Conductive polymer as a controlled microenvironment for the potentiometric high-throughput

ORIGINAL PAPERConductive polymer as a controlled microenvironment for the potentiometric high-throughput evaluationof ionic liquid cell toxicityWeilian Qiu&Xiangqun ZengReceived:2April2008/Revised:18June2008/Accepted:23June2008/Published online:19July2008#Springer-Verlag2008Abstract This paper presents both biological and potenti-ometric evaluations of the cell toxicity of a widely used ionic liquid,1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4),to Chinese hamster lung fibroblast cells(V79 cell line).The innovative potentiometric study takes advantage of the unique properties of conductive polymer polypyrrole(PPY)for the potentiometric evaluation of cell toxicity of[bmim]BF4to the V79cells in a real-time, noninvasive and high-throughput manner.The conductive polymer PPY provides a controlled microenvironment that allows the quantitative release of the anions of the ionic liquids into the cells being monitored in real time and noninvasively.Parallel biological assay results showed that V79cells exposed to[bmim]BF4usually grew in clusters, and that many small vacuoles could be seen in the cytoplasm.At the24th hour after the V79cells had been exposed to the ionic liquid(IL),the half inhibition concentration(EC50)of[bmim]BF4was around5mM. From a cell cycle study performed using a FACScan flow cytometer,it was found that the V79cells could be partially locked to the G1phase by[bmim]BF4,which extended the doubling time for cell paring with the EC50 values of cadmium chloride and mercury chloride,[bmim] BF4is not very toxic,but it may have a long-term toxic effect on mammalian pared to traditional biological in vitro assays,the use of a conductive polymer substrate in combination with a potentiometric sensor array is much more sensitive,faster,and enables a simpler evaluation of chemical cell toxicity.Additionally,it simplifies the study of the reversibility of cell toxicity, i.e.,cell recovery,because there is no need to refresh the culture medium since a finite amount of chemicals can be doped and released.We found that the cytotoxicity of [bmim]BF4at a concentration of less than6mM was reversible for the V79cell line,because cell morphology and proliferation rate returned to normal after the removal of the IL from the culture medium.This finding suggests that the IL[bmim]BF4could be used as a tool to control mammalian cell proliferation rate.Keywords Conductive polymer.Polypyrrole.Ionic liquid. V79cell.Cytotoxicity.PotentiometryIntroductionGaining a mechanistic understanding of the molecular biology of cells is an active area of research that is fundamental to all of the biological sciences.Many important advances in understanding living cells have directly followed the development of new materials, methods and tools that have opened novel avenues of investigation.The most commonly used tools in cell biology are microscopy(e.g.,light,confocal and electron microscopy)and cell culture,as well as numerous bio-chemistry and molecular biology assays[1].Those systems have had a profound impact on our understanding of the complex biological systems,especially cell biology and neurobiology.Yet,there is an increasing need for new tools that permit fast and controlled investigations of the complex biology and physiology inherent in cellular systems for well-defined scientific studies.Anal Bioanal Chem(2008)392:203–213DOI10.1007/s00216-008-2263-xElectronic supplementary material The online version of this article (doi:10.1007/s00216-008-2263-x)contains supplementary material, which is available to authorized users.W.Qiu:X.Zeng(*)Chemistry Department,Oakland University,Rochester,MI48309,USAe-mail:zeng@In the past decades,electric cell–substrate impedance sensing(ECIS),quartz crystal microbalance(QCM),optical wave guide lightmode spectroscopy(OWLS)and electro-chemical oncoprobes have been shown that can measure cell–substrate interactions quantitatively in a noninvasive and continuous manner[2].Even though these biophysical measurements are very sensitive,they are inherently nonselective.As a result,they provide an overall observa-tion of the cellular changes.In order to identify the precise nature of the signal,it is important to perform studies under well-controlled conditions.One of the important aspects of cell biology is that all cells receive and respond to signals from their surround-ings.It is well known that the transformation of cells in culture by oncogenic agents leads to a wide range of changes in cellular