CN112763558B - A kind of detection method of Staphylococcus aureus - Google Patents
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Abstract
Description
技术领域technical field
本发明涉及细菌检测技术领域，尤其涉及一种金黄色葡萄球菌检测方法。The invention relates to the technical field of bacterial detection, in particular to a method for detecting Staphylococcus aureus.
背景技术Background technique
食源性疾病指的是食品被致病菌污染后，通过摄食的方式进入到人体里，并致使人体感染或中毒，威胁人们身体健康健康和生命安全的一类疾病的统称。虽然现代科学技术的发展已到了一定的水平，但是不管在发达还是发展中国家，食源性疾病仍然严重地危害着人们的身体健康，近年来频频发生的食品安全事件也表明了食源性疾病尚未得到有效的控制，因此开发新型的准确检测食源性致病菌的检测技术至关重要，它对预防、控制食源性疾病，保障人们的健康有着重要的意义。Foodborne diseases refer to the general term for a class of diseases that enter the human body through ingestion after food is contaminated by pathogenic bacteria, and cause human infection or poisoning, threatening people's health, health and life safety. Although the development of modern science and technology has reached a certain level, no matter in developed or developing countries, food-borne diseases still seriously endanger people's health. The frequent food safety incidents in recent years also indicate that food-borne diseases It has not been effectively controlled, so it is very important to develop a new detection technology for accurate detection of food-borne pathogens. It is of great significance to prevent and control food-borne diseases and protect people's health.
金黄色葡萄球菌能引起新生儿脑膜炎、败血症和坏死性小肠结膜炎等疾病，对人们身体健康和生命安全构成严重的威胁，因此研究准确检测金黄色葡萄球菌的方法具有重要意义。现有的金黄色葡萄球菌检测方法一般采用化学检测法，存在操作繁琐，重复性差的不足Staphylococcus aureus can cause diseases such as neonatal meningitis, sepsis and necrotizing enteroconjunctivitis, which pose a serious threat to people's health and life safety. Therefore, it is of great significance to study the method for accurate detection of Staphylococcus aureus. The existing Staphylococcus aureus detection method generally adopts chemical detection method, which has the disadvantages of complicated operation and poor repeatability.
发明内容SUMMARY OF THE INVENTION
本发明为了解决上述技术问题，提供了一种金黄色葡萄球菌检测方法，其能够准确检测出金黄色葡萄球菌浓度，操作简单。In order to solve the above technical problems, the present invention provides a Staphylococcus aureus detection method, which can accurately detect the concentration of Staphylococcus aureus and is simple to operate.
为了解决上述问题，本发明采用以下技术方案予以实现：In order to solve the above problems, the present invention adopts the following technical solutions to realize:
本发明的一种金黄色葡萄球菌检测方法，包括以下步骤：A kind of Staphylococcus aureus detection method of the present invention comprises the following steps:
S1：配置不同浓度的金黄色葡萄球菌悬浮液，检测每个浓度的金黄色葡萄球菌悬浮液对应的特征值Y，将这些值进行拟合得到浓度计算公式：Y＝0.13×LgX-0.01，X为金黄色葡萄球菌悬浮液的浓度；S1: Configure different concentrations of Staphylococcus aureus suspension, detect the characteristic value Y corresponding to each concentration of Staphylococcus aureus suspension, and fit these values to obtain the concentration calculation formula: Y=0.13×LgX-0.01, X is the concentration of Staphylococcus aureus suspension;
S2：取待测金黄色葡萄球菌悬浮液，检测其对应的特征值Y，根据浓度计算公式：Y＝0.13×LgX-0.01，计算出待测金黄色葡萄球菌悬浮液的浓度。S2: Take the suspension of Staphylococcus aureus to be tested, detect its corresponding characteristic value Y, and calculate the concentration of the suspension of Staphylococcus aureus to be tested according to the concentration calculation formula: Y=0.13×LgX-0.01.
