CN101158638A - Nano-silver Spectrophotometry for Detecting Hydroxyl Radicals - Google Patents
Nano-silver Spectrophotometry for Detecting Hydroxyl RadicalsDownload PDFInfo
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Abstract
Translated fromChineseDescription
Translated fromChinese技术领域：Technical field:
本发明涉及羟基自由基的检测方法，具体是用纳米银作为羟基自由基的捕获剂，以分光光度计测定被测体系的吸光度的方法——纳米银分光光度法。The invention relates to a detection method for hydroxyl radicals, specifically a method for measuring the absorbance of a measured system with a spectrophotometer using nanometer silver as a capture agent for hydroxyl radicals—nano silver spectrophotometry.
背景技术：Background technique:
羟基自由基(·OH)是目前已知化学性质最活泼的一种活性氧分子，可以发生电子转移，夺取氢原子和羟基化等反应，使糖类、氨基酸、蛋白质、核酸和脂类发生氧化。由于·OH的氧化能力强、寿命短、对机体危害大，它与衰老、肿瘤、辐射损伤和细胞吞噬有关；此外，羟自由基清除率是反映药物抗氧作用的重要指标，因此，在基础医学、临床医学、预防医学、药学等领域，已对自由基开展了大量的研究。Hydroxyl radical ( OH) is the most active active oxygen molecule known chemically. It can undergo electron transfer, capture hydrogen atoms and hydroxylation reactions to oxidize sugars, amino acids, proteins, nucleic acids and lipids. . Due to the strong oxidation ability of OH, short lifespan and great harm to the body, it is related to aging, tumors, radiation damage and cell phagocytosis; in addition, the scavenging rate of hydroxyl radicals is an important indicator reflecting the anti-oxidation effect of drugs. Therefore, in the basic In the fields of medicine, clinical medicine, preventive medicine, and pharmacy, a lot of research has been carried out on free radicals.
此外，在已知的氧化剂中，·OH的氧化能力仅次于F2，是一种非选择性的氧化剂，能很容易的氧化各种有机物和无机物，氧化效率高，反应速度快。因而在大气化学、天然水体化学和废水深度氧化等的研究中，涉及许多自由基参与的化学反应，自由基与污染物的转化和清除密切相关。但是，由于自由基非常活泼，浓度低、存在的寿命短，因此建立一种灵敏快速测定·OH的方法尤为重要。。In addition, among the known oxidizing agents, the oxidation ability of ·OH is second only to F2 , and it is a non-selective oxidizing agent, which can easily oxidize various organic and inorganic substances, with high oxidation efficiency and fast reaction speed. Therefore, in the research of atmospheric chemistry, natural water chemistry and deep oxidation of wastewater, many chemical reactions involving free radicals are involved, and free radicals are closely related to the transformation and removal of pollutants. However, since free radicals are very active, low in concentration and short in lifetime, it is particularly important to establish a sensitive and rapid method for the determination of OH. .
目前国内外测定羟基自由基的方法主要有：电子自旋共振法(ESR)，高效液相色谱法(HPLC)，化学发光法(CL)，光度法，电化学法、荧光法等。其中，ESR法可直接用于检测和研究具有未成对电子的自由基与过渡金属离子及其化合物，但由于此方法是利用自旋捕捉剂与短寿命自由基结合生成相对稳定的自由基，即自旋加合物，然后进行ESR测定。由于自旋加合物的寿命仍然很短，只有几分钟或几十分钟，因此，必须捕捉自由基后立即进行EPR测定，故对样品的制备及检测有很大的限制。HPLC法也是一种常用的间接检测·OH的方法，该方法是必须先选择合适的化合物捕获体系中的自由基，使之生成具有一定稳定性，且能被液相色谱检测的产物，然后用HPLC液相色谱测定，其中，二甲基亚砜(DMSO)捕集自由基测定法所用的DMSO有毒性，能迅速透过皮肤进入体内；而水杨酸捕集自由基HPLC测定法是用电化学检测器测定·OH与水杨酸的生成物2，3-二羟基苯甲酸和2，5-二羟基苯甲酸，其检测限分别为10和5fmol。HPLC法选择性较好，能同时检测几种自由基，但所用仪器价格昂贵，不易普及推广。徐向荣等用酵母作发光底物，用化学发光法测定了Fenton反应产生的·OH，用于模拟染料废水处理中·OH的测定，重现性好，但所用试剂不易获得，且方法精密度较差。电化学分析法即利用脱氧核糖与Fenton体系产生·OH作用，在酸性条件下经过讲解生成丙二醛、丙二醛在于甲醛、氨反应生成电活性产物3，5-二乙酰-1，4-二氢吡啶，通过电化学分析法间接定量羟基自由基的产生量，但此法反应步骤较多且所用醛类试剂危害人体健康。荧光法是利用Fenton反应产生的·OH能将Ce3+氧化成Ce4+，测定Ce3+的荧光强度变化即可间接测定羟自由基的产生量，该方法重现性好、操作简便快速。此外，还有激光诱导荧光法、化学离子化质谱法、化学扩大法也可用来测定羟基自由基，但不易普及推广或仪器价格昂贵等。At present, the methods for the determination of hydroxyl radicals at home and abroad mainly include: electron spin resonance (ESR), high performance liquid chromatography (HPLC), chemiluminescence (CL), photometry, electrochemical methods, fluorescence methods, etc. Among them, the ESR method can be directly used to detect and study free radicals with unpaired electrons and transition metal ions and their compounds, but because this method uses spin trapping agents combined with short-lived free radicals to generate relatively stable free radicals, namely Spin adducts, followed by ESR measurements. Since the lifetime