流动注射技术是本领域技术人员公知技术)。Currently, glucose detection methods mainly include spectrophotometry, electrochemical method, surface-enhanced Raman scattering spectrometry and chemiluminescence [Wu ZS, Zhou GZ, Jiang JH, et al. Gold colloid-bienzyme conjugates for glucose detection utilizing surface-enhanced Raman scattering. Talanta, 2006, 70, 533-539.]. However, the spectrophotometric method has low accuracy and poor selectivity, while the electrochemical method and surface-enhanced Raman spectroscopy have harsh conditions and complicated instruments. Chemiluminescence method is the main analytical method for glucose detection in clinical medicine and food industry due to its advantages of simple instrument, low detection limit, wide linear range, high sensitivity and easy operation. Chemiluminescent detection of glucose is to use glucose to generate gluconic acid and hydrogen peroxide under the action of glucose oxidase. Hydrogen peroxide can oxidize luminol to produce chemiluminescence in the presence of a catalyst, and its luminous intensity is directly proportional to the concentration of hydrogen peroxide. Proportional, since the luminescence intensity of the Luminol-H 2 O 2 system in the current chemiluminescence method is relatively weak, a chemiluminescence instrument with light amplification is generally used for signal detection. Therefore, measuring the luminescence intensity with a chemiluminescence instrument can indirectly quantify the content of glucose in the sample. So far, in order to improve the detection limit of chemiluminescence, chemiluminescence is usually combined with enzymatic reaction and flow injection technology for the detection of biological samples (enzymatic reaction and flow injection technology are well known to those skilled in the art).
目前，化学发光反应通常采用金属络合物和过氧化物酶作催化剂来催化Luminol-H2O2体系实现葡萄糖的检测[Kricka LJ，Voyta JC，Bronstein I.Chemiluminescent methods for detecting and quantitating enzyme activity.Methods Enzymol，2000，305，370-390.]，然而由于金属络合物容易对化学发光信号产生干扰，而过氧化物酶价格昂贵、稳定性较差且不能回收[黄玉文，封满良，章竹君。反相流动注射化学发光法测定葡萄糖。分析化学1997，25，34-36]，因此需要寻找新的化学发光增强体系，实现葡萄糖简单、方便和高灵敏度检测。目前常用的检测方法有：At present, the chemiluminescent reaction usually uses metal complexes and peroxidases as catalysts to catalyze the Luminol-H 2 O 2 system to realize the detection of glucose [Kricka LJ, Voyta JC, Bronstein I. Chemiluminescent methods for detecting and quantitating enzyme activity. Methods Enzymol, 2000, 305, 370-390.], however, metal complexes are likely to interfere with the chemiluminescent signal, while peroxidase is expensive, has poor stability and cannot be recovered [Huang Yuwen, Feng Manliang, Mr. Zhang Zhu. Determination of glucose by reverse-phase flow injection chemiluminescence. Analytical Chemistry 1997, 25, 34-36], so it is necessary to find a new chemiluminescence enhancement system to realize the simple, convenient and high-sensitivity detection of glucose. Currently commonly used detection methods are:
1)金属络合物作催化剂1) metal complexes as catalysts
为了提高葡萄糖氧化酶的活性和酶的稳定性，研究人员采用固定化酶技术，即将葡萄糖氧化酶吸附在一定的基质上，以提高酶的寿命和活性。杨敏丽等将葡萄糖氧化酶固定在多孔的玻璃微珠上制成长寿命和高活性的固相酶，通过固相酶催化的葡萄糖氧化反应与流动注射化学发光体系(luminol-H2O2-K3Fe(CN)4)结合检测葡萄糖，通过优化反应温度、鲁米诺的浓度和葡萄糖溶液的流速，该方法的线性范围可达0.4～100μg/mL，检出限达0.08μg/mL[杨敏丽，熊楚明。固定化酶流动注射化学发光法测定葡萄糖。宁夏大学学报自然科学版，1995，16，28-31]。In order to improve the activity of glucose oxidase and the stability of the enzyme, the researchers adopted immobilized enzyme technology, that is, the glucose oxidase is adsorbed on a certain substrate to improve the life and activity of the enzyme. Yang Minli et al. immobilized glucose oxidase on porous glass microbeads to make a long-lived and highly active solid-phase enzyme. Through the glucose oxidation reaction catalyzed by the solid-phase enzyme and the flow injection chemiluminescence system (luminol-H 2 O 2 -K 3 Fe(CN) 4 ) combined to detect glucose, by optimizing the reaction temperature, the concentration of luminol and the flow rate of glucose solution, the linear range of this method can reach 0.4-100 μg/mL, and the detection limit can reach 0.08 μg/mL [Yang Minli, Xiong Chuming. Determination of glucose by flow injection chemiluminescence with immobilized enzyme. Ningxia University Journal Natural Science Edition, 1995, 16, 28-31].
