Volume Cytometry: Microfluidic Sensor for High-Throughput Screening in Real Time Daniel A. Ateya, † Frederick Sachs, ‡ Philip A. Gottlieb, ‡ Steve Besch, ‡ and Susan Z. Hua* ,†,‡ Bio-MEMS and Bio-Materials Laboratory, Department of Mechanical and Aerospace Engineering, SUNYsBuffalo, Buffalo, New York 14260, snf Center for Single Molecule Biophysics, Department of Physiology and Biophysics, SUNYsBuffalo, Buffalo, New York 14214 Regulation of cell volume was one of the earliest evolu- tionary demands for life and remains a universal measure of cell metabolism. Since conventional methods to mea- sure cell volume, such as microscopy, are complex and time-consuming, cell volume has not been used as the basis for cell-based screening. We have developed a microfabricated chip that can measure the volume of small numbers of cells in real time with unprecedented resolution. The method is applicable to adherent or suspended populations of cells and membrane-bound organelles. Our prototype device can detect volume changes in a monolayer of tissue-cultured astrocytes responding to anisotonic stimuli of <1mOsm. We deter- mined the sensitivity to antibiotics of different E. coli strains in <10 min at 24 °C. This time can be reduced at higher temperatures enabling on-site clinical testing of infectious agents. Using the chip to screen natural prod- ucts, we found a peptide in spider venom that inhibits eukaryotic volume regulation at ∼100pM. The prototype chip made in silicon is inexpensive, reusable, and runs on low-voltage electrical power. The technology can be readily transferred to large arrays in plastic. Cell volume and its physiological functions are intimately intertwined, 1-3 so that a real-time monitor of cell volume can serve as a screen for drugs or other environmental influences in the same manner as cell-based calcium assays. The perturbations that affect cell volume include excitability, 4,5 metabolism, 6 apoptosis, 7,8 necrosis, neurotransmitters, 9,10 environmental toxic agents, 11,12 and cell division and growth. 13,14 Conventional methods to measure cell volume include light or electron microscopy, 15 fluorescence microscopy, 16,17 electrophysiology, 18,19 atomic force microscopy, 20 or electrical impedance. 21 Among the electrical impedance meth- ods, Coulter counter technology 22,23 has been widely used for cell cytometry mainly due to its ease of use and relatively rapid sampling rate. The Coulter counter is capable of providing a histogram of cell sizes that can reveal heterogeneity of the population. While not a sufficiently fast method to measure the kinetics of cell volume regulation, the Coulter counter also requires free-floating cells. This is an abnormal condition for most cells, where isolation itself can produce significant physiological changes. We have developed a microfluidic/electrical sensor that is noninvasive and provides real-time measurement of changes in cell volume for both adherent and suspended cells. The small volume of the sensor enables rapid screening for natural products that are only available in small quantities. The principle of measuring cell volume in our sensor is based on the fact that cells are electrical insulators at low frequencies. With cells in a chamber of fixed cross section, a change of cell volume displaces the extracellular fluid, thereby changing the chamber conductance. Assuming a uniform monolayer of adherent cells, a first-order approximation of the relative cell volume change ∆V/V 0 ) (V - V 0 )/V 0 is given by * To whom correspondence should be addressed. Phone: (716) 645 2593, x2358. Fax: (716) 645 3875. E-mail: zhua@eng.buffalo.edu. † Department of Mechanical and Aerospace Engineering. ‡ Department of Physiology and Biophysics. (1) Barros, L. F.; Hermosilla, T.; Castro, J. Comp. 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Instrum. 1970, 41, 909-16. Anal. Chem. 2005, 77, 1290-1294 1290 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005 10.1021/ac048799a CCC: $30.25 © 2005 American Chemical Society Published on Web 01/22/2005