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Determination of Boron Content Using a Simple and Rapid Miniaturized Curcumin Assay
姜黄素法简单快速测定低浓度硼含量   

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本实验方案简略版
Journal of Plant Nutrition and Soil Science
Oct 2013

 

Abstract

To determine boron quantity in soil, water and biological samples, several protocols are available. Colorimetric assays are the simplest and cheapest methods which can be used to determine boron concentration. However, published protocols do not give straightforward guidance for beginners to adopt these protocols for routine use in the laboratory. Based on a previously published available procedure, we present a detailed and modified version of a curcumin based colorimetric protocol to determine boron concentration extracted from any sample. Our modified protocol is able to determine up to 0.2 nmole of Boron in a sample volume of 300 µl.

Keywords: Boron (硼), Curcumin method (姜黄素法), Protoplasts (原生质体), Arabidopsis (拟南芥), Yeast (酵母)

Background

Boron (B) can be quantified using spectrometric and colorimetric methods. Inductively coupled plasma mass spectrometry (ICP-MS) is the most sensitive method currently available having a detection limit of 0.01 mg/L (Kmiecik et al., 2016) but requires a sample volume of 5 ml. However, this technique requires sophisticated and expensive equipment which is not affordable for smaller laboratories. Alternatively, colorimetric based assays using curcumin or Azomethine-H dyes can be used for the routine analysis of boron in all laboratories with access to a spectrophotometer that can measure absorbance at 550 nm. Bingham (1982) reported that the curcumin assay is more efficient than the Azomethine-H based assay. Therefore, we present a modified version of simple and rapid curcumin assay to quantify B based on a protocol originally published by Wimmer and Goldbach (1999). We have used this protocol to determine the intracellular boron in yeast cells and Arabidopsis protoplasts. Additionally, this protocol can be used to study the uptake, root to shoot translocation-mechanisms of Boron in plants.

Materials and Reagents

Note: As boron leaches from lab glassware, all chemicals should be prepared in plastic bottles.

  1. Falcon tubes (15 ml and 50 ml) (SARSTEDT, catalog number: 62.547.254 )
  2. 1.5 ml tubes
  3. Pipette tips
  4. 96-Well UV Microplate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 8404 )
  5. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: 15663 )
  6. 0.1 N hydrochloric acid (HCl)
  7. 2-Ethyl-1,3-hexanediol (Acros Organics, catalog number: 118512500 )
  8. Chloroform (Fisher Scientific, catalog number: 10122190 )
  9. Concentrated sulfuric acid (H2SO4) (Fisher Scientific, catalog number: 10294300 )
  10. Concentrated acetic acid (Fisher Scientific, catalog number: 10304980 )
  11. Curcumin (Alfa Aesar, catalog number: B21573 )
  12. Methyl-Isobutyl-Ketone (MIBK) (Avantor Performance Materials, MACRON, catalog number: 6247-04 )
  13. Extraction solution (see Recipes)
  14. Acid mixture (see Recipes)
  15. Curcumin solution (see Recipes)

Equipment

  1. Centrifuge
  2. Chemical fume hood
  3. Pipette
  4. Microplate reader (BMG LABTECH, model: CLARIOstar )

Procedure

  1. Harvest the yeast/protoplasts samples in a 50 ml Falcon tube by centrifugation at 4,000 x g (for yeast)/100 x g (for protoplast) for 5 min. Wash the pellet with 25 ml ice cold water to remove boric acid from the pellet. Resuspend the pellet in 400 µl of Milli-Q water in a 1.5 ml tube. Heat the samples at 90 °C for 30 min and vortex to release intracellular B into solution. Centrifuge at maximum speed for 2 min at room temperature and collect 300 µl of supernatant for B quantification.
    For detailed boron uptake assay in yeast, users may refer to Takano et al., 2002 ; and to process the protoplasts from plant tissue please refer to Yoo et al., 2007.
    Note: We recommend that users test the assay with their own buffers/solvents if they do not use water to solubilise their samples.
  2. Prepare standards containing 0, 0.02, 0.05, 0.10, 0.19, 0.39, 0.77 mg boric acid/L using Milli-Q water in a 15 ml Falcon tube (refer to Table 1).

