The feasibilities of the Kelvin probe mode were shown to estimate a surface potential of lympho  cytes from the patients with leukemia. Having compared the values of the surface potential obtained by the traditional method of microelectrophoresis and by the Kelvin probe mode, it was established that the use of the atomic force microscope made it possible to obtain data which were of specified diagnostic importance while studying lymphocyte functional status at different stages in leucopoiesis and indicated progression of the pathological proces

Skorkina, M. Yu.

Sladkova, E. A.

DSpace at Belgorod State University

ISSN 00063509, Biophysics, 2014, Vol. 59, No. 2, pp. 254–256. © Pleiades Publishing, Inc., 2014.Original Russian Text © E.A. Sladkova, M.Yu. Skorkina, 2014, published in Biofizika, 2014, Vol. 59, No. 2, pp. 310–313.254INTRODUCTIONBioelectric processes exert substantial influence onthe activity of physiological systems [1]. On the cellular level they are realized at the expense of work of ionchannels of the membrane, participating in the formation of surface potential. The electric system of the cellplays the key role in the functioning of its receptors[2], maintenance of a state of equilibrium betweenintegrative physiological processes: proliferation, differentiation and programmed cell death [3]. In aseries of experimental works it is shown that achange of membrane surface potential is characteristic of activation of lymphocytes and may be immediately connected with processes of their blasttransformation [4].For studying bioelectric processes in single cells,use is made of a direct method of measuring the surface potential with the aid of a microelectrode technique, which is connected with certain methodicaldifficulties and great time expenditure [5]. Awhole series of advantages is possessed by optical indicators – probes reacting to changes of membranepotential by different fluorescence intensity of shiftingof the spectral maximum [5, 6]. The disadvantages ofsuch probes come to be their nonspecific binding withmembranes structures of the cell, fading, autofluorescence [7]. One of the promising methods that mayreplace the microelectrode technique and fluorescentprobes is presented by the Kelvin probe mode used inatomic force microscopy (AFM) technologies [8].The aim of the present work was to evaluate the surface potential of lymphocytes from leukemia patientsby the Kelvin probe mode.EXPERIMENTALInvestigations were executed on the basis of theChair of anatomy and physiology of living organisms,Belgorod National Research University. The object ofinvestigation was served by peripheral blood lymphocytes of patients with acute lymphoblastic leukemia,chronic lymphoblastic leukemia, acute myeloblasticleukemia and acute lymphoblastic leukemia in remission, 15 persons in each group. In the quality of control we used lymphocytes of 50 healthy donors.Preparation of samples of blood cells for investigation was conducted by the following scheme: bloodwas centrifuged 10 min at 1500 rpm, collecting thelower part of plasma rich in leukocytes and the leukocyte ring. Leukocytes were twice washed with isotonicbuffer solution (Dulbecco solution pH 7.4). A leukocyte suspension for measuring the surface potentialwas prepared by the following means (patentNo. 2027188) [9]. Blood white cells were washed withisotonic solution of sodium chloride for 5 min, thenfixation was conducted with 0.25% solution of glutaricaldehyde for 20 min. When the indicated time elapsed,the suspension of leukocytes was twice washed withisotonic solution of sodium chloride for 5 min eachand preparations were prepared on defatted metal support.Measurement of surface potential was actualizedon an AFM “INTEGRA Vita” (configuration on thebase of inverted optical microscope Olympus IX71)in the Kelvin probe mode, using a cantilever with currentconducting titanium coating of seriesNSG03/TiN (Nanoworld, USA). The currentlyapplied Kelvin probe mode is based on a twopass proCELL BIOPHYSICSEstimation of Surface Potential of Lymphocytes from Patients with Leukemia Using Kelvin Probe ModeE. A. Sladkova and M. Yu. SkorkinaBelgorod State National Research University, Belgorod, 308015 Russiaemail: evgenijasladkova00@rambler.ruReceived January 9, 2013; in final form, November 25, 2013Abstract—The feasibilities of the Kelvin probe mode were shown to estimate a surface potential of lymphocytes from the patients with leukemia. Having compared the values of the surface potential obtained by thetraditional method of microelectrophoresis and by the Kelvin probe mode, it was established that the use ofthe atomic force microscope made it possible to obtain data which were of specified diagnostic importancewhile studying lymphocyte functional status at different stages in leucopoiesis and indicated progression ofthe pathological process.Keywords: surface potential, lymphocytes, leukemia, atomic force microscopyDOI: 10.1134/S0006350914020250BIOPHYSICS  Vol. 59  No. 2  2014ESTIMATION OF SURFACE POTENTIAL USING KELVIN PROBE MODE 255tocol. In the first pass, the relief of sample surface isdetermined with the use of halfcontact mode of AFMscanning. In the second pass this relief is tracked during passing the probe over the sample at some heightfor determining the surface electric potential (see figure) [10] with consequent processing of the scans inthe Nova program (Zelenograd, Russia).For assessing the reliability of the new method inthe conducted investigation we collated the magnitudes of surface potential of lymphocytes from healthydonors measured by the Kelvin probe mode and thecommonly known method of cell microelectrophoresis in a horizontal