of the spin adduct is still very short, only a few minutes or tens of minutes, the EPR measurement must be carried out immediately after capturing the free radical, so there are great limitations on the preparation and detection of the sample. The HPLC method is also a commonly used method for indirectly detecting OH. In this method, a suitable compound must first be selected to capture the free radicals in the system to generate a product that has certain stability and can be detected by liquid chromatography, and then use HPLC liquid chromatography is measured, and wherein, the DMSO used in dimethyl sulfoxide (DMSO) capturing free radical assay method is toxic, can enter the body rapidly through the skin; The chemical detector detects 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid, the products of OH and salicylic acid, and the detection limits are 10 and 5 fmol, respectively. The HPLC method has good selectivity and can detect several kinds of free radicals at the same time, but the instruments used are expensive and difficult to popularize. Xu Xiangrong et al. used yeast as a luminescent substrate to measure the OH produced by the Fenton reaction by chemiluminescence, which was used to simulate the determination of OH in dye wastewater treatment. The reproducibility was good, but the reagents used were not easy to obtain, and the precision of the method was low. Difference. The electrochemical analysis method uses deoxyribose and Fenton system to produce OH interaction, and generates malondialdehyde after explanation under acidic conditions. Malondialdehyde is formed by the reaction of formaldehyde and ammonia to form an
已有的分光光度法如徐向荣等在《化学发光法测定Fenton反应中的羟基自由基及其应用》(环境科学，1998年19卷第2期)用DMSO捕集Fenton反应体系中的·OH，产生的甲基亚磺酸与坚牢蓝BB盐反应，生成的重氮化合物经甲苯∶正丁醇(3∶1)混合物萃取后，用分光光度法在420nm处进行比色测定，这种方法用来研究羟基自由基的产生与清除准确可靠，有较好的重现性。贾之慎等用水杨酸捕集Fenton反应体系中的·OH，生成的2，3-二羟基苯甲酸用乙醚萃取，用钨酸钠和亚硝酸钠显色，然后用分光光度计测定其510nm处的吸光度，结果显示吸光度随水杨酸浓度、Fe2+浓度及H2O2的浓度增加而上升，认为这体现了·OH生成的变化规律，这种方法是一般实验室可采用的简便实用的方法。此外还有细胞色素C光度法，金鸣等在《邻二氮菲-Fe2+氧化法检测H2O2/Fe2+产生的羟自由基》(生物化学与生物物理进展，1996年第23卷第6期)中的邻二氮菲-Fe2+-H2O2体系分光光度法，分光光度法测定·OH不需要昂贵的仪器，简便、快速，易推广普及，但测定过程中的干扰因素较多，准确性、灵敏度和选择性有待提高。Existing spectrophotometry such as Xu Xiangrong et al. in "Chemiluminescence Determination of Hydroxyl Free Radicals in the Fenton Reaction and Its Application" (Environmental Science, Volume 19, No. 2, 1998) traps OH in the Fenton reaction system with DMSO, The generated methanesulfinic acid reacts with the fast blue BB salt, and the generated diazo compound is extracted by a mixture of toluene:n-butanol (3:1), and then colorimetrically determined at 420nm by spectrophotometry. It is used to study the production and scavenging of hydroxyl radicals, which is accurate and reliable, and has good reproducibility. Jia Zhishen et al. captured OH in the Fenton reaction system with salicylic acid, extracted 2,3-dihydroxybenzoic acid with ether, developed color with sodium tungstate and sodium nitrite, and then measured its 510nm concentration with a spectrophotometer. The results show that the absorbance increases with the concentration of salicylic acid, Fe2+ and H2 O2 , which reflects the change rule of OH generation. This method is a simple method that can be used in general laboratories. practical approach. In addition, there is cytochrome C spectrophotometry, Jin Ming et al. in "Detection of Hydroxyl Free Radicals Produced by H2 O2 /Fe2+ by Ophenanthroline-Fe2+ Oxidation Method" (Progress in Biochemistry and Biophysics, 1996, p. The spectrophotometric method of o-phenanthroline-Fe2+ -H2 O2 system in volume 23, No. 6), the spectrophotometric determination of OH does not require expensive instruments, is simple, fast, and easy to popularize, but the determination process There are many interference factors, and the accuracy, sensitivity and selectivity need to be improved.