2)过氧化物酶作催化剂2) Peroxidase as a catalyst
Lin等在玻璃微珠(经硅烷预处理)上固定金纳米颗粒和壳聚糖作为葡萄糖氧化酶的固定基质，将葡萄糖液相酶制成固相酶。结合辣根过氧化物酶催化的鲁米诺发光体系(luminol-H2O2-HRP)，通过优化反应的pH值、反应时间和鲁米诺的浓度，在流动注射-化学发光仪上进行化学发光检测，此方法的线性范围可达到0.01～6.0mmol/L，检出限为5.0μmol/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]。Lin et al. immobilized gold nanoparticles and chitosan on glass microspheres (pretreated with silane) as the immobilization matrix of glucose oxidase, and made glucose liquid phase enzyme into solid phase enzyme. Combined with the luminol luminescence system catalyzed by horseradish peroxidase (luminol-H 2 O 2 -HRP), by optimizing the pH value of the reaction, reaction time and the concentration of luminol, it was carried out on a flow injection-chemiluminescence instrument Chemiluminescent detection, the linear range of this method can reach 0.01～6.0mmol/L, and the detection limit is 5.0μmol/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等用金纳米颗粒吸附葡萄糖氧化酶和辣根过氧化物酶，通过溶胶-凝胶法将吸附酶的金纳米颗粒固定在化学发光池的内表面，通过该方法可同时增强葡萄糖氧化酶和辣根过氧化物酶的活性，从而增强化学发光反应的信号，结合流动注射技术分析葡萄糖，该方法的线性范围可达1.0×10-5mol/L～1.0×10-3mol/L，检出限达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]。Lan et al. used gold nanoparticles to adsorb glucose oxidase and horseradish peroxidase, and immobilized the gold nanoparticles with adsorbed enzymes on the inner surface of the chemiluminescent cell by the sol-gel method. This method can simultaneously enhance glucose oxidase and horseradish peroxidase. Horseradish peroxidase activity, thereby enhancing the signal of chemiluminescent reaction, combined with flow injection technology to analyze glucose, the linear range of this method can reach 1.0×10 -5 mol/L～1.0×10 -3 mol/L, the detection The output limit reaches 5×10 -6 mol/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].
发明内容Contents of the invention
本发明的目的在于提供一种四氧化三铁纳米颗粒催化的化学发光检测葡萄糖含量的方法，本发明操作简单、成本低、线性范围宽、检出限低。The object of the present invention is to provide a method for detecting glucose content by chemiluminescence catalyzed by iron ferric oxide nanoparticles, which has simple operation, low cost, wide linear range and low detection limit.
为实现上述目的，本发明在化学发光体系中引进了磁性四氧化三铁纳米颗粒，用磁性四氧化三铁纳米颗粒作催化剂，替代了通常使用的过氧化物酶、金属离子或者金属络合物。In order to achieve the above object, the present invention introduces magnetic iron ferric oxide nanoparticles in the chemiluminescent system, and uses magnetic iron ferric oxide nanoparticles as a catalyst to replace the commonly used peroxidase, metal ions or metal complexes .