properties,including loss of growth control,altered morphology,increased mobility of surface lectin receptors,and changes in cytoskeletal organization [1].Substantial evidence suggests that alternations in the cell surface may be responsible for many of these observations[3].We demonstrated in this report that a functional conductive polymer biointerface can provide a controlled microenvironment for studying cell–substrate interactions that could significantly empower most current biophysical methods of studying cell biology.The process of transforming a polymer into its conduc-tive form via chemical or electrical oxidation or reduction of the nonconductive form with a variety of electron-accepting or electron-donating species is called doping.The doping and undoping processes have been used to directly incorporate various dopants into inherently conducting polymers for applications in chemical and biosensors, separation,bioreactors,and novel surfaces for cell culturing [4].For example,polypyrrole doped with biologically active molecules(i.e.,heparin)has been successfully used to support human umbilical vein endothelial cell growth [5].Oligonucleotides,salmon sperm DNA and even intact red blood cells have been incorporated directly as dopants. Among many conductive polymers,polypyrrole has been explored in the greatest depth for its biological applications due to its unique physicochemical and special character-istics,such as biological compatibility with cells,stability, durability and elasticity[5–9].In vitro studies by Wong et al.show that conductive polymer PPY is biocompatible and can noninvasively control the shape and growth of mammalian cells[6].PPY has high stability in aqueous solvents,and electrochemical polymerization allows the controlled synthesis and doping/undoping of PPY.There-fore,in this report,electrochemically synthesized conduc-tive polymer PPY was studied as a substrate for cell growth,attachment and toxic chemical incorporation. Potentiometric sensor arrays were used to probe and measure the dynamic changes in the cell–substrate interac-tion(i.e.,cell growth and attachment)during cell transforma-tion in a controlled and systematic manner.Electrochemical signals are manifested at the surfaces of all living cells.Subtle changes in cell behavior,for example those induced by exogenous factors such as cytokines,anticancer drugs,or other agents,result in a change in the electrochemical signal which is indicative of cell activation,suppression or cell detachment and death[10,11].There are two immediate advantages of using conductive PPY substrate for an electrochemical potentiomet-ric sensor array in vitro cytotoxicity assay.First,doped PPY has the properties of a reference electrode;it provides a stable baseline for electrochemical potentiometric monitoring.Sec-ond,doped PPY serves as an ion exchange membrane,so that the effects of the doped ions on cell adhesion and growth can be studied in the controlled microenvironment.We focus on the study of the cell toxicity of ionic liquids,since ionic liquids can be incorporated and released into or from the PPY conductive polymer matrix in a controlled manner.We also compared the IL-doped PPY as a substrate with several control substrates(i.e.,fibronectin,cysteine,and PPY/doped with glutamine)in terms of its cell growth and attachment properties.We first performed in vitro biological toxicity assays to obtain qualitative information on the toxicity of the ionic liquid to Chinese hamster lung fibroblast(V79)cells. Among four imidazolium ILs tested,1-butyl-3-methylimi-dazolium tetrafluoroborate([bmim]BF4)has the highest toxicity to V79cells.Additionally,[bmim]BF4was water-miscible,and the BF4anions are generally stable in the buffered system,such as that using phosphate buffer[21]. Thus,the studies reported here are centered on the cytotoxicity of[bmim]BF4to V79cells.V79cells were chosen for the toxicity assay because they are robust and grow rapidly,so that toxicity data can be obtained in a relatively short time and at a relatively low cost.The cytotoxicity of[bmim]BF4to Chinese hamster lung fibroblast cells(V79line)was studied as a model system to not only prove and validate our technology but also to obtain important information on the toxicity of ionic liquids to mammalian cells.Room-temperature ionic liquids containing organic cations and anions which melt at or close to room temperature have attracted significant interests over the past20years due to their“green”properties.In contrast to conventional organic solvents that are composed of molecular entities