Timber DNA release using focused ultrasound extraction (FUSE) for genetic species identification.

The use of genetic data for timber species and population assignment is a powerful tool for combating the illegal timber trade, but the challenges of extracting DNA from timber have prevented the routine use of genetics as a supply chain management tool. To overcome these challenges, we explored the feasibility of focused ultrasound extraction (FUSE) for rapid DNA release from timber. Using high-pressure ultrasound pulses, FUSE generates a cavitation bubble cloud that disintegrates samples into acellular debris, resulting in the mechanical release of DNA. In this work, FUSE was applied to white oak (Quercus alba) timber shavings to test the feasibility of using FUSE for timber DNA extraction for the first time. Results showed that FUSE processing disintegrated the tissue samples and released significant quantities of DNA. After five minutes of tissue processing DNA quantities of 0.21 ± 0.02 ng/mg, 0.99 ± 0.32 ng/mg, and 0.14 ± 0.01 ng/mg, were released from medium, coarse, and combination shaving groups, respectively. Amplification and sequencing of regions within the matK and rbcL chloroplast genes confirmed that the quality of DNA prepared with FUSE was suitable for PCR and short-read sequencing applications. Overall, these results show that FUSE can serve as a DNA sample preparation method capable of releasing high-quality DNA from timber in a fraction of the time required by conventional extraction methods. Based on the improved efficiency of DNA release with FUSE, ongoing work aims to develop this technology into portable systems that can be used to rapidly prepare timber samples for genetic species identification.

DNA release from plant tissue using focused ultrasound extraction (FUSE).

Premise: Sample preparation in genomics is a critical step that is often overlooked in molecular workflows and impacts the success of downstream genetic applications. This study explores the use of a recently developed focused ultrasound extraction (FUSE) technique to enable the rapid release of DNA from plant tissues for genetic analysis. Methods: FUSE generates a dense acoustic cavitation bubble cloud that pulverizes targeted tissue into acellular debris. This technique was applied to leaf samples of American chestnut (Castanea dentata), tulip poplar (Liriodendron tulipifera), red maple (Acer rubrum), and chestnut oak (Quercus montana). Results: We observed that FUSE can extract high quantities of DNA in 9-15 min, compared to the 30 min required for control DNA extraction methods. FUSE extracted DNA quantities of 24.33 ± 6.51 ng/mg and 35.32 ± 9.21 ng/mg from American chestnut and red maple, respectively, while control methods yielded 6.22 ± 0.87 ng/mg and 11.51 ± 1.95 ng/mg, respectively. The quality of the DNA released by FUSE allowed for successful amplification and next-generation sequencing. Discussion: These results indicate that FUSE can improve DNA extraction efficiency for leaf tissues. Continued development of this technology aims to adapt to field-deployable systems to increase the cataloging of genetic biodiversity, particularly in low-resource biodiversity hotspots.

Effects of frequency on bubble-cloud behavior and ablation efficiency in intrinsic threshold histotripsy.

Objective.Histotripsy is a non-thermal focused ultrasound ablation method that destroys tissue through the generation of a cavitation bubble cloud. Previous work studying intrinsic threshold histotripsy has shown that dense bubble clouds can be formed by a single-cycle pulse when the negative pressure exceeds an intrinsic threshold of ∼25-30 MPa, with the ablation efficiency dependent upon the size and density of bubbles within the cloud. This work investigates the effects of frequency on bubble-cloud behavior and ablation efficiency in intrinsic threshold histotripsy.Approach.A modular transducer was used to expose agarose tissue phantoms to 500 kHz, 1 MHz, or 3 MHz, histotripsy pulses. Optical imaging was used to measure the bubble-cloud dimensions, bubble density, and bubble size. The effects of frequency on ablation efficiency were also investigated by applying histotripsy to red blood cell (RBC) phantoms.Main results.Results revealed that the bubble-cloud size closely matched theoretical predictions for all frequencies. The bubble density, which is a measure of the number of bubbles per unit area, was shown to increase with increasing frequency while the size of individual bubbles within the cloud decreased at higher frequencies. Finally, RBC phantom experiments showed decreasing ablation efficiency with increasing frequency.Significance.Overall, results demonstrate the effects of frequency on histotripsy bubble-cloud behavior and show that lower frequency generates more efficient tissue ablation, primarily due to enhanced bubble expansion.