详细地说，本发明提供的方法，是于10-60℃，将葡萄糖溶液加入至pH为4.0-9.0的四氧化三铁纳米颗粒-鲁米诺-H2O2溶液体系中，用SpectraMaxM2酶标仪检测葡萄糖浓度；其中，四氧化三铁纳米颗粒的浓度为大于或者等于10ppm(优选10-30ppm)，鲁米诺浓度为1-2mmol/L。四氧化三铁纳米颗粒的平均粒径为25nm。In detail, the method provided by the present invention is to add the glucose solution to the iron ferric oxide nanoparticle-luminol-H 2 O 2 solution system with a pH of 4.0-9.0 at 10-60°C, and use SpectraMaxM2 enzyme The standard instrument detects the glucose concentration; wherein, the concentration of ferric oxide nanoparticles is greater than or equal to 10ppm (preferably 10-30ppm), and the concentration of luminol is 1-2mmol/L. The average particle diameter of ferric oxide nanoparticles is 25nm.
本发明利用了磁性四氧化三铁纳米颗粒的高吸附性、生物相容性、类酶的催化特性及可重复利用的特点，建立了操作简单、成本低、线性范围宽、检出限低的葡萄糖化学发光检测法，以实现临床和食品领域葡萄糖含量简单、方便和高灵敏度的检测。The present invention utilizes the characteristics of high adsorption, biocompatibility, enzyme-like catalytic properties and reusability of magnetic ferroferric oxide nanoparticles, and establishes a method with simple operation, low cost, wide linear range and low detection limit. Glucose chemiluminescence detection method to realize simple, convenient and highly sensitive detection of glucose content in clinical and food fields.
本发明与现有化学发光检测法的不同之处：The difference between the present invention and the existing chemiluminescence detection method:
(1)本发明在化学发光体系中加入了具有强催化活性的磁性四氧化三铁纳米颗粒，取代了现有的金属离子、金属络合物和过氧化物酶等催化剂。(1) The present invention adds magnetic iron ferric oxide nanoparticles with strong catalytic activity to the chemiluminescence system, replacing existing catalysts such as metal ions, metal complexes and peroxidase.
(2)该方法采用了更简单的广泛使用的酶标仪(带有附加化学发光测试功能)，无需专门的化学发光分析仪，为葡萄糖的检测提供了方便性。(2) The method adopts a simpler and widely used microplate reader (with additional chemiluminescence test function), without the need for a special chemiluminescence analyzer, which provides convenience for the detection of glucose.
附图说明Description of drawings
图1显示了本发明四氧化三铁纳米颗粒-鲁米诺-H2O2溶液体系于不同pH值时的发光曲线。Fig. 1 shows the luminescence curves of the ferric oxide nanoparticle-luminol-H 2 O 2 solution system of the present invention at different pH values.
图2显示了本发明四氧化三铁纳米颗粒-鲁米诺-H2O2溶液体系于不同温度时的发光曲线。Fig. 2 shows the luminescence curves of the ferric oxide nanoparticles-luminol-H 2 O 2 solution system of the present invention at different temperatures.
图3显示了本发明四氧化三铁纳米颗粒-鲁米诺-H2O2溶液体系中，不同浓度的四氧化三铁纳米颗粒发光曲线。Fig. 3 shows the luminescence curves of ferric oxide nanoparticles with different concentrations in the ferric oxide nanoparticle-luminol-H 2 O 2 solution system of the present invention.
图4为本发明四氧化三铁纳米颗粒-鲁米诺-H2O2溶液体系检测葡萄糖含量的线性范围。Fig. 4 is the linear range of detecting the glucose content of the ferric oxide nanoparticle-luminol-H 2 O 2 solution system of the present invention.