作为优选，所述检测某个浓度的金黄色葡萄球菌悬浮液对应的特征值Y的方法包括以下步骤：Preferably, the method for detecting the characteristic value Y corresponding to the Staphylococcus aureus suspension of a certain concentration comprises the following steps:
M1：制备培育了金黄色葡萄球菌抗原的工作电极，将未培育金黄色葡萄球菌抗原的工作电极标记为第一工作电极，将培育了金黄色葡萄球菌抗原的工作电极标记为第二工作电极，将第一工作电极、对电极、参比电极组成第一电化学传感器阵列，将第二工作电极、对电极、参比电极组成第二电化学传感器阵列；M1: Prepare the working electrode with culturing Staphylococcus aureus antigen, mark the working electrode without culturing Staphylococcus aureus antigen as the first working electrode, and mark the working electrode with culturing Staphylococcus aureus antigen as the second working electrode, The first working electrode, the counter electrode and the reference electrode form a first electrochemical sensor array, and the second working electrode, the counter electrode and the reference electrode form a second electrochemical sensor array;
M2：采用同样方法对该浓度的金黄色葡萄球菌悬浮液进行m次检测，每次检测的检测步骤如下：M2: Use the same method to perform m times of detection on the concentration of Staphylococcus aureus suspension, and the detection steps of each detection are as follows:
在未使用过的第一工作电极、未使用过的第二工作电极上都滴加2.5μl该浓度的金黄色葡萄球菌悬浮液，静置25分钟后，将第一电化学传感器阵列、第二电化学传感器阵列分别插入两个同样的缓冲溶液内并采用循环伏安法从小到大依次切换n种不同的扫描速率进行检测，得到n个还原峰电流差值ΔIpw1、ΔIpw2…ΔIpwn，ΔIpwn＝Ip1wn-Ip2wn，ΔIpwn为第w次检测中第n种扫描速率下的还原峰电流差值，Ip1wn为第w次检测中第n种扫描速率下第一电化学传感器阵列对应的还原峰电流值，Ip2wn为第w次检测中第n种扫描速率下第二电化学传感器阵列对应的还原峰电流值，1≤w≤m；2.5 μl of the suspension of Staphylococcus aureus at this concentration was dropped on both the unused first working electrode and the unused second working electrode, and after standing for 25 minutes, the first electrochemical sensor array, the second The electrochemical sensor arrays were inserted into two identical buffer solutions respectively and detected by switching n different scanning rates from small to large by cyclic voltammetry, to obtain n reduction peak current differences ΔIp w1 , ΔIp w2 ... ΔIp wn , ΔIp wn = Ip1 wn -Ip2 wn , ΔIp wn is the reduction peak current difference at the nth scan rate in the wth detection, and Ip1 wn is the first electrochemical sensor array at the nth scan rate in the wth detection The corresponding reduction peak current value, Ip2 wn is the reduction peak current value corresponding to the second electrochemical sensor array at the nth scan rate in the wth detection, 1≤w≤m;
M3：根据m次检测的检测数据构建出特征矩阵D，M3: Construct a feature matrix D according to the detection data of m times of detection,
M4：将特征矩阵D的每行数据进行二次样条插值，得到m个与每行数据对应的曲线x(t)，对m个曲线x(t)进行同样的数据处理，得到m个特征值F，对每个曲线x(t)进行的数据处理包括以下步骤：M4: Perform quadratic spline interpolation on each row of data in the feature matrix D to obtain m curves x(t) corresponding to each row of data, and perform the same data processing on m curves x(t) to obtain m features value F, the data processing for each curve x(t) consists of the following steps:
将x(t)带入非线性定向饱和共振模型
饱和势函数
检测信号载入分量ing(t)＝coS(kt+η)+x(t)，The detection signal load component ing(t)=coS(kt+η)+x(t),
其中，t为插值变量，x(t)为特定扫描速率下获得的还原峰电流差值经过二次样条插值后获得的曲线，k为实参数，η为实参数，nois(t)为功率谱密度函数不平坦的有色噪声，P为实参数，Q为实参数，Among them, t is the interpolation variable, x(t) is the curve obtained by quadratic spline interpolation of the reduction peak current difference obtained at a specific scan rate, k is a real parameter, η is a real parameter, and nois(t) is the power Colored noise with uneven spectral density function, P is a real parameter, Q is a real parameter,
绘制非线性定向饱和共振模型的共振曲线，取共振曲线中的最大峰值作为共振特征值E，Draw the resonance curve of the nonlinear directional saturated resonance model, and take the maximum peak in the resonance curve as the resonance eigenvalue E,
通过公式
M5：将m个特征值F取平均，得到的平均值即为特征值Y的值。M5: The m eigenvalues F are averaged, and the obtained average value is the value of the eigenvalue Y.
