海水养殖尾水总磷测定方法的比较与优化

刘雁; 佟飞; 陈丕茂; 袁华荣; 冯雪

doi:10.12131/20220056

海水养殖尾水总磷测定方法的比较与优化

doi: 10.12131/20220056

刘雁^{1, 2,},
佟飞²,
陈丕茂^2, ,,
袁华荣²,
冯雪²

1.
上海海洋大学海洋科学学院，上海 201306
2.
中国水产科学研究院南海水产研究所/农业农村部南海渔业资源环境科学观测试验站/中国水产科学研究院海洋牧场技术重点实验室/广东省渔业生态环境重点实验室/广东省海洋休闲渔业工程技术研究中心，广东广州 510300

基金项目: 广东省重点领域研发计划项目(2020B1111030002-2)；深圳市科技创新计划(JCYJ20160331141759795)

详细信息

作者简介:
刘雁：刘　雁 (1988—)，男，硕士研究生，研究方向为海洋生物学。E-mail: 287475306@qq.com

通讯作者:
陈丕茂 (1969—)，男，研究员，研究方向为渔业资源保护与利用。E-mail: chenpm@scsfri.ac.cn

中图分类号: S 912
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- 被引次数: 0
出版历程
- 收稿日期: 2022-03-08
- 修回日期: 2022-07-12
- 录用日期: 2022-08-01
- 网络出版日期: 2022-08-09

Comparison and optimization of total phosphorus determination method in mariculture tailwater

LIU Yan^{1, 2
,},
TONG Fei²,
CHEN Pimao^{2
, ,},
YUAN Huarong²,
FENG Xue²

1.
College of Marine Sciences, Shanghai Ocean University, Shanghai 201306, China
2.
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences/Scientific Observing and Experimental Station of South China Sea Fishery Resources and Environment, Ministry of Agriculture and Rural Affairs/Key Laboratory of Marine Ranching Technology, Chinese Academy of Fishery Sciences/Guangdong Provincial Key Laboratory of Fishery Ecology and Environment/ Guangdong Engineering Technology Research Center of Marine Recreational Fishery, Guangzhou 510300, China

摘要

摘要: 为提高海水养殖尾水中总磷 (Total phosphorus, TP) 的检测效率，使用酶标仪代替分光光度计进行海水养殖尾水TP的测定，并对TP的消解方法、酶标板类型与加样量等进行优化。结果表明，使用控温加热板消解60 min与高压灭菌器消解30 min的效果无显著差异 (P>0.05)；使用48孔板加样1 mL，检测的灵敏度优于其他实验组 (96孔板200 µL，96孔石英板200 µL，48孔板200和500 µL，24孔板500 µL和1 mL)，48孔板加样1 mL与96孔板加样200 µL，磷酸盐浓度与吸光度的线性相关性最高 (R²=0.999 9)；对养殖尾水TP测定的精确度和准确度验证结果显示，48孔板加样1 mL的相对误差 (Relative error, RE) 和相对标准偏差 (Relative standard deviation, RSD) 分别介于−2.97%~1.59%和0.42%~4.06%，对甘油磷酸二钠 (β-GLP) 和六偏磷酸钠 (SHMP) 的回收率分别介于98.2%~99.6%和93.4%~97.1%，TP的测定限 (Limit of quantification, LOQ) 为0.25 µmol·L⁻¹；96孔板加样200 µL的RE和RSD分别介于−14.03%~0.21%和2.63%~14.23%，对β-GLP和SHMP的回收率分别介于94.7%~99.0%和88.9%~97.3%，TP的LOQ为0.55 µmol·L⁻¹。在TP浓度介于0.55~6.4 µmol·L⁻¹内，两种优化法的准确度和精密度均符合测定要求，可用于海水养殖尾水TP的测定。
- 海水养殖尾水 /
- 总磷 /
- 消解 /
- 酶标仪
Abstract: In order to improve the efficiency of the determination of total phosphorus (TP) in the mariculture tailwater, we determined the TP content by microplate reader instead of spectrophotometer in mariculture tailwater, and optimized the digestion method of TP, the type of microwell plate and the amount of sample. The results show that there was no significant difference between 60 min of digestion using a temperature-controlled heating plate and 30 min of digestion using an autoclave (P>0.05). The sensitivity of the assay was better than those of the other experimental groups (200 µL for 96-well plate, 200 µL for 96-well quartz plate, 200 and 500 µL for 48-well plate, 500 µL and 1 mL for 24-well plate) when using a 48-well plate with 1 mL of sample. The linear correlation between phosphate concentration and absorbance was the best (R²=0.999 9) for the 48-well plate with 1 mL and the 96-well plate spiked with 200 µL. The accuracy and precision validation of TP determination in mariculture tailwater showed that the relative error (RE) and relative standard deviation (RSD) of the 48-well plate spiked with 1 mL were higher than those of the other experimental groups (96-well plate 200 µL, 96-well quartz plate 200 µL, 48-well plate 200 µL and 500 µL, 24-well plate 500 µL and 1 mL). The recoveries of disodium glycerophosphate (β-GLP) and sodium hexametaphosphate (SHMP) were 98.2%−99.6% and 93.4%−97.1%, respectively, and the limit of quantification (LOQ) of TP were 0.25 µmol·L⁻¹; the RE and RSD of 96-well ELISA plate spiked with 200 µL were −14.03%−0.21% and 2.63%−14.23%, respectively, and the recoveries for β-GLP and SHM were 94.7%−99.0% and 88.9%−97.3%, respectively, and the LOQ of TP was 0.55 µmol·L⁻¹. At TP concentration of 0.55−6.4 µmol·L⁻¹, the accuracy and precision of the two optimized methods meet the determination requirements and can be used for the determination of TP in mariculture tailwater.
- Mariculture tailwater /
- Total phosphorus /
- Digestion /
- Microplate reader

