Multiple-frequency impedance measurements in continuous flow for automated evaluation of yeast cell lysis

AbstractOptimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 103 CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.

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Bead beating-based continuous flow cell lysis in a microfluidic device

RSC Advances ◽

10.1039/c5ra01251a ◽

2015 ◽

Vol 5 (29) ◽

pp. 22350-22355 ◽

Cited By ~ 14

Author(s):

A. Berasaluce ◽

L. Matthys ◽

J. Mujika ◽

M. Antoñana-Díez ◽

A. Valero ◽

...

Keyword(s):

Microfluidic Device ◽

Continuous Flow ◽

Previous Treatment ◽

Cell Lysis ◽

Flow Cell ◽

Bead Beating

This paper describes a bead beating-based miniaturized cell lysis device that works in continuous flow allowing the analysis of large volumes of samples without previous treatment.

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Simultaneous cell lysis and bead trapping in a continuous flow microfluidic device

Sensors and Actuators B Chemical ◽

10.1016/j.snb.2005.04.018 ◽

2006 ◽

Vol 113 (2) ◽

pp. 944-955 ◽

Cited By ~ 36

Author(s):

Qasem Ramadan ◽

Victor Samper ◽

Daniel Poenar ◽

Zhu Liang ◽

Chen Yu ◽

...

Keyword(s):

Microfluidic Device ◽

Continuous Flow ◽

Cell Lysis

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A High-Throughput Screening Assay for Small Molecules That Disrupt Yeast Cell Integrity

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108320713 ◽

2008 ◽

Vol 13 (7) ◽

pp. 657-664 ◽

Cited By ~ 20

Author(s):

Damian J. Krysan ◽

Louis Didone

Keyword(s):

Yeast Cell ◽

Small Molecules ◽

High Throughput ◽

Adenylate Kinase ◽

High Throughput Screening ◽

Cell Lysis ◽

Kinase Assay ◽

Screening Assay ◽

Drug Concentrations ◽

High Throughput Screening Assay

Lead compounds for antifungal drug development are urgently needed because invasive fungal infections are an important cause of morbidity and mortality in immunocompromised patients. Here, a high-throughput screening assay for small molecules that cause yeast cell lysis is described. The assay is based on the detection of the intracellular enzyme adenylate kinase in the culture medium as a reporter of yeast cell lysis. Features of the assay protocol include 1) the ability to detect cell lysis at drug concentrations that cause no apparent growth defect, 2) specificity for fungicidal molecules, 3) a simple 1-plate, add-and-read protocol using a commercially available adenylate kinase assay kit, 4) short, 5-h incubation time, and 5) low cost. The assay is applicable to the model yeast Saccharomyces cerevisiae and to Candida albicans, the most common human fungal pathogen. The adenylate kinase assay is validated in a pilot screen of 4505 compounds. Consistent with its specificity for fungicidal molecules, the largest class of molecules identified in 2 libraries of known bioactive molecules targeted the plasma membrane. Fungistatic compounds are not detected by the assay. Adenylate kinase—based screening appears to be a useful approach to the direct identification of small molecules that kill yeast cells. ( Journal of Biomolecular Screening 2008:657-664)

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