Development of a novel compact sonicator for cell disruption

Abstract

Ultrasound microbial cell disrupters operating at around 20 kHz are often physically large and, due to significant heating, can be unsuitable for small sample volumes where biochemical integrity of the extracted product is required. Development of a compact device based on a 63.5-mm diameter, 6.5-mm thick tubular transducer for rapid cell disruption in small-volume samples in a high-intensity acoustic cavitation field with minimal temperature rises is described here. Suspensions of Saccharomyces cerevisiae were exposed to cavitation for various times in the compact device and a 20-kHz probe sonicator. Cell disruption was assessed by protein release and by staining. Yeast cell disruption was greater in the novel 267-kHz sonicator than in the 20-kHz probe sonicator for the same exposure time. A 1-dimensional (1-D) transfer matrix model analysis for piezoelectric resonators was applied to an axial cross-section of the tubular sonicator to predict frequencies of mechanical resonance in the sample volume associated with maximum acoustic pressure. Admittance measurements identified frequencies of electrical resonance. Ultrasonic cavitation noise peaks were detected by a hydrophone at both the mechanical and electrical resonances. Cell breakage efficiency was twice as great in terms of protein released per dissipated watt at the mechanical resonance predicted by the model, compared to those at the electrical resonance frequencies. The results form a basis for rational design of an ultrasound cell disruption technique for small-volume samples.

Introduction

Disruption of microorganisms by ultrasonic cavitation has been exploited extensively for many years. Commercially available cell disrupters typically consist of half wavelength probes about 15-cm long that are resonant at 20 kHz. The probes have titanium tips to minimise pitting damage by cavitation in the surrounding liquid. Due to the probe length, these systems are bulky and inconvenient for use outside of a laboratory. Cell disruption devices operating at higher frequencies are more compact because of the shorter wavelengths associated with the higher frequencies.

Ultrasonic disrupters operating at 20 and 40 kHz and at 1 MHz, respectively, have been described for treating small sample volumes as in the extraction of bacterial DNA for polymerase chain reaction (PCR) analysis (Fykse et al., 2003, Taylor et al., 2001, Chandler et al., 2001). The systems were (i) a 20-kHz disrupter transmitting energy through a 2-mm diameter horn into a 1-ml sample of bacterial suspension (Fykse et al., 2003), (ii) a 40-kHz ultrasonic horn coupled to a 100 μl sample through a flexible thin film interface (Taylor et al., 2001) and (iii) a 160° segment of a 1.5-mm walled (1.33-MHz thickness resonance) tubular transducer transmitting sound through water to a sample suspension in a 1-ml polypropylene sample tube positioned axially in the radially focussed field (Chandler et al., 2001). These devices effectively disrupt bacterial cells but can produce significant sample temperature increases of as much as 50 to 90 K (Taylor et al., 2001, Chandler et al., 2001). These temperature rises are tolerable when the purpose of disruption is to extract DNA for PCR analysis but can be deleterious to protein immunological targets. Protein toxins (Fox, 2002) and cell wall antigens provide useful targets for immuno-based cell detection methods. Exposure to ultrasound has the potential to release toxins and increase the number of cell fragments in suspension. Consequently, a disrupter with minimal temperature effects may reduce the cell concentration threshold required for immunological assay of cell numbers.

In the present work, a novel compact cell disrupter was developed based on a tubular transducer with a sample channel in the central axis. Tubular transducers driven in radial mode strongly focus acoustic energy at the axial region (Grundy et al., 1993, Sarvazyan and Ponamerev, 1996); in this case, high-intensity sound waves create a focussed cavitation zone in the sample channel located axially to the transducer. A 1-dimensional (1-D) transfer matrix model for piezoelectric multilayer resonators (Nowotny and Benes, 1987) based on the fundamental equations of piezoelectricity relates the piezoelectrically coupled properties of the active piezolayer and the properties of the nonpiezoelectric (passive) layers of the resonator system. The model then calculates electrical admittance as a function of frequency (Nowotny and Benes, 1987, Gröschl, 1998, Hawkes et al., 2002). Furthermore, electroacoustic field qualities, such as displacement and pressure amplitudes, acoustic energy density and energy flow, can be determined for each layer or for the whole resonator at specific frequencies (Gröschl, 1998, Hawkes et al., 2002). This model approach and an equivalent-circuit transducer model coupled with acoustic impedance transfer relationships (Hill et al., 2002) have been employed successfully to simulate plane acoustic wave propagation in planar multilayer systems (acoustic cell/particle filters). They have shown good agreement between predicted and measured resonator properties in terms of electrical admittance spectra (Gröschl, 1998, Hawkes et al., 2002, Nowotny et al., 1991) and frequencies of optimal filtration efficiency (Gröschl, 1998, Hawkes et al., 2002). No exact mathematical treatment is available to simulate acoustic wave propagation in piezoelectric multilayer resonators of cylindrical geometry. However, the similarity of the mathematical solutions of the equations of piezoelectricity for plane and cylindrical waves suggests the applicability of the 1-D transfer matrix model as a predictive tool for estimation of resonator properties in a tubular transducer system. By default, we applied the 1-D transfer matrix model without modification to the tubular system developed here. The properties of the different layers in a cross-section of the novel cell disrupter were taken as input to the model to calculate the electrical admittance of the system and to predict the operating frequencies at which best cell disruption results could be expected.

The electrical admittance was measured independently using a network analyser. Cavitation activity at different frequencies was assessed by aural and stereomicroscopic observations. In addition, a spectrum analyser was employed to monitor cavitation activity at the optimum operating frequency of the novel tubular cell disrupter. Disruption of Saccharomyces cerevisiae was assayed and compared to cell disruption in a standard commercial 20-kHz probe sonicator. The results show that the multilayer transfer matrix model correctly selects the sonication frequency at which efficient cell disruption is achieved under conditions where temperature rise is contained to levels that are tolerable for retention of protein function.