我们承认研究增长倡议 (101X340)、国家科学基金会、主要研究仪器项目 (批准号:) 提供的财政支持。PHY-1626450), 大密尔沃基基金会 (肖奖) 和威斯康星大学系统 (应用研究补助金)。

1. 试剂溶液的制备
<ol><li>用三个缓冲器 [20 毫米三 (羟甲基) 氨基甲烷和150毫米氯化钠, pH 7.4], 将感兴趣的蛋白质溶解/稀释至所需浓度, 制备起动蛋白溶液。 注: 交联导致水凝胶的最小蛋白质浓度取决于所使用的蛋白质, 通常 > 1 毫米。</li>
	<li>通过溶解 ap 和 [汝 (bpy)3]2 +粉末在三个缓冲器中, 准备过硫酸铵 (APS) (1 米) 和三 (吡啶) 钌 (II) 氯化物 ([汝 (bpy)3]2 +) (6.67 毫米) 溶液。</li>
</ol>2. 蛋白质基水凝胶的合成
<ol><li>用压柱塞固定1毫升注射器上的23克针。</li>
	<li>用刀片切割10厘米聚四氟乙烯 (聚四氟乙烯) 管 (内径为0.022 英寸, 外径为0.044 英寸)。将针头和注射器连接到聚四氟乙烯管的一端。</li>
	<li>将管的第二端插入硅烷溶液中, 并通过缩回注射器柱塞来填充管子。把管子放在30分钟左右。</li>
	<li>除去硅烷溶液, 用压缩空气干燥管。 注意: 确保所有的硅烷溶液都是干的, 并且没有残留在管子里。</li>
	<li>将蛋白质溶液与 APS 和 [汝 (bpy) 3]2 +在1.5 毫升管中使用恒定容积比 [例如, 15:1: 1 或 15:0. 5:0. 5 (v: v: v)]。</li>
	<li>涡感光溶液, 直到它完全混合。</li>
	<li>离心混合以最大速度 (例如, 1.4万 x g), 以消除任何气泡从解决方案。</li>
	<li>将经处理的聚四氟乙烯管的开口端插入感光混合物中, 并通过缩回注射器柱塞将溶液拉入管内。</li>
	<li>把装满的管子从 100 W 水银灯放到10厘米, 以防加热, 并在室温下保持30分钟 (图 1B)。 注: 在某些情况下, 曝光时间可低至三十年代. 在这里使用较短的凝胶时间用于荧光凝胶, 以限制漂白。</li>
	<li>把管子从针头上取下, 用剃刀刀片把水凝胶末端的管子边缘切开。</li>
	<li>使用钝24克针将水凝胶挤出到三溶液中 (图 1C)。 注: 钝针用于避免任何凹槽或损坏的水凝胶样品。</li>
	<li>目视检查凝胶是否有任何可能在挤出过程中形成的缺陷, 或由于气泡而丢弃有缺陷的凝胶。</li>
</ol>3. 基于蛋白质的水凝胶附着体和力钳式流变仪的建立
<ol><li>启动仪表控制程序。打开声音线圈马达。将线圈位置设置为指向范围末尾的值 (例如, 7.5 mm)。 注: 建议将语音线圈位置朝向最大运动范围的末尾, 最大限度地扩大水凝胶的可能延伸。</li>
	<li>在z方向偏移钩子, 并将它们对准x方向的折弯 (即拉动坐标; 请参见图 2B)。记录x方向的千分尺螺钉的值。</li>
	<li>将2条无菌缝线切成等长的股 (2-3 厘米; 见图 3A和B)。</li>
	<li>绑一个松散的双上手结进入每股, 并把2环路连接到力传感器 (图 3C和3D) 挂钩。</li>
	<li>用三缓冲器填充实验室, 用医用镊子将水凝胶样品输送到填充腔内。</li>
	<li>将声音线圈和强制传感器挂钩靠近解决方案表面, 并使用x/y/z定位机械手将钩子对准所有方向。</li>
	<li>使用医用镊子, 将蛋白质水凝胶样品的两侧挂在连接到语音线圈和力传感器的挂钩上 (图 3C)。</li>
