作者感谢加拿大新前沿研究基金 （NFRF）、加拿大自然科学与工程研究委员会 （NSERC） 发现计划以及魁北克自然与技术研究基金会 （FRQNT） 硕士培训奖学金的资金支持。作者还要感谢McGill的Yaoyao Zhao教授团队对某些组件的3D打印提供的技术支持。

用于该方案的蚊子种类是未感染的成年雌性 埃及伊蚊 （A. aegypti），冷冻接收并储存在-20°C度的冰箱中。该物种由NIH/NIAID丝虫病研究试剂资源中心提供，通过BEI Resources，NIAID，NIH分发：未感染的 埃及伊蚊，菌株黑眼利物浦（冷冻），NR-48920。用于研究的试剂和设备列在 材料表中。
1. 从长鼻中解剖唇
<ol><li>使用镊子，将死蚊子放在显微镜下的载玻片上，并确保在长鼻的末端有一个锥形的尖端（图2A）。</li>
	<li>在将蚊子保持在载玻片上的同时，将手术刀刀片轻轻地放在蚊子头部附近的阴唇上（图2B）。</li>
	<li>继续在阴唇的整个上半部分（约 80 μm 的切口）做一个切口，在阴唇厚度上有一个较浅的穿透深度。确保对刀片施加轻微的压力，以便只切开阴唇，而不切开位于下方的束。</li>
	<li>用一把镊子牢牢握住蚊子的头部，然后用另一把精密镊子轻轻捏住锥形尖端和切口位置之间的任何位置的阴唇（图2B）。<ol><li>将夹住唇的镊子拉向锥形尖端的方向（图2C）。继续拉动镊子，直到阴唇脱离并完全从束中取出。</li>
	</ol></li>
	<li>将蚊子放在显微镜下，并确认盂唇的尖端是否存在。这可以通过束上存在锥形尖端来识别（图2D）。</li>
</ol>2. 将盂唇尖端与其他盂唇成员分开
<ol><li>夹住并闭合一组精密镊子的尖端，并将镊子的尖端放在靠近其尖端的盂唇旁边。</li>
	<li>使用镊子尖端在垂直于束长度的方向上对盂唇施加轻轻的力（图3A）。</li>
	<li>继续将盂唇推过载玻片，直到盂唇与其他束成员分离。</li>
	<li>在显微镜下检查样品，以验证盂唇和其他束成员之间已实现正确分离（图3，左）。如果分离不成功，请返回步骤 2.1。</li>
</ol>3. 切开盂唇尖端
<ol><li>当盂唇仍在载玻片上时，将手术刀刀片放在盂唇上，距离盂唇尖端约~200μm（图4A）。轻轻地施加足够的压力，将盂唇的尖端完全切开。虽然理想情况下，盂唇尖端应尽可能短，但 ~200 μm 是当前方法可以处理的最佳方法。</li>
	<li>使用任何数字测量软件测量切割盂唇的长度，以确保其长度不超过300μm（图4B）。在该协议中，使用ImageJ14。</li>
</ol>4.抓住盂唇的尖端
<ol><li>用一把精密镊子在载玻片上找到并隔离盂唇的尖端。丢弃载玻片上剩余的所有部分，但盂唇尖端除外。</li>
	<li>用同样精密的镊子，缓慢而轻微地捏住盂唇，使切割端自由且不受镊子的阻碍。此外，确保盂唇的方向平行于镊子长度的方向，并且盂唇的切割端远离镊子的身体。</li>