发明内容：Invention content:
本发明的目的是提供一种新的纳米银为捕获剂的检测羟基自由基的纳米银分光光度法。这种方法简单、灵敏度高、稳定性好、检测费用低，且所用试剂安全无毒。The purpose of the present invention is to provide a new nano-silver spectrophotometric method for detecting hydroxyl radicals using nano-silver as a trapping agent. This method is simple, high sensitivity, good stability, low detection cost, and the reagents used are safe and non-toxic.
本发明通过以下步骤实现对羟基自由基的检测：The present invention realizes the detection of hydroxyl radicals through the following steps:
(1)取定量的纳米银，与已知浓度的含有或可产生羟基自由基的被测物混匀，用水定容后形成被测体系；(1) Take quantitative nano-silver, mix it with known concentration containing or capable of producing hydroxyl radicals, and form the measured system after distilling to volume with water;
(2)将被测体系转移至比色皿，使用分光光度计于460nm处测定其吸光度A；(2) Transfer the measured system to a cuvette, and use a spectrophotometer to measure its absorbance A at 460nm;
(3)取纳米银作空白对照，测定吸光度A0；(3) Take nano-silver as a blank control, and measure the absorbance A0 ;
(4)求得ΔA＝A0-A；(4) Obtain ΔA=A0 -A;
(5)依照不同浓度的被测体系中羟基自由基的浓度C与ΔA值的对应关系，作出标准曲线；(5) According to the corresponding relationship between the concentration C of hydroxyl radicals and the value of ΔA in the measured system of different concentrations, a standard curve is made;
(6)按上述(1)～(4)的方法，测得某未知浓度被测体系的ΔA，依据(5)的标准曲线计算出未知浓度被测体系中羟基自由基的浓度。(6) According to the method (1)-(4) above, measure the ΔA of a test system with an unknown concentration, and calculate the concentration of hydroxyl radicals in the test system with an unknown concentration according to the standard curve in (5).
为了与目前的分光光度计的检测范围和灵敏度相适应，确保和提高检测的准确度，本发明将被测体系中纳米银的浓度规定为0.004～0.04m mol/L。In order to adapt to the detection range and sensitivity of the current spectrophotometer, to ensure and improve the accuracy of detection, the present invention regulates the concentration of nano-silver in the measured system to be 0.004～0.04m mol/L.
本发明在被测体系形成后需在5min后将被测体系移入比色皿测定吸光度，这样能提高测定方法的稳定性，保证检测结果的可靠性和准确性。In the present invention, after the formation of the measured system, the measured system needs to be moved into the cuvette to measure the absorbance after 5 minutes, which can improve the stability of the measuring method and ensure the reliability and accuracy of the detection result.
纳米银是一种新型功能材料，存在表面等离子共振吸收，共振散射效应，已用于共振散射光谱分析，并获得了较好的效果。其在杀菌消毒等方面的应用也取得了很大的成功。纳米银制备简单，可利用微波高压快速制得纳米银微粒，然后采用高速离心法纯化。Nano-silver is a new type of functional material, which has surface plasmon resonance absorption and resonance scattering effect. It has been used in resonance scattering spectroscopy analysis and achieved good results. Its application in sterilization and disinfection has also achieved great success. Nano-silver is easy to prepare, and nano-silver particles can be quickly prepared by microwave high pressure, and then purified by high-speed centrifugation.