Vibration Induced Transport of Enclosed Droplets.

The droplet response to vibrations has been well characterized on open substrates, but microfluidic applications for droplets on open systems are limited by rapid evaporation rates and prone to environmental contamination. However, the response of enclosed droplets to vibration is less understood. Here, we investigate the effects of a dual-plate enclosure on droplet transport for the anisotropic ratchet conveyor system. This system uses an asymmetric pattern of hydrophilic rungs to transport droplets with an applied vibration. Through this work, we discovered that the addition of a substrate on top of the droplet, held in place with a 3D printed fixture, extends the functional frequency range for droplet transport and normalizes the device performance for droplets of different volumes. Furthermore, we found that the edge movements are anti-phasic between top and bottom substrates, providing a velocity profile that is correlated to vibration frequency, unlike the resonance-dependent profiles observed on open systems. These results expand the capabilities of this system, providing avenues for new applications and innovation, but also new insights for droplet mechanics in response to applied vibration.

Control of cellular adhesion and myofibroblastic character with sub-micrometer magnetoelastic vibrations.

The effect of sub-cellular mechanical loads on the behavior of fibroblasts was investigated using magnetoelastic (ME) materials, a type of material that produces mechanical vibrations when exposed to an external magnetic AC field. The integration of this functionality into implant surfaces could mitigate excessive fibrotic responses to many biomedical devices. By changing the profiles of the AC magnetic field, the amplitude, duration, and period of the applied vibrations was altered to understand the effect of each parameter on cell behavior. Results indicate fibroblast adhesion depends on the magnitude and total number of applied vibrations, and reductions in proliferative activity, cell spreading, and the expression of myofibroblastic markers occur in response to the vibrations induced by the ME materials. These findings suggest that the subcellular amplitude mechanical loads produced by ME materials could potentially remotely modulate myofibroblastic activity and limit undesirable fibrotic development.

Transport velocity of droplets on ratchet conveyors.

Anisotropic ratchet conveyors (ARC) are a type of digital microfluidic system. Unlike electrowetting based systems, ARCs transport droplets through a passive, micro-patterned surface and applied orthogonal vibrations. The mechanics of droplet transport on ARC devices has yet to be as well characterized and understood as on electrowetting systems. In this work, we investigate how the design of the ARC substrate affects the droplet response to vibrations and perform the first characterization of transport velocity on ARC devices. We discovered that the design of the ARC device has a significant effect on both the transport efficiency and velocity of actuated droplets, and that the amplitude of the applied vibration can modulate the velocity of transported droplets. Finally, we show that the movement of droplet edges is not continuous but rather the sum of quantized steps between features of the ARC device. These results provide new insights into the behavior of droplets vibrated on asymmetric surface patterns and will serve as the foundation for the design and development of future lab-on-a-chip systems.

Converting Vertical Vibration of Anisotropic Ratchet Conveyors into Horizontal Droplet Motion.

An anisotropic ratchet conveyor is an asymmetric, periodic, micropatterned surface that propels droplets when vibrated with a sinusoidal signal at certain frequencies and amplitudes. For each input frequency, there is a threshold amplitude beyond which the droplet starts to move. In this paper, we study the parameters that initiate droplet motion and the relationship between the input frequency and threshold amplitude among droplets with different volume, density, viscosity, and surface tension. Through this investigation we demonstrate how nondimensionalization reveals consistent behavior for droplets of different volumes. Finally, we propose a compact model that captures the essential features of the system to describe how a pure vertical vibration results in horizontal droplet motion. This model provides an intuitive understanding of the underlying physics and explains how the surface asymmetry is the key for lateral droplet motion.

