Development of a Bio-Hybrid Mosquito Stinger-Based Atomic Force Microscopy Probe
April 26th, 2024
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.
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 singing 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.
To begin, place a dead mosquito on the glass slide. Then gently place a scalpel blade over the labium, near the mosquito's head. Make an incision across the entire upper half of the labium with a shallow penetration depth.
Next, with a pair of tweezers, firmly hold the mosquito's head. And with another pair of precision tweezers, lightly pinch the labium between the tapered tip and the incision. Then pull the tweezers holding the labium towards the tapered tip.
Place the mosquito under the microscope to verify if the labrum's tip is present. Now clamp and close the tips of a set of precision tweezers and place the tip of the tweezers right beside the labrum's tip. Use the tweezer tip to apply a gentle force on the labrum perpendicular to the length of the fascicle.
Continue pushing the labrum across the glass slide until separation from the other fascicle members is achieved. Finally, inspect the sample under a microscope to verify the separation. To begin, take the glass slide containing the mosquito labrum.
Position a scalpel blade approximately 200 micrometers from the labrum's tip. Then apply gentle pressure to cut the tip of the labrum all the way through. With precision tweezers, locate and isolate the labrum's tip on the glass slide.
Then using the same tweezers, gently pinch the labrum. Once the specimen is firmly pinched, gradually release the clamping force. Under a microscope, examine the tweezers tips to confirm the presence of the labrum's tip on one of them.
Begin by placing a small amount of epoxy on the edge of a new glass slide. Then position the glass slide under the probe station and focus on it. Mount a tipless AFM cantilever to the clamp assembly.
Secure the base of the cantilever, leaving its end free and suspended in space. Next, attach the manipulator to the probe station. Adjust the z-axis of the manipulator to elevate the cantilever a few millimeters above the glass slide containing the epoxy.
Then using the manipulator, adjust the position of the AFM cantilever along the x and y direction over the epoxy on the glass slide. Carefully align the cantilever's tip directly above the epoxy. After that, slowly lower the tipless cantilever in the z direction toward the epoxy on the glass slide until it touches the epoxy.
Afterward, gently move the cantilever in the x and y direction away from the epoxy, ensuring it is separated from the slide. Raise the cantilever in the z direction using the manipulator. Rotate the manipulator 90 degrees around the cantilever's long axis, and place it sideways on the probe station.
Then place the precision tweezers with the labrum's tip under the probe station camera so that the entire length of it is visible. Afterward, position the manipulator assembly with the clamp and cantilever under the camera so that the entire length of the cantilever is visible. Rotate the manipulator to adjust the cantilever perpendicular to the labrum's tip.
Using the degrees of freedom of the manipulator, slowly move the tip less cantilever in the x-y directions, such that the glue on the cantilever contacts the cut end of the labrum's tip. Then cure the epoxy to solidify the connection between the cantilever and the labrum. Gently move the cantilever away from the tweezers, ensuring the labrum's tip is attached to the cantilever beam.
Overview
This study introduces a novel method for fabricating bio-hybrid atomic force microscopy (AFM) probes to quantitatively investigate insect biting behaviors, particularly in mosquitoes. The protocol aims to enhance the accuracy and reliability of studying animal biting processes, addressing limitations of current methodologies.
Key Study Components
Area of Science
- Neuroscience
- Biophysics
- Vector-borne disease research
Background
- Insect biting behaviors are critical for understanding vector-borne diseases.
- Current methodologies for studying these behaviors are often unreliable.
- AFM technology can provide insights into the mechanics of biting.
- There is a need for improved fabrication methods for AFM probes.
Purpose of Study
- To develop a protocol for creating custom bio-hybrid AFM cantilevers.
- To enable accurate simulation of animal biting processes.
- To facilitate reproducible and controllable experimental setups.
Methods Used
- Development of a new AFM cantilever fabrication protocol.
- Testing the reliability and accuracy of the probes.
- Simulating animal biting processes in controlled environments.
- Providing guidelines for other researchers to replicate the method.
Main Results
- The new protocol allows for the creation of accurate AFM probes.
- Probes demonstrated improved control and reproducibility in experiments.
- Facilitated realistic testing scenarios for studying biting mechanisms.
- Enabled further development of protocols merging biological materials with testing systems.
Conclusions
- The study presents a significant advancement in AFM technology for biological research.
- Custom bio-hybrid AFM cantilevers can enhance the study of insect behaviors.
- This work lays the groundwork for future research in vector-borne disease mechanisms.
Frequently Asked Questions
Executive Industry Relevance
Quantitative understanding of mosquito biting mechanics is critical for developing next-generation barriers and interventions against vector-borne diseases. The bio-hybrid AFM probe enables controlled, reproducible simulation of mosquito penetration, supporting predictive confidence in early-stage discovery and target validation. This capability strengthens the translational bridge from mechanistic insight to applied vector control solutions in biopharma R&D portfolios.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables precise interrogation of mosquito biting mechanisms for target validation.
- Supports biological de-risking by replicating authentic penetration scenarios.
- Facilitates predictive confidence in evaluating candidate repellents or barriers.
Screening & Assay Development
- Prepares validated biological systems for quantitative assessment of intervention efficacy.
- Standardizes assay conditions for reproducibility and cross-study comparability.
- Enables reliable measurement of penetration forces and barrier performance.
Translational & Preclinical Research
- Aligns experimental outputs with disease-relevant vector behaviors.
- Supports continuity from mechanistic discovery to preclinical validation of anti-vector strategies.
- Provides quantitative endpoints for risk-adjusted advancement decisions.
Pipeline & Workflow Integration
This bio-hybrid AFM probe method integrates at the interface of early discovery and preclinical evaluation, enabling mechanistic de-risking and quantitative screening of candidate interventions.
- Discovery Biology: Supports hypothesis testing on mosquito penetration and barrier disruption mechanisms.
- Screening: Delivers reproducible, quantitative outputs for compound or material evaluation.
- Analytics: Provides force measurements and penetration profiles for comparative analysis.
- Translational Research: Bridges mechanistic findings to preclinical anti-vector product development.
- Enterprise Reuse: Establishes a reusable platform for diverse vector and barrier studies.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in vector control research.
- Operational Value: Enhances standardization, reproducibility, and scalability of penetration assays.
- Strategic Value: Informs go/no-go decisions and optimizes resource allocation in early-stage portfolios.
- Portfolio Impact: Supports risk-adjusted prioritization of anti-vector candidates and technologies.
Implementation Considerations
- Requires expertise in AFM operation and biological sample handling.
- Needs access to precision instrumentation and microscopy infrastructure.
- Demands cross-team standardization for reproducible probe fabrication.
- Adaptable to other vector or animal models with protocol modifications.
- Dependent on quality of biological material and operator skill for consistency.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:59
Dissection of the Labium and Separation of the Labrum's Tip for Fabricating an Atomic Force Microscopy Probe
2:02
Isolation of the Desired Labrum Tip for Preparing the Atomic Force Microscopy Probe
2:40
Mounting and Bonding the Labrum's Tip to the Atomic Force Microscopy (AFM) Tipless Cantilever Beam
