Name: colorimetric detection of bacteria using litmus test
Uploaded: 2016-09-17T13:00:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Colorimetric Detection of Bacteria Using Litmus Test at JoVE.com

The funding for this research project was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) via a Discovery Grant to YL.

1.试剂和缓冲液的制备<ol><li>的0.5M乙二胺四乙酸（EDTA）的<ol><li>在2L烧杯中，186.1克EDTA添加到800毫升去离子蒸馏水的（DDH 2 O的）。调节溶液至8.0用NaOH粒料的pH值。添加DDH 2 O的以1.0升的最终体积，并在4℃下将溶液转移到一个高压灭菌玻璃瓶高压灭菌和储存。 </li></ol></li><li> 10×Tris-硼酸盐-EDTA（10×TBE） <ol><li>在一个4升的塑料烧杯中，使用搅棒，直到所有的组分432克Tris-碱将200克硼酸中，加入80ml的0.5M EDTA（pH 8.0）中和DDH 2 O的的添加4 L.混合的最终体积溶解。在4℃下将溶液转移至1升的玻璃瓶高压灭菌和储存。 </li></ol></li><li> 10％的变性聚丙烯酰胺股票<ol><li>在4升的塑料烧杯中，加入1681.7克尿素，加入400ml 10X TBE，1升40％的丙烯酰胺/双丙烯酰胺（29:1）解决方案和DDH 2 O的，直到在FINAL量为4 L.混合使用搅拌棒直到尿素溶解。在4℃下将溶液转移至1L的琥珀色玻璃瓶和存储。 </li></ol></li><li> 2X凝胶缓冲液（2X GLB） <ol><li>在200毫升玻璃烧杯中，加入44克尿素8克蔗糖，10毫克溴酚蓝，10毫克二甲苯青FF，400微升10％十二烷基硫酸钠的，和4毫升10×TBE的。添加DDH 2 O的以40 ml的终体积，并在50℃下溶解有轻度加热的部件，同时用磁力搅拌棒混合。传输1毫升一份到1.5 ml离心管和储存在4℃。 注意:使用前需要在90℃加热简要重新溶解固体的任何。 </li></ol></li><li>的1M Tris盐酸（Tris-盐酸）（pH7.5）中<ol><li>在玻璃瓶中，添加12.1克Tris-碱和70ml DDH 2 O的的并混合直到固体溶解。调整使用1M盐酸（HCl）将pH值至7.5。添加DDH 2 O的至100ml，高压釜和STO终体积再在4℃ </li></ol></li><li>的5M氯化钠（NaCl）中<ol><li>在玻璃瓶中，溶解58.4克NaCl的150毫升的DDH 2 O的调节音量至200毫升的DDH 2 O保存在4℃。 </li></ol></li><li> DNA洗脱缓冲液<ol><li>在玻璃瓶中，混合2.0毫升的1M的Tris-HCl（pH为7.5），8.0毫升5摩尔NaCl和0.4ml的0.5M的EDTA（pH8.0）中的。调节音量到200毫升的DDH 2 O，高压灭菌器和储存在4℃。 </li></ol></li><li> 1M的4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）（pH 7.4）中<ol><li>在玻璃瓶中，溶解2.38克HEPES的80ml的DDH 2 O的调整用5N氢氧化钠将pH至7.4，并添加DDH 2 O的至100ml的最终体积。 </li></ol></li><li> 1 1M氯化镁（ 氯化镁 ） <ol><li>在玻璃瓶中，添加2.03克的MgCl 2 -6H 2 O和使体积为100毫升的DDH 2 O </li></ol></li><li>反应缓冲液（RB） <ol><li&gt;在50ml锥形管中，加入50微升的1M HEPES（pH7.4）中，将1.5ml 5M的NaCl，0.75毫升的1摩尔的 MgCl 2，和5μl吐温-20组成。添加的DDH 2 O至50ml终体积。混合溶液中，并使用注射器驱动的滤器（0.22μm），并储存在4℃直至使用过滤到另一个锥形管中。 </li></ol></li><li>结合缓冲液（BB） <ol><li>在一个50毫升锥形管，加入500微升的1M的Tris-HCl（pH7.5）中的，8.8克NaCl，50微升的1M 的 MgCl 2，和5μl吐温20的添加DDH 2 O的至最终体积为50毫升。混合溶液中，并使用注射器驱动的滤器（0.22μm），并储存在4℃直至使用过滤到另一个锥形管中。 </li></ol></li><li>底物溶液（SS） <ol><li>在50ml锥形管中，加入5.8克NaCl的3毫升的1M MgCl 2的，1.5克尿素，和40毫升的DDH 2 O的调节溶液至5.0用10mM的HCl的pH值。由于该解决方案不进行缓冲，使用盐酸调节pH小心通过添加盐酸小等分。添加的DDH 2 O至50ml终体积。 </li><li>混合溶液中，并使用注射器驱动的滤器（0.22μm），并储存在4℃直至使用过滤到另一个锥形管中。 </li></ol></li><li>卢里亚BERTANI（LB）肉汤<ol><li>在烧杯中，溶解20.0克LB粉1升的DDH 2 O的转移到玻璃瓶中，高压釜中，并储存在4℃。 </li></ol></li><li> 1.5％LB琼脂<ol><li>在250毫升烧瓶中，添加1.5克琼脂和100ml LB肉汤中。高压灭菌器和储存在4℃。 </li></ol></li><li>琼脂平板<ol><li>再溶解在微波的LB琼脂并冷却溶液至〜50℃。将溶液倒入皮氏培养皿，使得〜5块板，并允许它们固化。 </li></ol></li></ol> 2.合成和E的纯化coli-响应核酶EC1 <ol><li>通过模板介导的酶连接EC1的合成<ol><li>净化COMMercially合成的寡核苷酸BS1，DE1，和T1 10％，根据标准方法变性聚丙烯酰胺凝胶电泳（dPAGE）（ 表1中提供了序列）。 </li><li>准备BS1，DE1，和T1的100微米的股票。储存在-20℃直到使用。 </li><li>在1.5ml微量离心管中，添加38.5微升DDH 2 O的，5微升DE1和5微升10×T4多核苷酸激酶反应缓冲液中由酶的供应商（500毫摩尔Tris盐酸（pH值7.6），100mM的MgCl 2的设置，的50mM二硫苏糖醇（DTT），1.0mM的亚精胺）。 </li><li>加入1μl三磷酸腺苷（ATP）（100毫米）的。加入5单位T4多核苷酸激酶（10U /微升）。通过仔细吹打反应混合物混合。 </li><li>孵育在37℃下进行30分钟的反应。 </li><li>淬火加热5分钟，将反应至90℃。 </li><li>加入118微升的DDH 2 O，5微升BS1和5微升T1的。加热反应至90℃2英里n和然后在10分钟内冷却至室温。 </li><li>加入20μl的酶的供应商提供的10×T4 DNA连接酶反应缓冲液（400毫摩尔Tris盐酸（pH值7.8），100mM的MgCl 2的，100毫摩尔DTT，5毫摩尔ATP）。加10单位T4 DNA连接酶（5U /微升）和通过吸液仔细混合。 </li><li>在室温下孵育2小时。 </li><li>加入20μl的3M乙酸钠（NaOAc）（pH为5.2），500微升冷的100％乙醇。混合通过涡旋该溶液，然后将管在-20℃下冷冻30分钟。 </li><li>离心机离心在20000 XG在4℃下20分钟。小心通过移液除去上清液。 </li><li>在4℃下以20,000 xg离心再次洗用冷70％乙醇，离心沉淀10分钟。再次去除上清液并干燥在真空下将沉淀10分钟。 </li><li>重悬DNA与15微升的DDH 2 O，然后加入15微升2×GLB的。 </li><li>涡大力并加热至90℃下2分钟。如下所述通过10％dPAGE纯化全长DNA。 </li></ol></li><li>设置10％dPAGE <ol><li>清洁两块玻璃板（一个完整的板和一个切口），两个0.75毫米厚间隔物，和一个12孔的梳子。放置在平坦的表面上的一个玻璃板上每侧的两个间隔件和在顶部的切口板。剪辑在一起使用由供应商提供的四个片段的两大板块。 </li><li>在150毫升的塑料烧杯中，倒入40含10％dPAGE的，四甲基乙二胺的40微升（TEMED）和400微升10％过硫酸铵（APS）的。混合组分和倾慢慢板之间的溶液。 </li><li>插入梳子，然后使凝胶聚合在10分钟内。一旦凝胶聚合，慢慢取出梳子，用的DDH 2 O冲洗井</li><li>安装在板上的凝胶电泳装置。使用金​​属板来消散所产生，以防止过热的热量。 </li><li>倾1×TBE上的顶部和底部腔室并确保井在缓冲器井淹没。冲洗使用吸液管或注射器用1×TBE的孔中。 </li><li>设置装载样品前15分钟在35马润的设备和预运行。 </li></ol></li><li>从10％dPAGE结扎EC1的溶出<ol><li>以下步骤2.2.6，运行在35毫安直到底部染料（溴酚蓝）凝胶到达玻璃板的底部。这大约需要1.5小时。关闭电源，并从该装置中删除所述板。 </li><li>躺在纸巾几片板并小心地从玻璃板取下隔离物。小心取出顶部玻璃板并用保鲜膜包住凝胶。 </li><li>翻转凝胶上以除去第二玻璃板，并再次用保鲜膜覆盖。请注意，以避免保鲜膜的皱纹。 </li><li>通过使用UV遮蔽（260纳米），这将产生4个不同的DNA条带（完全连接的EC1，DE1，BS1和T1）可视化的连接产物。 </li><li&gt;消费用无菌刀片并转移到1.5ml的微离心管的顶部带（EC1）。粉碎用无菌枪头（200微升针尖大小），凝胶，直到它变成一个精膏。加入550微升DNA洗脱缓冲液，覆盖铝箔管，以保护嵌入荧光团，振摇15分钟。 </li><li>离心以20,000 xg离心凝胶溶液5分钟，并小心地加入400μl的上清液转移至另外的150 ml离心管。避免在移液吸胶块。 </li><li>为了与转移的上清液的离心管中添加40微升的3M醋酸钠（pH值5.2）和1.0冷100％乙醇的溶液中。混合通过涡旋该溶液，然后将管在-20℃下冷冻30分钟。 </li><li>离心机离心在20000 XG在4℃下20分钟。小心通过移液除去上清液。 </li><li>在4℃下以20,000 xg离心再次洗用冷70％乙醇，离心沉淀10分钟。再次，删除上清液并干燥在真空下将沉淀10分钟。 </li><li>通过在260nm处测量UV吸光度确定EC1的浓度。使10μM的库存和样品储存在-20℃直到使用。 </li></ol></li></ol> 3.尿素酶的DNA的共轭<ol><li>琥珀酰亚胺4-（N-马来酰亚胺甲基）环己烷-1-羧酸酯（SMCC）股票的制备<ol><li>溶于676微升DMSO的1毫克SMCC。涡置于冰上直至使用。 </li></ol></li><li>股票脲酶的制备<ol><li>在1毫升1×PBS（无镁离子和钙离子 ）的溶解1毫克尿素酶。在冰上放置直至使用。 注:结晶尿素酶溶解缓慢且需要温和的混合，以避免变性。 </li></ol></li><li>脲酶DNA的合成（UrDNA） <ol><li>加10μl100μMLD1至2.5 ml离心管。加入140微升的DDH 2 O，40微升10X的PBS，旋涡。 </li><li>添加80.5微升SMCC的S滴答，DMSO，旋涡，短暂离心的159.5微升使用台式离心机。 </li><li>孵育在37℃下60分钟。避免离心帽下冷凝。 </li><li>加入200μl1×PBS中的60微升的3M醋酸钠（pH值5.2）和1.5冷100％乙醇的溶液中。混合通过涡旋该溶液并在-20℃下孵育30分钟。 </li><li>离心在4℃下以20,000 xg离心该溶液20分钟。除去上清液，并在真空下干燥沉淀。避免过度干燥。 </li><li>向干燥的沉淀中添加400μl的脲酶的库存中，并在室温下孵育5小时。 </li><li>转移200μl的粗共轭物的到预先洗涤100k的MWCO离心过滤柱。在14000 xg离心离心柱5分钟。 </li><li>传输剩余200μl的粗共轭到柱和离心机在14,000rpm xg离心5分钟。删除列，把它倒挂在一个新的2.0 ml离心管（"收集管"）。 </li><li> CentrifUGE的收集管（与倒置列）在1000 xg离心2分钟。取下收集管，并添加30微升1×PBS中的离心柱来洗膜对偶联物的额外的恢复。 </li><li>再次反转柱放回收集管。在1000 xg离心离心收集管（与倒置列）2分钟。移走和处置列。 </li><li>存储UrDNA在4℃直至使用。 </li></ol></li></ol> 4. EC1和UrDNA装配到磁珠<ol><li>混合磁珠（MB）的股票以及100微升MB悬挂转移到1.5 ml离心管中。放置离心管上用于隔离所述MB的磁性架子保持部。 </li><li>通过移液除去上清液，并添加150微升的结合缓冲液（BB）的管中。从保持器中取出管，并仔细挖掘管悬浮MB到均匀的溶液。避免飞溅悬挂到T管或盖的运算。如果发生这种情况，可使用台式离心机，以旋转简要残余物中回悬浮液中。 </li><li>重复步骤4.2两次。 </li><li>在此悬浮液加入10微升10μMEC1的。小心地敲打管子混用。 </li><li>孵育温和振摇30分钟的溶液中。挖掘管每隔2-3分钟，以避免对MB的沉淀。 </li><li>放置离心管背面上的磁性机架隔离MB和通过移液除去上清液。用的BB 150微升（如在步骤4.2中所述）洗MB的三倍。 </li><li>一旦在洗涤完成后，暂停所述MB在总共150微升的BB。向此溶液中，添加15微升UrDNA和加热该溶液至45℃持续2分钟。冷却该溶液至室温，并孵育2小时。 </li><li>放置离心管背面上的磁性机架隔离MB和通过移液除去上清液。 </li><li>加入100微升反应缓冲液（RB）的。除去叔他从磁架离心管，小心地悬浮MB。 </li><li>通过重复步骤4.9洗MB三次。 注意:洗涤上清液可以快速测试，以确定未杂交UrDNA仍然存在，这可能会导致假阳性信号。测试可以通过加入50mM的尿素的10微升和10微升0.04％酚红的洗涤溶液来完成。继续洗MB直到洗涤液中不引起颜色变化。在100微升的悬浮液贮存于4℃直至使用。 </li></ol> 5.制备细菌细胞20的<ol><li> E.培养大肠杆菌从股票<ol><li> E.板大肠杆菌 K12（MG1655）上从一个甘油下火焰或在生物安全柜的LB琼脂平板上。 </li><li>使用无菌移液管尖端，轻轻触摸甘油和轻轻条纹的琼脂平板的表面上，以避免刺穿LB琼脂。 </li><li>反转挑染板孵育在37℃下14小时。 </li><li>密封在4℃下用封口膜板和存储最多3周。 </li></ol></li><li> E.培养大肠杆菌的细胞计数<ol><li>在无菌14毫升培养管，取2毫升LB肉汤。 </li><li>使用无菌移液管尖端，从在步骤5.1中制备的划线琼脂平板挑取单菌落，并将其转移到培养管中。 </li><li>孵育培养在37℃和以230rpm 14小时摇动。 </li><li>连续稀释细菌培养的10倍区间。 </li><li>对于每个稀释样品，均匀地镀上分开的LB琼脂平板个100微升等分试样。倒置平板，并且在37°C孵育14小时。 </li><li>算每个样品的细胞，以获得每个稀释的平均细胞浓度。 注:10 7个细胞经常被用来设置为E.基准石蕊试大肠杆菌作为这个级别的E.大肠杆菌可引发快速的陈色GE。然而，正确执行试金石可检测低至50​​0个细胞，如结果部分讨论。 </li></ol></li><li> E.准备大肠杆菌细胞试验<ol><li>对于所需的细胞悬液，转移1毫升培养股票到1.5 ml离心管。 </li><li>离心细胞以6000 xg离心在4℃下10分钟。小心取出上清液，而不会干扰细胞沉淀。 </li><li>添加10微升反应缓冲液中的细胞沉淀重悬细胞。超声处理的细胞悬浮液5分钟。细胞悬液转移到冰盒5分钟。 </li><li>声波处理另一个5分钟的细胞悬浮液。 </li><li>离心细胞悬浮液以13,000rpm xg离心在4℃下10分钟。用于测试的上清液（10微升）。 </li></ol></li></ol> 6.试金石<ol><li>在1.5 ml离心管中，通过在离心管中添加和涡旋100微升反应缓冲液（RB）的的预洗管弃去缓冲液。 </li><li>转移15微升组装EC1（协议4）向洗涤微量离心管。 </li><li>通过将离心管上的磁性机架洗涤磁珠。通过移液除去上清液。从机架上卸下的离心管中，添加的RB的100微升，并小心地重悬磁珠。 </li><li>通过重复步骤6.3洗MB两次以上。 </li><li>放置离心管放回磁架，除去上清液，并添加10微升大肠杆菌从步骤5.3编写的大肠杆菌样本。 </li><li>通过在离心管轻轻拍打仔细混合样品和磁性珠粒。 </li><li>在室温下孵育1小时的反应。 </li><li>向反应中，添加90微升DDH 2 O的和离心管放置到一个磁架。 </li><li>后大约3分钟磁选，仔细转移85微升上清液至0.5 ml离心管。慢慢拔出上清避免收集任何磁珠。 </li><li>向上述离心管加入15微升的0.04％酚红，和100微升的底物溶液。 </li><li>采取以特定时间间隔的照片以记录颜色变化。 注:在pH值的变化也可以使用pH计用微电极监测。起始pH值应为大约5.2-5.5（溶液是黄色）。如果不是，则溶液可以通过添加1mM醋酸缓冲液pH 5.0）进行调整。 </li></ol>