对金黄色葡萄球菌悬浮液进行m次检测过程中，每次检测都采用新的未使用过的第一工作电极、第二工作电极。During m times of detection of the Staphylococcus aureus suspension, new and unused first and second working electrodes are used for each detection.
作为优选，所述步骤M3还包括以下步骤：Preferably, the step M3 also includes the following steps:
计算稳定系数λij：Calculate the stability coefficient λ ij :
如果λij≤0.8，则判断数据异常，跳转至步骤M2重新检测，If λ ij ≤ 0.8, judge that the data is abnormal, jump to step M2 to re-detect,
如果λij＞0.8，则判断数据正常，执行步骤M4。If λ ij >0.8, it is judged that the data is normal, and step M4 is executed.
作为优选，所述n种不同的扫描速率包括50mV/s、100mV/s、200mV/s、300mV/s、400mV/s、500mV/s。Preferably, the n different scan rates include 50mV/s, 100mV/s, 200mV/s, 300mV/s, 400mV/s, and 500mV/s.
作为优选，所述缓冲溶液的配置方法如下：将0.5mmol/1的H2O2溶液与1.0mol/1的Thi/HaC-NaAc溶液按体积比1∶2混合均匀。Preferably, the configuration method of the buffer solution is as follows: 0.5 mmol/1 H 2 O 2 solution and 1.0 mol/1 Thi/HaC-NaAc solution are uniformly mixed in a volume ratio of 1:2.
作为优选，所述第一工作电极为铜膜电极，所述第二工作电极的制备方法如下：Preferably, the first working electrode is a copper film electrode, and the preparation method of the second working electrode is as follows:
N1：将银浆SL和羧甲基壳聚糖CMC按照质量比1∶3比例混合制成10ml溶胶水溶液，接着超声分散15min，称取5mg的氧化石墨烯GO溶解于溶胶水溶液中，超声震荡25min，得到GO/SL-CMC混合液，取1.5μL的GO/SL-CMC混合液滴加到铜膜电极表面，于室温干燥5h，在铜膜电极上形成一层GO/SL-CM薄膜；N1: Mix the silver paste SL and carboxymethyl chitosan CMC according to the mass ratio of 1:3 to make 10ml of sol aqueous solution, then ultrasonically disperse for 15min, weigh 5mg of graphene oxide GO and dissolve it in the sol aqueous solution, and ultrasonically vibrate for 25min , to obtain a GO/SL-CMC mixture, 1.5 μL of the GO/SL-CMC mixture was added dropwise to the surface of the copper membrane electrode, dried at room temperature for 5 hours, and a layer of GO/SL-CM thin film was formed on the copper membrane electrode;
N2：将铜膜电极置入1mmol/1的硫堇Thi溶液里浸泡约4min，将硫堇Thi组装在铜膜电极表面，用蒸馏水冲洗铜膜电极，洗掉多余的硫堇Thi，将铜膜电极静置干燥；N2: Immerse the copper membrane electrode in a 1mmol/1 thionine Thi solution for about 4 minutes, assemble the thionine Thi on the surface of the copper membrane electrode, rinse the copper membrane electrode with distilled water, wash off the excess thionine Thi, put the copper membrane The electrode is left to dry;
N3：用PBS缓冲液将金黄色葡萄球菌多克隆抗体溶液稀释300倍后取4μl修饰在干燥后的铜膜电极上，在干燥皿中静置干燥；N3: Dilute the Staphylococcus aureus polyclonal antibody solution by 300 times with PBS buffer, then take 4 μl to modify it on the dried copper membrane electrode, and let it dry in a drying dish;
N4：取3.5μl金黄色葡萄球菌抗原溶液滴涂在铜膜电极上，在30℃条件下培育25min，用蒸馏水洗掉铜膜电极上未结合的金黄色葡萄球菌抗原；N4: Take 3.5 μl of Staphylococcus aureus antigen solution and drop it on the copper membrane electrode, incubate at 30°C for 25 minutes, and wash off the unbound Staphylococcus aureus antigen on the copper membrane electrode with distilled water;
N5：将纳米TiO2与水以0.1∶20的重量比混合后超声均质，然后取5μl滴涂在铜膜电极上，晾干，制成第二工作电极。N5: Mix the nano-TiO 2 with water at a weight ratio of 0.1:20, then ultrasonically homogenize, and then take 5 μl drop-coated on the copper membrane electrode, and dry it to make the second working electrode.
