Research Article Explorations of ABO-Rh antigen expressions on erythrocyte dielectrophoresis: Changes in cross-over frequency A quadrupole dielectrophoretic microdevice was utilized to examine the ABO-Rh dependencies on erythrocyte polarizations. This important step toward medical micro- device technology would transform key clinical blood tests from the laboratory into the field. Previous work in dielectrophoretic microdevices demonstrated that the large number of ABO antigens on erythrocyte membranes impacts their dielectrophoretic signature at 1 MHz. This work explores the dielectrophoretic behavior of native human erythrocytes categorized by their ABO-Rh blood types and directly compares these responses to the same erythrocyte sample modified to remove the A and B antigens. A b(1–3)-galactosidase enzyme was utilized to cleave the ABO polysaccharide backbone at the galactosidase bonds. The enzymatic reaction was optimized by comparing aggluti- nation of the native and modified blood cells in addition to UV–Vis and HPLC analysis of the reaction effluent for saccharide residues. Next, the dielectrophoretic behaviors of the native and modified erythrocytes were visually verified in a quadrupole electrode microdevice over a frequency range from 100 kHz to 80 MHz. The lower cross-over frequency (COF), which transitions from negative to positive dielectrophoresis, for ABO blood types tested (A1,A,B1,B, AB1,O1 and O) differed over the range from 17 to 47 MHz. The COFs of the corresponding enzyme-modified erythrocytes were also determined and the range narrowed to 29–41 MHz. A second COF in the 70–80 MHz range was observed and was reduced in the presence of the transmembrane Rhesus factor. These results suggest that antigen expression on erythrocyte membrane surfaces influence cell polarizations in nonuniform AC fields. Keywords: ABO-Rh / Antigen modification / Cross-over frequency / Dielectrophoresis / Erythrocytes DOI 10.1002/elps.201100077 1 Introduction 1.1 Dielectrophoresis Dielectrophoresis (DEP) has been shown to distinguish native and altered cells based on infection, live/dead status, and treatments with membrane alterations [1–6]. Cells infected with malaria were separated from healthy cells due to changes induced in the host cell by the parasite. An additional cytosol and membrane develop within the host cell changing the permittivity and conductivity thus leading to a change in Clausius–Mossotti (CM) factor and dielec- trophoretic force [4]. Healthy blood cells were shown to have a different cross-over frequency (COF) than HL-60 cells and T-lymphocytes as well as different electrorotation spectra [1]. It has also been seen that erythrocytes infected with Babesia bovis virus have significantly different dielectrophoretic responses than cells without viral infection [2]. Live versus dead yeast cells also exhibit different COFs especially as medium conductivity increases. Non-viable yeast cells were observed to have a much higher low COF and a much lower high COF than viable cells [3]. Finally, electroporated cells were separated from non-electroporated cells at a medium conductivity of 0.174 S/m and a frequency of 2 MHz [6]. Our group has focused on the dielectrophoretic responses of erythrocytes categorized by their ABO-Rh antigens. Erythrocytes are a unique system because both surface (ABO) and transmembrane (Rh) antigens are expressed in combinations with each other; further the cells demonstrate genetic diversity via a plethora of other anti- gens. The ABO-Rh antigen’s influence on dielectrophoretic polarizability is more complex than the present/not present differences demonstrated in Refs. [1–6]. Prior results in a Kaela M. Leonard Adrienne R. Minerick Chemical Engineering Department, Michigan Technological University, Houghton, MI, USA Received January 26, 2011 Revised June 9, 2011 Accepted June 9, 2011 Colour Online: See the article online to view Figs. 1,2 and 6 in colour. Abbreviations: AC, alternating current; AMT, ammonium transporters; CM, Clausius–Mossotti factor; COF, cross-over frequency; DEP, dielectrophoresis; nDEP, negative dielectrophoresis; pDEP, positive dielectrophoresis Correspondence: Dr. Adrienne R. Minerick, 202H Chemical Sciences & Engineering, 1400 Townsend Drive, Houghton, MI 49931, USA E-mail: minerick@mtu.edu Fax: 11-906-487-3213 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2011, 32, 2512–2522 2512