	<li>收紧1缝合环水凝胶样品的声音线圈钩通过持有两端的缝合环与医疗镊子, 并拉他们同时 (图 3D)。</li>
	<li>对连接到力传感器的循环重复步骤 3.8 (图 3D)。 注意: 避免对缝线进行极端收紧, 以防止水凝胶样品的结构损伤和横向切割。</li>
	<li>拧紧每个钩的弯曲上的缝合环, 以防止任何滑动;使用这些折弯作为参考点, 以查找步骤3.2 中挂钩之间的零分隔。用医用剪刀剪掉缝合线的多余长度 (图 3D)。</li>
	<li>将所附的水凝胶沿z轴的z形机械手移动到实验室, 将水凝胶浸入实验溶液中。</li>
	<li>使用机械手将水凝胶样品在yz上对齐, 这样凝胶就不会受到任何压力。</li>
	<li>零力传感器, 并使用x微米级分隔两个挂钩, 直到凝胶开始体验力。一旦发生这种情况, 在x方向稍微向后旋转千分尺螺钉。</li>
	<li>记录两个机械手的位置, 为语音线圈马达和传感器, 并利用这些值之间的差异和测量的步骤 3.2, 以计算在实验开始的挂钩钩之间的精确分离。</li>
	<li>将松弛曲线的范围设置为 ~ 1.5-2 毫米, 并测量凝胶松弛度 (图 4A)。 注意: 对于每个时差测量, 尝试保持松弛系统的开始, 靠近初始的声音线圈位置, 允许最佳的数据点数以适应2种制度 (图 4A)。凝胶长度可以用微米的分辨率来确定, 方法是使用钩子与松弛曲线中2个体制之间的交集 (请参见步骤 5.1)。由于实验条件的变化, 力传感器可能会随时间漂移, 所以在不受力的情况下, 松弛曲线的一部分会在这个可能的漂移上报告。在将设置点命令发送到 PID 回路时, 控制仪表的程序自动补偿此差异 (图 4A插入)。</li>
</ol>4. 基于蛋白质的水凝胶特性的控制力-坡道和恒定力测量
<ol><li>
力坡道实验
	<ol>
<li>通过增加所需加载速率的力 (如0.01 锰/秒) 来执行力-坡道循环, 将启动和最终的力量以及协议的持续时间输入为翻转的 "V"。然后, 保持凝胶在0锰 (或低力) > 200s, 使蛋白质领域再折叠餐巾和凝胶弹性恢复。</li>
		<li>保存跟踪。</li>
	</ol>
</li>
	<li>
恒力实验
	<ol>
<li>执行恒定力协议, 通过应用低力 (例如, 0.1 锰) 在三十年代, 然后增加的力量, 以恒定的力量 (例如, 1 锰), 在规定的时间 (例如, 120s), 然后淬火的力量回到相同的低价值 (如0.1 锰) > 300s 允许蛋白质领域再折叠餐巾和凝胶弹性恢复。</li>
		<li>在第一个脉冲之后, 调整 PID 设置以最大化反馈回路的响应时间 (见图 2D)。 注: 对于僵硬的凝胶和小的力量变化, 回路的响应时间受力传感器的电子和线圈的响应时间的限制, 并且可以低至5毫秒7。对于软凝胶和力的大变化, 响应时间取决于水凝胶的弹性 (图 2D)。</li>
		<li>保存跟踪。</li>
	</ol>
</li>
</ol>5. 数据分析
<ol><li>利用在挂钩和计算线圈位置之间的测量的分离, 当凝胶开始体验力 (Δx 在图 4A插入), 计算凝胶长度L使用等式: l =0 + ∆x 这里, L0 是钩子之间的分离, 测量从测微螺钉的位置在实验之前 (步骤 3.14)。 注: 对于不导致完全交联的低蛋白浓度凝胶, 测量的长度将从痕量变化。而且, 在长时间内, 水凝胶内的蛋白质可能会经历衰老效应25, 从而导致凝胶的整体延长。</li>
	<li>将测量到的扩展到凝胶长度正常化以获得应变。</li>
	<li>使用用于聚合的管内径将所测力正常化为横向表面积。</li>
</ol>