such as CH2Cl2and CHCl3,ionic liquids(ILs)have insignificant vapor pres-sures,thus allowing chemical processes to be carried out with essentially zero emission of toxic organic solvents into the air.Consequently,they have been considered to be potentially environmental friendly,recyclable media for synthetic organic chemistry,separation sciences and other chemical sciences[12].However,toxic effects of ionic liquids on enzymes[13],bacteria[14],algae[15],mam-malian cells[16,17],snails[18],zebra fishes[19]and204W.Qiu,X.Zengfreshwater fleas[20],etc.,have been observed.Because many ionic liquids are water-soluble,there is the potential for human beings and animals to be exposed to them during their use and after they have been discarded as chemical wastes.As a result,toxicity studies of ILs are important and are needed since they enable us to predict toxicity among ILs and to perform accurate risk assessment.Studying the responses of V79cells to ILs can provide general cytotoxicity information on imidazolium ILs,because different kinds of mammalian cells have many features in common.1-Alkyl-3-methyl imidazolium salts are the most commonly used ionic liquids.They remain in the liquid state over a broad temperature range and have been shown to be poorly biodegradable[21].In vitro cytotoxicity data for[bmim]BF4could be very useful,since they could provide an example of the ranking of chemicals with similar exposure potentials according to their potential toxicities,and they could possibly confirm other predictions based on considering their chemical structures. Experimental proceduresMaterials The ionic liquid[bmim]BF4was purchased from Fluka(Buchs,Switzerland)with a purity of over98%. Solid24K gold plates were purchased from Hoover& Strong Inc.(Richmond,V A,USA).Ag/AgCl reference electrodes were purchased from CH Instrument Co. (Austin,TX,USA).Poly(styrene sulfonic acid)sodium salt (PS)(MW70,000)was purchased from Alfa Aesar (Karlsruhe,Germany).All other chemicals were purchased from Aldrich(reagent grade;St.Louis,MO,USA)and used as received.Chinese hamster lung fibroblast cell line (V79)was purchased from American Type Culture Collec-tion(ATCC,Manassas,V A,USA).Living cells were stained by Vybrant®DyeCycle™Orange Stain,purchased from Invitrogen Corporation(Carlsbad,CA,USA),and cell cycles were measured with a FACScan flow cytometer (Beckton Dickinson,Franklin Lakes,NJ,USA)and analyzed by the software CellFit®.Electrochemical polymerization of pyrrole and dopant incorporation The doped PPY thin films of various thickness were prepared in100mM pyrrole in various solvents and electrolytes by electrochemical oxidation of pyrrole monomer at a constant current of10μA for200s, 1000s or1500s,respectively.Their thicknesses were about 4μm,20μm and30μm,respectively,assuming that the density of PPY film was1.5g/cm3and the apparent n value was2.25according to the literature[22–24].PPY-PS was formed using0.1M poly(styrene sulfuric acid)sodium salt (PS)as the electrolyte.PPY-IL was prepared in pure [bmim]BF4liquid as the electrolyte.PPY-Glu was prepared from0.1M glutamine aqueous solution as the electrolyte.A three-electrode setup consisting of a gold working electrode(area:0.22cm2),a platinum wire counter electrode and a Ag/AgCl reference electrode was used for the electrochemical synthesis of PPY using an EG&G (Wellesley,MA,USA)283potentiostat.The same three-electrode setup was used in all of the above PPY film formations.Cyclic voltammetry experiments were used to characterize each PPY film made above.We also studied V79cell growth and attachment on three control substrates.In one,the gold electrode was immobi-lized with fibronectin.In another one,the gold electrode was immobilized with cysteine.The gold electrode was cleaned and autoclaved and then immersed in the0.1mg/ mL fibronectin solution or0.05M cysteine aqueous solution overnight.Then the immobilized gold plate electrodes were rinsed with DI water and dried by air until further use for cell culture experiments.The third one was a PPY-PS modified electrode that was further modified by adding various precise volumes of1M[bmim]BF4ethanol solution directly onto the PPY-PS modified gold electrode and dried by air.Electrochemical potentiometric sensor array The potentio-metric sensor array system had four components:a sixteen-channel Lawson(Malvern,PA,USA)EMF-16precision electrochemistry EMF interface instrument,a cell culture incubator and a sixteen-channel micro cell teflon block (Fig.1).Rather than use one reference electrode for the whole sixteen-channel system,we used sixteen