Nanomechanical Characterization of Bacillus anthracis Spores by Atomic Force Microscopy
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2016-03-12 |
| Journal | Applied and Environmental Microbiology |
| Authors | Alex G. Li, Larry W. Burggraf, Yun Xing |
| Institutions | Oak Ridge Institute for Science and Education, U.S. Air Force Institute of Technology |
| Citations | 14 |
| Analysis | Full AI Review Included |
Nanomechanical Characterization of Bacillus anthracis Spores by Atomic Force Microscopy: Technical Analysis for 6CCVD
Section titled âNanomechanical Characterization of Bacillus anthracis Spores by Atomic Force Microscopy: Technical Analysis for 6CCVDâExecutive Summary
Section titled âExecutive SummaryâThe analyzed research details the development and application of a novel nanosurgical Atomic Force Microscopy (AFM) methodology, leveraging the extreme hardness of diamond, to characterize the internal mechanical and thermal properties of microbial spores. This technique sets a high standard for ultrastructural analysis, requiring specialized, high-ststiffness diamond components.
- Novel Methodology: A two-step nanosurgical AFM technique was developed, using a stiff diamond tip for sectioning individual spores followed by a soft silicon/silicon nitride tip for high-resolution imaging and nanomechanical mapping.
- Diamond Component Criticality: The method relies entirely on the extreme stiffness (186 N/m) and precision of the diamond cube-corner tip to cut through the highly cross-linked protein coat and peptidoglycan layers of the spore.
- Nanomechanical Differentiation: Significant variations were observed in properties across the spore layers: elastic modulus decreases in the order Ycortex > Ycoat > Ycore.
- Thermal Stability: The spore core showed significant mechanical stability when heated transiently up to 230°C, consistent with a glassy state, before undergoing changes resembling a phase transition at higher temperatures (335°C).
- Ultrastructure Revealed: High-resolution AFM revealed the peptidoglycan (PG) cortex consists of rod-like structures (10 nm wide, 60 nm long) oriented perpendicular to the spore surface.
- Application Relevance: This work demonstrates the necessity of high-stiffness, low-wear diamond materials for precise biological nanosurgery and nanothermal sensing required for bio-security and extreme environment research.
Technical Specifications
Section titled âTechnical SpecificationsâData extracted from the methodology and results sections regarding material properties and experimental parameters.
| Parameter | Value | Unit | Context |
|---|---|---|---|
| Nanosurgical Tip Material | Diamond | N/A | Cube-corner profile |
| Nanosurgical Tip Spring Constant (k) | 186 | N/m | Stiff diamond tip required for cutting |
| Nanosurgical Tip Average Radius (R) | 40 | nm | Determines cutting precision |
| Probing Tip Material | Si or SiN | N/A | Used for nanomechanical characterization |
| Probing Tip Radius (R) | <30 | nm | Used for high-resolution mapping |
| AFM Modulation Frequency | 2 | kHz | Intermittent contact mode operation |
| Applied Load Range (Peak Force) | 0.3 to 50 | nN | Force applied during mechanical mapping |
| Max Tip Temperature (Ttip) | 335 | °C | Thermal analysis maximum |
| Heating Rate (Ramp) | ~1,000 | °C/s | Used for transient localized heating |
| Core Modulus (Ycore, Cooled) | ~0.9 | GPa | Measured at 20°C after heating cycle |
| Heated Core Modulus (Ycore) | 1.2 to 1.6 | GPa | Measured between 265°C and 335°C |
| Heated Coat/Cortex Modulus (Y) | ~2 | GPa | Measured at high temperatures |
| Cortex Rod Structure Dimensions | 10 (width) x 60 (length) | nm | Observed perpendicular ultrastructure |
| Core Adhesion (High T) | <2 | nN | Reduced significantly between 265°C and 335°C |
| Calibration Melting Points | 55, 116, 235 | °C | PCL, HDPE, and PET, respectively |
Key Methodologies
Section titled âKey MethodologiesâThe experiment utilized a complex two-step AFM procedure, critically relying on high-quality diamond material for the nanosurgery step.
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Sample Preparation:
- Bacillus anthracis spores (Sterne strain) were grown at 35°C on nutrient agar.
- Spores were heat-inactivated (65°C for 30 min) and purified.
- A drop of 10 ”l spore suspension (~105 spores) was placed on freshly cleaved mica and air-dried for 12 hours.
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Nanosurgical Sectioning (Cutting Phase):
- A stiff, calibrated diamond cube-corner AFM tip (k = 186 N/m) was used.
- The tip was initially used to scratch the native spore surface, creating identifiable zig-zag patterns to distinguish the outer coat from the freshly exposed section.
- The tip then performed single or multiple indentations (point-cut or line-cut methods) with loads varying from tens of nanonewtons (nN) to micronewtons (”N), successfully cutting through the spore layers (Coat, Cortex, Core).
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Nanomechanical Characterization (Probing Phase):
- A soft, sharp silicon or silicon nitride AFM tip (R < 30 nm, k = 0.5 N/m to 3 N/m) was used for imaging the exposed cross-section.
- The AFM operated in intermittent contact mode (modulation frequency 2 kHz) with small applied loads (0.3 nN to 50 nN).
- Surface properties (Modulus, Adhesion, Deformation, Dissipation) were measured simultaneously and fitted using the Derjaguin-Muller-Toporov (DMT) model.