    Table1. Boric acid standards presented in different units


  3. Aliquot 300 µl (which is equivalent to 0.1, 0.2, 0.5, 0.9, 1.9, 3.7 nmole B) of each standard and samples into 1.5 ml tubes.
  4. Acidify the samples and standards by adding 100 µl of 0.1 N HCl. Mix well by vortex and incubate for 5 min at room temperature.
    Note: From Step 3 onwards perform all the reactions in chemical fume hood.
  5. Add 70 µl extraction solution (see Recipes), mix vigorously by vortexing for 30 sec. After 2 min, repeat the vortex for another 30 sec.
  6. Centrifuge the samples at 15,000 x g speed for 5 min to facilitate clear separation of organic phase.
  7. Pipette 50 µl of lower organic phase (Figure 1A) into a new 1.5 ml tube containing 200 µl of acid mix (see Recipes) and mix well by vortexing.
    Notes:
    1. Be aware that the lower phase contains chloroform and that chloroform leaks out from plastic pipette tips. To avoid pipetting errors, make sure that the tip is tightly attached to pipette and transfer liquids quickly.
    2. The mixture of sulphuric acid and acetic acid is a viscous solution. Therefore, care must be taken while pipetting to avoid pipetting errors and hazards to users from corrosive solutions. We recommend cutting the tip of the 1 ml tip and pipette slowly in a fume hood.


    Figure 1. Steps showing phase separation and colour development in the absence (-B) or presence (+B) of boron

  8. Add 250 µl of curcumin solution (see Recipes) and shake well until it forms homogenous dark purple colour (Figure 1B).
  9. Centrifuge the tubes at 15,000 x g for 1 min and incubate the reaction mixture at room temperature for 1 h.
  10. After 1 h, stop the reaction by adding 500 µl of Milli-Q water and mix well by inverting the tubes.
  11. Centrifuge the tubes at 15,000 x g for 1 min to facilitate clear phase separation.
  12. Carefully pipette 200 µl of upper phase (Figure 1C) into a 96-well UV Microplate and measure the absorbance at 550 nm using a microplate reader.
    Note: Chloroform in the reaction mixture melts many types of plastic microplates and interferes in absorbance reading. Therefore, it is very important to use microplates resistant to organic solvents (for example, Thermo ScientificTM 96-Well UV Microplate or a quartz microplate).
  13. Plot the absorbance against the quantity of B and prepare the calibration curve as shown in Figure 2. To obtain calibration curve, plot the absorbance values (A550 nm) against the quantity of boron (nmole B) using a spreadsheet (left panel of Figure 2).
    Note: Jennings et al., 2007 have used both curcumin method and ICP-MS to quantify boron in yeast cells. They found that using the two methods the results were indistinguishable.


    Figure 2. Calibration curve for the determination of boron concentration. Absorbance measured at 550 nm (A550 nm).

  14. Use the obtained calibration curve to determine the quantity of B in unknown samples.

Recipes

Note: Every time make fresh solutions in a falcon tube.

  1. Extraction solution
    Dissolve 2-ethyl-1,3-hexanediol 10% (v/v) in chloroform
  2. Acid mixture
    Mix sulphuric acid (conc.) and acetic acid (conc.) in 1:1 (v/v) ratio in a Falcon tube
  3. Curcumin solution
    Dissolve curcumin (2 mg/ml) powder in MIBK (Methyl-Isobutyl-Ketone)

Acknowledgments

This protocol is adapted from the original paper by Wimmer and Goldbach (1999). This work is supported by Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/N017765/1), United Kingdom. Authors declare no conflict of interest.

References

  1. Bingham, F. T. (1982). Boron. In: Page, A. L. (Ed.). Methods of soil analysis Part-2 chemical and mineralogical properties. American Society of Agronomy pp: 431-448.
  2. Jennings, M. L., Howren, T. R., Cui, J., Winters, M. and Hannigan, R. (2007). Transport and regulatory characteristics of the yeast bicarbonate transporter homolog Bor1p. Am J Physiol Cell Physiol 293(1): C468-76.
  3. Kmiecik, E., Tomaszewska, B., Wator, K. and Bodzek, M. (2016). Selected problems with boron determination in water treatment processes. Part I: comparison of the reference methods for ICP-MS and ICP-OES determinations. Environ Sci Pollut Res Int 23(12): 11658-11667.
  4. Takano, J., Noguchi, K., Yasumori, M., Kobayashi, M., Gajdos, Z., Miwa, K., Hayashi, H., Yoneyama, T. and Fujiwara, T. (2002). Arabidopsis boron transporter for xylem loading. Nature 420(6913): 337-40.
  5. Yoo, S. D., Cho, Y. H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7): 1565-72.
  6. Wimmer, M. A. and Goldbach, H. E. (1999). A miniaturized curcumin method for the determination of boron in solutions and biological samples. J Plant Nutr Soil Sci 162: 15-18.