选取氧化石墨烯GO作为工作电极修饰材料，以羧甲基壳聚糖CMC和银浆SL作为分散剂将不溶于水的氧化石墨烯GO进行分散使其稳定地固定于工作电极表面，再浸泡于生物染料硫堇Thi中，提高此工作电极表面的电子传递速率，并采用纳米二氧化钛TiO2掺杂并以光照射，达到增强响应信号的作用，然后将金黄色葡萄球菌抗体固定于修饰好的工作电极上，在制备好的工作电极上孵育金黄色葡萄球菌抗原，在优化检测条件下，用循环伏安法(CV)检测其还原峰电流值。Selecting graphene oxide GO as the working electrode modification material, using carboxymethyl chitosan CMC and silver paste SL as dispersants to disperse the water-insoluble graphene oxide GO to make it stably fixed on the surface of the working electrode, and then soaking in In the biological dye thionine Thi, the electron transfer rate on the surface of the working electrode was increased, and nano-titanium dioxide TiO 2 was doped and irradiated with light to enhance the response signal, and then the Staphylococcus aureus antibody was immobilized on the modified work On the electrode, the Staphylococcus aureus antigen was incubated on the prepared working electrode, and its reduction peak current value was detected by cyclic voltammetry (CV) under optimized detection conditions.
作为优选，所述步骤M2中采用循环伏安法从小到大依次切换n种不同的扫描速率进行检测的过程中，将可见光照射在第一工作电极、第二工作电极上。Preferably, in the step M2, the first working electrode and the second working electrode are irradiated with visible light in the process of sequentially switching n different scanning rates from small to large by cyclic voltammetry for detection.
作为优选，所述金黄色葡萄球菌抗原溶液的制备方法如下：将浓度为2×108cfu/ml～2×109cfu/ml的金黄色葡萄球菌用8％～12％的福尔马林在25℃～39℃灭活，灭活后离心去除福尔马林，涂布平板进行无菌检验，确定无菌后，沉淀用等体积无菌生理盐水重新悬浮，从而得到金黄色葡萄球菌抗原溶液。Preferably, the preparation method of the Staphylococcus aureus antigen solution is as follows: using 8% to 12% formalin for Staphylococcus aureus with a concentration of 2×10 8 cfu/ml～2×10 9 cfu/ml Inactivated at 25°C to 39°C, centrifuged to remove formalin after inactivation, coated with a plate for sterility test, after confirming sterility, the precipitate was resuspended with an equal volume of sterile saline to obtain Staphylococcus aureus antigen solution.
作为优选，所述金黄色葡萄球菌多克隆抗体溶液的制备方法如下：As preferably, the preparation method of described Staphylococcus aureus polyclonal antibody solution is as follows:
兔饲养2周后，耳静脉采血15ml，取出血清作为阴性血清样品；金黄色葡萄球菌抗原免疫兔，间隔3天进行第二次免疫，再间隔6天加强免疫，加强免疫后第4天颈动脉一次性采血，室温放置45min，转入4℃过夜，次日4℃、5000rpm离心45min得抗血清保存；After the rabbits were raised for 2 weeks, 15 ml of blood was collected from the ear vein, and the serum was taken out as a negative serum sample; the rabbits were immunized with Staphylococcus aureus antigen, and the second immunization was performed at 3-day intervals, and then the booster immunization was performed at 6-day intervals. One-time blood collection, placed at room temperature for 45 min, transferred to 4 °C overnight, and centrifuged at 4 °C for 45 min at 5000 rpm the next day to obtain antiserum for preservation;
将1.5ml亲和层析柱固定于蛋白纯化仪上，用去离子水洗出保护剂溶液，接着用PBS缓冲液平衡柱子，然后将1.5ml抗血清样品上柱，用PBS缓冲液洗脱杂质，最后用柠檬酸缓冲液洗脱金黄色葡萄球菌多克隆抗体，得到金黄色葡萄球菌多克隆抗体溶液。Fix 1.5ml affinity chromatography column on the protein purifier, wash out the protective agent solution with deionized water, then equilibrate the column with PBS buffer, then put 1.5ml antiserum sample on the column, and use PBS buffer to elute impurities, Finally, the Staphylococcus aureus polyclonal antibody was eluted with a citric acid buffer to obtain a Staphylococcus aureus polyclonal antibody solution.
