Analytica Chimica Acta 857 (2015) 39–45

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Analytica Chimica Acta

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Droplet electrochemical study of the pH dependent redox behavior ofnovel ferrocenyl-carborane derivatives and its application in specificcancer cell recognition

Changyu Wua, Afzal Shah c, Hongde Ye b, Xiao Chen a, Jing Ye a, Hui Jiang a,Baoan Chen d, Xuemei Wang a,*, Hong Yan b,*a State Key Laboratory of Bioelectronics (Chien-Shiung Wu Laboratory), Southeast University, Nanjing 210096, PR Chinab State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, PR ChinacDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320, PakistandDepartment of Hematology, the Affiliated Zhongda Hospital, Clinical Medical School, Southeast University, Nanjing 210009, PR China

H I G H L I G H T S

* Corresponding authors. Tel.: +86 25 83792177; faE-mail addresses: xuewang@seu.edu.cn (X. Wang)

(H. Yan).

http://dx.doi.org/10.1016/j.aca.2014.12.0190003-2670/ã 2014 Elsevier B.V. All rights reserved.

G R A P H I C A L A B S T R A C T

� Electrochemical behaviors of novelferrocenyl based carboranes (FcCB)were exploredwith a droplet system.

� The shifts of peak potentials withchanges of pH values indicated theinvolvement of proton during elec-tron transfer reaction.

� Normal cells and cancer cells couldbe specifically recognized by usingFcCB as probe.

� This electrochemical method in adroplet shows great potential appli-cation for relevant diagnostics ofclinical samples.

A R T I C L E I N F O

Article history:

Received 5 November 2014Received in revised form 5 December 2014Accepted 9 December 2014Available online 16 December 2014

Keywords:Ferrocenyl based carboranesDroplet electrochemistryLeukemiaCancer cell recognition

A B S T R A C T

Novel ferrocenyl based carboranes (FcCBs) and their distinguish behavior for cancer cell recognition havebeen explored in this contribution. The voltammetric study in a droplet of 10mL placed on the surface of aglassy carbon electrode demonstrates the excellent electrochemical behavior of FcCBs, which could befurther exploited for establishing the promising and sensitive biosensors. The FcCBs’ redox behavior isexamined in a wide pH range, and square wave voltammetry revealed the reversible and irreversiblenature of first and second anodic peaks. The obvious shifts in peak potentials corresponding with thechange of pH values demonstrate the abstraction of electrons to be accompanied with the transfer ofprotons. By using the droplet electrochemical technique, FcCBs can be employed to distinguish normaland cancer cells with a linear range from 1.0�103 to 3.0�104 cellsmL�1 and the limit of detection at800 cellsmL�1. The novel carborane derivatives could be utilized as important potentialmolecular probesfor specific recognition of cancer cells like leukemia cells from normal cells.

ã 2014 Elsevier B.V. All rights reserved.

x: +86 25 83792177., hyan1965@nju.edu.cn

[(Scheme_1)TD$FIG]

Scheme 1. Chemical structures of FcCB (A) and Fc2CB (B).

Table 1Compositions of pH buffers with 0.1M ionic strength.

pH value Composition

3.0 NaAcO–HAcO4.0 NaAcO–HAcO5.0 NaAcO–HAcO6.0 NaAcO–HAcO7.0 Na2HPO4–HCl8.0 Na2HPO4–HCl9.0 Na2HPO4–HCl

10.0 Na2HPO4–NaOH11.0 Na2HPO4–NaOH

40 C. Wu et al. / Analytica Chimica Acta 857 (2015) 39–45

1. Introduction

Recently scientistshavepaidmuchattention to the synthesis andapplication of organometallic compounds in biomedical field due tothe success of cisplatin in chemotherapy [1–3]. Ferrocene is the firstknown prototypical metallocene and has unique structure, robust-ness, and redox property. Its numerous derivatives have beenendowed with broad range activities, such as anticancer [4–6],antimalarial [5,7], antibacterial [8], antiviral [9], anti-inflammatory[10] agents,andcentralnervoussystemactivedrugs[11].Carboraneshave been used as a source of neutron in boron neutron capturetherapy (BCNT) for their high boron content and stability tocatabolism[12]. Carboraneshavealsobeenreported tobeapotentialpharmacophore. It allows the bioisosteric replacement for phenylrings, adamantine or steroid structures as rigid scaffolding inbioactive molecules and pharmacological agents [8,13–15].

