CN114397418A - 一种基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法 - Google Patents
一种基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法 Download PDFInfo
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Abstract
本发明公开了一种基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,包括将费氏弧菌试剂分别加入阴性质控试剂和多个梯度稀释浓度的待测水样得到发光强度,并计算出多个梯度稀释浓度待测水样的发光抑制率;采用最小二乘法拟合得到稀释倍数与发光抑制率的待测水样logstic曲线,将EC50处水样稀释倍数作为待测水样的综合毒性强度;构建已知毒性物质的logstic曲线库,通过动态时间规整算法将待测水样logstic曲线与已知毒性物质的logstic曲线进行相似度比较通过softmax算法得到疑似毒性物质的概率分布确定为待测水样的疑似毒性物质。该方法能够快速准确的检测水质综合毒性强度和疑似毒性物质。
Description
技术领域
本发明属于水质毒性检测技术领域,具体涉及一种基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法。
背景技术
发光细菌毒性试验是根据细菌发光强度与毒性物质之间的负相关性,该方法已被广泛应用。发光细菌是一类含有lux基因,能够发出可见光,且分布非常广泛,多数菌种分布在海洋中,少数菌种分布在陆地上。现在已知的发光细菌有弧菌属(Vibrio)、发光杆菌属(Photobacterium)、希瓦氏菌属(Shewanella)和光杆菌属(Photorhabdus)四种。发光细菌在正常生理条件下能发出450~490nm蓝绿色可见光,在一定的环境条件下发光强度比较稳定。其发光机理为特异性荧光酶(LE)、还原性黄素单核苷酸(FMNH2)、八碳以上长链脂肪醛(RCHO)、氧分子(O2)所参与的复杂反应,FMN为黄素单核苷酸,大致反应历程如下:
FMNH2+LE→FMNH2·LE+O2→LE·FMNH2·O2+RCHO
→LE·FMNH2·O2·RCHO
→LE+FMN+H2O+RCOOH+光
发光细菌发光是细菌体内正常生理氧化反应的结果。发光细菌的发光过程极易受到外界条件的影响,凡是干扰细菌呼吸或生理过程的因素均能使细菌发光强度发生改变,这就是发光细菌用于毒性检测的基本原理。培养至正常发光的发光细菌与毒性物质接触后,由于其与发光相关的代谢途径受阻,短时间内即可表现出发光强度减弱,且发光强度的减弱量与毒性物质的浓度呈良好的相关性,可以此来检测物质的毒性。本发明的水质综合毒性测试方法采用费氏弧菌,其在正常的生理条件发光强度是恒定的。但是,与外来受试物接触后,由于毒物具有抑制发光的作用,发光细菌的发光强度减弱,光强变化的程度与受试物的浓度在一定范围内呈正相关关系,同时与该物质的毒性大小有关。即毒物的浓度越大,毒性越强,光强变化程度越大,检测到的光强越弱。常见的发光细菌有明亮发光杆菌、费氏弧菌和青海弧菌。发光抑制率曲线公式下所示,该曲线为4参数logistc公式,其中max和min表示曲线的最大值和最小值,slope为斜率参数,EC50为该曲线的半数抑制浓度,变量C表示浓度,effect表示C浓度下的发光抑制率。
目前,废水生物毒性的检测方法有:发光菌毒性试验,藻类毒性试验,蚤类毒性试验,鱼类毒性试验,鸟类毒性试验,化学毒性试验,现有发光菌急性毒性试验具有以下不足:1.检测往往只得出定性结果,即阳性(有毒,发光抑制率≥20%)和阴性(低毒,发光抑制率<20%),无法进行定量测试。2.自动化程度较低,需要耗费大量时间精力。
发明内容
