Name: capillary electrophoresis to monitor peptide grafting onto chitosan films in real time
Uploaded: 2016-10-26T13:00:00.000+00:00
Duration: 11 min 5 s
Description: Watch this Scientific Journal Video about Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time at JoVE.com

MG, MO'C and PC thank the Molecular Medicine Research Group at WSU for Research Seed Funding, as well as Michele Mason (WSU), Richard Wuhrer (Advanced Materials Characterisation Facility, AMCF, WSU) and Herv&#233; Cottet (Montpellier) for discussions.

1.壳聚糖膜的制备<ol><li>称输出2克冰醋酸的，完整的100毫升的超纯水。 </li><li>称出1.7克壳聚糖粉末，加入100毫升的2％M / M乙酸水溶液。搅拌5天，在室温下或用铝箔覆盖或在黑暗中搅拌棒和磁力搅拌板。 </li><li>离心脱乙酰壳多糖分散液在1,076 xg离心在23℃1小时。收集用注射器上清液并丢弃沉淀物。 </li><li>为每部影片，等分试样的10ml脱乙酰壳多糖的悬浮液成在室温下具有9厘米塑料培养皿中。离开覆盖干燥至少7天的电影。 </li><li>用剪刀剪断干膜成1×1cm的正方形。注意:实验可在此阶段被暂停。 </li></ol> 2.制备磷酸盐缓冲盐水的（PBS）中<ol><li>称出8克氯化钠将0.2g氯化钾，1.44克氢二钠磷酸phate及0.24g的磷酸二氢钾。 </li><li>溶解这些称量的化学品在800毫升的超纯水，用浓盐酸将溶液滴定至pH 7.4。 注意:实验可在此阶段被暂停。 </li></ol>在pH 9.2的75mM硼酸钠缓冲液3.制备<ol><li>称取3.0915克硼酸。将其溶解于75ml超纯水中。 </li><li>滴定硼酸溶液与氢氧化钠溶液的pH为9.2，以10 M或更高的浓度。 注意:浓缩的氢氧化钠溶液有腐蚀性，应带手套处理。 </li><li>完全用超纯水，得到100毫升溶液中。这产生在pH 9.2的500mM硼酸钠缓冲液。 </li><li>稀释用超纯水的500mM的硼酸钠缓冲液至75mM的硼酸钠缓冲液。注意:实验可在此阶段被暂停。 </li></ol> 4.壳聚糖网络连接的研制LMS的接枝反应<ol><li>冲洗在5ml PBS中进行2小时10平方壳聚糖膜（1×1厘米）在室温下在培养皿中。 </li><li>在此期间，准备和验证的毛细管电泳仪（步骤5）。 </li></ol> 5.编写和验证的毛细管电泳仪的<ol><li>制备43.5厘米裸熔融石英毛细管50μm的内径（43.5厘米是总长度，有效长度的检测窗口通常为35厘米）通过在与所述设定的长度弱化毛细管的聚合物外涂层钝具则贴紧毛细管。 <ol><li>用打火机烧在从入口8.5厘米聚合物涂层创建毛细管一个窗口，它冷却后擦拭干净用乙醇。烧毛细管的涂层在每一端为用打火机几毫米，并且它冷却后擦拭干净，用乙醇。 </li><li>地方毛细血管INSI德检测窗口，并将其在毛细管盒通过将它放置在该入口和出口相等的长度和卷绕它围绕带盒的转轴安装。然后安装在毛细管电泳仪的纸盒。 </li></ol></li><li>设置每个分离的方法的参数。在软件菜单中选择"方法"，然后"编辑整个方法"。设定温度，时间，电压，和用于分离（例如25℃，10分钟，30千伏）的小瓶中。 <ol><li>在预调节部分，设置连续刷新:10分钟，用1M氢氧化钠（在水中），5分钟用0.1M氢氧化钠（在水中），5分钟，用超纯水和5分钟，75毫硼酸钠缓冲液在一系列分析的第一方法pH为9.2。 </li><li>用于后续的方法中，设置集在预处理部分连续刷新:1分钟用1M氢氧化钠（在水中），5分钟，75毫硼酸钠缓冲液在pH为9。2。 </li><li>在注入部分，用于高压注射为10秒，所有的方法30毫巴压力设定参数。在分离部分中，9分钟为所有方法设置分离条件至30千伏在25℃。 注:请具体CE设备的用户手册，作为程序操作CE仪器制造商之间可能会有所不同。制备的当天1M氢氧化钠溶液。 </li></ol></li><li>注入和分离出中性内标（10％体积/体积的二甲基亚砜10微升（DMSO）中，在稀释到450微升的75mM硼酸钠缓冲液的水）。然后注入并以同样的方式分离的低聚丙烯酸酯标准（以10g∙L-1溶解在超纯水中;见材料清单）检查毛细管的有效性。在这里暂停序列，直到接枝反应准备开始。 </li></ol> 6.嫁接到RGDS壳聚糖膜的<ol><li>称出所述肽（1毫克RGDS）和偶联剂（3毫克EDC·HCl和2毫克NHS）。 </li><li>在PBS中的脱乙酰壳多糖膜浸泡开始后2小时，溶解肽，并在5毫升的PBS的偶联剂。 <ol><li>取该溶液的50微升等分试样。在水中添加2微升的10％体积/体积的DMSO作为内部中性标准的等分试样。分析与CE等分（见第7步）。 </li></ol></li><li>除去5毫升的PBS用于冲洗从培养皿的脱乙酰壳多糖膜。 5 ml的溶液肽和偶联剂添加到含有脱乙酰壳多糖膜的培养皿。 </li><li>覆盖培养皿用石蜡膜，并将其放置在室温下轨道摇床。以反应介质的50微升等分在设定的时间。 注:总的分析时间与CE为15分钟，从而将等分试样可以采取每15分钟（或每30分钟，如果两个反应并行监视， 等 ）。 <ol><li>在水中添加2微升的10％体积/体积的DMSO作为内部中性标准对每个人iquot。 注:等分应该符合CE，尽快为他们取来分析（见第7步）。 </li></ol></li><li>后摇和分装去除4小时，取出从振动筛的培养皿。除去从培养皿反应介质中。加入5毫升的PBS冲洗壳聚糖薄膜。 </li><li>从培养皿中取出PBS，漂洗用超纯水的脱乙酰壳多糖膜，并允许他们干燥过夜。除去超纯水，并在-20℃下的薄膜储存于塑料培养皿。 </li></ol> 7.接枝反应监测使用CE <ol><li>注射以及使用的分析条件，在部分5.2培养皿中取出后，立即反应介质的单独分装。 </li><li>一旦分离完成漂洗用超纯水毛细管10分钟。干它通过与10分钟的空药瓶（空气）冲洗。 注:实验可以在此阶段被暂停。 </li></ol> 8.数据TREA<html> tment为CE <ol><li>检查每个分离的有效性，通过检查这两个分离期间的电流和电渗流动性标记（DMSO在这种情况下）的迁移时间是类似的低聚丙烯酸酯标准分离所观察到的那些。 注:最多10-15％的变化是从约50微安和1.3分钟迁移时间值的预期电流值上可接受的（如需要更高的可重复性的电泳迁移率的值应该被用来代替迁移时间）。 </li><li>对于每个成功的分离，通过选择一个特定的数据集，右键点击出口，并选择适当的信号输出从毛细管电泳软件的原始数据。 </li><li>转换由CE记录的原始数据（表示为UV吸收为迁移时间的函数）。转换X轴（迁移时间t 米 ）到下面的等式1的电泳迁移率μ: N + 1"SRC ="/文件/ ftp_upload / 54549 / 54549eq1.jpg"/&gt;（1） 其中，L d是长度到检测器中，L t是毛细管的总长度，V是电压，而t EO为中性物种（在此情况下，DMSO的内标）27的迁移时间。 转换的原始数据（吸光度在AU）的Y轴的电泳迁移率W（μ）的分布下列公式2:28 <img alt="公式（2）" src="/files/ftp_upload/54549/54549eq2.jpg" /> （2） </li></ol> 9.肽接枝膜18的附加特性<ol><li>将肽接枝壳聚糖膜，在自己周围转了转，采用4 mm固态核磁共振转子。填充转子用磷酸盐缓冲盐水溶胀膜，并关闭转子。等待了几个小时。 </li><li> 13分析了电影&lt;/ SUP&gt; C-NMR光谱18。 </li></ol>