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本文描述了一种力钳 rheometry 技术来研究低体积蛋白质基水凝胶的生物力学响应。此外, 还提供了一种合成均匀柱状低体积蛋白质水凝胶样品的协议。文中还介绍了如何将不同类型的蛋白质基水凝胶与各种弹性材料结合在一起, 而不会对蛋白质基水凝胶样品或钩上的凝胶的滑移造成任何机械变形或损伤。模拟 PID 系统, 连同线性的声音线圈和力传感器, 使应用的控制力的协议, 如力坡道和恒定的力量?...

除了具有独特的物理特性外, 基于蛋白质的水凝胶具有革命性的力量光谱学的希望, 能够在一个 ' 拉力 ' 中测量数亿的分子, 从而使在拥挤的环境中对蛋白质进行研究,与皮肤和其他组织中遇到的类似。蛋白质领域仍然折叠在水凝胶内, 允许研究其生物力学反应的力量, 结合合作伙伴, 和化学条件。此外, 水凝胶内蛋白质领域的生物力学反应类似于单分子力谱技术所看到的反应。例如, 化学变性剂和氧化剂降低了折叠状态的稳定性, 无论是在单蛋白领域1,2,3和在宏观水平4,5,6,7. 同样, 渗透调节物质增加了单蛋白8、9的稳定性, 导致水凝胶的粘弹性反应减少, 同一力条件为7、10。
通过使用物理相互作用11、12或共价键交联4、13, 已经实施了几种合成蛋白质基水凝胶的方法。共价反应允许固定的交联位置和这些水凝胶可以恢复初始状态后的机械或化学扰动的去除。一种成功的共价交联方法依赖于用过硫酸铵 (APS) 作为氧化剂和钌 (II) 盐作为引发剂 (图 1)14, 在暴露的酪氨酸氨基酸之间形成共价键碳碳键。当接触到白光时, 浓缩蛋白质的溶液可以转化为水凝胶。通过控制反应开始时, 蛋白质 APS 组合可以注入任何铸件形式, 如聚四氟乙烯 (PFTE) 管 (图 1B和1C), 允许使用一个非常小的解决方案体积15。此外, 利用白光触发交联反应导致荧光蛋白的有限漂白, 并允许用荧光标记来配制复合水凝胶 (图 1)。其他基于蛋白质的水凝胶形成方法采用基于 SpyTag-SpyCatcher 共价相互作用的交联16、胺交联通过戊二醛13或生物素-链亲和素相互作用17。
动态力学分析 (DMA) 是目前广泛用于研究聚合物基水凝胶13,18的技术。虽然 DMA 可以将恒定的力量协议应用于生物材料, 它需要年轻的模数超过10帕, 和大样本量超过200µL19。由于这些局限性, 蛋白质水凝胶一般太软, 不能用这种方法来研究。由于工程 polyproteins 比聚合物更难合成, 因为它们需要一个活的系统来生产, 这样的高容量是低效的, 充其量是4,15。此外, 大多数生物组织的软比10帕。为生物样品开发了几种方法, 特别是研究肌肉弹性20、21。这些技术也可以在反馈作用下运行恒定力, 但为小直径 (在微米范围内) 暴露在非常短的时间 (通常少于1秒) 的样品优化。
采用改进的 rheometry 技术, 成功地研究了蛋白质基水凝胶。例如, 在环形形状中浇铸水凝胶, 可以利用伸展 rheometry 来测量有经验的力的变化, 作为扩展4,22的函数。其他研究蛋白质基水凝胶流变特性的方法是采用受控剪切应力 rheometry。这些技术也可以达到低样本量和容忍软材料。然而, 这些方法缺乏模仿导致蛋白质在体内展开的拉动力的能力, 并且根据需要各种假设和修正23的复杂理论计算杨氏模量。
我们最近报告了一种新的方法, 利用了小体积的蛋白质, 聚合管内与直径 < 1 毫米。我们的第一个实施这一技术是在长度钳模式, 其中凝胶是延长后, 预期的协议15。在这种方法中, 当域展开时, 蛋白质在扩展和力方面都经历了不断的变化, 使得数据解释变得繁琐。最近, 我们报告了一种新的力钳 rheometry 技术, 其中反馈回路可以揭露低容量的蛋白质水凝胶到预定义的力量协议7 (图 2)。模拟 PID 系统将力传感器测量的力与计算机发送的设定点进行比较, 并通过移动语音线圈来调整凝胶的扩展, 以最小化两个输入之间的差异。这种 "夹紧" 的力量现在允许新的实验, 以测量蛋白质水凝胶的生物力学。
在力-坡道模式下, 栓蛋白水凝胶的受力随时间的推移不断增加和减少。根据蛋白质和水凝胶配方的变化, 采用非线性的方法, 对任何粘弹性变形进行补偿。力坡道的主要优点是, 由于蛋白质领域的展开和复用, 它允许量化标准参数, 如杨氏模量和能量耗散。
在恒力模式下, 应用力的变化是一种类似于步进的方式。在这种模式下, 当力增加或减小时, 胶凝体扩展和收缩弹性, 其次是时变变形。这种粘弹性变形发生在凝胶经历恒定力的情况下, 与域展开/复用直接相关。在简化的方法中, 这种扩展可以被看作是相当于几个亿个单一分子的痕迹平均在一起, 并测量所有一次。恒力协议可以用来研究蛋白质水凝胶的蠕变和松弛, 作为一种力和时间的函数。作为一种力的功能, 对 BSA 为基础的蛋白质水凝胶, 我们最近表明, 有一个线性依赖性之间的弹性和粘弹性延长和反冲与应用应变7。
在这里, 我们详细介绍了一种使用由蛋白质 l (8 域24, 描述为 L8) 和蛋白质 l-eGFP 构造 (l-eGFP) 组成的复合凝胶的力钳流变仪的操作, 这使得整个水凝胶荧光和易于证明。