本发明提供一种基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,该方法能够快速准确的检测水质综合毒性强度和疑似毒性物质。
一种基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,包括:
(1)将费氏弧菌试剂分别加入阴性质控试剂,以及多个梯度稀释浓度的待测水样,摇匀后,分别检测摇匀后的阴性质控试剂和多个梯度稀释浓度待测水样的发光强度,将摇匀后的阴性质控试剂的发光强度作为参考发光强度计算出摇匀后的多个梯度稀释浓度待测水样的发光抑制率;
(2)采用最小二乘法,当总的拟合误差达到误差阈值时,拟合得到稀释倍数与发光抑制率的待测水样logstic曲线,其中,待测水样logstic曲线的EC50点的水样稀释倍数作为待测水样的水质综合毒性强度;
(3)将已知毒性物质水样替换待测水样,通过步骤(1)和(2)构建已知毒性物质的logstic曲线库,通过动态时间规整算法将待测水样logstic曲线与已知毒性物质的logstic曲线进行相似度比较,将比较结果代入softmax回归算法得到疑似毒性物质的概率分布,将达到疑似毒性物质概率阈值的疑似毒性物质确定为待测水样的疑似毒性物质。
阴性质控试剂为NaCl溶液。
阴性质控试剂为NaCl溶液,NaCl溶液与费氏弧菌试剂的体积比为1~9。
在检测摇匀后的阴性质控试剂和多个梯度稀释浓度待测水样的发光强度之前,将费氏弧菌试剂加入到阳性质控试剂,并检测加入费氏弧菌试剂后的阳性质控试剂的发光强度,以筛选出高于阳性质控试剂的发光强度的摇匀后的多个梯度稀释浓度待测水样。
阳性质控试剂为ZnCl2溶液,ZnCl2溶液的浓度为2.2mg/L~4mg/L(以Zn离子计)。
待测水样为冶金废水、造纸废水、金属酸洗废水、纺织印染废水、制革废水、含铬废水、含镉废水、含汞废水。
多个梯度稀释浓度待测水样的稀释倍数为1、2、4、8、16、32、64、128倍稀释浓度待测水样。
摇匀时间为15~30分钟。
已知毒性物质为Cu、Pb、Zn、Ni、Cd、Ag、V、Cr、As、Mn、Fe、Co、亚硝酸盐、苯乙酮、对硝基苯酚、富里酸、3-羟基吡啶、腐殖酸、4-甲基咪唑、腐殖酸钠、4-羟基吡啶、2-羟基吡啶。
与现有技术相比,本发明的有益效果为:
(1)本发明采用最小二乘法构建待测水样的稀释倍数与发光抑制率的logstic曲线,将拟合结果的样品的半数抑制浓度的稀释倍数作为水质综合毒性表征数据,能够直观的快速测试不同样品的毒性值,以及具有较高的检测灵敏度以及准确度。
(2)本发明采用最小二乘法构建已知毒物的稀释倍数与发光抑制率的logstic曲线建立标准数据库,即已知毒性物质的logstic曲线库,能够实现样品的疑似毒性物质的检测判定。
附图说明
图1为本发明具体实施例提供的待测96孔板试剂、样品加样示意图;
图2为本发明具体实施例提供的水质综合毒性及疑似毒性物质测试方法流程图;
图3为本发明具体实施例提供的待测水样的稀释倍数与发光抑制率的logstic曲线图;
图4为本发明具体实施例提供的待测水样的疑似毒性物质的概率分布图;
图5为本发明具体实施例提供的自研水质综合毒性检测仪器功能示意图。
具体实施方式
为了更为具体地描述本发明,下面采用苏州某工业区水样结合附图及具体实施方式对本发明的技术方案进行详细说明。
本发明一种基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法通过自研的水质综合毒性检测仪进行加样,详细的加样方式如图1所示,101a、101b、101c为阴性对照组加入180uL的2%的NaCl溶液和20uL的费氏弧菌试剂,102a、102b、102c为阳性对照组加入180uL的2mg/L的ZnCl2溶液和20uL的费氏弧菌试剂,103~110为苏州某工业区水样进行梯度稀释后依次加入,从103~110分别为1倍浓度待测水样、2倍稀释待测水样、4倍稀释待测水样、8倍稀释待测水样、16倍稀释待测水样、32倍稀释待测水样、64倍稀释待测水样、128倍稀释待测水样,共8组。