Electrochemical techniques are simple, fast, highly sensitive andpowerful tools for the determination of physicochemical param-eters, understanding redox mechanism and quantitative analysis ofelectroactive compounds [16]. Electrochemical methods are furtherutilized as a promising diagnostic approach in clinical analysis[17,18]. Usually, electrochemical cytosensors are based on thechanges incurrentor resistanceatcell–electrodeinterface.However,current and impedance based cytosensors are much more sensitiveto and easily affected by cell concentrations, incubation time andother environmental factors [19]. Based on these observations, wehave developed a potential based cytosensor to overcome thesedisadvantages for cell specific identification [20,21].

Droplet electrochemical technique provides more detailedinformation comparedwith conventional large-scale electrochem-ical systems, and only small volumes of electrolyte are required[22]. Usually, the working, reference and counter electrodes areemployed by touching onto or immersing within the droplet.Moreover, the droplet can also be placed onto the surface ofinvertedworking electrodewhile reference and counter electrodesare ahead of it [23]. The electrochemistry carried out in a droplethas achieved great success in analytical application, while themostfamous application is in self-monitoring of blood glucose [24,25].In addition, droplet electrochemistry has also been widely used incorrosion [22], capillary detection [22,26], theoretical study[23,27] and cell analysis [28]. In our previous work, dropletelectrochemistry combined with contact angle has been employedto study biological molecules and cells [20,29].

In this study, we have designed and synthesized the electro-chemical active ferrocenyl based carboranes, abbreviated as FcCBand Fc2CB, by taking the advantages of ferrocenyl and carboranes.We have further investigated the electrochemical properties ofthese compounds in a droplet system. Meanwhile, we haveexplored the potential application of these novel ferrocenyl basedcarboranes in specific recognition of cancer cells for monitoringrelevant biological process or treatment. The results demonstratethat cancer cells like leukemia cells and normal cells could bereadily distinguished through the relevant electrochemical probe.This raises the possibility for the rapid and highly sensitive analysisof clinical samples by using electrochemistry in a droplet systemand thus monitoring the process of cancer diagnosis and therapy.

2. Experimental section

2.1. Materials and reagents

FcCB and Fc2CB (Scheme 1) were dissolved in ethanol and storedin freezer as stock solutions. All supporting electrolyte buffers(Table 1) were freshly prepared using analytical grade reagents anddeionized water from Milli-Q water purification system (Millipore,USA). The desired concentration of FcCB orFc2CB was obtained by

diluting the stock solutions with buffers of known pH values.Lymphocyte separation medium (human) was purchased fromSinopharmChemicalReagentCo., Ltd. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)was bought fromAmresco,USA. Other reagents were of analytical grade.

2.2. Isolation of leucocytes

Leucocyteswere isolated fromperipheral blood by using Ficoll–Hypaque gradient method [30]. Briefly, lymphocyte separationmediumwas added to EDTA-anticoagulated blood diluted with anequal volume of PBS and centrifuged 15min at 2000 rpm 20 �C. Theupper layer containing the plasma and most of the remaining cell,platelet fraction was removed. The lymphocyte cell layer waswashed with PBS twice to remove the supernatant and anyremaining platelets. The relevant studywas approved by the EthicsCommittee of Southeast University, Nanjing, China and adhered tothe Declaration of Helsinki.

2.3. Equipment and measurement

All electrochemical experiments were performed in a droplet(10mL) by utilizing CHI 660B electrochemical workstation (CHInstruments Inc., USA) with a glassy carbon electrode (K 3mm) asthe working electrode, a platinumwire as the counter and a silverwire as a quasi-reference electrode [16,31]. CAM 200 contact angle(KSV Instrument Ltd., Finland) with video camera was applied tomonitor the droplet (Fig. 1). The UV–vis absorption spectra wererecorded on BioMate 3S UV–vis spectrophotometer (ThermoFisher Scientific Inc., USA). The pH values were adjusted by PHS-3C pH meter (Leici Corporation, China).