HTML全文

图 1 消解百分比与消解时间关系

注：不同字母之间表示同一样品在不同消解时间的消解百分比差异显著 (P<0.05)。

Figure 1. Relationship between digestion percentage and digestion time

Note: Different letters indicate that the digestion percentage of the same sample at different digestion time is significantly different (P<0.05).

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图 2 酶标仪测定法和常规方法测定的TP标准曲线

Figure 2. TP standard curve determined by microplate reader and conventional method

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表 1 不同酶标板与加样量和常规方法测定的回归方程

Table 1. Regression equations of different types of microwell plates, amount of sample and conventional method

测定方式 Determination method	回归方程 Regression equation	相关性 R²
常规方法 Conventional method	A=0.019 1c+0.001 2	0.999 9
48孔板1 mL 48-well plate 1 mL	A=0.019 1c+0.002 1	0.999 9
96孔板 200 µL 96-well plate 200 µL	A=0.010 8c+0.002 3	0.999 9
96孔石英板 200 µL 96-well quartz plate 200 µL	A=0.009 7c+0.007 5	0.999 3
24孔板1 mL 24-well plate 1 mL	A=0.010 0c+0.001 4	0.999 7
48孔板500 µL 48-well plate 500 µL	A=0.009 4c+0.001 2	0.999 0
24孔板500 µL 24-well plate 500 µL	A=0.004 7c+0.000 6	0.999 1
48孔板200 µL 48-well plate 200 µL	A=0.003 5c+0.000 4	0.995 5

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表 2 96孔板加样200 µL与常规方法测定水样TP的结果比较

Table 2. Comparison of results of measuring TP of samples by 96-well plate with 200 µL of sample and conventional method

样品号 Sample No.	优化法 Optimized method/(µmol·L⁻¹)	常规方法 Conventional method/(µmol·L⁻¹)	相对误差 RE/%	相对平均偏差 RAD/%	相对标准偏差 RSD/%
1	1.50±0.091	1.60±0.106	−6.25	5.33	6.04
2	3.00±0.137	3.10±0.079	−3.28	3.41	4.57
3	4.26±0.157	4.28±0.078	−0.39	3.15	3.69
4	1.48±0.128	1.43±0.102	3.73	7.04	8.61
5	4.74±0.118	4.73±0.067	0.21	2.32	2.49
6	2.44±0.115	2.49±0.097	−1.94	3.75	4.73
7	0.87±0.115	1.01±0.116	−14.03	10.56	13.19
8	3.17±0.117	3.32±0.082	−4.57	3.42	3.70
9	0.96±0.137	1.05±0.098	−8.41	12.65	14.23
10	1.87±0.116	1.96±0.086	−4.59	4.28	4.84
11	0.90±0.096	1.04±0.093	−13.46	10.00	11.55
12	3.05±0.139	3.11±0.074	−2.09	2.63	3.15
13	5.30±0.125	5.33±0.065	−0.59	2.35	2.63
14	3.88±0.156	3.82±0.073	1.53	2.95	3.24
15	2.18±0.093	2.28±0.080	−4.53	3.52	4.57
16	2.03±0.147	2.09±0.090	−3.11	3.95	4.74
17	5.85±0.204	5.64±0.056	3.72	3.42	3.49
18	1.19±0.100	1.26±0.118	−5.42	6.9	8.37
注：以常规方法的测定均值为真值，相对偏差 (%) 是优化法与常规方法的偏差，测定浓度是尾水稀释20倍后的浓度；表4同此。 Note: The measured mean value of the conventional method is considered to be the true value, and the relative deviation (%) is the deviation between the optimized method and the conventional method, and the measured concentration is the concentration after tail water dilution of 20 times. The same case in Table 4.