本发明的有益效果是：能够准确检测出金黄色葡萄球菌浓度，操作简单。The beneficial effects of the invention are: the concentration of Staphylococcus aureus can be accurately detected, and the operation is simple.
附图说明Description of drawings
图1是本发明的流程图；Fig. 1 is the flow chart of the present invention;
图2是非线性定向饱和共振模型的共振曲线示意图。FIG. 2 is a schematic diagram of the resonance curve of the nonlinear directional saturated resonance model.
具体实施方式Detailed ways
下面通过实施例，并结合附图，对本发明的技术方案作进一步具体的说明。The technical solutions of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings.
实施例：本实施例的一种金黄色葡萄球菌检测方法，如图1所示，包括以下步骤：Embodiment: A kind of Staphylococcus aureus detection method of the present embodiment, as shown in Figure 1, comprises the following steps:
S1：配置不同浓度的金黄色葡萄球菌悬浮液，检测每个浓度的金黄色葡萄球菌悬浮液对应的特征值Y，将这些值进行拟合得到浓度计算公式：Y＝0.13×LgX-0.01，X为金黄色葡萄球菌悬浮液的浓度；S1: Configure different concentrations of Staphylococcus aureus suspension, detect the characteristic value Y corresponding to each concentration of Staphylococcus aureus suspension, and fit these values to obtain the concentration calculation formula: Y=0.13×LgX-0.01, X is the concentration of Staphylococcus aureus suspension;
S2：取待测金黄色葡萄球菌悬浮液，检测其对应的特征值Y，根据浓度计算公式：Y＝0.13×LgX-0.01，计算出待测金黄色葡萄球菌悬浮液的浓度。S2: Take the suspension of Staphylococcus aureus to be tested, detect its corresponding characteristic value Y, and calculate the concentration of the suspension of Staphylococcus aureus to be tested according to the concentration calculation formula: Y=0.13×LgX-0.01.
在本方案中，以金黄色葡萄球菌悬浮液浓度为x轴，特征值Y为y轴建立直角坐标系，将每个金黄色葡萄球菌悬浮液浓度及对应的特征值Y在直角坐标系中标出对应的点，将这些点拟合得到浓度计算公式：Y＝0.13×LgX-0.01。In this scheme, a Cartesian coordinate system is established with the concentration of Staphylococcus aureus suspension as the x-axis and the characteristic value Y as the y-axis, and the concentration of each Staphylococcus aureus suspension and the corresponding characteristic value Y are marked in the Cartesian coordinate system Corresponding points, these points are fitted to obtain the concentration calculation formula: Y=0.13×LgX-0.01.