简介

为了确定土壤,水和生物样品中的硼含量,可以使用几种方法。 比色测定是可用于测定硼浓度的最简单且最便宜的方法。 然而,出版的协议并没有给初学者在实验室常规使用这些协议提供直接的指导。 基于以前发布的可用程序,我们提出了一个姜黄素比色协议的详细和修改版本,以确定从任何样品中提取的硼浓度。 我们修改的协议是能够确定在300微升的样本量为0.2纳摩尔的硼。


【背景】硼(B)可以用光谱和比色法进行定量。电感耦合等离子体质谱法(ICP-MS)是目前可用的最灵敏的方法,其检测极限为0.01mg / L(Kmiecik等人,2016),但需要5ml的样品体积。然而,这种技术需要复杂和昂贵的设备,这对小实验室来说是不可承受的。或者,使用姜黄素或偶氮甲碱-H染料的基于比色的测定法可以用于所有实验室中的硼的常规分析,可以使用可以测量550nm处的吸光度的分光光度计。 Bingham(1982)报道,姜黄素测定法比基于甲亚胺-H的测定法更有效。因此,我们提出了一个简单和快速的姜黄素检测方法的修改版本,以Wimmer和Goldbach(1999)最初发表的方案来定量B。我们已经使用这个协议来确定酵母细胞和拟南芥原生质体中的细胞内硼。此外,该协议还可用于研究硼在植物中的摄取,根和芽的易位机制。

关键字:硼, 姜黄素法, 原生质体, 拟南芥, 酵母

材料和试剂

注意:由于硼从实验室玻璃器皿中渗出,所有化学品都应该用塑料瓶包装。

  1. 猎鹰管(15毫升和50毫升)(SARSTEDT,目录号:62.547.254)
  2. 1.5毫升管
  3. 移液器提示
  4. 96孔UV微孔板(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:8404)
  5. 硼酸(H 3 BO 3)(Sigma-Aldrich,目录号:15663)
  6. 0.1 N盐酸(HCl)
  7. 2-乙基-1,3-己二醇(Acros Organics,目录号:118512500)
  8. 氯仿(Fisher Scientific,目录号:10122190)
  9. 浓硫酸(H 2 SO 4)(Fisher Scientific,目录号:10294300)
  10. 浓缩乙酸(Fisher Scientific,目录号:10304980)
  11. 姜黄素(Alfa Aesar,产品目录号:B21573)
  12. 甲基异丁基酮(MIBK)(Avantor Performance Materials,MACRON,目录号:6247-04)
  13. 提取解决方案(见食谱)
  14. 酸混合物(见食谱)
  15. 姜黄素溶液(见食谱)

设备

  1. 离心机
  2. 化学通风橱
  3. 移液器
  4. 酶标仪(BMG LABTECH,型号:CLARIOstar)

程序

  1. 通过在4,000×g(对于酵母)/ 100×g(对于原生质体)中离心5分钟,将酵母/原生质体样品收获在50ml Falcon管中。用25毫升冰冷的水清洗沉淀,以去除沉淀中的硼酸。在一个1.5毫升的管中重悬在400微升Milli-Q水中的沉淀。将样品在90℃加热30分钟并涡旋以将细胞内B释放到溶液中。在室温下以最大速度离心2分钟并收集300μl上清液用于B定量。
    对于酵母中的详细的硼摄取测定,用户可以参考Takano et al。,2002;并处理来自植物组织的原生质体,请参阅Yoo et al。,2007。
    注意:如果用户不使用水溶解样品,我们建议用户使用自己的缓冲液/溶剂进行测试。
  2. 准备含有0,0.02,0.05,0.10,0.19,0.39,0.77mg硼酸/ L的标准品,使用Milli-Q水在15ml Falcon管中(参见表1)。

    的表1。硼酸标准不同单位


  3. 将300μl(相当于0.1,0.2,0.5,0.9,1.9,3.7nmole B)的各标准品和样品分装到1.5ml管中。
  4. 通过加入100μl0.1N HCl酸化样品和标准品。通过涡旋混匀,室温孵育5分钟。
    注意:从第3步开始,在化学通风柜中进行所有的反应。
  5. 加入70μl提取液(见食谱),剧烈搅拌30秒。 2分钟后,重复旋涡30秒。
  6. 将样品以15,000×g的速度离心5分钟以促进有机相的清晰分离。
  7. 吸取50μL较低的有机相(图1A)到一个新的1.5毫升含200μl酸混合物的管中(见食谱),并通过涡旋混匀。
    注意:
    1. 请注意,较低的相含有氯仿,氯仿会从塑料吸头漏出。为避免吸移误差,请确保吸头紧密连接到移液器并快速转移液体。
    2. 硫酸和乙酸的混合物是粘性溶液。因此,在移液过程中必须小心,以避免由于腐蚀性的解决方案而导致的吸液错误和对使用者的危害。我们建议切割1毫升尖端的尖端,并在通风橱中慢慢吸取。