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R., Gaborieau, M., Castignolles, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+poly(acrylic+acid)+salts+according+to+topology+using+capillary+electrophoresis+in+the+critical+conditions.">Separation of poly(acrylic acid) salts according to topology using capillary electrophoresis in the critical conditions.</a> Anal. Bioanal. Chem. 405, 9009-9020 (2013).</li><li>Chung, T. W., Lu, Y. F., Wang, S. S., Lin, Y. S., Chu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+endothelial+cells+on+photochemically+grafted+Gly-Arg-Gly-Asp+(GRGD)+chitosans.">Growth of human endothelial cells on photochemically grafted Gly-Arg-Gly-Asp (GRGD) chitosans.</a> Biomaterials. 23, 4803-4809 (2002).</li></ol>

作者宣称，他们没有竞争的经济利益。

这里所描述的协议的简单性使得它非常适合于广泛的应用。但是，需要特别注意支付给下面的关键步骤。 正确的CE仪器准备 它以分离已知的标准之前立即未知样品的分离（以及在一系列分离的结束时），检查在毛细管和仪器在当天的有效性是重要的。这个标准可以是低聚丙烯酸酯27或已知在宽范围的迁移时间，得到多个峰的任何样品...

自由溶液的毛细管电泳（CE）是分隔在根据它们的电荷对摩擦比1,2解化合物的技术。电荷-尺寸比通常在文献中所提到的，但这种简化并不适用于聚电解质，其中包括在这项工作中的多肽，并且也显示出不适合小有机分子3。 CE不同于其它分离技术，因为它不具有固定相，仅一个背景电解质（通常是缓冲液）。这使得该技术将在其分析大范围样本的复杂基质4，如植物纤维5，发酵酿造6接枝到合成聚合物7，食品样品8和难溶肽9无需繁琐的样品制备和能力强劲纯化。这是一个复杂的聚电解质具有的溶解问题（S特别显著UCH壳聚糖10和结冷胶11），因此，存在如凝集或在溶液中沉淀，并已成功地分析没有样品过滤。此外，早餐谷类食品糖的分析涉及的早餐麦片样品颗粒注入样品中水8沉淀。这还延伸至支链聚电解质或共聚物12,13的分析。大量的工作也已在CE技术的发展专门为蛋白质的蛋白质组学14，天然或合成的肽15和蛋白质和肽16的微芯片的分离的手性分离的分析完成。由于分离和分析发生在毛细管，样品只有小体积和溶剂被用于使CE为具有比其它分离技术包括色谱法5,6,17较低的运行成本。由于通过CE分离速度快，它允许monito反应动力学的环。这表现在肽的接枝改进细胞粘附18脱乙酰壳多糖膜的情况下。 脱乙酰壳多糖是从几丁质的N- -deacetylation衍生的多糖。脱乙酰壳多糖膜可用于各种生物医学应用，例如生物粘合剂19和细胞生长基板18,20，由于脱乙酰壳多糖的生物相容性21。细胞附着到特定的细胞外基质蛋白，如纤连蛋白，胶原和层粘连蛋白，直接关系到细胞22的存活。值得注意的是，不同的细胞类型往往需要连接到不同的细胞外基质蛋白生存和正常功能。结果表明细胞附着于壳聚糖膜，通过纤维连接蛋白23的嫁接得到加强;然而，制备，纯化和这样大蛋白质的接枝是在经济上不可行。或者一系列小肽甲肝È被证明能够模仿大胞外基质蛋白的性质。例如，肽如纤连蛋白模拟物的RGD（精氨酸-甘氨酸-天冬氨酸）和RGDS（精氨酸-甘氨酸-天冬氨酸-丝氨酸）已经被用于促进和增加细胞附着24。 RGDS到壳聚糖膜的共价嫁接导致已知附加到纤维连接蛋白在体内细胞18提高细胞附着。代较大的蛋白质喜欢和较小的肽具有相同的功能提供了一个显著成本降低纤维连接蛋白。 这里，如先前发表18进行肽接枝到壳聚糖。正如以前证明，这种方法通过使用偶联剂EDC盐酸（1-乙基-3-（3-二甲氨基丙基）碳化二亚胺）和NHS（N-羟基）官能的RGDS的羧酸是提供简单而有效的接枝接枝到壳聚糖薄膜。这种接枝方法的两个优点是，它不需要壳聚糖或肽的任何修改，并且在水性介质中进行的，以最大程度地与未来细胞培养应用18,20的兼容性。作为耦合剂和所述肽可以充电，CE是用于反应动力学的分析的合适方法。重要的是，经由CE中的反应动力学的分析，可以在接枝反应进行实时监控，并且因此能够既优化和量化接枝度。 虽然它不是常规必要，在CE分析的结果可以被离线由肽接枝到利用固体NMR（核磁共振）光谱25,26的脱乙酰壳多糖膜的直接测量验证以证明共价接枝的的肽到薄膜18。然而，随着固态NMR光谱法相比，实时分析由设置CE能够实时肽消耗，从而以评估反应的动力学的能力的定量。 上述方法是简单，并且允许肽的接枝到与接枝的程度的间接定量脱乙酰壳多糖膜的实时分析。所表现出的方​​法可以扩展到不同的化学反应，只要反应物或待分析可充电产品的实时定量评估。