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The authors have nothing to disclose.

的细菌响应核酶到石蕊试验的RNA裂解活性的作用的翻译是通过使用脲酶和磁分离的成为可能，由图1所示。虽然改性石蕊试验的细菌检测示范是与E.完成大肠杆菌依赖性的RNA切割核酶，5,19,20设计通常可延伸为任何的RNA切割核酶。定为不同的分析物，并且各种方法的RNA切割DNA核酶的极大可用性来隔离从新的目标随机序列池新的RNA切割DNA核酶，我们预计，改性石蕊测?...

Bacterial pathogens are one of the major causes of global morbidity and mortality. Outbreaks from hospital-acquired infections, food-borne pathogens, and bacterial contaminants in the environment pose serious and on-going threats to public health and safety. To prevent these outbreaks, effective tools are needed that permit pathogen detection in a timely fashion under a variety of settings. Simple but still effective tests that are portable and cost-effective are greatly coveted, especially in regions that are susceptible to outbreaks but cannot afford expensive testing facilities.1-3 Although there exists a multitude of methods to detect bacteria, many of them are not suitable as screening or on-site testing tools because they require long test times, expensive instruments and complicated testing procedures. 
Colorimetric tests are particularly attractive for point-of-care or field applications as color changes can be easily detected by the naked eye. The litmus test for pH is simple, quick, and effective. Although it is a very old technology, it is still widely used today because of its simplicity and effectiveness. Surprisingly, this simple test had never been modified to achieve the detection of other analytes before we recently developed an approach of modifying this test for E. coli testing.4
The expanded litmus test for E. coli employs three additional components: an E. coli activated RNA-cleaving DNAzyme (EC1), 5 urease, and magnetic beads. DNAzymes refer to synthetic single-stranded DNA molecules with catalytic activity.6 They can be isolated from random-sequence DNA pools using in vitro selection.7,8 They are highly stable and can be produced cost-effectively using high-efficiency automated DNA synthesis.9 For these reasons, DNAzymes, particularly RNA-cleaving DNAzymes, have been widely examined for biosensing applications.6,10,11 RNA-cleaving DNAzyme sensors have been developed to detect metal ions,12-16 small molecules,17,18 bacterial pathogens5,19-21 and cancer cells.22 Given the great availability of target-induced RNA-cleaving DNAzymes, any assay that utilizes a DNAzyme can be potentially expanded to detect a diverse range of analytes.
Urease is chosen for its ability to hydrolyze urea into ammonia,23,24 resulting in a pH increase. Urease is also highly efficient, stable and amenable for conjugation to other biomolecules. Therefore, we postulated that a conjugate of an RNA-cleavage DNAzyme with urease would allow the use of litmus test for the detection of other targets.5
The action of the RNA-cleaving DNAzyme is relayed to urease-mediated increase of pH through the use of magnetic beads that are immobilized with the DNAzyme-urease conjugate. Because the activity of the DNAzyme under investigation is strictly dependent on E. coli, the presence of this bacterium in the test solution will result in the release of urease from the magnetic beads to the solution, which is then taken and used to hydrolyze urea in a reporter solution that contains a pH-sensitive dye. The final outcome of this procedure is a color change that can be conveniently reported by the dye or pH paper.