    图1.在硼不存在(-B)或存在(+ B)的情况下,显示相分离和颜色发展的步骤

  8. 加250μL姜黄素溶液(见食谱),摇匀,直到形成均匀的深紫色(图1B)。
  9. 在15,000×gg离心管1分钟,并将反应混合物在室温下孵育1小时。
  10. 1小时后,加入500微升Milli-Q水,停止反应,并倒置管混匀。

  11. 15,000×g g离心管1分钟,以促进清晰的相分离。
  12. 小心吸取200μL的上层相(图1C)到96-孔紫外微孔板中,并使用酶标仪在550nm处测量吸光度。
    注意:反应混合物中的氯仿溶解许多类型的塑料微孔板并干扰吸光度读数。因此,使用耐有机溶剂的微孔板是非常重要的(例如Thermo Scientific TM 96孔UV微孔板或石英微孔板)。
  13. 绘制吸光度与B的量的关系曲线,并如图2所示制备校准曲线。为了获得校准曲线,使用相对于硼量(nmole B)的吸光度值(A505nm)绘图一个电子表格(图2的左图)。
    注:詹宁斯等人,2007年使用姜黄素法和ICP-MS来量化酵母细胞中的硼。他们发现使用这两种方法的结果是难以区分的。


    图2.用于测定硼浓度的校准曲线。在550nm测量的吸光度(A505nm)。

  14. 使用获得的校准曲线来确定未知样品中的B的数量。

食谱

注意:每次在猎鹰管中制作新鲜的解决方案。

  1. 提取解决方案
    将2-乙基-1,3-己二醇10%(v / v)溶于氯仿中
  2. 酸混合物
    在Falcon管中以1:1(v / v)的比例混合硫酸(浓)和乙酸(浓)
  3. 姜黄素溶液
    将姜黄素(2毫克/毫升)粉末溶于MIBK(甲基异丁基酮)

致谢

本议定书改编自Wimmer和Goldbach(1999)的原始论文。这项工作得到了英国生物技术和生物科学研究委员会(BBSRC)的资助(BB / N017765 / 1)的支持。作者声明不存在利益冲突。

参考

  1. Bingham,F. T.(1982)。硼。在:Page,A.L。(编)。 土壤分析方法第2部分化学和矿物学性质 < 美国农艺学会 pp:431-448。
  2. Jennings,M.L.,Howren,T.R。,Cui,J.,Winters,M。和Hannigan,R。(2007)。 酵母碳酸氢盐转运蛋白同系物Bor1p的运输和调控特征 Am J Physiol Cell Physiol 293(1):C468-76。
  3. Kmiecik,E.,Tomaszewska,B.,Wator,K.和Bodzek,M.(2016)。 在水处理过程中选定的硼测定问题。第一部分:比较ICP-MS和ICP-OES测定的参考方法。


    环境科学Pollut Res Int 23(12):11658-11667。

  4. Takano,J.,Noguchi,K.,Yasumori,M.,Kobayashi,M.,Gajdos,Z.,Miwa,K.,Hayashi,H.,Yoneyama,T。和Fujiwara,T。(2002)。 拟南芥用于木质部负载的硼转运蛋白 自然 420(6913):337-40。
  5. Yoo,S.D。,Cho,Y.H。和Sheen,J。(2007)。 拟南芥叶肉原生质体:用于瞬时基因表达分析的多功能细胞系统。


    Nat Protoc 2(7):1565-72。

  6. Wimmer,M.A。和Goldbach,H.E。(1999)。 (a)(a) “目标=”_ blank“>微型姜黄素测定溶液和生物样品中硼的方法。 J植物营养土壤科学 162:15-18。
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引用:TC, M. and Jones, A. M. (2018). Determination of Boron Content Using a Simple and Rapid Miniaturized Curcumin Assay. Bio-protocol 8(2): e2703. DOI: 10.21769/BioProtoc.2703.
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