<table><tbody><tr><td>Water</td><td>Millipore</td><td></td><td>All water used in the experiment has to be of Milli-Q quality</td></tr><tr><td>Chitosan powder (medium molecular weight)</td><td>Sigma-Aldrich</td><td>448877</td><td>lot MKBH1108V was used. Significant batch-to-batch variations occur with natural products such as polysaccharides</td></tr><tr><td>Acetic acid - Unilab</td><td>Ajax Finechem</td><td>2-2.5L GL</td><td>laboratory reagent</td></tr><tr><td>Dimethylsulfoxide</td><td>Sigma-Aldrich</td><td>D4540</td><td>laboratory reagent, slightly hazardous to skin, hazardous if ingested</td></tr><tr><td>Sodium hydroxide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>221465&amp;nbsp;</td><td>laboratory reagent, corrosive&amp;nbsp;</td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl)carbodiimide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D80002</td><td>Irritant to skin&amp;nbsp;</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-hydroxysuccinimide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>130672</td><td>Irritant to skin</td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Ajax Finechem</td><td>466-500G</td><td>laboratory reagent</td></tr><tr><td>Potassium chloride - Univar</td><td>Ajax Finechem</td><td>384-500G</td><td>analytical reagent, slight skin irritant</td></tr><tr><td>Disodium hydrogen phosphate - Unilab</td><td>Ajax Finechem</td><td>1234-500G</td><td>laboratory reagent, slight skin irritant</td></tr><tr><td>Potassium dihydrogen phosphate - Univar</td><td>Ajax Finechem</td><td>4745-500G</td><td>analytical reagent, slight skin irritant</td></tr><tr><td>Oligoacrylate standard</td><td>custom made</td><td></td><td>See reference for synthetic protocol: Castignolles, P.; Gaborieau, M.; Hilder, E. F.; Sprong, E.; Ferguson, C. J.; Gilbert, R. G. &lt;em&gt;Macromol. Rapid Commun.&lt;/em&gt; 2006, &lt;strong&gt;27&lt;/strong&gt;, 42-46</td></tr><tr><td>Boric acid&amp;nbsp;</td><td>BDH AnalR, Merck Pty Ltd</td><td>10058</td><td>Corrosive</td></tr><tr><td>Hydrochloric acid - Unilab</td><td>Ajax Finechem</td><td>A1367-2.5L</td><td>laboratory reagent, corrosivie</td></tr><tr><td>Fused silica tubing</td><td>Polymicro (Molex)</td><td>TSP050375</td><td>Flexible fused silica capillary tubing with standard polyimide coating, 50 &amp;micro;m internal diameter, 363 &amp;micro;m outer diameter&amp;nbsp;</td></tr><tr><td>Agilent 7100 CE</td><td>Agilent Technologies</td><td>G7100CE</td><td>Capillary electrophoresis instrument</td></tr><tr><td>Orbital shaker&amp;nbsp;</td><td>IKA</td><td>KS260</td><td></td></tr><tr><td>Electronic balance</td><td>Mettler Toledo</td><td>MS204S</td><td></td></tr><tr><td>Milli-Q Synthesis&amp;nbsp;</td><td>Millipore</td><td>ZMQS5VF01</td><td>Ultrapure water filtration system</td></tr><tr><td>Parafilm&amp;nbsp;</td><td>Labtek</td><td>PM966</td><td>Parrafin wax</td></tr></tbody></table>

capillary electrophoresis to monitor peptide grafting onto chitosan films in real time

