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with the separation of bioparticles by Over the years, humans have harnessed capillary electromigration techniques,” the beneficial properties of fungi to make bread and cheese, to ferment alco- says Horka´. “But whole microscopic filamentous fungi are too large to sepahol, and to produce life-saving medications such as penicillin. However, fungi can also cause serious human illness and death, particularly in immunocompromised individuals. To quickly identify strains of microscopic filamentous fungi that are harmful to humans, Marie Horka´ and colleagues at the Academy of Sciences of the Czech Republic developed CE methods that discriminate the asexual spores, or conidia, of various fungal strains. They report their results in a new Conidia, or asexual fungal spores, were separated AC paper (DOI 10.1021/ and identified by CE. ac900374v). Microscopic filamentous fungi, rate in a capillary.” For this reason, the which are prevalent in both indoor and investigators analyzed conidia of filaoutdoor environments, produce mycmentous fungi, which are sufficiently otoxins that can contaminate food, small for CE. cause allergies, and have carcinogenic The researchers released conidia by effects in humans. “Microscopic filavortexing five strains of filamentous fungi mentous fungi cause serious human inthat had been cultured from collection fections, present problems for agriculstrains or clinical samples. Then, they optiture, and may be used in bioterrorism,” mized protocols to separate the conidia says Horka´. Strains of filamentous fungi with the two CE techniques. In CIEF, an differ in pathogenesis and sensitivity to electric current pulls charged particles, antifungal agents; therefore, rapid, relisuch as conidia, through a buffer-filled able identification is important for diagcapillary tube that contains a pH gradient. nosis and therapy. Most conventional methods of fungal Each molecule migrates through the gradient until it reaches its isoelectric point identification rely on the microscopic (pI), the pH at which the molecule has no examination of morphological or physinet charge. Unknown pIs are estimated by ological characteristics. However, these evaluations usually require growth of the comparison with co-electrophoresed pI markers. In CZE, molecules are separated fungus for several days to several weeks, in a buffer-filled capillary on the basis of and the results often depend on varielectrophoretic mobility, which depends ables such as media or culture condion the charge-to-mass ratio of the particle tions. Newer methods, such as the dein the presence of background electrolytes. tection of fungal nucleic acids by PCR or of fungal proteins by immunoassay or Once separated by CIEF or CZE, conidia can be detected in-column either by UV MS, are more reliable but still relatively absorbance or with fluorescent dyes that laborious and expensive. bind to the conidia’s surface. That’s why Horka´’s team optimized Conidia pose challenges for CE becapillary isoelectric focusing (CIEF) and cause they are coated with hydrophobic capillary zone electrophoresis (CZE) techniques to identify filamentous fungi. proteins that cause the spores to aggregate or adsorb onto the capillary wall. “At our institute we have experience 10.1021/AC900921S 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 05/21/2009
CENTERS FOR DISEASE CONTROL AND PREVENTION
CE of fungal conidia Therefore, the researchers experimented with different buffer additives (e.g., ethanol, PEG 1000, the detergent Brij 35) to achieve a narrow zone of separated conidia for each fungal strain. Under the optimized conditions, CZE and CIEF with UV detection were used to differentiate conidia in a mixture from the five filamentous fungal strains. In CIEF, the pIs of conidia from medical samples were not host-specific, which supports the clinical applicability of the method. To increase the detection sensitivities of the capillary electrophoretic techniques, the team added poly(ethylene glycol) pyrenebutanoate (PB-PEG) to the electrophoresis buffers to dynamically modify conidia during separation. PB-PEG, which was developed by Horka´ and colleagues in a previous study (DOI 10.1021/ac061200h), binds noncovalently to surface proteins and fluoresces upon excitation with UV light. Importantly, labeling with PBPEG did not alter the pIs of bioanalytes, a common problem with other fluorescent probes. After optimizing the labeling conditions for both CIEF and CZE, the investigators could detect as few as 10 conidia from each of the five examined strains. “We established that conidia can be separated by CIEF and CZE,” says Horka´. “The knowledge of isoelectric points and other electromigration properties of conidia will help in the identification of fungi.” Compared with previous methods for fungal identification, CE is simple, rapid, and relatively inexpensive. Horka´ says that lab-on-a-chip CE devices could allow the identification of filamentous fungi in a variety of settings. However, determination of the electromigration properties of conidia from additional strains of filamentous fungi is necessary. This paper appears in the May 15, 2009, issue of AC. —Laura Cassiday
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