Ag/AgCl reference electrodes that were connected together,since each gold plate electrode was assembled in its individual cell.This setup can prevent cross-contamination and minimize the noise.Each Ag/AgCl reference electrode purchased from CH Instruments has two components,an AgCl-coated Ag wire and fritted glass tubing.Since the AgCl film on the Ag wire is not stable after2–3days at37°C in the incubator and can peel off,we removed the AgCl coatings and remade each Ag/AgCl reference electrode for each experiment. Briefly,the Ag wire was immersed into the ammonium hydroxide for five minutes and then rinsed with DI water. The Ag wire was coated with a brown AgCl coating by applying0.2V potential for200s on the Ag wire in0.1M hydrochloric acid(HCl)solution.Then the Ag/AgCl wire was rinsed with DI water and autoclaved by Tuttnauer 2450M autoclave steam sterilizer(Brinkmann Inc.,West-bury,NY,USA)at a temperature of121°C for60min. Autoclaving the reference electrode components helps to prevent any biocontamination for the freshly reassembled Ag/AgCl reference electrode.Since the stability of the reference electrode is critical for the accurate measurement of the indicator electrode potential,we tested Ag/AgCl electrodes with various filling solutions.Our results showConductive polymer as a controlled microenvironment205that Ag/AgCl with PBS buffer as the filling solution provides a highly stable potential for an extended period of time.The fritted glass tubing was filled with PBS buffer as internal solution rather than the saturated potassium chloride (KCl).PBS internal solution not only provides a constant chloride concentration but it also prevents the formation of a liquid junction potential when the reference electrode is used in the culture medium.The fritted glass tubing was also autoclaved following the same procedure as above.Finally,the Ag/AgCl reference electrode was reassembled in the laminar flow hood with care.The freshly assembled reference electrode was then soaked in the DMEM cell culture medium overnight before being used for the cell culture experiments.This procedure enables the freshly assembled reference electrode to reach a stable potential baseline.The above procedures were repeated after each use of the Ag/AgCl reference electrode for cell culture experiments.The electrochemical potential of the substrate electrode was measured against this Ag/AgCl reference electrode.Each gold plate electrode was cleaned with freshly mixed concentrated sulfuric acid and 20%hydrogen peroxide solution (3:1V/V).Then it was mounted onto the Teflon cell block.The whole sensor array on the Teflon block was then autoclaved.After that,each gold plate electrode was modified with various substrate films.After the completion of all of the above steps,the 16-channel array block was put into the incubator and connected with the EMF 16Lawson analyzer,and then it was ready for cell culture.The Lawson software allows the potentials of the16potentiometric sensors to be monitored simultaneously in real time.Cell culture for biological assays V79cells (105<N 0<1.5×106)inoculated in a T25or T75flask were incubated in an incubator at 37°C containing 5%CO 2for pH and humidity control.Dulbecco ’s modified Eagle ’s medium (DMEM;ATCC,Manassas,V A,USA),used for the cell culture,was supplemented with 5%fetal bovine serum (FBS,Sigma,St.Louis,MO,USA).At harvest time (22h<t <80h),cell suspensions were prepared using 0.25%Trypsin/0.53mM EDTA in Hanks Balanced Salt Solution without calcium or magnesium (ATCC,Manassas,V A,USA).Cell viability was assessed using trypan blue exclusion technique,and the cell number N (t )was counted with a hemocytometer under an inverted phase contrast microscope (Nikon TMS,Tokyo,Japan).Counting cells of V79subjected to [bmim]BF 4Usually cells are counted by hemocytometers,but cell culture processes must be interrupted.We used a digital imaging technique to count cells continually during cell culture without interrupting the cell culture.Ionic liquid [bmim]BF 4at 100mM was prepared in culture medium (DMEM +5%FBS)as a stock solution.In a batch toxicity assay,two six-well cell culture clusters (Corning,New York,USA)were used for cell culture.Each well bottom had an area of 9.5cm 2.A cross mark (“+”)was drawn on the bottom of each well (outside)to mark where photos should be taken.