<table><tbody><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>VWR AMRESCO</td><td>0105</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH) pellets</td><td>BIO BASIC CANADA INC.</td><td>SB6789</td><td></td></tr><tr><td>Tris-base</td><td>VWR AMRESCO</td><td>0497</td><td></td></tr><tr><td>Boric acid</td><td>AMRESCO</td><td>0588</td><td></td></tr><tr><td>Urea</td><td>VWR AMRESCO</td><td>M123</td><td></td></tr><tr><td>40% acrylamide/bisacrylamide (29:1) solution</td><td>BIO BASIC CANADA INC.</td><td>A0007</td><td></td></tr><tr><td>Sucrose</td><td>Bioshop Canada inc.</td><td>SUC507</td><td></td></tr><tr><td>Bromophenol blue</td><td>Bioshop Canada inc.</td><td>BRO777</td><td></td></tr><tr><td>Xylenecyanol FF</td><td>SIGMA-ALDRICH</td><td>X-4126</td><td></td></tr><tr><td>10% sodium dodecyl sulfate</td><td>Bioshop Canada inc.</td><td>SDS001</td><td></td></tr><tr><td>Hydrochloric Acid (HCl)</td><td>CALEDON LABORATORIES LTD</td><td>6026</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Bioshop Canada inc.</td><td>SOD001</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Bioshop Canada inc.</td><td>HEP001</td><td></td></tr><tr><td>Magnesium Chloride (II) hexahydrate</td><td>VWR AMRESCO</td><td>0288</td><td></td></tr><tr><td>Tween 20</td><td>Bioshop Canada inc.</td><td>TW508</td><td></td></tr><tr><td>Adenosine Triphospahte (ATP)</td><td>AMRESCO</td><td>0220</td><td></td></tr><tr><td>Sodium Acetate trihydrate (NaOAc)</td><td>SIGMA-ALDRICH</td><td>S8625</td><td></td></tr><tr><td>Ethanol</td><td>Commercial Alcohols</td><td>P016EAAN</td><td></td></tr><tr><td>Tetramethyleneethylenediamine (TEMED)</td><td>AMRESCO</td><td>0761</td><td></td></tr><tr><td>10% Ammonium persulfate (APS)</td><td>BIO BASIC CANADA INC.</td><td>AB0072</td><td></td></tr><tr><td>Succinimidyl 4-(&lt;em&gt;N&lt;/em&gt;-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)</td><td>ThermoFisher SCIENTIFIC</td><td>22360</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>CALEDON LABORATORIES</td><td>803540</td><td></td></tr><tr><td>Urease</td><td>SIGMA-ALDRICH</td><td>U0251</td><td></td></tr><tr><td>1x Phosphate Buffered Saline (PBS)</td><td>ThermoFisher SCIENTIFIC</td><td>70011-069</td><td></td></tr><tr><td>0.04% Phenol red</td><td>SIGMA-ALDRICH</td><td>P3532</td><td></td></tr><tr><td>10x T4 polynucleotide kinase reaction buffer</td><td>Lucigen</td><td>30061-1</td><td></td></tr><tr><td>10x T4 DNA ligase reaction buffer</td><td>Bio Basics Canada</td><td>B1122-B</td><td></td></tr><tr><td>T4 DNA ligase (5 U/&amp;#956;l)</td><td>Thermo Fischer Scientific</td><td>B1122</td><td></td></tr><tr><td>Luria Bertani (LB) Broth</td><td>AMRESCO</td><td>J106</td><td></td></tr><tr><td>Agar</td><td>AMRESCO</td><td>J637</td><td></td></tr><tr><td>T4 polynucleotide kinase (10 U/&amp;#956;l)</td><td>Lucigen</td><td>30061-1</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; K12 (MG1655)</td><td>ATCC</td><td>ATCC700926</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter, Inc.</td><td>392187</td><td></td></tr><tr><td>Glass plates</td><td>CBS scientific</td><td>ngp-250nr</td><td></td></tr><tr><td>0.75 mm thick spacers</td><td>CBS scientific</td><td>VGS-0725r</td><td></td></tr><tr><td>12-well comb</td><td>CBS scientific</td><td>VGC-7512</td><td></td></tr><tr><td>UV Lamp</td><td>UVP</td><td>95-0017-09</td><td></td></tr><tr><td>Spectrophotometer (NanoVue)</td><td>GE Healthcare</td><td>N/A</td><td></td></tr><tr><td>Metal plate</td><td>CBS scientific</td><td>CPA165-250</td><td></td></tr><tr><td>Vortex</td><td>VWR International</td><td>58816-123</td><td></td></tr><tr><td>Gel electrophoresis apparatus</td><td>CBS scientific</td><td>ASG-250</td><td></td></tr><tr><td>Petri dishes</td><td>VWR International</td><td>25384-342</td><td></td></tr><tr><td>100 kDa MWCO centrifugal filters</td><td>EMD Millipore</td><td>UFC510024</td><td></td></tr><tr><td>Magnetic Bead (BioMag)</td><td>Bangs Laboratories Inc</td><td>BM568</td><td></td></tr><tr><td>Magnetic Seperation Rack</td><td>New England BioLabs</td><td>S1506S</td><td></td></tr><tr><td>Microfuge tubes</td><td>Sarstedt</td><td>72.69</td><td></td></tr><tr><td>Syringe filter (0.22 &amp;#956;m)</td><td>VWR International</td><td>28145-501</td><td></td></tr><tr><td>14 ml culture tube</td><td>VWR International</td><td>60818-725</td><td></td></tr><tr><td>Cell culture incubator</td><td>Eppendorf Scientific</td><td>M13520000</td><td></td></tr><tr><td>Branson Ultrasonic cleaner</td><td>Branson</td><td>N/A</td><td></td></tr><tr><td>Camera (Canon Powershot G11)</td><td>Canon</td><td>N/A</td><td></td></tr><tr><td>50 ml conical tube</td><td>VWR International</td><td>89004-364</td><td></td></tr></tbody></table>

colorimetric detection of bacteria using litmus test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

细菌石蕊试验的原理在图1中说明的测试使用了三个关键材料:由一特定细菌酶，脲酶和磁珠活化的RNA切割核酶。所述核酶被用作分子识别元件，以实现所关注的细菌具有高度特异性的检测。脲酶和磁性珠粒用来实现DNA核酶的RNA的切割活性的信号转导。这涉及含有尿素酶结合物核酶磁珠的创建。在目标细菌的存在，所述核酶裂解其RNA的联动。这个动作引起脲酶从...