microchamber [11]. Electrophoresiswas actualized in phosphate buffer solution, pH 7.4 ata dilution of lymphocyte suspension 1 : 800. In thebasis of the electrophoretic method there lies measurement of the velocity of mobility of blood cells in anelectric field with subsequent calculation of ξpotential by the Helmholtz–Smoluchowski formula [12].We have measured the ξpotential of lymphocytes of10 healthy donors (50 cells in every sample). Statisticaltreatment of the obtained results was actualized withthe aid of the Student’s ttest.RESULTS AND DISCUSSIONIn a preliminary series of tests we have measuredwith the aid of microelectrophoresis the magnitudes ofsurface potential of lymphocytes from healthy donors(–47.43 ± 2.31 mV). They turned out to be close tomagnitudes (–46.14 ± 6.42 mV) measured with theKelvin probe mode (see Table 1). In this way, the values of surface potential obtained with application ofAFM can be deemed adequate.By the Kelvin probe mode it is established that forlymphocytes of patients with various forms of leukemia, characteristic is an increase of surface potential,and consequently, of hyperpolarization of the cellmembrane. For lymphocytes of acute lymphoblasticleukemia patients, characteristic was the greatestincrease in surface potential, while for cells of acutemyeloblastic leukemia patients – the smallest as compared with the values of surface potential of the lymphocytes of healthy donors (Table 2). The revealedpeculiarity is consistent with literature data and, probably, comes as a result of growth of K+conductivityduring blasttransformation of lymphocytes [4]. Thedistinction of values of the surface potential off cellsfrom patients with various forms of leukemia is connected with peculiarities of the course of disease.Probably, growth of the surface potential of lymphocytes from acute myeloblastic leukemia patientscomes as a consequence of stimulatory impact oftumor factors released by transformed cells of themyeloid lineage [13, 14].µm151050 20 25 30 35 40 45µm(a)15105µm151050 20 25 30 35 40 45µm(b)15105mV2000–200A (40.1, 6.3, –11)(a) Scan of lymphocytes (first pass), (b) distribution of surface potential of the surface of lymphocytes (second pass).256BIOPHYSICS  Vol. 59  No. 2  2014SLADKOVA and SKORKINAIn this way, the simplicity of technical execution,reduction to an minimum of factors capable of lowering the reliability of obtained results, analysis of a largemass of cell at one time and the possibility of evaluating the surface potential of a single cell allow supposing that the Kelvin probe mode may find broad application in experimental biological investigations in thequality of indicator of the degree of expression of apathological process during investigation of the functional status of lymphocytes at different step of leukemigenesis.ACKNOWLEDGMENTSThe work was supported by the Ministry of Education and Science of the Russian Foundation, agreement 14.132.21.1320.REFERENCES1. A. A. Yarygin, Patol. Fisiol. Eksperim. Terapiya 2, 38(1998).2. O. V. Tarnopol’skaya, Candidate’s Dissertation inPhysics & Math (MGU, Moscow, 1994).3. M. F. Nikonova, M. M. Litvina, M. I. Varfolomeeva,et al., Immunologiya 2, 20 (1999).4. A. A. Vereninov, E. V. Gusev, O. M. Kazakova, et al.,Tsitologiya 33 (11), 78 (1991).5. N. S. Kavok, M. Yu. Malyukina, I. A. Borovoi, andN. L. Pogrebnyak, Vestn. Biofiziki 21 (2), 45 (2008).6. L. V. Johuson, M. L. Walsh, and L. B. Chen, Proc. Natl.Acad. Sci. USA 77, 990 (1980).7. S. Salvioli and A. Ardizroni, FEBS Lett., 77–82 (1997).8. D. V. Allison, N. P. Mortensen, K. J. Sulivan, andM. J. Doktycz, Adv. Rev. 2, 619 (2010).9. Yu. A. Sheremetev, G. I. Makin, and F. Yu. Suslov, RFPatent 2027188 G01N33/49. Appl. No. 4947820/14 of26.04.1991.10. User Manual “Probe Nanolaboratory Integra Vita”(NTMDT, Zelenograd, 2006).11. S. G. Karasev, O. V. D’yachenko, O. V. Popova, et al.,Klin. Lab. Diagnost. 11, 16 (1997).12. G. M. Stolyar, Vopr. Eksperim. Biofiz. 6, 117 (1975).13. O. V. Lebedinskaya, Doctoral Dissertation in Medicine(St. Petersburg, 2006).14. O. V. Lebedinskaya, Sovrem. Naukoemkie Tekhnol. 7,49 (2005).Table 1. Magnitudes of surface potential of lymphocytes measured by method of microelectrophoresis and in the Kelvinprobe modeMeasured values Method of microelectrophoresis Kelvin probe modeSurface potential, mV –47.43 ± 2.31 –46.14 ± 6.42Time of lymphocyte motion, s 60.00 –Distance passed by lymphocytes, µm 4.31 ± 0.52 –Table 2. Surface potential of peripheral blood lymphocytesExperimental group Surface potential, mVHealthy donors –46.13 ± 6.41Patients with acute lymphoblastic leukemia –17.72 ± 1.64*Patients with chronic lymphoblastic leukemia –27.63 ± 1.17*, **Patients with acute myeloblastic eukemia –34.72 ± 2.37*, **, ***Patients with acute lymphoblastic leukemia in remissiona –39.91 ± 3.10**** Statistically reliable differences of surface potential values compared with healthy donors by Student’s test at p < 0.05, ** statisticallyreliable differences of values compared with acute lymphoblastic leukemia patients by Student’s test at p < 0.05, *** statistically reliable differences of values compared with chronic lymphoblastic leukemia patients by Student’s test at p < 0.05.

Estimation of surface potential of lymphocytes from patients with leukemia using kelvin probe mode

E. A. Sladkova

M. Yu. Skorkina

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Estimation of surface potential of lymphocytes from patients with leukemia using Kelvin probe mode

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Estimation of surface potential of lymphocytes from patients with leukemia using kelvin probe mode

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