	<li>一旦试样被牢固地夹住，就移除将镊子尖端固定在一起的夹紧力。盂唇的尖端会粘在镊子的一个尖端上。</li>
	<li>在显微镜下，检查镊子的尖端，并确保盂唇的尖端位于镊子的一个尖端上（图5）。如果盂唇的尖端不在镊子上，请返回步骤 4.2，如果盂唇的尖端不在镊子上也不在载玻片上，请返回步骤 1。</li>
</ol>5. 在无尖悬臂梁上涂上环氧树脂
<ol><li>将一滴 （~0.05 mL） 环氧树脂滴在新载玻片的边缘，方法是直接从原来的瓶子/容器中滴铸粘合剂。将含环氧树脂的载玻片放在探针台下方并聚焦在其上。</li>
	<li>通过固定底座（即较大的端），将无尖 AFM 悬臂安装到夹具组件上，使悬臂端保持自由并悬挂在空间中。确保悬臂梁的底部朝下。</li>
	<li>将机械手安装到探针台上。</li>
	<li>将机械手的 Z 轴升高到无尖悬臂位于含环氧树脂玻璃载玻片上方几毫米的位置。</li>
	<li>手动移动机械手，使无尖端悬臂在探针台上的摄像机的视野视图中可见。</li>
	<li>使用机械手，沿 X 和 Y 方向移动悬臂梁，直到悬臂的尖端直接位于载玻片边缘的环氧树脂上方。</li>
	<li>再次使用机械手，将无尖悬臂沿 Z 方向缓慢降低到载玻片边缘上方。</li>
	<li>当悬臂降低并接近载玻片时，请继续非常缓慢地降低悬臂，直到它第一次接触环氧树脂。不要再降低悬臂。</li>
	<li>小心地接合机械手，在 X 或 Y 方向上缓慢移动悬臂，并通过沿选定方向连续移动悬臂，将悬臂从环氧树脂池中取出，直到悬臂与载玻片上的环氧树脂完全分离。无尖悬臂的尖端应有一个微型环氧树脂气泡，在探针台下方可见。</li>
	<li>使用机械手向 Z 方向升高悬臂。</li>
</ol>6. 将盂唇的尖端粘合到无尖端悬臂梁上
<ol><li>将机械手绕悬臂的长轴旋转 90 度，然后将机械手侧放在探针台上。在这种配置中，沿悬臂梁长度的厚度在垂直方向上。</li>
	<li>将包含盂唇尖端的精密镊子放在探针台摄像头下方，使盂唇尖端的整个长度在计算机显示器上可见。</li>
	<li>将握住夹具和无尖端悬臂的机械手组件放置在探针台摄像头下方，使无尖端悬臂的整个长度在计算机显示器上可见。</li>
	<li>将探针台显微镜聚焦在盂唇的尖端和无尖端悬臂上。</li>
	<li>通过小心和手动旋转操纵器，将悬臂垂直于盂唇的尖端（图6A）。</li>
	<li>利用机械手的自由度，沿XY方向缓慢移动无尖悬臂，使悬臂上的胶水接触盂唇尖端的切割端（图6B）。</li>
	<li>固化环氧树脂，固化悬臂和蚊子盂唇之间的交叉点。</li>
	<li>环氧树脂固化后，轻轻地在XY方向上接合机械手，并将悬臂从镊子上移开，确认盂唇的尖端现在位于无尖端悬臂梁上（图6C）。</li>
</ol>