具体实施方式Detailed ways
四氧化三铁纳米颗粒由于具有变价的特性，已被证明具有类过氧化物酶的生物活性，并且颗粒尺寸越小，该类过氧化物酶的生物活性越高[Gao L，Zhuang J，Nie L，et al.Intrinsic peroxidase-like activity of ferromagnetic nanoparticles.Nature Nanotech，2007，2，577-58]。Ferroferric oxide nanoparticles have been shown to have peroxidase-like biological activity due to their valence-variable properties, and the smaller the particle size, the higher the biological activity of this type of peroxidase [Gao L, Zhuang J, Nie L, et al. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nature Nanotech, 2007, 2, 577-58].
本发明结合葡萄糖的酶促反应，利用磁性四氧化三铁纳米颗粒的这种性质催化Luminol-H2O2化学发光体系，采用带有化学发光测试功能的SpectraMaxM2酶标仪(美国分子仪器公司)测量葡萄糖的浓度。并且，本发明对用四氧化三铁纳米颗粒作催化剂的化学发光反应的条件(pH值、温度和四氧化三铁纳米颗粒浓度)进行了优化。参照文献5中研究了鲁米诺浓度对化学发光强度的影响，考虑到随鲁米诺浓度的增加虽然发光信号增大，本底值也随之增加，本发明选用的鲁米诺浓度为2mmol/L。本发明采用的四氧化三铁纳米颗粒的平均粒径为25nm。The present invention combines the enzymatic reaction of glucose, utilizes this property of magnetic iron ferric oxide nanoparticles to catalyze the Luminol-H 2 O 2 chemiluminescence system, and adopts SpectraMaxM2 microplate reader with chemiluminescence test function (Molecular Instruments, Inc., USA) Measure the concentration of glucose. Moreover, the present invention optimizes the conditions (pH value, temperature and concentration of iron ferric oxide nanoparticles) of the chemiluminescent reaction using ferric oxide nanoparticles as catalyst. The influence of luminol concentration on the intensity of chemiluminescence has been studied with reference to
实施例：Example:
1)pH值优化1) pH optimization
配制四氧化三铁纳米颗粒-鲁米诺-H2O2溶液体系，在恒定四氧化三铁纳米颗粒、鲁米诺和H2O2浓度以及反应温度(四氧化三铁纳米颗粒的浓度为10ppm；鲁米诺浓度为2mmol/L；H2O2浓度为50μmol/L；反应温度为25℃)的条件下，通过向溶液中添加HCl和NaOH的方法调节体系的pH值(pH值范围为4～10)，用pH计测量pH值后，用SpectraMaxM2酶标仪测量各体系的发光强度，结果如图1所示。可以看出：在pH值范围为4～9时，发光强度较大，并且在pH＝7.0时，发光强度最大。Preparation of iron ferric oxide nanoparticles-luminol-H 2 O 2 solution system, at constant concentration of iron ferric oxide nanoparticles, luminol and H 2 O 2 concentration and reaction temperature (the concentration of iron ferric oxide nanoparticles is 10ppm; luminol concentration is 2mmol/L; H 2 O 2 concentration is 50μmol/L; reaction temperature is 25 ℃) conditions, by adding HCl and NaOH method to the solution to adjust the pH value of the system (
2)温度优化2) Temperature optimization
配制四氧化三铁纳米颗粒-鲁米诺-H2O2溶液体系，在恒定四氧化三铁纳米颗粒、鲁米诺和H2O2浓度以及pH值(四氧化三铁纳米颗粒的浓度为10ppm；鲁米诺浓度为2mmol/L；H2O2浓度为50μmol/L；pH值为7.0)的条件下，用恒温振荡培养箱对体系进行不同温度控制，温度控制范围为4～70℃，并用SpectraMaxM2酶标仪测量各体系的发光强度，结果如图2所示。可以看出：在反应温度为10～60℃时，发光强度较大，并且在反应温度为40℃时，发光强度最大。Preparation of iron ferric oxide nanoparticles-luminol-H 2 O 2 solution system, in constant iron ferric oxide nanoparticles, luminol and H 2 O 2 concentration and pH value (the concentration of iron ferric oxide nanoparticles is 10ppm; the concentration of luminol is 2mmol/L; the concentration of H2O2 is 50μmol/L; the pH value is 7.0). , and the luminescence intensity of each system was measured with a SpectraMaxM2 microplate reader, and the results are shown in Figure 2. It can be seen that: when the reaction temperature is 10-60°C, the luminous intensity is higher, and when the reaction temperature is 40°C, the luminous intensity is the largest.
