A Minisonicator To Rapidly Disrupt Bacterial Spores for DNA Analysis

Belgrader, Phillip; Hansford, D; Kovacs, Gab; Venkateswaran, K.; Mariella, Raymond P.; Milanovich, Fred P.; Nasarabadi, Shanavaz; Okuzumi, Masayo; Pourahmadi, F.; Ma, Northrup

doi:10.1021/ac990347o

Cited by 165 publications

(140 citation statements)

References 18 publications

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“…1,2 Chemical lysis uses lytic agents such as sodium dodecyl sulfate to dissolve the cell membrane or react with the membrane lipids. [3][4][5] Mechanical lysis uses nanoscale filtrations, 6 spherical particles, 7 or microscale sonication 8 to break down cells with shear and/or frictional forces. Thermal lysis disrupts cells at high temperatures.…”

Section: Introductionmentioning

confidence: 99%

Integrated electrical concentration and lysis of cells in a microfluidic chip

et al. 2010

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Lysing cells is an important step in the analysis of intracellular contents. Concentrating cells is often required in order to acquire adequate cells for lysis. This work presents an integrated concentration and lysis of mammalian cells in a constriction microchannel using dc-biased ac electric fields. By adjusting the dc component, the electrokinetic cell motion can be precisely controlled, leading to an easy switch between concentration and lysis of red blood cells in the channel constriction. These two operations are also used in conjunction to demonstrate a continuous concentration and separation of leukemia cells from red blood cells in the same microchannel. The observed cell behaviors agree reasonably with the simulation results.

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Section: Introductionmentioning

confidence: 99%

Integrated electrical concentration and lysis of cells in a microfluidic chip

et al. 2010

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“…However, lysis is a significant challenge for thick-walled microorganisms such as Bacillus anthracis spores and Mycobacterium tuberculosis cells (13,18,22). The multilayer structure of Bacillus spores includes an outer cortex and coat that is resistant to chemical and physical treatments (5,23). Similarly, mycobacteria have a thick, waxy cell wall that is difficult to disrupt for the extraction of nucleic acids (9,17).…”

mentioning

confidence: 99%

“…Lysis by sonication has been attributed to cavitation, where the rapid formation and shrinkage of gas bubbles creates high pressures and temperatures (5). Lysis of thick-walled organisms by bead beating typically involves high-frequency oscillation of a closed tube containing a suspension of the target organism and beads.…”

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confidence: 99%

Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a Miniature, Low-Power, Disposable Device

et al. 2011

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Molecular detection of microorganisms requires microbial cell disruption to release nucleic acids. Sensitive detection of thick-walled microorganisms such as Bacillus spores and Mycobacterium cells typically necessitates mechanical disruption through bead beating or sonication, using benchtop instruments that require line power. Miniaturized, low-power, battery-operated devices are needed to facilitate mechanical pathogen disruption for nucleic acid testing at the point of care and in field settings. We assessed the lysis efficiency of a very small disposable bead blender called OmniLyse relative to the industry standard benchtop Biospec Mini-BeadBeater. The OmniLyse weighs approximately 3 g, at a size of approximately 1.1 cm 3 without the battery pack. Both instruments were used to mechanically lyse Bacillus subtilis spores and Mycobacterium bovis BCG cells. The relative lysis efficiency was assessed through real-time PCR. Cycle threshold (C T ) values obtained at all microbial cell concentrations were similar between the two devices, indicating that the lysis efficiencies of the OmniLyse and the BioSpec Mini-BeadBeater were comparable. As an internal control, genomic DNA from a different organism was spiked at a constant concentration into each sample upstream of lysis. The C T values for PCR amplification of lysed samples using primers specific to this internal control were comparable between the two devices, indicating negligible PCR inhibition or other secondary effects. Overall, the OmniLyse device was found to effectively lyse tough-walled organisms in a very small, disposable, batteryoperated format, which is expected to facilitate sensitive point-of-care nucleic acid testing.

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“…Routine laboratory methods for cell lysis include freeze/thaw, proteinase K, lysozyme, and guanidium salt treatments, ethanol or 2-propanol precipitation, DNA; ballistic disintegration; or sonication at low frequencies (e.g. kilohertz) after pretreatment with other chemicals [1]. A key aspect is that the extraction format must be highly scalable to benefit many types of biodetection systems already in place.…”

Section: Introductionmentioning

confidence: 99%

“…Often the altered pH and chemical background adds additional steps that can otherwise be avoided. Recently, large-scale acoustic transducers have proven powerful for disrupting cell membranes and spores for subsequent DNA analysis [1,3]. Thin-film based ultrasonic actuators have also been used lyse cellular samples and have proven more effective for microsystem applications [4,5].…”

Section: Introductionmentioning

confidence: 99%

Intelligent front-end sample preparation tool using acoustic streaming.

Cooley¹,

McClain²,

Murton³

et al. 2009

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We have successfully developed a nucleic acid extraction system based on a microacoustic lysis array coupled to an integrated nucleic acid extraction system all on a single cartridge. The microacoustic lysing array is based on 36º Y cut lithium niobate, which couples bulk acoustic waves (BAW) into the microchannels. The microchannels were fabricated using Mylar laminates and fused silica to form acoustic-fluidic interface cartridges. The transducer array consists of four active elements directed for cell lysis and one optional BAW element for mixing on the cartridge. The lysis system was modeled using one dimensional (1D) transmission line and two dimensional (2D) FEM models. For input powers required to lyse cells, the flow rate dictated the temperature change across the lysing region. From the computational models, a flow rate of 10 µL/min produced a temperature rise of 23.2 ºC and only 6.7 ºC when flowing at 60 µL/min. The measured temperature changes were 5 ºC less than the model. The computational models also permitted optimization of the acoustic coupling to the microchannel region and revealed the potential impact of thermal effects if not controlled. Using E. coli, we achieved a lysing efficacy of 49.9 ± 29.92 % based on a cell viability assay with a 757.2 % increase in ATP release within 20 seconds of acoustic exposure. A bench-top lysing system required 15-20 minutes operating up to 58 Watts to achieve the same level of cell lysis. We demonstrate that active mixing on the cartridge was critical to maximize binding and release of nucleic acid to the magnetic beads. Using a sol-gel silica bead matrix filled microchannel the extraction efficacy was 40%. The cartridge based magnetic bead system had an extraction efficiency of 19.2 %. For an electric field based method that used Nafion films, a nucleic acid extraction 4 efficiency of 66.3 % was achieved at 6 volts DC. For the flow rates we tested (10 -50 µL/min), the nucleic acid extraction time was 5-10 minutes for a volume of 50 µL. Moreover, a unique feature of this technology is the ability to replace the cartridges for subsequent nucleic acid extractions.5

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A Minisonicator To Rapidly Disrupt Bacterial Spores for DNA Analysis

Cited by 165 publications

References 18 publications

Integrated electrical concentration and lysis of cells in a microfluidic chip

Integrated electrical concentration and lysis of cells in a microfluidic chip

Mechanical Disruption of Lysis-Resistant Bacterial Cells by Use of a Miniature, Low-Power, Disposable Device

Intelligent front-end sample preparation tool using acoustic streaming.

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