带有蚊唇的生物杂交 AFM 探针，用于研究叮咬行为

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作者无需声明任何利益冲突。

该协议的第 1 步旨在清洁不需要的阴唇的生物样本。为了实现这一点，在阴唇上做一个切口，但不在位于阴唇正下方的束上做一个切口（图1）。由于束和唇在其界面处没有连接在一起（即，唇可以沿着束自由滑动，并且仅通过其附着在蚊子头上保持到位），因此进行的切口旨在将唇的一部分与蚊子的头部分开，从而促进外罩的移除。当唇形物从样品中拉出时，技术人员应该...

世界卫生组织 （WHO） 报告说，病媒传播疾病 （VBD） 占所有传染病的 17% 以上，每年导致全球超过 7,00,000 人死亡。例如，作为世界上最致命的动物，蚊子通过吸血节肢动物传播登革热、疟疾和寨卡病毒等多种病原体，每年导致 7 亿例感染1。探索制定有效措施预防VBDs至关重要，包括模仿蚊子的侵入行为以研究其叮咬机制，以及研究潜在障碍以证明其预防侵入的有效性。一个关键的挑战是制定适当的方法来进行这种调查。在文献中已经做出了努力，包括开发类似于蚊子毒刺几何形状的微尺度针头;然而，许多用于制造这些微针的材料（即粘弹性材料2、硅 （Si）、玻璃、陶瓷3 等）与蚊子长鼻的生物材料具有不同的机械性能。工程材料可能很脆，容易断裂和屈曲 3,4，而蚊子的长鼻可以更好地承受断裂或屈曲4.使用蚊子的盂唇而不是工程材料的生物混合探针的好处是，它可以更准确地表示蚊子的穿刺机制。此外，必须将专用工具与微针集成以执行定量研究，例如力5 的准确测量，这对于使用工程微针的定制设置来说不容易实现。
基于原子力显微镜 （AFM） 的方法很有前途的，因为它通过使用带有超细尖端的悬臂来操作，该悬臂被小心地放置在靠近样品表面的位置。尖端既可以扫描表面，也可以被压向/压入表面，由于其与样品的相互作用，会受到不同的吸引力或排斥力6.这些相互作用导致悬臂的偏转，通过激光束从悬臂顶部反射到光电探测器6 上来跟踪偏转。AFM 对系统运动的超强灵敏度使 AFM 能够进行各种测量，包括但不限于皮米精度的形态映射、从皮牛顿到微牛顿的力测量以及全面的多物理场研究7。例如，可以执行 AFM 压痕以精确评估对样品施加力的响应，并通过与适当的分析模型耦合来测量样品的硬度、弹性和其他机械性能8。AFM 的探针通常由硅 （Si） 或氮化硅 （Si3N4）8 制成，长度为 20-300 μm9 ，尖端半径约为几到几十纳米10。纳米级针尖半径是高分辨率成像等应用的理想选择;然而，对于试图在刚度、半径、形状和纵横比方面模仿穿透行为的研究，它不具备生物毒刺的特征。例如，蚊子的微针结构是束，其纵横比为 ~6011 （长度 ~1.5 毫米至 2 毫米;直径 ~30 μm）12。虽然可以假设传统的AFM探针类似于盂唇等生物毒刺，但其独特的材料特性和尺寸并不能反映咬合过程中的真实情况。
为了能够对模仿昆虫或其他动物用毒刺的生物叮咬的穿透行为进行定量研究，这里开发了一种制造生物混合AFM悬臂的工艺，该工艺以生物毒刺为尖端。作为一个案例研究，成功地展示了一种带有蚊子盂唇尖端的AFM悬臂。利用文献中关于蚊子用来刺穿受害者皮肤的典型插入力的现有信息12,13，这种生物混合AFM悬臂有可能在常规AFM下实现蚊子叮咬的近乎真实的模拟。利用微生物毒刺制造生物混合 AFM 悬臂的方案也可应用于开发其他基于 Sharp Stinger 的生物混合 AFM 悬臂，用于对各种咬合机制进行定量研究。
术语 长鼻及其相关组件的示意图如图 1 所示，其定义是 （1） 长鼻：蚊子口中的身体部位，允许蚊子自我进食，其核壳结构由束（核心）和唇（壳）组成，（2） 唇：长鼻的深色和钝的外壳2，（3）束：一组细长的针头包含在唇内，包括两个上颌骨、两个下颌骨、一个下咽部和一个盂唇2、（4）下咽部：负责将唾液分泌到宿主的血液中2、（5）上颌骨：协助摄食机制的锯齿状构件2、（5）下颌骨：类似于上颌骨，它们帮助蚊子摄食机构，并有一个锋利的尖端2，（6）盂唇：穿透受害人皮肤的主要构件，比上颌骨、下颌骨和下咽部大得多。它还具有感觉结构，使其能够在皮肤下找到血管和内部通道2，（7） 操纵器：具有三个自由度和微米级定位精度的组件，允许在 XYZ 方向上移动，（8） 夹具组件：安装在机械手上的定制 2 部分夹具，用于在实验过程中夹紧无尖端 AFM 悬臂。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;5-SA-SE Straight Tapered Ultra Fine-Pointed Tweezers</td>
			<td>Excelta</td>
			<td>N/A</td>
			<td>For manipulating/dissecting the proboscis.</td>
		</tr>
		<tr>
			<td>C-4D Probe station</td>
			<td>Everbeing Int&#8217;l Corp&#160;</td>
			<td>N/A</td>
			<td>Used for AFM assembly.</td>
		</tr>
		<tr>
			<td>Tipless Tapping Mode Cantilever</td>
			<td>NanoAndMore USA</td>
			<td>TL-NCH</td>
			<td>AFM cantilever used for mounting the labrum. 
			Specs are shown here: 
			 