3)四氧化三铁纳米颗粒浓度优化3) Optimization of the concentration of ferroferric oxide nanoparticles
配制四氧化三铁纳米颗粒-鲁米诺-H2O2溶液体系，在恒定鲁米诺和H2O2浓度以及pH值和温度(四氧化三铁纳米颗粒的浓度范围为3～60ppm；鲁米诺浓度为2mmol/L；H2O2浓度为50μmol/L；pH值为7.0；反应温度为40℃)的条件下。用SpectraMaxM2酶标仪测量各体系的发光强度，结果如图3所示。可以看出：当四氧化三铁纳米颗粒的浓度为3～10ppm时，随着四氧化三铁纳米颗粒浓度的增加，发光强度迅速增强，当四氧化三铁纳米颗粒的浓度大于10ppm时，发光强度较大，并且随着四氧化三铁纳米颗粒浓度的增加也逐渐增强；而当四氧化三铁纳米颗粒的浓度大于25ppm时，随着四氧化三铁纳米颗粒浓度的增加，发光强度基本保持不变，因此四氧化三铁纳米颗粒的优化浓度为25ppm。Preparation of iron ferric oxide nanoparticles-luminol-H 2 O 2 solution system, under constant luminol and H 2 O 2 concentration and pH value and temperature (concentration range of ferric iron tetroxide nanoparticles is 3～60ppm; The concentration of luminol is 2 mmol/L; the concentration of H 2 O 2 is 50 μmol/L; the pH value is 7.0; the reaction temperature is 40° C.). The luminescence intensity of each system was measured with a SpectraMaxM2 microplate reader, and the results are shown in Figure 3. It can be seen that: when the concentration of ferric oxide nanoparticles is 3-10ppm, along with the increase of the concentration of ferric oxide nanoparticles, the luminous intensity increases rapidly; when the concentration of ferric oxide nanoparticles is greater than 10ppm, the luminous intensity The intensity is large, and gradually increases with the increase of the concentration of ferric oxide nanoparticles; while when the concentration of ferric oxide nanoparticles is greater than 25ppm, the luminous intensity basically maintains with the increase of the concentration of ferric oxide nanoparticles unchanged, so the optimal concentration of Fe3O4 nanoparticles is 25ppm.
因此，本发明最优的化学发光条件为：pH＝7，反应温度为40℃，四氧化三铁纳米颗粒的浓度为25ppm。Therefore, the optimal chemiluminescence conditions of the present invention are: pH=7, reaction temperature is 40° C., and the concentration of iron ferric oxide nanoparticles is 25 ppm.
