The objective of this research is to establish the technology for electrical control of droplet-ejection direction for coalescence of multiple directionally-ejected droplets (up to 21) in air. Coalescence of such a large number of nanoliter droplets midair is unprecedented, and is transformative as it enables midair micromixing, synthesis, analysis, etc. Fundamental studies will be performed to elucidate the underlying physics of directional droplet ejections by a focused acoustic beam, to eject nanoliter droplets with electrically controllable directions, and to merge a large number of ejected droplets in air. The intellectual merit is in the electrical controllability of the direction in which the droplets are ejected, its usage in merging simultaneously-ejected droplets in air, and the novel applications of the midair coalescence. The electrical controllability of the droplet direction will allow fine control of the direction, droplet-coalescence, and ensuing event after the coalescence. Array of multi-directional droplet ejectors capable of varying their ejection direction over a wide angle will be developed and used for automated screening of protein crystallization. The broader impacts are on cell separation and/or lysis, protein synthesis and/or crystallization, complex combinatory analyses at high throughput and great precision, etc. The results of the proposed fundamental studies will be incorporated into the interdisciplinary curriculum at the investigators' institution. Also, several undergraduate students, particularly from underrepresented groups, will be included in the research. Moreover, local inner-city high schools in south Los Angeles will be offered some droplet ejectors with which the high school students can experiment in their science labs.
The research established the acoustic ejector technology for electrical control of droplet-ejection direction for inking a spot with multiple directionally-ejected droplets without moving the ejectors. Fundamental studies were performed to elucidate the underlying physics of directional droplet ejections by a focused acoustic beam, to eject nanoliter droplets with electrically controllable directions, and to merge a large number of ejected droplets in air. Array of multi-directional droplet ejectors capable of varying their ejection direction over a wide angle was developed. The following three types of Self-Focusing Acoustic Transducer (SFAT) ejectors were designed, microfabricated and tested for electrical control of droplet ejection direction: the phase-varied ejectors, the dual-frequency ejectors, and the ejectors with series resistance control circuit. Among them, the phase-varied ejectors produced the largest direction change. The ejectors with series resistance control circuit had the smallest direction change range, but the direction of the droplet ejection could be linearly controlled over the range. Adjustment of the pulse width was needed for all of the three types to get a stable ejection as the directional angle was electrically varied. SFAT ejectors with silicon acoustic lens and silicon-nitride acoustic lens were fabricated for dense packing of a large number of SFAT ejectors, and shown to be able to eject nanoliter liquid droplets of 70 - 80 microns in diameter, which are similar to the droplets generated by the conventional ejector with parylene acoustic lens. For driving a large number of SFAT ejectors integrated on a single chip, a miniature electronic driver was developed on a printed circuit board with commercial off-the-shelf electronic components including power transistors. The driver was able to produce about 20 MHz sinusoidal pulses with controllability of the pulse width, pulse repetition frequency, and voltage amplitude for SFAT ejectors. Also designed and fabricated was SFAT with 10-mm-long focal length for delivering focused acoustic beam (of submillimeter in diameter) deep inside a living tissue. The SFAT with 10 mm focal length and 160-μm focus beam width (-6 dB) was used to demonstrate localized cytolysis with long penetration depth using 3D cell spheroids assays in 3D Matrigel environment. Acoustic intensity thresholds (AITs) of cell spheroids in various 3D extracellular environments were investigated. The experimental results showed variations of AITs of cell spheroids between malignant MCF-7 spheroids and benign MCF-10A spheroids in 3D Matrigel environments. This difference of AITs shows potential for cancer-specific treatment by ultrasonic radiations. The results confirmed that a long penetration depth of an acoustic energy was feasible in a tissue-like environment without raising temperature and without cavitation. With an appropriate level of acoustic intensity, the SFAT was shown to be effective for selective cell cytolysis in 3D cell spheroids assays, purely based on radiant pressure effect apart from heat or temperature effects. Thus, the results have demonstrated the feasibility of killing cancerous cells without damaging nearby benign cells or connective tissues. In addition, synthesis of glycine peptides with various molecular lengths (1 - 9 mers) was demonstrated on a modified glass surface with an SFAT and SPOTTM peptide synthesis protocol. Usage of an acoustic droplet ejector (based on silicon Fresnel lens) reduced the spot size for peptide synthesis through dispensing nanoliter droplets of pre-activated amino acid solution on photo-lithographically defined active spots (1 mm in diameter) on a glass substrate where the dispensed liquid keeps its spot boundary with minimal spreading. The experiment results on the protein synthesis showed that SFATs were ideally suited for synthesizing proteins on a glass slide with SFAT-ejected peptide droplets. Since SFAT does not use a droplet-defining nozzle (unlike the commercial droplet ejectors), SFAT can easily be made to eject droplets at any oblique angle through electrical control. A large number of SFATs (e.g., 8 SFAT ejectors) has already been integrated on a single chip, and shown to ink a spot on a glass substrate without having to align the spot with respect to the SFATs. Thus, the results have laid a solid foundation for droplet-based protein microarray fabrication.