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表 3 96孔板优化法的样品加标回收率

Table 3. Sample spiked recovery by 96-well plate optimized method

样品浓度 Sample concentration/ (µmol·L⁻¹)	加入浓度 (β-GLP) Added concentration (β-GLP)/(µmol·L⁻¹)	加标后浓度 Found concentration/ (µmol·L⁻¹)	平均回收率 Average recovery/%	加入浓度 (SHMP) Added concentration (SHMP)/(µmol·L⁻¹)	加标后浓度 Found concentration/ (µmol·L⁻¹)	平均回收率 Average recovery/%
3.05	1.50	4.53±0.04	98.4	1.50	4.51±0.04	97.1
3.05	3.00	6.01±0.04	98.7	3.00	5.97±0.04	97.3
2.42	1.50	3.91±0.06	98.0	1.50	3.84±0.03	93.6
2.42	3.00	5.41±0.03	99.0	3.00	5.34±0.04	96.6
2.21	1.50	3.64±0.05	97.3	1.50	3.58±0.04	93.1
2.21	3.00	5.14±0.04	98.6	3.00	5.08±0.04	96.8
1.19	1.50	2.61±0.07	94.7	1.50	2.52±0.07	88.9
1.19	3.00	4.10±0.04	97.1	3.00	4.02±0.07	94.2

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表 4 48孔板加样1 mL与常规方法测定水样TP的结果比较

Table 4. Comparison of results of measuring TP of samples by 48-well plate with 1 mL of sample and conventional method

样品号 Sample No.	优化法 Optimized method/(µmol·L⁻¹)	常规方法 Conventional method/(µmol·L⁻¹)	相对误差 RE/%	相对平均偏差 RAD/%	相对标准偏差 RSD/%
1	1.55±0.029	1.60±0.106	−2.50	1.9	1.10
2	3.12±0.024	3.10±0.079	0.65	0.8	0.77
3	4.31±0.025	4.28±0.078	0.70	0.5	0.58
4	1.39±0.043	1.43±0.102	−2.80	2.1	3.09
5	4.75±0.021	4.73±0.067	0.42	0.3	0.44
6	2.45±0.032	2.49±0.097	−1.61	1.0	1.31
7	0.98±0.037	1.01±0.116	−2.97	3.3	3.78
8	3.36±0.024	3.32±0.082	1.20	0.7	0.71
9	1.04±0.031	1.05±0.098	−0.95	2.3	2.98
10	1.95±0.027	1.96±0.086	−1.52	1.6	1.38
11	1.01±0.041	1.04±0.093	−2.88	2.9	4.06
12	3.12±0.029	3.11±0.074	0.32	0.8	0.93
13	5.37±0.034	5.33±0.065	0.75	0.6	0.63
14	3.79±0.033	3.82±0.073	−0.79	0.8	0.87
15	2.24±0.030	2.28±0.080	−1.75	1.2	1.34
16	2.11±0.035	2.09±0.090	−1.40	1.5	1.66
17	5.68±0.024	5.64±0.056	0.71	0.4	0.42
18	1.28±0.033	1.26±0.118	1.59	2.1	2.58

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表 5 48孔板优化法的样品加标回收率

Table 5. Sample spiked recovery by 48-well plate optimized method

样品浓度 Sample concentration/ (µmol·L⁻¹)	加入浓度 (β-GLP) Added concentration (β-GLP)/(µmol·L⁻¹)	加标后浓度 Found concentration/ (µmol·L⁻¹)	平均回收率 Average recovery/%	加入浓度 (SHMP) Added concentration (SHMP)/(µmol·L⁻¹)	加标后浓度 Found concentration/ (µmol·L⁻¹)	平均回收率 Average recovery/%
3.05	1.50	4.59±0.04	98.2	1.50	4.56±0.04	95.8
3.05	3.00	6.11±0.02	99.6	3.00	5.98±0.03	95.2
2.42	1.50	3.93±0.04	98.7	1.50	3.91±0.03	97.1
2.42	3.00	5.42±0.03	99.4	3.00	5.35±0.03	96.8
2.21	1.50	3.73±0.04	99.1	1.50	3.66±0.02	94.7
2.21	3.00	5.22±0.02	99.3	3.00	5.12±0.03	95.9
1.19	1.50	2.76±0.06	98.4	1.50	2.69±0.04	94.2
1.19	3.00	4.23±0.04	98.4	3.00	4.08±0.04	93.4

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