[bmim]BF 4stock solutions of 0,20,40,Fig.1Schematic diagram of the potentiometric sensor array system setup206W.Qiu,X.Zeng60,80,100and120μL were injected into the seven wells of the two plates,respectively,and then culture mediums of 1000,980,960,940,920,900and880μL were injected into these wells,respectively.At time t=0,105/mL×1mL V79cell suspension was added to each of the seven wells, so that the final[bmim]BF4concentrations in these wells were0,1,2…6mM respectively,and the cell number per square millimeter was around50.From t=3h and then in at about every12h interval,each well was put under the inverse microscope with a digital camera(SPOT,1600×1200pixels,Diagnostic Instrument A)in order to take four photos at the four corners of the“+”mark of the bottom of each well.The plates were then taken back to the incubator as soon as possible.Since each photo only covered1175×880μm2(∼1mm2)of the bottom of a well, even with the cross marks it was difficult to take photos at exactly the same location but at different times during cell culture.Photos were taken at randomly chosen locations close to the corners of each cross mark.The number of cells in each photo were counted manually on the computer screen[25].The cell numbers in the four photos of a well were added to N(t,C),where t was the time for which the V79cells were exposed to the ionic liquid with molar concentration C.A mathematical model of the cell growth and the toxicity assay Normal V79cells proliferate exponentially according to the equation:N tðÞ¼N02tÀt T t!tðÞð1Þwhere T is the doubling time of the cell line andτis the time taken to go from the inoculation process to normal cell growth.In each passage,the number of cells initially N0 and the number of cells harvested at time t could be directly measured using a ing N0and N(t),T and τcould be directly obtained from the slope and the intercept of the linear relationship between ln(N(t)/N0)and t.lnN tðÞN0¼ln2ðÞTtÀln2ðÞTt t!tðÞð2ÞWhen the cells are exposed to a chemical with aconcentration C,both T andτcan change with the concentration of the chemical,so the cell number N(C,t) will be less than the cell number of the control N(t).The half-inhibition concentration of[bmim]BF4for the V79is defined as EC50:1ÀN EC50;tðÞ=N EC50;0ðÞN tðÞ=N0ðÞð3ÞThe relationship between EC50and the time t showed thedynamic cytotoxicity of[bmim]BF4to V79.According toEq.2,the doubling time T of V79cells subjected to[bmim]BF4at concentration C could be obtained from the slope ofthe linear relationship between ln(N(t))and time t bycounting cell numbers in photos at different times.Therelationship between T and C also showed the cytotoxicityof[bmim]BF4to the V79cell line.Results and disscussionThe goal of this work is to demonstrate and validate thatconductive polymers can be used as a controlled microen-vironment for the in vitro potentiometric high-throughputevaluation of ionic liquid cell toxicity.Since few ionicliquid cell toxicity studies have been reported in theliterature,we first performed a systematic study of ionicliquid toxicity using various traditional biological assays.The traditional biological assays help us to understand theionic liquid cell toxicity and provide qualitative informationthat can be used to validate our later potentiometric sensorarray results.Reversible toxicity of[bmim]BF4The toxicity of[bmim]BF4to the V79cell line was first studied using variousbiological assays.To be able to use the doubling time as anindex for cytotoxicity assay,we must first know themultiplication rate of the cell line under normal conditions.Figure2shows the exponential relationship between N(t)/N0and time obtained from twelve passages.At thebeginning of each passage(t=0),an initial number ofV79cells N0was inoculated into a flask;at time t,the cellswere harvested and counted as N(t).The inset of Fig.1shows a linear relationship between ln(N(t)/N0)and time:ln N tðÞN0¼0:0535tÀ0:151;the coefficient of determi-20304050607080Time (h)N/NoFig.2Curve of the ratio of the number of harvested cells to the numberof initial cells plotted against time.Normal V79grew exponentially.Theinset shows the linear relationship between ln(N(t)/N0)and timeConductive polymer as a controlled microenvironment207nation (r 2)of the linear relationship is >97%.From the slope and the intercept of the equation,we found that the doubling time of V79was 13.0h and the recovery time was 2.8h.ATCC reports that the doubling time of V79is between 12and 14h.Our measurement of the V79doubling time agreed well with the parameter from ATCC (the cell line provider).The recovery time of 2.8h was also close to the previous measurement (2.7h)[25].The relationships between the cell number (N (t ))and cell culture time with and without the presence of 4mM [bmim]BF 4are shown in Fig.3.The solid curve with the