<table><tbody><tr><td>SI-KG4A force transducer</td><td>World Precision Instruments (WPI)</td><td>SI-KG4A</td><td></td></tr><tr><td>Linear Voice Coil Motor</td><td>Equipement Solutions</td><td>LFA2010</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Rocky Mountain Biologicals (RMBIO)</td><td>BSA-AAF-1XG / 100 G</td><td></td></tr><tr><td>Trizma</td><td>Sigma-Aldrich</td><td>T1503-1KG</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653-1KG</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Sigma-Aldrich</td><td>248614-100G</td><td></td></tr><tr><td>Tris(bipyridine)ruthenium(II) chloride</td><td>Sigma-Aldrich</td><td>544981-1G</td><td></td></tr><tr><td>EXPRESS MEDICAL SUPPLIES&amp;nbsp;6-0 NYLON SUTURE 12/PK</td><td>Fisher Scientific</td><td>NC0395626</td><td></td></tr><tr><td>1mL Syringe Only, Luer-Lok Tip</td><td>BD</td><td>309628</td><td></td></tr><tr><td>Silane, Sigmacote</td><td>Sigma-Aldrich</td><td>SL2-25ML</td><td></td></tr><tr><td>Microbore PTFE Tubing, 0.022&quot;ID x 0.042&quot;OD, 100 ft/roll</td><td>Cole-Parmer</td><td>EW-06417-21</td><td></td></tr><tr><td>Hypodermic Needle, 23 Gauge</td><td>Healthcare Supply Pros</td><td>305194</td><td></td></tr><tr><td>Jensen Global JG24-1.5X Red IT Dispensing Tips - 24 gauge</td><td>KIMCO</td><td>JG24-1.5X</td><td></td></tr><tr><td>USH-103D USHIO 100W Short Arc Mercury Lamp</td><td>ALB</td><td>USH-103D USHIO</td><td></td></tr><tr><td>Medical Tweezers</td><td></td><td></td><td></td></tr><tr><td>Medical scissors</td><td></td><td></td><td></td></tr><tr><td>Olympus</td><td></td><td></td><td></td></tr><tr><td>The computer code and CAD design of the custom parts can be made available on request to the corresponding author.</td><td></td><td></td><td></td></tr></tbody></table>