本发明发现Fenton反应产生的羟基自由基可以氧化纳米银微粒而使其成为银离子，导致体系的吸光度降低，据此建立了测定羟基自由基的纳米银分光光度法——以纳米银为羟基自由基捕获剂，采用分光光度计测定体系的吸光度。The present invention finds that the hydroxyl free radicals produced by the Fenton reaction can oxidize nano-silver particles to become silver ions, resulting in a decrease in the absorbance of the system. Based on this, a nano-silver spectrophotometric method for measuring hydroxyl free radicals is established—using nano-silver as the hydroxyl free radical The absorbance of the system was measured with a spectrophotometer.
Ag+·OH→Ag++OH-Ag+·OH→Ag+ +OH-
银虽然是重金属，但本发明的方法中，用量极少，且本发明只使用纳米银这一种试剂，因此，不会增加检测费用。银性质稳定，不容易与其它一般物质发生反应，所以能避免检测过程中各种干扰，表现出高度选择性，减少检测误差，确保数据的稳定和检测结果的可靠，而且银安全无毒，也不对环境产生污染。Although silver is a heavy metal, in the method of the present invention, the amount used is extremely small, and the present invention only uses nano-silver as a reagent, so the detection cost will not be increased. Silver is stable in nature and does not easily react with other common substances, so it can avoid various interferences in the detection process, exhibit high selectivity, reduce detection errors, ensure stable data and reliable detection results, and silver is safe and non-toxic, and also No pollution to the environment.
本发明发现羟基自由基对纳米银的氧化反应灵敏，不可逆、速度快，从而使得检测过程快速，所得结果稳定，方法的重现性好、灵敏度高，检出限可达0.25μmol/L(H2O2)，超出现有的分光光度法。The present invention finds that the oxidation reaction of hydroxyl radicals to nano-silver is sensitive, irreversible, and fast, so that the detection process is fast, the obtained results are stable, the method has good reproducibility, high sensitivity, and the detection limit can reach 0.25 μmol/L (H2 O2 ), beyond existing spectrophotometric methods.
纳米银分光光度法与一般的分光光度法一样，所用的分光光度计是一种传统的检测仪器，品种型号很多，价格便宜，操作简单，被普遍使用，一般实验人员在一般的实验室中即可完成对羟基自由基的检测。Nano-silver spectrophotometry is the same as general spectrophotometry. The spectrophotometer used is a traditional detection instrument with many varieties and models. It is cheap and easy to operate. It is widely used. The detection of hydroxyl radicals can be completed.
本发明将被测体系中纳米银的浓度规定为0.004～0.04m mol/L，使得吸光度的数值及其变化均能被目前的分光光度计可靠地检测出来，从而确保和提高了检测准确性。考虑到氧化反应的时间因素，本发明规定需在被测体系形成后5min之后再进行检测，预留出氧化反应过程所需要的时间，等待体系反应停止，状态稳定，这样的检测结果才是可靠的、准确的。In the present invention, the concentration of nano-silver in the measured system is specified as 0.004-0.04m mol/L, so that the value and change of absorbance can be reliably detected by the current spectrophotometer, thereby ensuring and improving the detection accuracy. Considering the time factor of the oxidation reaction, the present invention stipulates that the detection should be carried out 5 minutes after the formation of the tested system, and the time required for the oxidation reaction process should be set aside, waiting for the reaction of the system to stop and the state to be stable. Only such detection results are reliable. Yes, exactly.
根据本发明的试验，液相纳米银仅在460nm处产生一个表面等离子体共振吸收峰，所以本发明的检测羟基自由基的纳米银分光光度法选取460nm作为检测波长，这样就保证了检测结果的最大程度的可靠。According to test of the present invention, liquid phase nano-silver only produces a surface plasmon resonance absorption peak at 460nm place, so the nano-silver spectrophotometry method of detection hydroxyl radical of the present invention chooses 460nm as detection wavelength, has thus just guaranteed the accuracy of detection result. Maximum reliability.