diamond symbols was obtained for cells that grew in the complete culture medium.The solid curve with cross symbols was obtained for cells that were grown in culture mediums in which 4mM [bmim]BF 4was present.The cell growth rate was significantly reduced when they were exposed to 4mM [bmim]BF 4.At t =48h (points A and A ′in Fig.3),the wells with 4mM [bmim]BF 4in the culture medium were washed and refilled with 2mL of the complete culture medium without any [bmim]BF 4,and the cells were then cultured for an additional 24hours.The inset in Fig.3showed that ln(N )had a linear relationship to time.As shown in Eq.2,we can obtain the doubling time of the cell from the slope of the straight line.The V79cell doubling times for the control and the cells exposed to 4mM [bmim]BF 4were 12.2h and 27.5±1.3h,respectively.According to a t -test performed by setting α=0.05,the difference between these two doubling times was very significant.The [bmim]BF 4at a concentration of 4mM decreased the proliferation rate of the V79cells and was toxic to them.When the [bmim]BF 4was removed (at t =48–72h)and the V79cells were grown in pure culture medium,their doubling time decreased to 13.0±0.7h,which was not significantly different from 12.2h,as verified by a t -test.Consequently,the toxicity of the 4mM [bmim]BF 4to V79cells was observed to be reversible.The most striking toxic effect of [bmim]BF 4observed was the change in the morphology of the V79cells.At the beginning of the inoculation,the V79cells floated in the culture medium.They appeared as round dots under an optical ter,the cells attached to and spread over the bottom of the flask.Normal V79cells are usually triangular in shape or rhombic.The length of a normal V79cell is around 120μm,and the width of these cells is around 30μm.Under an optical microscope,many small vacuoles could be seen in the cytoplasm of V79cells exposed to [bmim]BF 4.These cells were usually present in clusters.According to Pretti ’s report [19],some ionic liquids produce intracytoplasmic vacuolation in zebrafish skin cells,and they are erosive to fish ’s skin and gill cells.Figure 4shows three photos taken at the same location at the bottom of a well at different times.Each of the photos is only a small part of a bigger photo covering 1mm 2.The white oval-shaped object with a black core in the lower left corner of each photo is a defect of the plastic Petri dish cluster.This defect was used as a marker to find the same location at the bottom of the well.At t =3h,two V79cells could be seen in photo A in Fig.4.At that moment they had touched the bottom of the well but had not spread,so they looked round in shape.The two cells in photo A that were subjected to 4mM [bmim]BF 4became two clusters of cells at t =36h,as shown in photo B,Fig.4.One cluster had eight cells and another had just two cells.Small vacuoles were seen in the cytoplasma of cells exposed to the [bmim]BF 4,but normal V79cells did not have these vacuoles.The toxic effect of the [bmim]BF 4could usually only be discerned from the cell morphology changes after the cells were exposed to the [bmim]BF 4for 12h or longer.At t =48h,the culture medium with [bmim]BF 4was removed and washed,and then the wells were refilled with complete culture medium without the [bmim]BF 4.The vacuoles inside the V79cells disappeared after those cells were grown in the complete culture medium without [bmim]BF 4for an additional 12hours.Photo C shows the situation 24h after the culture medium had been changed;here,the cell morphology has returned to normal:cells are separated from each other and no vacuoles appear to be present inside the cells now.This result again shows that the toxicity of the 4mM [bmim]BF 4to V79is reversible.Tuning the V79doubling time using [bmim]BF 4A biolog-ical assay was also performed to study the effect of the IL concentration on the V79cell line growth rate.According244872Time (H)01 0 0 2 0 0 3 0 0 4 0 0 N (t )Fig.3Number of cells as a function of time for cells exposed in thecomplete culture medium (curve a ),and those that were initially incubated in the culture medium with 4mM [bmim]BF 4(the first part of curve b before point A )and then exposed to fresh culture medium (the second part of curve b after point A ).Curve c and curve d in the inset show the natural log of the cell number as a function of time,and correspond to curve a and curve b ,respectively.The points A and A ′indicate the times at which the cells were placed in fresh culture medium without [bmim]BF 4after 48hours of exposure to the culture medium with [bmim]BF 4208W.Qiu,X.Zeng。