Enabling Droplet Functionality on Anisotropic Ratchet Conveyors.

Anisotropic ratchet conveyors (ARCs) are a recently developed microfluidic platform that transports liquid droplets through a passive, microfabricated surface pattern and applied orthogonal vibrations. In this work, three new functionalities are presented for controlling droplet transport on the ARC system. These devices can pause droplet transport (ARC gate), decide between two pathways of droplet transport (ARC switch), and pass droplets between transport tracks (ARC delivery junction). All devices function solely through the modification of pinning forces acting on the transported droplet and are the first reported devices that can selectively control droplet timing and directionality without active (e.g., thermal, electrical, or magnetic) surface components.

Application of sub-micrometer vibrations to mitigate bacterial adhesion.

As a prominent concern regarding implantable devices, eliminating the threat of opportunistic bacterial infection represents a significant benefit to both patient health and device function. Current treatment options focus on chemical approaches to negate bacterial adhesion, however, these methods are in some ways limited. The scope of this study was to assess the efficacy of a novel means of modulating bacterial adhesion through the application of vibrations using magnetoelastic materials. Magnetoelastic materials possess unique magnetostrictive property that can convert a magnetic field stimulus into a mechanical deformation. In vitro experiments demonstrated that vibrational loads generated by the magnetoelastic materials significantly reduced the number of adherent bacteria on samples exposed to Escherichia coli, Staphylococcus epidermidis and Staphylococcus aureus suspensions. These experiments demonstrate that vibrational loads from magnetoelastic materials can be used as a post-deployment activated means to deter bacterial adhesion and device infection.

S-nitroso-N-acetylpenicillamine (SNAP) derivatization of peptide primary amines to create inducible nitric oxide donor biomaterials.

An S-nitroso-N-acetylpenicillamine (SNAP) derivatization approach was used to modify existing free primary amines found in fibrin (a natural protein-based biomaterial) to generate a controlled nitric oxide (NO) releasing scaffold material. The duration of the derivatization reaction affects the NO release kinetics, the induction of controlled NO-release, hydrophobicity, swelling behavior, elastic moduli, rheometric character, and degradation behavior. These properties were quantified to determine changes in fibrin hydrogels following covalent attachment of SNAP. NO-releasing materials exhibited minimal cytotoxicity when cultured with fibroblasts or osteoblasts. Cells maintained viability and proliferative character on derivatized materials as demonstrated by Live/Dead cell staining and counting. In addition, SNAP-derivatized hydrogels exhibited an antimicrobial character indicative of NO-releasing materials. SNAP derivatization of natural polymeric biomaterials containing free primary amines offers a means to generate inducible NO-releasing biomaterials for use as an antimicrobial and regenerative support for tissue engineering.

Fabrication of biocompatible, vibrational magnetoelastic materials for controlling cellular adhesion.

This paper describes the functionalization of magnetoelastic (ME) materials with Parylene-C coating to improve the surface reactivity to cellular response. Previous study has demonstrated that vibrating ME materials were capable of modulating cellular adhesion when activated by an externally applied AC magnetic field. However, since ME materials are not inherently biocompatible, surface modifications are needed for their implementation in biological settings. Here, the long-term stability of the ME material in an aqueous and biological environment is achieved by chemical-vapor deposition of a conformal Parylene-C layer, and further functionalized by methods of oxygen plasma etching and protein adsorption. In vitro cytotoxicity measurement and characterization of the vibrational behavior of the ME materials showed that Parylene-C coatings of 10 µm or greater could prevent hydrolytic degradation without sacrificing the vibrational behavior of the ME material. This work allows for long-term durability and functionality of ME materials in an aqueous and biological environment and makes the potential use of this technology in monitoring and modulating cellular behavior at the surface of implantable devices feasible.