Watch this Scientific Journal Video about Colorimetric Detection of Bacteria Using Litmus Test at JoVE.com

Colorimetric Detection of Bacteria Using Litmus Test

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

细菌利用试金石比色法检测

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

colorimetric-detection-of-bacteria-using-litmus-test

Research

JoVE Journal

Biology

9.1K 次观看.  McMaster University. We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads. 

视频: Colorimetric Detection of Bacteria Using Litmus Test

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

比色纸为基础的检测<em&gt;大肠杆菌</em&gt;<em&gt;沙门氏菌</em&gt;属，和<em&gt;单核细胞增生李斯特氏菌</em&gt;从农业用水大容量

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

科研

环境科学

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

免疫与感染

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

生物工程

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Visualizing Bacterial Motility Based on a Color Reaction

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

基于颜色反应可视化细菌运动

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

生物学

Water Quality Analysis via Indicator Organisms

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba -The University of Arizona 
Demonstrating Author: Luisa Ikner
Water quality analysis monitors anthropogenic influences such as pollutants, nutrients, pathogens, and any other constituent that can impact the water&#8217;s integrity as a resource. Fecal contamination contributes microbial pathogens that threaten plant, animal, and human health with disease or illness. Increasing water demands and strict quality standards require that water being supplied for human or environmental resources be monitored for low pathogen levels. However, monitoring each pathogen associated with fecal pollution is not feasible, as laboratory techniques involve extensive labor, time, and costs. Therefore, detection for indicator organisms provides a simple, rapid, and cost effective technique to monitor pathogens associated with unsanitary conditions.

Microbial Indicators and Most Probable Number Analysis for Water Quality

通过指示生物水质分析

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba -The University of Arizona
Demonstrating Author: Luisa Ikner
Water quality analysis monitors ...