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Thermal Analysis:
- Localized heating was performed using a heated thermal AFM tip (AN2-200) controlled via Joule heating.
- The tip was rapidly heated (1,000°C/s) to preset temperatures ranging from 55°C up to 335°C.
- Thermal calibration was performed using reference polymers (PET, HDPE, PCL) to correlate heating voltage with tip temperature.
6CCVD Solutions & Capabilities
Section titled â6CCVD Solutions & Capabilitiesâ6CCVD provides the specialized diamond materials necessary to replicate and advance this cutting-edge nanosurgical and nanomechanical research. Our MPCVD diamond offers unparalleled stiffness, thermal stability, and customizable geometries, making it the ideal substrate and tool material for extreme-condition engineering applications.
Applicable Materials for Nanosurgery and Sensing
Section titled âApplicable Materials for Nanosurgery and SensingâTo ensure high-fidelity replication and extension of this research, 6CCVD recommends materials that guarantee maximum wear resistance and mechanical stability:
| Research Requirement | 6CCVD Solution & Material Grade | Key Capabilities Met |
|---|---|---|
| Stiff Cutting Indenter | Optical Grade SCD (Single Crystal Diamond) | SCD offers the highest purity and stiffness for ultra-low wear nanosurgical tips, ensuring repeatable cutting force profiles over prolonged use. |
| Thermal Stability & Sensing | BDD (Boron-Doped Diamond) Films & Wafers | The localized thermal analysis utilized Joule heating. 6CCVD provides heavily boron-doped diamond, allowing precise control over resistivity for integration into custom micro-heater elements (MEMS cantilevers), significantly improving sensor lifetime and stability at temperatures exceeding 335°C. |
| Large-Area Substrates | High-Purity PCD (Polycrystalline Diamond) Plates | We supply PCD substrates up to 125 mm diameter, suitable for hosting large arrays of spore samples or integrated sensors for high-throughput characterization. |
Customization Potential
Section titled âCustomization PotentialâThe success of the nanosurgical method relies on precise tip geometry (cube-corner) and specific cantilever specifications. 6CCVDâs advanced processing capabilities allow researchers to rapidly customize diamond components:
- Custom Dimensions and Shaping: While the paper used a 40 nm radius tip, 6CCVD provides the necessary bulk SCD and PCD material for engineering micro- and nano-indenters. We offer laser cutting and precision grinding for creating custom plates and wafers in geometries required for integration into specialized AFM heads or cantilever designs.
- Ultra-Smooth Polishing: For applications requiring precise mechanical contact modeling (like the DMT model used here), 6CCVD guarantees surface finishes with Ra < 1 nm on SCD and Ra < 5 nm on inch-size PCD, minimizing extraneous surface roughness effects.
- Advanced Metalization: The development of integrated thermal sensors, like the heated tip used in the experiment, often requires thin-film electrodes. 6CCVD offers in-house metalization services, including deposition of Ti, Pt, Au, Pd, W, and Cu, crucial for creating robust, thermally stable contacts on BDD or SCD sensors.
Engineering Support
Section titled âEngineering SupportâThis study falls directly into the domain of high-performance diamond applications in biological and material science under extreme conditions. 6CCVDâs in-house PhD engineering team possesses deep expertise in the mechanical, thermal, and electrical performance of MPCVD diamond. We are prepared to assist researchers with:
- Optimizing diamond material selection (SCD vs. PCD vs. BDD) based on required tip stiffness, wear resistance, and thermal operating range.
- Designing and fabricating customized diamond components for nanosurgery and advanced AFM techniques, including specialized tip arrays or micro-cantilever prototypes.
- Consultation on material integration for similar biothreat/spore characterization projects requiring precision nanomechanical or thermal stress analysis.
For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.
View Original Abstract
ABSTRACT The study of structures and properties of bacterial spores is important to understanding spore formation and biological responses to environmental stresses. While significant progress has been made over the years in elucidating the multilayer architecture of spores, the mechanical properties of the spore interior are not known. Here, we present a thermal atomic force microscopy (AFM) study of the nanomechanical properties of internal structures of Bacillus anthracis spores. We developed a nanosurgical sectioning method in which a stiff diamond AFM tip was used to cut an individual spore, exposing its internal structure, and a soft AFM tip was used to image and characterize the spore interior on the nanometer scale. We observed that the elastic modulus and adhesion force, including their thermal responses at elevated temperatures, varied significantly in different regions of the spore section. Our AFM images indicated that the peptidoglycan (PG) cortex of Bacillus anthracis spores consisted of rod-like nanometer-sized structures that are oriented in the direction perpendicular to the spore surface. Our findings may shed light on the spore architecture and properties. IMPORTANCE A nanosurgical AFM method was developed that can be used to probe the structure and properties of the spore interior. The previously unknown ultrastructure of the PG cortex of Bacillus anthracis spores was observed to consist of nanometer-sized rod-like structures that are oriented in the direction perpendicular to the spore surface. The variations in the nanomechanical properties of the spore section were largely correlated with its chemical composition. Different components of the spore materials showed different thermal responses at elevated temperatures.
Tech Support
Section titled âTech SupportâOriginal Source
Section titled âOriginal SourceâReferences
Section titled âReferencesâ- 1989 - Regulation of prokaryotic development