force-clamp rheometry for characterizing protein-based hydrogels

在这里, 我们描述了一种力钳 rheometry 方法来表征蛋白质基水凝胶的生物力学特性。该方法采用模拟比例积分-导数 (PID) 系统, 在圆柱蛋白基水凝胶样品上应用受控力协议, 并将其与线性音圈电机和力传感器拴在一起。在操作过程中, PID 系统调整水凝胶样品的延伸以遵循预定义的力协议, 通过最小化测量和设定点力之间的差异。这种独特的蛋白质基水凝胶方法能够使极低体积的水凝胶样品 (< 5 µL) 与不同的蛋白质浓度结合在一起。在力-匝道协议中, 当应用应力随时间线性增加和减小时, 系统能够研究与蛋白质 (un) 折叠相关的弹性和滞后行为, 以及标准弹性的测量和粘弹性参数。在恒定力的情况下, 力脉冲具有一步状形状, 由于力的变化, 弹性响应与粘弹性响应是分离的, 这是由蛋白质领域展开和复用而来的。由于其体积小, 在应用各种机械扰动时具有通用性, 因此, 采用大体积法对力钳 rheometry 进行了优化, 以研究蛋白质在受力下的力学响应。

图 1A显示了用于合成 l-感光8水凝胶的反应方案。图 1B显示了 photoactivation 前后聚四氟乙烯管中的水凝胶混合物。图 1C给出了在三溶液中挤压的 l-eGFP8水凝胶。水凝胶样品没有凹槽等结构缺陷。应丢弃明显可见损伤的水凝胶。
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Watch this Scientific Journal Video about Force-Clamp Rheometry for Characterizing Protein-based Hydrogels at JoVE.com

Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

基于蛋白质的水凝胶的力钳 Rheometry

采用一种新的力钳 rheometry 技术, 研究了声线圈电机与力传感器之间的低体积蛋白质基水凝胶样品的力学性能。模拟比例积分-导数 (PID) 系统允许 "夹紧" 的力量体验到所需的协议。

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog ...

force-clamp-rheometry-for-characterizing-protein-based-hydrogels

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

6.9K 次观看.  University of Wisconsin-Milwaukee. 采用一种新的力钳 rheometry 技术, 研究了声线圈电机与力传感器之间的低体积蛋白质基水凝胶样品的力学性能。模拟比例积分-导数 (PID) 系统允许 "夹紧" 的力量体验到所需的协议。

视频: Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

投诉矩阵量化细胞收缩的准备工作

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

科研

生物工程

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

生物化学

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is &#181;2rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. &#181;2rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. &#181;2rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.

We demonstrate the fabrication and use of a microfluidic device that enables multiple particle tracking microrheology measurements to study the rheological effects of repeated phase transitions on soft matter.

微流体和 Microrheology 相结合测定软物质在重复相变过程中的流变特性

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement ...

工程学

Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of the data and determines which properties of the film can be quantified. The quartz crystal microbalance offers a uniquely sensitive platform for measuring fine changes in mass and/or mechanical properties of an applied film by observing the changes in mechanical resonance of a quartz crystal oscillating at high frequency. The advantages of this approach include its experimental versatility, ability to study changes in properties over a wide range of experimental time lengths, and the use of small sample sizes. We demonstrate that, based on the thickness and shear modulus of the layer deposited on the sensor, we can acquire different information from the material. Here, this concept is specifically exploited to display experimental parameters resulting in mass and viscoelastic calculations of adsorbed collagen on gold and polyelectrolyte complexes during swelling as a function of salt concentration.