自由溶液的毛细管电泳（CE）在溶液中，通过电场的应用分离分析物，通常电荷的化合物。相对于其他的分析分离技术，如色谱法，CE是便宜，耐用和有效不需要样品制备（对于一些复杂的天然基质或聚合物样品）。 CE是快速和可用于实时跟踪（ 例如 ，化学反应动力学）的混合物的演进，作为用于分离的化合物中观察到的信号是直接正比于它们在溶液中的数量。 这里，CE的效率表现出用于监测肽的共价接枝到用于随后的生物医学应用的壳聚糖薄膜。脱乙酰壳多糖的抗微生物和生物相容特性使其用于生物医学应用，如细胞生长底物的有吸引力的材料。共价嫁接肽RGDS（精氨酸 - 甘氨酸 -天冬氨酸 - 丝氨酸）到脱乙酰壳多糖膜的表面旨在改善细胞附着。从历史上看，色谱法和氨基酸分析已经被用于提供接枝肽的量的直接测量。然而，通过CE提供样品制备的快速分离和不存在下使肽接枝过程同样准确但实时监控。 CE是能够分离和量化反应混合物的不同组成部分:（非接枝）肽和化学偶联剂。在这种方式中使用的CE导致下游应用改进的薄膜。 通过固态NMR（核磁共振）光谱的壳聚糖膜进行了表征。这种技术是比较耗时，并且不能在实时应用，但产生的肽的直接测量，从而验证了CE技术。

CE非常适合监测肽（ 例如 ，RGDS）接枝到壳聚糖膜。合适的偶联剂包括EDC·HCl和NHS其中激活肽接枝到壳聚糖（ 图1）。 CE是能够与感兴趣的不同的分子从反应介质中分离。要分配的电泳峰，纯RGDS，EDC-HCl和NHS溶解，注入并单独分开。峰值分配后，将反应介质注入和各种反应物进行鉴定（ 图2）。的EDC·HCl反应成副产物EDH盐酸（3 - （（（乙基氨?...

Watch this Scientific Journal Video about Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time at JoVE.com

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

毛细管电泳监视肽接枝壳聚糖膜实时

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

capillary-electrophoresis-to-monitor-peptide-grafting-onto-chitosan

Research

JoVE Journal

Chemistry

9.0K 次观看.  Western Sydney University. Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

视频: Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

科研

生物工程

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

生物电子界面衔接与Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

荧光肽酶谱法检测蛋白酶活性

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

癌症研究

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

生物学

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

界面聚电解质纤维的生物分子在时间控释复合支架

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

免疫与感染

A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these systems are limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading and drug release capacity), which depend on the freezing conditions and the kind and ratio of polymers. This protocol describes how to prepare hydrogels from chitosan and poly(vinyl alcohol) at 50/50 w/w % of polymer composition and varying the freezing temperature (-4 &#176;C, -20 &#176;C, -80 &#176;C) and freeze-thawing cycles (4, 5, 6 freezing cycles). FT-IR spectra, SEM micrograph and porosimetry data of hydrogels were obtained. Also, the swelling capacity and drug loading and release of diflunisal were assessed. Results from SEM micrographs and porosimetry show that the pore size decreases, while the porosity increases at lower temperatures. The swelling percentage was higher at the minor freezing temperature. The release of diflunisal from the hydrogels has been studied. All the networks maintain the drug release for 30 h and it has been observed that a simple diffusion mechanism regulates the diflunisal release according to Korsmeyer-Peppas and Higuchi models.

A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

一种冷冻-解冻方法，用于制备奇托桑-波里（乙烯基酒精）水凝胶，无交联剂和二分释放研究

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

用于组织工程应用的尺寸控制和无乳化壳聚糖 -染料木酚微凝胶的制备

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

毛细管电泳监视肽接枝壳聚糖膜实时

摘要

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此视频中的章节

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

生物电子界面衔接与Biofabrication

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

荧光肽酶谱法检测蛋白酶活性

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装

界面聚电解质纤维的生物分子在时间控释复合支架

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

一种冷冻-解冻方法，用于制备奇托桑-波里（乙烯基酒精）水凝胶，无交联剂和二分释放研究

用于组织工程应用的尺寸控制和无乳化壳聚糖 -染料木酚微凝胶的制备

毛细管电泳监视肽接枝壳聚糖膜实时

摘要

探索更多视频

此视频中的章节

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

生物电子界面衔接与Biofabrication

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

荧光肽酶谱法检测蛋白酶活性

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

界面聚电解质纤维的生物分子在时间控释复合支架

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

一种冷冻-解冻方法，用于制备奇托桑-波里（乙烯基酒精）水凝胶，无交联剂和二分释放研究

用于组织工程应用的尺寸控制和无乳化壳聚糖 -染料木酚微凝胶的制备

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装