总之，本发明的检测羟基自由基的纳米银分光光度法，不使用昂贵的仪器设备，不需要种类繁多的试剂，也不需要严格的检测条件和环境，由一般的实验员在很普通的实验室中即可快速简单地完成检测，而且数据稳定，结果可靠，灵敏度高，重现性好，非常适宜于测定羟基自由基，可应用于抗氧化研究中检测离体实验体系中的羟基自由基的氧化、清除效应，特别是对于筛选抗氧化剂具有重要的应用价值。本发明的方法也可用于快速简单灵敏地检测臭氧浓度和其它自由基。In a word, the nano-silver spectrophotometric method for detecting hydroxyl radicals of the present invention does not use expensive instruments and equipment, does not require a wide variety of reagents, and does not require strict detection conditions and environments. The detection can be completed quickly and simply in the laboratory, and the data is stable, the result is reliable, the sensitivity is high, and the reproducibility is good. It is very suitable for the determination of hydroxyl radicals, and can be applied to the detection of hydroxyl radicals in in vitro experimental systems in antioxidant research. Oxidation and scavenging effects, especially for screening antioxidants have important application value. The method of the present invention can also be used for fast, simple and sensitive detection of ozone concentration and other free radicals.
附图说明：Description of drawings:
图1是本发明的一个实施例的标准曲线；Fig. 1 is the calibration curve of an embodiment of the present invention;
图2是纳米银的吸收光谱图。其中：Figure 2 is the absorption spectrum diagram of nano-silver. in:
曲线1：pH 4.0-0.12mmol/L FeSO4-0.016mmol/L Ag；Curve 1: pH 4.0-0.12mmol/L FeSO4 -0.016mmol/L Ag;
曲线2：pH4.0-0.12mmol/L FeSO4-0.016mmol/L Ag-1.25μmol/L H2O2；Curve 2: pH4.0-0.12mmol/L FeSO4 -0.016mmol/L Ag-1.25μmol/L H2 O2 ;
曲线3：pH4.0-0.12mmol/L FeSO4-0.016mmol/LAg-2.5μmol/L H2O2；Curve 3: pH4.0-0.12mmol/L FeSO4 -0.016mmol/LAg-2.5μmol/L H2 O2 ;
曲线4：pH4.0-0.12mmol/L FeSO4-0.016mmol/LAg-5.0μmol/L H2O2。Curve 4: pH4.0-0.12mmol/L FeSO4 -0.016mmol/LAg-5.0μmol/L H2 O2 .
具体实施方式：Detailed ways:
下面的实施方式描述了在酸性条件下，Fe2+催化H2O2产生羟基自由基，羟基自由基具有较强的氧化性，可将银纳米微粒氧化为银离子，吸光度降低，据此建立的测定羟基自由基的方法。主要反应式如下，The following embodiment describes that under acidic conditions, Fe2+ catalyzes H2 O2 to generate hydroxyl radicals. Hydroxyl radicals have strong oxidizing properties, and can oxidize silver nanoparticles into silver ions, and the absorbance decreases. Based on this, it is established method for the determination of hydroxyl radicals. The main reaction formula is as follows,
Fe2++H2O2→Fe3++OH-+·OH (1)Fe2+ +H2 O2 →Fe3+ +OH- + OH (1)
Fe3++H2O2→Fe2++HO2·+H+ (2)Fe3+ +H2 O2 →Fe2+ +HO2 +H+ (2)
(Ag)n+·OH→Ag++OH- (3)(Ag)n + OH→Ag+ +OH- (3)
羟基自由基的测定方法的描述如下：在5mL的具塞刻度试管中，依次加入pH3.0～6.0 HAc-NaAc缓冲溶液0.1～1.0mL和0.2～1.0mL的浓度为2.0mmol/L的FeSO4溶液以及一定量的H2O2溶液(取标定过的储备液稀释)，然后再加入0.1～1.0mL浓度为0.20mmol/L的纳米银微粒，用水稀释到刻度，混匀，5～10min后将溶液转移到比色皿，测定其460nm波长处的A，不加H2O2溶液做空白，测定其吸光度A0，求得ΔA＝A0-A，以ΔA与形成被测体系时加入的过氧化氢量所成的浓度C作出标准曲线。由反应原理可知，羟基自由基的浓度与过氧化氢浓度成正比，为研究方便，这里用过氧化氢浓度代替羟基自由基的浓度。当然这里过氧化氢的用量应能全部反应完全，全部转化成羟基自由基，且羟基自由基又全部用来氧化银纳米微粒。The description of the method for the determination of hydroxyl radicals is as follows: In a 5 mL graduated test tube with stoppers, add 0.1 to 1.0 mL of pH 3.0 to 6.0 HAc-NaAc buffer solution and 0.2 to 1.0 mL of FeSO4 with a concentration of 2.0 mmol/L in sequence. solution and a certain amount of H2 O2 solution (take the calibrated stock solution to dilute), then add 0.1-1.0mL nano-silver particles with a concentration of 0.20mmol/L, dilute to the mark with water, mix well, and wait for 5-10min Transfer the solution to a cuvette, measure its A at the wavelength of 460nm, do not add H2 O2 solution as a blank, measure its absorbance A0 , obtain ΔA=A0 -A, and use ΔA to form the measured system when adding Concentration C formed by the amount of hydrogen peroxide to make a standard curve. It can be seen from the reaction principle that the concentration of hydroxyl radicals is directly proportional to the concentration of hydrogen peroxide. For the convenience of research, the concentration of hydrogen peroxide is used here instead of the concentration of hydroxyl radicals. Certainly the consumption of hydrogen peroxide here should be able to all react completely, and all change into hydroxyl radicals, and hydroxyl radicals are all used to oxidize silver nanoparticles again.