教育

JoVE Science Education

环境微生物学

Photometric Protein Determination

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more a sample contains light-absorbing substances, the less the light will transmit through it. Since the relationship between concentration and absorption is linear, this phenomenon can be used to measure the concentration in samples where it is unknown.
This video describes the basics of photometric protein determination and introduces the Bradford Assay and the Lowry Method. The procedure in the video will cover a typical Bradford assay. Applications covered include direct measurement of very small volumes of nucleic acids to characterize concentration and purity, determination of coupling efficiency of a biomimetic material, and another variation of photometric protein determination using Remazol dye.
<div class="chapter_text" id="script_0">
	Determining the concentration of a protein in samples is a fundamental step in many biochemical assays. Photometric determination can be done with small sample sizes. The more a sample contains light-absorbing substances, the less the light will transmit through it. This provides a quantitative measurement of the absorbing substances. These concepts are so fundamental to science that the articles that introduced two of the techniques are in the three most cited papers of all time. This video will show the concepts behind some of the most common photometric protein determination techniques, how they are performed, and how the gathered data is analyzed.
</div>
<div class="chapter_text" id="script_1">
	Photometric protein determination is based on the relationship between concentration and light absorbency. This is known as the Beer-Lambert Law, which states that the concentration of a light-absorbing species is proportional to its absorbance.
	This principle underlies all photometric protein determination methods. 
	For direct absorption analysis, the absorbance values of unaltered protein samples are measured. Because of their aromatic side chains, tryptophan and tyrosine residues give the highest absorbance readings at a wavelength of 280 nm.
	However, these amino acids-which are two of the least frequently found in proteins-are present in different amounts in every protein, so each determination is unique. To overcome this limitation, more complex assays-that are not dependent on these amino acids-were developed.
	One example is the Bradford Assay, where colored dye is added to the sample. The dye, known as Coomassie Blue, responds proportionally-the more protein present, the more binding events with the dye.
	Then, protein concentration is determined by measuring the absorbance of the bound Coomassie Blue dye, which absorbs light at 594 nm. However, the Bradford assay is linear over a short range of concentrations, so dilutions are often required before analysis.
	The Lowry Method combines the Biuret reagent, an alkaline solution of copper ions that react with peptide bonds, and the Folin-Cioc&#226;lteu reagent, which oxidizes aromatic protein residues. The resulting color change of the sample is proportional to the protein concentration.
	The absorbance of the reduced Folin reagent can be determined at 750 nm. Like direct absorption, each protein has a unique response, and must be calibrated for the protein of interest. Now that we've reviewed the basic principles behind some of the most common assays, let's look at how direct absorption and the Bradford assay are performed.
</div>
<div class="chapter_text" id="script_2">
	To begin a direct absorption analysis, the spectrophotometer is calibrated with a blank to determine zero absorbance. Standard solutions are prepared for use in creating the calibration curve. Then, an aliquot of the first standard is added to a cuvette, and placed into the spectrophotometer.
	The absorbance value at 280 nm is then recorded. This process is repeated for each standard, using a clean cuvette for each run. Once complete, a calibration curve is created by plotting the absorbance versus concentration. The slope of this line is the molar attenuation coefficient, which relates absorbance to concentration.
	Next, the unknown sample is added to a cuvette, and the absorbance value is recorded. As the data analysis for the different photometric determination methods is similar, we will cover that after we look at the Bradford assay.
	Here, the Bradford protein assay is performed with a BSA standard on a 96-well plate. To begin, BSA stock solutions are prepared.
	The unknown solutions are diluted with deionized water to ensure that the concentrations are within the assay's range. Depending on the kit, the Coomassie dye may also require dilution. Then, the calibration curve is set up by adding the BSA standards to the 96-well plate.
	Deionized water is added to reach the needed concentration to generate a standard curve. The unknown sample should be added to the plate in triplicates to ensure an accurate measurement is taken. Coomassie dye is next added to each well, mixing with the pipette.
	Deionized water is added to an empty well as a blank, to measure the absorbance. After waiting 5 min for the dye to bind, the absorbance is measured in a plate-reader at 590 nm.
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	Now that we've performed a few assays, let's look at how to analyze the data. Each photometric protein determination method is based on the Beer-Lambert Law.
	The measured absorbance of the standards is used to create a calibration curve, which is then used to determine the concentration of unknown samples. This curve can be manually plotted, though newer spectrophotometric tools will create the calibration curve once all standards have been measured. These systems will also calculate protein concentration as unknown samples are analyzed.
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	Now that we've reviewed how to analyze photometric protein determination data, let's look at some of the ways these procedures are utilized.
	The principles of photometric protein determination can also be used to directly measure nucleic acid concentration. The nanodrop spectrophotometer accepts samples of very small volume onto an optically active pedestal. The absorbance is then measured, and the system automatically determines the nucleic acid concentration. Because proteins and other sources can interfere with measurements, sample purity is determined by analyzing the 260 to 280 nm and 260 to 230 nm absorbance ratios. Pure nucleic acids typically yield ratios of approximately 1.8 and approximately 2.0 for DNA and RNA, respectively.
	Photometric protein determination can also be used in the production of biomimetic materials, which are inspired from nature to elicit specific cellular responses. Recombinant adhesins are bound to polystyrene beads to simulate bacterial attachment to host cells. The Bradford assay is used to determine the coupling efficiency of the recombinant adhesion to the beads in the production of the biomimetic material.
	Alternative photometric protein assays can be used in the detection and characterization of protein antimicrobials. Remazol brilliant blue R dye is covalently bonded to heat-killed bacteria. The protein antimicrobial is incubated in the dyed solution. Then, the sample is centrifuged, and the absorbance of the supernatant at 595 nm is measured using a microplate spectrophotometer. Increased absorbance, by the soluble dye released into the supernatant from the labeled bacteria, is a quantitative measurement of enzymatic activity.
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	You've just watched JoVE's video on photometric protein determination. This video described the underlying principles of photometric determination, went over general procedures for some common assays, and covered some new advances in techniques. Thanks for watching!
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Photometric Protein Determination: Bradford Assay

光度法蛋白的测定

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more ...

化学

生物化学

Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test

After preparing E. coli cells according to the text protocol, pre-wash a 1.5-milliliter microfuge tube by adding and vortexing 100 microliters of RB. Then, discard the buffer.
Add 15 microliters of assembled EC1 to the washed tube, then, place the tube on the magnetic rack, and aspirate the supernatant. Remove the tube from the rack, add 100 microliters of RB, and carefully resuspend the magnetic beads.
After using RB to wash the beads two more times and removing the RB, add 10 microliters of the E. coli sample to the tube, and gently mix the contents by tapping on the tube. Then, incubate the reaction at room temperature for one hour.
After the incubation, add 90 microliters of doubly-distilled water, and place the tube on the magnetic rack. Following approximately three minutes of magnetic separation, carefully transfer 85 microliters of the supernatant to a 0.5-milliliter microfuge tube.
It is very important to pipette the solution carefully without taking up any of the magnetic beads. These beads are loaded with urease and will generate a false positive signal.
Add 15 microliters of 0.04% phenol red and 100 microliters of substrate solution. Then, take photographs at specific time intervals to record a color change.

细菌利用试金石比色法检测

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比色纸为基础的检测