如图2所示,为本发明水质综合毒性及疑似毒性物质测试方法流程图,仪器开机并在试剂区分别放入待检测的水样、费氏弧菌试、阴性质控试剂和阳性质控试剂后可以开始检测流程,201为加样模块机械臂自动将阴性质控试剂即180uL的2%NaCl和阳性质控试剂180uL的2.2mg/L ZnCl2分别放入上述101和102,之后会进入水样加样流程,该流程由加样模块自动实现了样品的8梯度稀释并将相应的稀释样品分别取180uL加入到上述103~110各个孔位。203为费氏弧菌试剂的分别取20uL菌液试剂加入到上述101~110各个孔位,试剂加入后需要等待30分钟反应时间,期间需要对96孔板进行持续摇匀,待反应结束后进行205发光强度检测,206计算不同稀释倍数的样品的发光抑制率,如表1所示,每组中三个平行样用于减小加样和检测误差。通过发光强度计算每组稀释浓度的发光抑制率,第一组发光抑制率计算公式如下(101i,103i表示图一中的样品孔位检测数值):
表1待测水样不同稀释倍数下的发光抑制率
通过上一步计算得到的发光抑制率数组207采用最小二乘法通过最小化误差的平方和寻找数据的最佳函数匹配,其拟合曲线的标准为,始终使Min最小化,如下最小二乘法公式所示:
其中n为数组,f1为待拟合的函数表达式,S为实际样品抑制率数组,xi为第i个实际样品稀释倍数的平均发光抑制率数。
208通过拟合得到待测水样的稀释倍数与发光抑制率的logstic曲线,如图3所示,该曲线的EC50点的水样稀释倍数可以表征该水样的毒性强度,实际测得该水样EC50的水样稀释倍数为13.07倍,该水样的水质综合毒性强度换算成Zn离子毒性当量为26.15mg/L。209通过动态时间规整算法通过动态时间规整算法将待测水样logstic曲线与已知毒性物质的logstic曲线进行相似度行相似度计算得到已知毒性物质在待测水样的疑似概率。
其中表示Softmax的输入向量,其中输入向量为22种已知毒性物质毒性当量分别与待测毒性当量构成的22维度向量,zi表示输入向量的第i个单个元素值,K表示向量空间的维度即分类种类,表示待测水样中疑似毒性物质与第i个已知毒性物质相似度的概率分布,结算结果如表2所示。
表2待测水样中疑似毒性物质与已知毒性物质在待测水样的疑似概率:
物质类型 | 概率 | |
1 | Pb | 0.723 |
2 | Cu | 0.103 |
3 | Ag | 0.061 |
4 | Zn | 0.034 |
5 | Cr | 0.021 |
6 | Ni | 0.01 |
7 | HA-Na | 0.008 |
8 | Cd | 0.006 |
9 | V | 0.006 |
10 | Fe | 0.005 |
11 | 2-Hydroxypyridine | 0.005 |
12 | Co | 0.003 |
13 | 4-Hydroxypyridine | 0.003 |
14 | Mn | 0.003 |
15 | NO2 | 0.002 |
16 | Fulvic Acid | 0.001 |
17 | As | 0.001 |
18 | 4-Nitrophenol | 0.001 |
19 | 3-Hydroxypyridine | 0.001 |
20 | Humic Acid | 0.001 |
21 | 1-Phenylethan-1-one | 0.001 |
22 | 4-MEI | 0.001 |
最后为210,如上所示为Softmax计算公式,通过Softmax算法最终得出该样品中存在的疑似毒性物质的概率分布,可以看到该水样的最大疑似污染物Pb元素其疑似概率为72.3%,如图4所示。