检测某个浓度的金黄色葡萄球菌悬浮液对应的特征值Y的方法包括以下步骤：The method for detecting the characteristic value Y corresponding to a certain concentration of Staphylococcus aureus suspension comprises the following steps:
M1：制备培育了金黄色葡萄球菌抗原的工作电极，将未培育金黄色葡萄球菌抗原的工作电极标记为第一工作电极，将培育了金黄色葡萄球菌抗原的工作电极标记为第二工作电极，将第一工作电极、对电极、参比电极组成第一电化学传感器阵列，将第二工作电极、对电极、参比电极组成第二电化学传感器阵列；M1: Prepare the working electrode with culturing Staphylococcus aureus antigen, mark the working electrode without culturing Staphylococcus aureus antigen as the first working electrode, and mark the working electrode with culturing Staphylococcus aureus antigen as the second working electrode, The first working electrode, the counter electrode and the reference electrode form a first electrochemical sensor array, and the second working electrode, the counter electrode and the reference electrode form a second electrochemical sensor array;
M2：采用同样方法对该浓度的金黄色葡萄球菌悬浮液进行m次检测，每次检测的检测步骤如下：M2: Use the same method to perform m times of detection on the concentration of Staphylococcus aureus suspension, and the detection steps of each detection are as follows:
在未使用过的第一工作电极、未使用过的第二工作电极上都滴加2.5μl该浓度的金黄色葡萄球菌悬浮液，静置25分钟后，将第一电化学传感器阵列、第二电化学传感器阵列分别插入两个同样的缓冲溶液内并采用循环伏安法从小到大依次切换n种不同的扫描速率进行检测，检测时将可见光照射在第一工作电极、第二工作电极上，得到n个还原峰电流差值ΔIpw1、ΔIpw2…ΔIpwn，ΔIpwn＝Ip1wn-Ip2wn，ΔIpwn为第w次检测中第n种扫描速率下的还原峰电流差值，Ip1wn为第w次检测中第n种扫描速率下第一电化学传感器阵列对应的还原峰电流值，Ip2wn为第w次检测中第n种扫描速率下第二电化学传感器阵列对应的还原峰电流值，1≤w≤m；2.5 μl of the suspension of Staphylococcus aureus at this concentration was dropped on both the unused first working electrode and the unused second working electrode, and after standing for 25 minutes, the first electrochemical sensor array, the second The electrochemical sensor arrays are inserted into two identical buffer solutions respectively, and cyclic voltammetry is used to switch n different scanning rates from small to large for detection. When detecting, visible light is irradiated on the first working electrode and the second working electrode. Obtain n reduction peak current differences ΔIp w1 , ΔIp w2 … The reduction peak current value corresponding to the first electrochemical sensor array at the nth scan rate in the wth detection, Ip2 wn is the reduction peak current value corresponding to the second electrochemical sensor array at the nth scan rate in the wth detection , 1≤w≤m;
M3：根据m次检测的检测数据构建出特征矩阵D，M3: Construct a feature matrix D according to the detection data of m times of detection,
计算稳定系数λij：Calculate the stability coefficient λ ij :
如果λij≤0.8，则判断数据异常，跳转至步骤M2重新检测，If λ ij ≤ 0.8, judge that the data is abnormal, jump to step M2 to re-detect,
如果λij＞0.8，则判断数据正常，执行步骤M4；If λ ij > 0.8, it is judged that the data is normal, and step M4 is executed;
M4：将特征矩阵D的每行数据进行二次样条插值，得到m个与每行数据对应的曲线x(t)，对m个曲线x(t)进行同样的数据处理，得到m个特征值F，对每个曲线x(t)进行的数据处理包括以下步骤：M4: Perform quadratic spline interpolation on each row of data in the feature matrix D to obtain m curves x(t) corresponding to each row of data, and perform the same data processing on m curves x(t) to obtain m features value F, the data processing for each curve x(t) consists of the following steps:
将x(t)带入非线性定向饱和共振模型
饱和势函数
检测信号载入分量ing(t)＝coS(kt+η)+x(t)，The detection signal load component ing(t)=coS(kt+η)+x(t),
其中，t为插值变量，x(t)为特定扫描速率下获得的还原峰电流差值经过二次样条插值后获得的曲线，k为实参数，η为实参数，nois(t)为功率谱密度函数不平坦的有色噪声，P为实参数，Q为实参数，Among them, t is the interpolation variable, x(t) is the curve obtained by quadratic spline interpolation of the reduction peak current difference obtained at a specific scan rate, k is a real parameter, η is a real parameter, and nois(t) is the power Colored noise with uneven spectral density function, P is a real parameter, Q is a real parameter,
如图2所示，绘制非线性定向饱和共振模型的共振曲线，取共振曲线中的最大峰值作为共振特征值E，As shown in Figure 2, the resonance curve of the nonlinear directional saturated resonance model is drawn, and the maximum peak in the resonance curve is taken as the resonance eigenvalue E,
通过公式
M5：将m个特征值F取平均，得到的平均值即为特征值Y的值。M5: The m eigenvalues F are averaged, and the obtained average value is the value of the eigenvalue Y.