			Shape: Beam 
			Force Constant: 42 N/m (10 - 130 N/m) 
			Resonance Frequency: 330 kHz (204 - 497 kHz) 
			Length: 125 &#181;m (115 - 135 &#181;m) 
			Width: 30 &#181;m (22.5 - 37.5 &#181;m) 
			Thickness: 4 &#181;m ( 3 - 5 &#181;m)</td>
		</tr>
		<tr>
			<td>UV Expoxy</td>
			<td>Let&#39;s resin</td>
			<td>ALR00146</td>
			<td>For stinger attachment.</td>
		</tr>
	</tbody>
</table>

development of a bio-hybrid mosquito stinger-based atomic force microscopy probe

蚊子因其传播疾病的能力而臭名昭著，是对人类最致命的动物，对公共卫生构成持续挑战。目前使用的一级预防策略涉及化学驱虫剂，由于蚊子迅速产生抗药性，化学驱虫剂往往被证明是无效的。因此，发明新的预防方法至关重要。这种发展取决于对蚊子叮咬行为的透彻理解，因此需要一种实验装置，该实验装置能够通过可控的测试参数和定量测量准确复制实际叮咬场景。为了弥合这一差距，设计了一种生物混合原子力显微镜（AFM）探针，其尖端是生物毒刺 - 特别是蚊子盂唇。这种生物混合探针与标准AFM系统兼容，可以对蚊子的渗透行为进行近乎真实的模拟。这种方法标志着在叮咬机制的定量研究方面向前迈出了一步，有可能为对抗病媒传播疾病（VBDs）建立有效的屏障，并为对抗蚊媒传播疾病开辟新的途径。

World Health Organization (WHO) reported that vector-borne diseases (VBDs) account for over 17% of all infectious diseases, which cause more than 7,00,000 deaths per year globally. For example, as the deadliest animal in the world, mosquitoes spread numerous pathogens, such as dengue, malaria, and Zika, through blood-feeding arthropods, resulting in 700 million infections each year1. Explorations toward the development of effective measures to prevent VBDs are of crucial importance, including mimicking the penetration behaviors of mosquitoes to investigate their biting mechanisms and studies of potential barriers to prove their efficacy in preventing penetration. One key challenge is to develop proper approaches to perform such investigations. Efforts have been made in the literature, including the development of micro-scale needles that resemble the geometry of a mosquito stinger; however, many of the materials used to make these microneedles (i.e., viscoelastic materials2, silicon (Si), glass, ceramic3, etc.) have different mechanical properties than the biological material of the mosquito&#39;s proboscis. The engineered materials can be brittle and prone to fracture and buckling3,4, whereas the mosquito&#39;s proboscis can withstand fracture or buckling better4. The benefit of having a bio-hybrid probe using the labrum of a mosquito instead of engineered materials is that it can be a more accurate representation of the piercing mechanism of mosquitoes. Also, specialized tools must be integrated with micro needles to perform quantitative studies, such as the accurate measurement of force5, which is not easily achievable with customized setups using engineered microneedles.
Atomic force microscopy (AFM)-based approach is promising in that it operates by employing a cantilever with an ultra-fine tip that is carefully positioned close to a sample&#39;s surface. The tip can either scan across or be pressed towards/into a surface, experiencing varying attractive or repulsive forces due to its interactions with a sample6. These interactions lead to the cantilever&#39;s deflection, which is tracked by the reflection of a laser beam from the cantilever&#39;s top onto a photodetector6. The exceptional sensitivity to movement of the system enables AFM to conduct a diverse range of measurements, including but not limited to morphological mapping with picometer accuracy, force measurements ranging from piconewtons to micronewtons, and comprehensive multiphysics investigations7. For example, AFM indentations can be performed to precisely assess the response to the applied force of a sample and also to measure the hardness, elasticity, and other mechanical properties of a sample by coupling with appropriate analytical models8. The probe of the AFM is most commonly made of silicon (Si) or silicon nitride (Si3N4)8 with a length of 20-300 &#181;m9 and a tip radius on the order of several to tens of nanometers10. The nanometer scale tip radius can be ideal for applications such as high-resolution imaging; however, it does not possess the characteristics of biological stingers for studies that try to mimic penetration behaviors in terms of stiffness, radius, shape, and aspect ratio. For example, the microneedle structure of a mosquito is the fascicle, which has an aspect ratio of ~6011 (length ~1.5 mm to 2 mm; diameter ~30 &#181;m)12. While a conventional AFM probe can be assumed to resemble a biological stinger like a labrum, its distinct material properties and dimensions will not reflect the real situation during a bite.
To enable quantitative investigations of penetration behaviors mimicking biological bites of insects or other animals with stingers, here, a process to fabricate bio-hybrid AFM cantilevers with a biological stinger as its tip is developed. As a case study, an AFM cantilever with a mosquito labrum&#39;s tip attached was successfully demonstrated. Harnessing existing information from literature on the typical insertion forces that a mosquito uses to pierce through a victim&#39;s skin12,13, this bio-hybrid AFM cantilever can potentially allow near-real mimicry of mosquito bites under a regular AFM. The protocol of leveraging micro biological stingers to fabricate bio-hybrid AFM cantilevers can also be applied to the development of other sharp stinger-based biohybrid AFM cantilevers for quantitative investigations of a variety of biting mechanisms.
Terminologies 
A schematic of a proboscis and its components of interest are shown in Figure 1, and their definitions are (1) Proboscis: a body part from the mouth of a mosquito that allows the mosquito to feed itself, with a core-shell structure composed of the fascicle(core) and the labium(shell), (2) Labium: the dark and blunt outer cover of a proboscis2, (3) Fascicle: a group of slender needles contained inside of the labium, including two maxillae, two mandibles, a hypopharynx, and a labrum2, (4) Hypopharynx: responsible for secreting saliva into the host&#39;s bloodstream2, (5) Maxillae: serrated member assisting in the feeding mechanism2, (5) Mandibles: similar to the maxilla, they help the mosquito in the feeding mechanism and have a sharp tip2, (6) Labrum: the main member for penetrating the skin of a victim, which is much bigger than the maxillae, mandibles and hypopharynx. It also has sensory structures that allow it to find blood vessels and internal channels under the skin2, (7) Manipulator: an assembly with three degrees of freedom and micron-scale accuracy for positioning, allowing movement in XYZ directions, (8) Clamp assembly: a custom-made 2-part clamp mounted to the manipulator used to clamp the tipless AFM cantilever during the experiment.

