海洋生态与环境科学重点实验室知识库

    本论文提出了基于微流控液液萃取法对海水中的超痕量磷酸盐进行检测的微流控芯片，以奠定超痕量磷酸盐实时监测的技术基础，反应和检测的原理为磷钼蓝萃取分光光度法。通过实验对磷钼蓝方法中试剂的保存方法进行了改进，设计微反应芯片和微萃取芯片并对芯片的参数进行了确定，最终通过样品检测初步验证了方法的可行性。

    钼酸盐混合溶液在4℃~25℃范围内，避光条件可保存半年以上。抗坏血酸溶液随着温度的升高，有效性降低；在每100 mL 0.1 g/mL的抗坏血酸溶液中，加入1 g EDTA和2 ml冰乙酸作为络合剂掩盖金属离子催化氧化的效果；通入氮气去氧减缓抗坏血酸氧化；溶液过滤除菌降低细菌对抗坏血酸的消耗作用；在4℃~25℃范围内，抗坏血酸溶液的有效使用期限延长2.7~5倍以上。

    微流控反应芯片的主要目的是通过芯片中的反应生成磷钼蓝，实现对海水中常量磷酸盐的检测，并为萃取阶段持续的提供样品；芯片材质选用PDMS，采用十字进样通道和连续半圆形反应通道，通道宽度250 μm，深度100 μm，总长2000 mm；通道出口由圆形储液池承接反应后的试样，储液池的半径为2.52 mm，深度为2 mm；试样流量比为样品：钼酸盐混合溶液：抗坏血酸溶液=5:1:1。

    微流控液液萃取芯片中磷钼蓝试剂和萃取剂形成稳定的双相层液液萃取体系。萃取反应通道宽250 μm，深100 μm，长1000 mm，均为高度50 μm的引导结构；除萃取通道外，其余通道宽125 μm，深度100 μm；利用蠕动泵和三通阀，设计循环萃取流程，实现磷钼蓝的多倍富集。芯片材质为PDMS，萃取剂为正己醇，样品和萃取剂的流量为100 μL/min，样品富集10倍需要2.5分钟。

    将微流控反应芯片和微流控液液萃取芯片串联使用，验证了微流控液液萃取法在μmol/L级和nmol/L级的磷酸盐检测方面重现性好、准确度高、线性关系好、试剂消耗量小、反应耗时短；方法检出限为3.92 nmol/L，在对海水样品的实际检测中也显示了良好的性能。 

    This paper mainly studied the microfluidic chip based on microfluidic liquid-liquid extraction method for the determination of ultra-trace phosphate in seawater, which lays the technical foundation for real-time monitoring of ultra-trace phosphate. The principle of reaction and detection is phosphomolybdenum blue spectrophotometric method. Through experiments, the reagents preservation method in the phosphomolybdenum blue spectrophotometric method was improved, the microfluidic reaction chip and microfluidic extraction chip were designed and the parameters of the chips were determined. Finally, the feasibility of the method was preliminarily verified through sample detection.

    The molybdate mixed solution can be preserved in darkness over half a year within 4℃~25℃ ranges. The validity of ascorbic acid solution is shortened as the temperature rises; in per 100 mL of 0.1 g/mL ascorbic acid solution,adding 1 g of EDTA and 2 mL of glacial acetic acid as a complexing agents , removing oxygen by means of high purity nitrogen, followed by filtration sterilization reduces the effect of bacteria ,and seal preserved in darkness can prolong the validity of the ascorbic acid solution to over 2.7~5 times within 4℃~25℃ ranges.

    The main purpose of the microfluidic reaction chip is to generate phosphomolybdenum blue solution through the reaction in the chip, so as to detect the constant phosphate in seawater and provide samples for continuous extraction. The chip material is PDMS with cross injection channel and continuous semicircular reaction channel. The channel width is 250 μm, the depth is 100 μm, and the total length is 2000 mm. The outlet of the channel is taken over by the circular liquid storage tank after the reaction. The radius of the liquid storage tank is 2.52 mm and the depth is 2 mm. Flow ratio is sample: molybdate mixed solution: ascorbic acid solution =5:1:1.

    A stable two-phase liquid-liquid extraction system was formed by the phosphomolybdenum blue solution and the extractant in the microfluidic liquid-liquid extraction chip. The extraction reaction channel is 250 μm wide, 100 μm deep and 1000 mm long, with a guide structure of 50 μm in height. Except the extraction channel, the width of other channels is 125 μm, the depth is 100 μm. The peristaltic pump and three-way valve were used to design the cycle extraction process. The chip material was PDMS, the extractant was n-hexanol, the flow rate of the sample and extractant was 100 μL/min, and the concentration of sample 10 times needed 2.5 minutes.

    The microfluidic reaction chip and microfluidic liquid-liquid extraction chip were used in series to verify that the microfluidic liquid-liquid extraction method has good reproducibility, high accuracy, good linear relationship, low reagent consumption and short reaction time in the detection of phosphate at μmol and nmol/L levels. The detection limit of the method was 3.92 nmol/L, which also showed good performance in the actual detection of seawater samples.