实施例：配制H2O2溶液：取30％H2O2 1.0ml，加水稀释成250ml，混匀；取此溶液用高锰酸钾溶液标定其浓度为0.0395mol/L；临用前取适量，用水稀释成54.0μmol/L的H2O2溶液。Example: preparation of H2 O2 solution: take 1.0ml of 30% H2 O2 , add water to dilute to 250ml, and mix well; take this solution and calibrate its concentration to 0.0395mol/L with potassium permanganate solution; take it before use Appropriate amount, diluted with water to form a 54.0 μmol/L H2 O2 solution.
在5 mL的具塞刻度试管中，依次加入0.10mL pH 4.0 HAc-NaAc缓冲溶液和0.30mL的浓度为2.0mmol/L的FeSO4溶液以及46.3μL、92.5μL、185μL、370μL、555μL、740μL上述已配制好的浓度为54.0μmol/L的H2O2溶液，然后再加入0.40mL浓度为0.20mmol/L的纳米银微粒，用水稀释到刻度，混匀，5min后将溶液转移到比色皿，置于UV1900PC型分光光度计(上海亚研电子公司)测定其460nm波长处的A，不加H2O2溶液做空白，测定其吸光度A0，求得ΔA＝A0-A。有关数据见表一。Into a 5 mL graduated test tube with stopper, add 0.10 mL pH 4.0 HAc-NaAc buffer solution and 0.30 mL FeSO4 solution with a concentration of 2.0 mmol/L and 46.3 μL, 92.5 μL, 185 μL, 370 μL, 555 μL, 740 μL of the above The prepared H2 O2 solution with a concentration of 54.0 μmol/L, and then add 0.40 mL of nano-silver particles with a concentration of 0.20 mmol/L, dilute to the mark with water, mix well, and transfer the solution to a cuvette after 5 minutes , placed in a UV1900PC spectrophotometer (Shanghai Yayan Electronics Co., Ltd.) to measure A at the wavelength of 460nm, without adding H2 O2 solution as a blank, measure the absorbance A0 , and obtain ΔA=A0 -A. See Table 1 for relevant data.
表一Table I
由表一得出标准曲线如图1。图1表明随吸光度的变化值随着过氧化氢浓度C的增大呈线性关系，线性范围为0.50～8.0μmol/L，回归方程为ΔA460nm＝0.0424C+0.002，相关系数为0.9997，检出限为0.25μmol/L。The standard curve obtained from Table 1 is shown in Figure 1. Fig. 1 shows that the change value along with the absorbance is linearly related with the increase of hydrogen peroxide concentration C, and the linear range is 0.50～8.0 μ mol/L, and the regression equation is ΔA460nm =0.0424C+0.002, and the correlation coefficient is 0.9997, and the detection The limit is 0.25μmol/L.
取上述配制好的H2O2溶液550μl，按上述的方法测出A、A0、ΔA，重复三次，结果如表二。Take 550 μl of the above-prepared H2 O2 solution, measure A, A0 , and ΔA according to the above-mentioned method, repeat three times, and the results are shown in Table 2.
表二Table II
依据图1的标准曲线，可知被测体系中原有的H2O2溶液为5.925μmol/L，产生的羟基自由基的浓度平均值亦为5.925μmol/L，检测结果的RSD＝1.0％。According to the standard curve in Figure 1, it can be seen that the original H2 O2 solution in the tested system is 5.925 μmol/L, and the average concentration of the produced hydroxyl radicals is also 5.925 μmol/L, and the RSD of the test result is 1.0%.
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