Microsonicators

Identification of microorganisms requires exposing their nucleic acids to a detection mechanism. Especially for robust cells such as spores, the means of detection may never interact with the nucleus of the cell unless they are lysed. Currently, electrokinetic, ultrasonic and chemical lysing methods are employed. The ultrasonic methods yield the maximum efficiency. However, these devices are bulky and difficult to integrate with microfluidic systems. We propose the use of high frequency acoustic waves to solve this problem.

In the experiments, we used a similar channel that is described in the previous section. The channel dimensions are larger to be able to accommodate high volumes of fluids to be required for PCR analysis. The channel is shown in Fig. 1.

 

 

 

FIGURE 1. The channel used in lysing experiments

 

 

In lysing experiments, ATCC 9372 B. subtilis var. niger spores were used. They were in water suspension at a concentration of 1.8x106 spores/ml. We first washed the minisonicator channel with 1 ml of 99% ethanol and 5 ml of DI water. We then shook the spore vial thoroughly to obtain an even spore distribution in suspension and loaded a 250 µl, 2.304 mm diameter Gastight #1725 syringe (Hamilton Co) with 450,000 spores. A PHD 2000 programmable syringe pump (Harvard Instruments) pushed the spores through the channel at a constant flow of 5 µl/min corresponding to 150 spores/s, i.e. 2 spores/ml). Hence each spore remained in the channel for 30 s, corresponding to 13.5 s over the ultrasonic transducers or 1.5 s per transducer. The spores thus theoretically covered less than 0.5% the channel floor.

To ensure maximum electric power coupling to the piezo-elements, we added in these experiments a matching series inductor of 0.66 mH to the minisonicator. This made the transducers resonate at 367.377 ±0.173 MHz (3 dB bandwidth) with a 0.2% power-loss due to electrical mismatch. In this experiment the same signal generator and amplifier setup as in the HL-60 experiment powered the minisonicator. At the resonance frequency a total of 30 dBm of power was delivered. This corresponds to 10.0 Vpeak across each of the matched 50 W PZT elements (1W), resulting in an M.I. of 0.06. The maximum acoustic intensity in front of a transducer was thus 44.4 W/cm2 corresponding to a pressure po of 1.15 MPa.

We performed RTPCR analysis on a BioRad iCycler (AME Bioscience) calibrated with a proprietary well-factor plate mix (Biorad) to determine the sporocidal effect of sonication. Each 20 µl reaction aliquot contained 10 µl sample DNA and 10 µl master mix so that the final reaction conditions included 1x Buffer II with Stoffel Fragments, 3 mM MgCl2, 0.1 units/ml AmpliTaq Polymerase (Applied Biosystems) 0.2 mM of each primer (Left Primer 5’T GATCTTAGTTGCCAGCAT TCAGTT3’, Right Primer 5’TCTGTCCATTGTAGCACG TGTGTAG3’), 0.125 mM dNTP, 1%vol phenyl-methyl-sulfonyl-fluoride (PMSF, Sigma Aldrich), and 0.25x Syber Green (Molecular Probes). Thermal cycling started with a 5-minute hold phase at 95oC followed by 40 cycles of denaturation at 94oC, annealing at 64oC and extension at 72oC, each step lasting 30 s.

The master mix was prepared afresh before every RTPCR experiment. We undertook three consecutive experiments, one with four replications per sample and two with eight replications per sample. In the first experiment we tested three positive controls: 1:4, 1:40 and 1:400 serial dilutions of the spores treated with the macro-sonicator. The estimated number of spores in the samples were 4.5x105, 4.5x104 and 4.5x103 spores/ml, respectively. We also included a negative control using a 1:10 dilution of untreated spores in water, and a 1:10 dilution of spores treated with the minisonicator.

In the second and third experiments we tested three positive controls. These were 1:10, 1:40 and 1:100 serial dilutions of spores treated with the macro-sonicator, estimated to correspond to 1.8x105, 4.5x104 and 1.8x104 spores/ml, respectively. We also tested three negative controls. These were 1:10 dilution of untreated spores, 1:10 dilution of untreated spores and containing 1%vol PMSF in water, and a 1:10 dilution of a sample treated with the minisonicator, Fig. 2.

In two separate experiments, using identical settings as in the macrosonicator experiments except that no ultrasound was administered, we weighed the effects of flow on our positive and negative controls. To measure the effect of flow-induced shear stress damage on the spores we compared a 1:10 dilution of untreated spores to a similar solution that we had flowed through the channel at 5 µl/min. To verify that we recovered all of the sample, we compared a 1:10 dilution of sonicated spores that had not been flowed through the channel to one that we allowed to flow through the channel at 5 µl/min.

A comparison by means of a two-sided heteroscedastic t-test of sonicated spores to ones that unsonicated have passed through the channel gave a t-value of 0.126 for CtLow and 0.169 for CtHigh. For untreated (no sonication) spores the corresponding t-values were 0.214 and 0.581. Similarly, the CtLow and CtHigh for spores with and without PMSF were 0.8785 and 0.8021 for the second experiment and 0.7021 and 0.4563 for the third experiment. All eight t-values were significant on the 0.05 level that was chosen for the test. Therefore the means were indistinguishable and neither flow, nor the addition of PMSF had an appreciable effect on our controls by either inducing lysis or reducing the available sample.

The linear model based on the PCR equation, of the second experiment predicted 58.2% ±11.87% lysis (p-value <0.0001) while the third experiment predicted 50.2% ±6.05% (p-value <0.0001). The logarithmic model of the first experiment predicted 36.1% ±9.8% lysis, the second experiment 39.8% ±11.3%, and the third 33.0% ±4.6%.

 

 

 

FIGURE 2. Averaged Real Time PCR fluorescence signals of positive controls, negative controls and spores treated with the minisonicator obtained in the second experiment.

 

 

 

FIGURE 3. Relationship between number of lysed spores and threshold cycle (A). and between threshold cycle and fractional release of DNA due to lysing (B). CtLow of positive control serial dilutions 1:10, 1:40 and 1:100 from fig. 4 are plotted versus their respective concentrations and curvefitted to produce a mathematical relation used to determine the percent of available DNA for minisonicated and untreated spores. Concentrations normalized so that the 1:10 dilution corresponds to 100% lysis.