对金黄色葡萄球菌悬浮液进行m次检测过程中，每次检测都采用新的未使用过的第一工作电极、第二工作电极。During m times of detection of the Staphylococcus aureus suspension, new and unused first and second working electrodes are used for each detection.
n种不同的扫描速率包括50mV/s、100mV/s、200mV/s、300mV/s、400mV/s、500mV/s。n different scan rates including 50mV/s, 100mV/s, 200mV/s, 300mV/s, 400mV/s, 500mV/s.
缓冲溶液的配置方法如下：将0.5mmol/l的H2O2溶液与1.0mol/l的Thi/HaC-NaAc溶液按体积比1∶2混合均匀。The preparation method of the buffer solution is as follows: 0.5 mmol/l H 2 O 2 solution and 1.0 mol/l Thi/HaC-NaAc solution are uniformly mixed in a volume ratio of 1:2.
第一工作电极为铜膜电极，对电极为Pt电极、参比电极为Ag/AgCl电极。The first working electrode is a copper film electrode, the counter electrode is a Pt electrode, and the reference electrode is an Ag/AgCl electrode.
第二工作电极的制备方法如下：The preparation method of the second working electrode is as follows:
N1：将银浆SL和羧甲基壳聚糖CMC按照质量比1∶3比例混合制成10ml溶胶水溶液，接着超声分散15min，称取5mg的氧化石墨烯GO溶解于溶胶水溶液中，超声震荡25min，得到GO/SL-CMC混合液，取1.5μL的GO/SL-CMC混合液滴加到铜膜电极表面，于室温干燥5h，在铜膜电极上形成一层GO/SL-CM薄膜；N1: Mix the silver paste SL and carboxymethyl chitosan CMC according to the mass ratio of 1:3 to make 10ml of sol aqueous solution, then ultrasonically disperse for 15min, weigh 5mg of graphene oxide GO and dissolve it in the sol aqueous solution, and ultrasonically vibrate for 25min , to obtain a GO/SL-CMC mixture, 1.5 μL of the GO/SL-CMC mixture was added dropwise to the surface of the copper membrane electrode, dried at room temperature for 5 hours, and a layer of GO/SL-CM thin film was formed on the copper membrane electrode;
N2：将铜膜电极置入1mmol/1的硫堇Thi溶液里浸泡约4min，将硫堇Thi组装在铜膜电极表面，用蒸馏水冲洗铜膜电极，洗掉多余的硫堇Thi，将铜膜电极静置干燥；N2: Immerse the copper membrane electrode in a 1mmol/1 thionine Thi solution for about 4 minutes, assemble the thionine Thi on the surface of the copper membrane electrode, rinse the copper membrane electrode with distilled water, wash off the excess thionine Thi, put the copper membrane The electrode is left to dry;
N3：用PBS缓冲液将金黄色葡萄球菌多克隆抗体溶液稀释300倍后取4μl修饰在干燥后的铜膜电极上，在干燥皿中静置干燥；N3: Dilute the Staphylococcus aureus polyclonal antibody solution by 300 times with PBS buffer, then take 4 μl to modify it on the dried copper membrane electrode, and let it dry in a drying dish;
N4：取3.5μl金黄色葡萄球菌抗原溶液滴涂在铜膜电极上，在30℃条件下培育25min，用蒸馏水洗掉铜膜电极上未结合的金黄色葡萄球菌抗原；N4: Take 3.5 μl of Staphylococcus aureus antigen solution and drop it on the copper membrane electrode, incubate at 30°C for 25 minutes, and wash off the unbound Staphylococcus aureus antigen on the copper membrane electrode with distilled water;
N5：将纳米TiO2与水以0.1∶20的重量比混合后超声均质，然后取5μl滴涂在铜膜电极上，晾干，制成第二工作电极。N5: Mix the nano-TiO 2 with water at a weight ratio of 0.1:20, then ultrasonically homogenize, and then take 5 μl drop-coated on the copper membrane electrode, and dry it to make the second working electrode.
