mounting and bonding the labrum&#39;s tip to the atomic force microscopy (afm) tipless cantilever beam

Ponta de prova Bio-híbrida do AFM com o Mosquito Labrum para estudar comportamentos de mordida

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.

Autor em destaque: Desenvolvimento de balanços AFM bio-híbridos para análise quantitativa de mecanismos de picada de mosquito

Desenvolvimento de uma sonda de microscopia de força atômica baseada em ferrão de mosquito bio-híbrido

Montagem e colagem da ponta do labrum ao feixe cantilever sem ponta da microscopia de força atômica (AFM)

Comece colocando uma pequena quantidade de epóxi na borda de uma nova lâmina de vidro, depois posicione a lâmina de vidro sob a estação de sonda e concentre-se nela. Monte um cantilever sem ponta do AFM ao conjunto da braçadeira. Prenda a base do balanço, deixando sua extremidade livre e suspenda-o no espaço.Em seguida, conecte o manipulador à estação de sonda. Ajuste o eixo Z do manipulador para elevar o cantilever alguns milímetros acima da corrediça de vidro que contém o epóxi. Em seguida, usando o manipulador, ajuste a posição do cantilever AFM ao longo da direção x e Y sobre o epóxi na corrediça de vidro.Alinhe cuidadosamente a ponta do cantilever diretamente acima do epóxi. Depois disso, abaixe lentamente o cantilever sem ponta na direção Z em direção ao epóxi na corrediça de vidro até que ele toque o epóxi. Depois, mova suavemente o cantilever nas direções X e Y para longe do epóxi, certificando-se de que esteja separado da corrediça.Levante o cantilever na direção Z usando o manipulador. Gire o manipulador 90 graus em torno do eixo longo do balanço e coloque-o de lado na estação da sonda. Em seguida, coloque as pinças de precisão com a ponta do labrum sob a câmera da estação de sonda para que todo o comprimento dela fique visível.Em seguida, posicione o conjunto do manipulador com a braçadeira e o cantilever sob a câmera de forma que todo o comprimento do cantilever fique visível. Gire o manipulador para ajustar o cantilever perpendicularmente à ponta do labrum. Usando os graus de liberdade do manipulador, mova lentamente o cantilever sem ponta nas direções X, Y, de modo que a cola no cantilever entre em contato com a extremidade cortada da ponta do labrum.Em seguida, cure o epóxi para solidificar a conexão entre o cantilever e o labrum. Afaste suavemente o cantilever das pinças, garantindo que a ponta do labrum esteja presa à viga do cantilever.

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Research

JoVE Journal

Engineering

Mounting and Bonding the Labrum&#39;s Tip to the Atomic Force Microscopy (AFM) Tipless Cantilever Beam

86 visualizações. Comece colocando uma pequena quantidade de epóxi na borda de uma nova lâmina de vidro, depois posicione a lâmina de vidro sob a estação de sonda e concentre-se nela. Monte um cantilever sem ponta do AFM ao conjunto da braçadeira. Prenda a base do balanço, deixando sua extremidade livre e suspenda-o no espaço.Em seguida, conecte o manipulador à estação de sonda. Ajuste o eixo Z do manipulador para elevar o cantilever alguns milímetros acima da corrediça de vidro que contém...

Vídeo: Montagem e colagem da ponta do labrum ao feixe cantilever sem ponta da microscopia de força atômica (AFM)

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

Mosquitoes, notorious as the deadliest animals to humans due to their capacity to transmit diseases, pose a persistent challenge to public health. The primary prevention strategy currently in use involves chemical repellents, which often prove ineffective as mosquitoes rapidly develop resistance. Consequently, the invention of new preventive methods is crucial. Such development hinges on a thorough understanding of mosquito biting behaviors, necessitating an experimental setup that accurately replicates actual biting scenarios with controllable testing parameters and quantitative measurements. To bridge this gap, a bio-hybrid atomic force microscopy (AFM) probe was engineered, featuring a biological stinger - specifically, a mosquito labrum - as its tip. This bio-hybrid probe, compatible with standard AFM systems, enables a near-authentic simulation of mosquito penetration behaviors. This method marks a step forward in the quantitative study of biting mechanisms, potentially leading to the creation of effective barriers against vector-borne diseases (VBDs) and opening new avenues in the fight against mosquito-transmitted illnesses.

Quantitative and controlled investigations into insect biting behaviors are crucial for devising effective strategies to combat vector-borne diseases. In this context, a method for fabricating a bio-hybrid atomic force microscopy (AFM) probe is introduced.

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