作者聚焦：用于定量分析蚊子叮咬机制的生物混合 AFM 悬臂的开发

基于生物杂化蚊虫叮咬的原子力显微镜探针的研制

We're looking to develop an AFM cantilever to potentially quantitatively study the biting mechanisms of the mosquito. Our protocol offers a new method for creating accurate, controllable, and reliable AFM probes in order to study animal biting or stinging processes. Our protocol aims at accurately simulating the animal biting process, causing current methodologies to either be uncontrollable, unreproducible in a reliable manner, or in certain cases completely inaccurate.The impact of our work is twofold. The first, we create a protocol that enables other researchers to fabricate their own custom bio-hybrid AFM cantilevers. Secondly, our work paves the way for creating other protocols that merge testing systems with biological material for creating more realistic testing scenarios.

制备的生物杂化AFM探针的扫描电子显微镜（SEM）图像如图 7所示。盂唇的末端成功地粘在无尖端悬臂梁上。由于蚊子毒刺的自然曲率和所提出协议的手动操作，因此获得具有完全垂直于悬臂的毒刺尖端的悬臂是极其困难的。毒刺与垂直于悬臂的假想中心线之间的偏心角通常为 ~10 度。虽然将探针安装到 AFM 悬臂15 上似乎是一个常见问题，但在执行力/应力分析...

Watch this Scientific Journal Video about Author Spotlight: Development of Bio-Hybrid AFM Cantilevers for Quantitative Analysis of Mosquito Biting Mechanisms at JoVE.com

对昆虫叮咬行为进行定量和受控调查对于制定有效的策略来对抗病媒传播疾病至关重要。在此背景下，介绍了一种制备生物杂化原子力显微镜（AFM）探针的方法。

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The ...

我们正在开发一种AFM悬臂，以潜在地定量研究蚊子的叮咬机制。我们的协议提供了一种新方法，用于创建准确、可控和可靠的 AFM 探针，以研究动物咬伤或刺痛过程。我们的协议旨在准确模拟动物咬伤过程，导致当前的方法要么不可控，无法以可靠的方式重现，要么在某些情况下完全不准确。我们工作的影响是双重的。首先，我们创建了一个协议，使其他研究人员能够制造他们自己的定制生物混合AFM悬臂。其次，我们的工作为创建其他协议铺平了道路，这些协议将测试系统与生物材料相结合，以创建更真实的测试场景。

development-bio-hybrid-mosquito-stinger-based-atomic-force-microscopy

Research

JoVE Journal

Engineering

Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe

423 次观看.  McGill University. 我们正在开发一种AFM悬臂，以潜在地定量研究蚊子的叮咬机制。我们的协议提供了一种新方法，用于创建准确、可控和可靠的 AFM 探针，以研究动物咬伤或刺痛过程。我们的协议旨在准确模拟动物咬伤过程，导致当前的方法要么不可控，无法以可靠的方式重现，要么在某些情况下完全不准确。我们工作的影响是双重的。首先，我们创建了一个协议，使其他研究人员能够?...