4)优化条件下葡萄糖含量的测量4) Measurement of glucose content under optimized conditions
将不同浓度的葡萄糖溶液分别与葡萄糖氧化酶作用，在优化的发光条件下用SpectraMaxM2酶标仪对其化学发光强度进行测试，作出化学发光强度随萄糖葡浓度变化的标准曲线。如图4所示。可以看出在该优化发光条件下，萄糖葡浓度检测的线性范围可达2×10-6mol/L～1×10-3mol/L(相当于0.36mg/L～180mg/L)，相关系数为0.9990。通过对优化条件下(2mmol/L鲁米诺，25ppm Fe3O4，pH＝7和反应温度为40℃)的空白本底进行测试，以3σ(空白平行测定11次的标准偏差)对应的浓度计算检出限，可得到该方法检测葡萄糖的检出限为0.4μmol/L。Glucose solutions with different concentrations were reacted with glucose oxidase, and the chemiluminescence intensity was tested with a SpectraMaxM2 microplate reader under optimized luminescence conditions, and a standard curve of chemiluminescence intensity versus glucose concentration was made. As shown in Figure 4. It can be seen that under the optimized luminescent conditions, the linear range of glucose concentration detection can reach 2×10 -6 mol/L～1×10 -3 mol/L (equivalent to 0.36mg/L～180mg/L), The correlation coefficient is 0.9990. By testing the blank background under optimized conditions (2mmol/L luminol, 25ppm Fe 3 O 4 , pH=7 and
本发明技术方案的有益效果Beneficial effects of the technical solution of the present invention
(1)本发明优化条件下检测葡萄糖的线性范围为2×10-6mol/L～1×10-3mol/L(相当于0.36mg/L～180mg/L)，检出限达到0.4μmol/L。与现有相关技术的对比如下表1所示。可以看出：本发明不仅具有检测葡萄糖的线性范围宽的特点，而且检出限较现有技术降低了5倍，能够实现更低浓度的葡萄糖含量检测。(1) The linear range of glucose detection under the optimized conditions of the present invention is 2× 10-6 mol/L～1× 10-3 mol/L (equivalent to 0.36mg/L～180mg/L), and the detection limit reaches 0.4μmol /L. The comparison with the existing related technologies is shown in Table 1 below. It can be seen that the present invention not only has the characteristics of a wide linear range for detecting glucose, but also has a detection limit that is 5 times lower than that of the prior art, and can realize the detection of glucose content at a lower concentration.
(2)需要说明的是，本发明使用的检测仪器为SpectraMaxM2酶标仪。同化学发光法专门使用的检测仪器-化学发光分析仪相比，SpectraMaxM2酶标仪不具备光放大功能，因此，如果使用化学发光分析仪测量本发明涉及的四氧化三铁纳米颗粒-鲁米诺-H2O2-葡萄糖溶液体系中的葡萄糖含量，其检出限将更低。同时，由于本发明无需专门的化学发光分析仪，只需更简单的广泛使用的酶标仪(带有附加化学发光测试功能)，因此为葡萄糖含量的检测提供了便利。(2) It should be noted that the detection instrument used in the present invention is a SpectraMaxM2 microplate reader. Compared with the detection instrument-chemiluminescence analyzer that is specially used in the chemiluminescence method, the SpectraMaxM2 microplate reader does not have the light amplification function. Therefore, if the chemiluminescence analyzer is used to measure the iron ferric oxide nanoparticles-luminol -H 2 O 2 -glucose content in the glucose solution system, the detection limit will be lower. At the same time, since the present invention does not need a special chemiluminescence analyzer, it only needs a simpler and widely used microplate reader (with additional chemiluminescence testing function), thus providing convenience for the detection of glucose content.
(3)由于四氧化三铁纳米颗粒具有磁性，因此利用磁场作用，如磁铁等，即可实现四氧化三铁纳米颗粒的分离，从而实现可重复利用，克服了用过氧化物酶、金属离子和金属络合物作催化剂的难以重复利用的缺点，更有利于降低检测成本。(3) Since ferric oxide nanoparticles are magnetic, the separation of ferric oxide nanoparticles can be realized by using the magnetic field, such as magnets, etc., so as to realize reusability and overcome the problem of using peroxidase and metal ions. The disadvantage of being difficult to recycle with metal complexes as catalysts is more conducive to reducing detection costs.
表1Table 1
注：标注*栏为使用SpectraMaxM2酶标仪获得的检测结果；未标注栏为使用化学发光分析仪获得检测结果。Note: The marked * column is the detection result obtained by using the SpectraMaxM2 microplate reader; the unmarked column is the detection result obtained by using the chemiluminescence analyzer.
文献[5]为：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.The literature [5] is: 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.
文献[6]为：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.The literature [6] is: 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.
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