The quartz crystal microbalance can provide accurate mass and viscoelastic properties for films in the micron or submicron range, which is relevant for investigations in biomedical and environmental sensing, coatings, and polymer science. The sample thickness influences which information can be obtained from the material in contact with the sensor.

石英晶体微平衡测量中的蛋白质吸附和聚合物力学的样品制备

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of ...

化学

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi River basin due to its highly efficient filter feeding mechanism. The characteristic organs called gill rakers (GRs), found in many such filter feeders, facilitate the efficient filtration of food particles such as phytoplankton that are of a few microns in size.
The motivation to investigate the rheology of the GR mucus stems from our desire to understand its role in aiding the filter feeding process in the silver carp. The mucus-rich fluid, in a &#8216;thick and sticky&#8217; state may facilitate the adhesion of food particulates. The permeation and transport through the GR membrane are&#160;facilitated by the action of external shear forces that induce varying shear strain rates. Therefore, mucus rheology can provide a vital clue to the tremendous outcompeting nature of the silver carp within the pool of filter feeding fish. Based on this it was posited that GR mucus may provide an adhesive function to food particles and act as a transport vehicle to assist in the filter feeding process.
The main objective of the protocol is to determine the yield stress of the mucus, attributed to the minimum shear stress required to initiate flow at which irreversible plastic deformation is first observed across a structured viscoelastic material. Accordingly, rheological properties of the GR mucus, i.e., viscosity, storage, and loss moduli, were investigated for its non-Newtonian, shear-thinning nature using a rotational rheometer. &#160;
A protocol presented here is employed to analyze the rheological properties of mucus extracted from the gill rakers of a silver carp, fished at Hart Creek location of the Missouri River. The protocol aims to develop an effective strategy for rheological testing and material characterization of mucus assumed to be a structured viscoelastic material.

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

This protocol presents a method to perform rheology characterization of mucus that resides on gill rakers (GRs) of the silver carp. Viscoelastic characteristics of GR-mucus, obtained by measuring viscosity, storage and loss moduli, are evaluated for the apparent yield stress to understand the filter feeding mechanism in GRs.

银鲤鱼中吉尔 · 拉克 · 穆库斯的宏观流变特征， 低语种米希里斯 · 莫利特里克斯

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

神经科学

<meta charset="utf8"></head><body> 肽水凝胶:ECF-5 的环境响应性凝胶化方法

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

<meta charset="utf8"></head><body>作者聚焦:改进自组装纤维和肽水凝胶的生产以获得卓越的生物相容性

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

机械稳定的自组装肽水凝胶的制备

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...

基于蛋白质的水凝胶的力钳 Rheometry

摘要

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此视频中的章节

投诉矩阵量化细胞收缩的准备工作

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

微流体和 Microrheology 相结合测定软物质在重复相变过程中的流变特性

石英晶体微平衡测量中的蛋白质吸附和聚合物力学的样品制备

银鲤鱼中吉尔 · 拉克 · 穆库斯的宏观流变特征，低语种米希里斯 · 莫利特里克斯

RGD功能化水凝胶作为治疗应用的工具合成

DNA 纳米管作为研究半聚合物的万能工具

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

机械稳定的自组装肽水凝胶的制备

机械稳定的自组装肽水凝胶的制备

基于蛋白质的水凝胶的力钳 Rheometry

摘要

探索更多视频

此视频中的章节

投诉矩阵量化细胞收缩的准备工作

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

微流体和 Microrheology 相结合测定软物质在重复相变过程中的流变特性

石英晶体微平衡测量中的蛋白质吸附和聚合物力学的样品制备

银鲤鱼中吉尔 · 拉克 · 穆库斯的宏观流变特征， 低语种米希里斯 · 莫利特里克斯

RGD功能化水凝胶作为治疗应用的工具合成

DNA 纳米管作为研究半聚合物的万能工具

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

机械稳定的自组装肽水凝胶的制备

机械稳定的自组装肽水凝胶的制备

银鲤鱼中吉尔 · 拉克 · 穆库斯的宏观流变特征，低语种米希里斯 · 莫利特里克斯