如图5所示为自研的水质综合毒性检测仪功能示意图,本发明的测试方法基于改仪器实现,该仪器主要实现自动加样及水质综合毒性检测,301为仪器清洗模块,实现了加样钢针的清洗功能,主要为了防止样品和试剂之间的交叉污染,从而提升检测精度。302为仪器的加样模块,其结构主要包括XYZ三轴机械臂及注射泵和加样钢针,加样精度可以精确到1uL。303为试剂和样品区该区用于存放费氏弧菌试剂、实际水样用于加样模块取样加入到检测模块的96孔板中,304为仪器的温控模块,该温控模块用于支持试剂区的冷藏温度设定及检测区的特定检测温度设定。305为仪器的检测区,采用光子计数系统作为采集器,其速度快、脉宽小的光电倍增管作为检测元件,同时采用合适的磁场屏蔽材料,设计绝对暗室及快速时间分辨检测电路,具有高灵敏度的生物荧光检测性能。
Claims (9)
1.一种基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,其特征在于,包括:
(1)将费氏弧菌试剂分别加入阴性质控试剂和多个梯度稀释浓度的待测水样,摇匀后,分别检测阴性质控试剂和多个梯度稀释浓度待测水样的发光强度,将阴性质控试剂的发光强度作为参考发光强度,以计算出多个梯度稀释浓度待测水样的发光抑制率;
(2)采用最小二乘法,当总的拟合误差达到误差阈值时,拟合得到稀释倍数与发光抑制率的待测水样logstic曲线,其中,待测水样logstic曲线的EC50点的水样稀释倍数作为待测水样的水质综合毒性强度;
(3)将已知毒性物质水样替换待测水样,通过步骤(1)和(2)构建已知毒性物质的logstic曲线库,通过动态时间规整算法将待测水样logstic曲线与已知毒性物质的logstic曲线进行相似度比较,将比较结果代入softmax回归算法得到疑似毒性物质的概率分布,将达到概率阈值的疑似毒性物质确定为待测水样的疑似毒性物质。
2.根据权利要求1所述的基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,其特征在于,阴性质控试剂为NaCl溶液。
3.根据权利要求1所述的基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,其特征在于,阴性质控试剂为NaCl溶液,NaCl溶液与费氏弧菌试剂的体积比为1~9。
4.根据权利要求1所述的基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,其特征在于,在检测摇匀后的阴性质控试剂和多个梯度稀释浓度待测水样的发光强度之前,将费氏弧菌试剂加入到阳性质控试剂,并检测加入费氏弧菌试剂后的阳性质控试剂的发光强度,以筛选出高于阳性质控试剂的发光强度的摇匀后的多个梯度稀释浓度待测水样。
5.根据权利要求4所述的基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,其特征在于,阳性质控试剂为ZnCl2溶液,ZnCl2溶液的Zn离子浓度为2.2mg/L~4mg/L。
6.根据权利要求1所述的基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,其特征在于,待测水样为冶金废水、造纸废水、金属酸洗废水、纺织印染废水、制革废水、含铬废水、含镉废水、含汞废水。
7.根据权利要求6所述的基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,其特征在于,多个梯度稀释浓度待测水样的稀释倍数为1、2、4、8、16、32、64、128倍稀释浓度待测水样。
8.根据权利要求1所述的基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,其特征在于,摇匀时间为15~30分钟。
9.根据权利要求1所述的基于Logistic拟合的水质综合毒性及疑似毒性物质测试方法,其特征在于,已知毒性物质为Cu、Pb、Zn、Ni、Cd、Ag、V、Cr、As、Mn、Fe、Co、亚硝酸盐、苯乙酮、对硝基苯酚、富里酸、3-羟基吡啶、腐殖酸、4-甲基咪唑、腐殖酸钠、4-羟基吡啶、2-羟基吡啶。
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