选取氧化石墨烯GO作为工作电极修饰材料，以羧甲基壳聚糖CMC和银浆SL作为分散剂将不溶于水的氧化石墨烯GO进行分散使其稳定地固定于工作电极表面，再浸泡于生物染料硫堇Thi中，提高此工作电极表面的电子传递速率，并采用纳米二氧化钛TiO2掺杂并以光照射，达到增强响应信号的作用，然后将金黄色葡萄球菌抗体固定于修饰好的工作电极上，在制备好的工作电极上孵育金黄色葡萄球菌抗原，在优化检测条件下，用循环伏安法(CV)检测其还原峰电流值。Selecting graphene oxide GO as the working electrode modification material, using carboxymethyl chitosan CMC and silver paste SL as dispersants to disperse the water-insoluble graphene oxide GO to make it stably fixed on the surface of the working electrode, and then soaking in In the biological dye thionine Thi, the electron transfer rate on the surface of the working electrode was increased, and nano-titanium dioxide TiO 2 was doped and irradiated with light to enhance the response signal, and then the Staphylococcus aureus antibody was immobilized on the modified work On the electrode, the Staphylococcus aureus antigen was incubated on the prepared working electrode, and its reduction peak current value was detected by cyclic voltammetry (CV) under optimized detection conditions.
金黄色葡萄球菌抗原溶液的制备方法如下：将浓度为2×108cfu/ml～2×109cfu/ml的金黄色葡萄球菌用8％～12％的福尔马林在25℃～39℃灭活，灭活后离心去除福尔马林，涂布平板进行无菌检验，确定无菌后，沉淀用等体积无菌生理盐水重新悬浮，从而得到金黄色葡萄球菌抗原溶液。The preparation method of Staphylococcus aureus antigen solution is as follows: the concentration of Staphylococcus aureus is 2×10 8 cfu/ml～2×10 9 cfu/ml with 8%～12% formalin at 25℃～39 Inactivated at °C, centrifuged to remove formalin after inactivation, coated on a plate for sterility test, and after confirming sterility, the precipitate was resuspended with an equal volume of sterile physiological saline to obtain a Staphylococcus aureus antigen solution.
金黄色葡萄球菌多克隆抗体溶液的制备方法如下：The preparation method of Staphylococcus aureus polyclonal antibody solution is as follows:
兔饲养2周后，耳静脉采血15ml，取出血清作为阴性血清样品；金黄色葡萄球菌抗原免疫兔，间隔3天进行第二次免疫，再间隔6天加强免疫，加强免疫后第4天颈动脉一次性采血，室温放置45min，转入4℃过夜，次日4℃、5000rpm离心45min得抗血清保存；After the rabbits were raised for 2 weeks, 15 ml of blood was collected from the ear vein, and the serum was taken out as a negative serum sample; the rabbits were immunized with Staphylococcus aureus antigen, and the second immunization was performed at 3-day intervals, and then the booster immunization was performed at 6-day intervals. One-time blood collection, placed at room temperature for 45 min, transferred to 4 °C overnight, and centrifuged at 4 °C for 45 min at 5000 rpm the next day to obtain antiserum for preservation;
将1.5ml亲和层析柱固定于蛋白纯化仪上，用去离子水洗出保护剂溶液，接着用PBS缓冲液平衡柱子，然后将1.5ml抗血清样品上柱，用PBS缓冲液洗脱杂质，最后用柠檬酸缓冲液洗脱金黄色葡萄球菌多克隆抗体，得到金黄色葡萄球菌多克隆抗体溶液。Fix 1.5ml affinity chromatography column on the protein purifier, wash out the protective agent solution with deionized water, then equilibrate the column with PBS buffer, then put 1.5ml antiserum sample on the column, and use PBS buffer to elute impurities, Finally, the Staphylococcus aureus polyclonal antibody was eluted with a citric acid buffer to obtain a Staphylococcus aureus polyclonal antibody solution.
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