[(Fig._1)TD$FIG]

Fig. 1. Scheme of relevant droplet electrochemical system for investigation of new ferrocenyl-carborane derivative. Right corner: images of droplet electrochemical system,recorded by contact angle video camera.Working electrodewas placed at the bottom; counter electrode and reference electrodewere immersed into the electrolyte droplet.

[(Fig._3)TD$FIG]

C. Wu et al. / Analytica Chimica Acta 857 (2015) 39–45 41

Before each experiment, the surface of glassy carbon electrodewas polished with alumina powder (50nm in diameter) followedby thoroughly rinsingwith doubly distilled water. For reproducibleexperimental results, the clean glassy carbon electrode was placedin buffers and various squarewave voltammogramswere recordeduntil the achievement of steady state baseline voltammogram.

The experimental parameters for square wave voltammetrywere 25Hz frequency and 4mV potential increments correspond-ing to an effective scan rate of 100mVs�1. Differential plusvoltammetry was performed at a scan rate of 5mVs�1. For thepolarity of currents, cathodic currents were taken as positive [16].

The cells were re-suspended with PBS to a certain concentra-tion. FcCB solution (0.5mL, 5mM) was added to 10mL cellsuspension incubated for a period of time (5, 15, 30 and 60min)at room temperature. Then the electrochemical behaviors of themixturewere investigatedwith differential pulse voltammetry in adroplet system. Different cell concentration (1000, 5000, 30,000,150,000 and 700,000 cellmL�1) was also investigated to evaluatethe effects of cell concentrations and to determine the limit ofdetection and linear range for cancer cells.

[(Fig._2)TD$FIG]

Fig. 2. CVs of 1.0mM FcCB in pH 4.0 (—), 7.0 (- - -) and 10.0 (� � �) buffers at glassycarbon electrode at 100mVs�1. Inlets show the peak potentials and currents atvarying pH.

2.4. Data processing and analysis

The differential pulse and square wave voltammograms werebaseline-fitted and subtracted by using the “Baseline Fitting andSubtraction” function in CHI 660B software. The baseline was fitted

Fig. 3. (A) The net (—), forward (- - -) and reverse (� � �) SWV of 1.0mM FcCB in pH7.0 buffer on glassy carbon electrode at 100mVs�1. (B) SWV of 1.0mM FcCB in 0.1Mphosphate buffer (pH 7.0): five scans (from a to e) on glassy carbon electrode at100mVs�1. The inlet shows the percentage of decrease in the peak currents.

42 C. Wu et al. / Analytica Chimica Acta 857 (2015) 39–45

with orthogonal least squares baseline fitting algorithm and in ninefittingorders.However, thevalues forpeakcurrents inallfigures andtables were calculated from the original voltammograms.

Statistical analyses were carried out using IBM SPSS Statisticsversion 21. One-way ANOVA was used for significance testing andP<0.05 is considered as statistically significant difference.

3. Results and discussion

3.1. Electrochemical behavior of new ferrocenyl-carborane derivative

3.1.1. Cyclic voltammetryThe electrochemical behavior of FcCB was initially investigated

by cyclic voltammetry (CV) at a glassy carbon electrode in bufferswith different pH values (Fig. 2). Peak potentials had shifts withchanges of pH values. It indicated that proton was involved duringthe electron transfer reaction. As shown in Fig. 2, in acidicconditions of pH 4.0 (straight line in Fig. 2), the anodic peaks in theforward scan were found at Epa1 = 0.608V and Epa2 = 0.930V,respectively, and no cathodic peakwas found in the reverse scan. Inneutral medium of pH 7.0 (dash line in Fig. 2), the oxidation peaksappeared in the forward scan at Epa1 = 0.690V and Epa2 = 1.085V,respectively, and a single reduction wave, 1c was registered atEpc1 = 0.544V in the backward scan. Like the voltammetricbehavior in neutral conditions, FcCBwitnessed two steps oxidationand one step reduction of the oxidation product in basicmediumofpH 10.0 (dot line in Fig. 2), but with peaks located at more positivepotentials, peak 1a at Epa1 = 0.693V, peak 2a at Epa2 = 1.129V, and

[(Fig._4)TD$FIG]

Fig. 4. (A) 3D plot of DPV of 1.0mM FcCB at 100mVs�1 vs.

the reduction peak 1c at Epc1 = 0.562V. Peaks 1a and 1c correspondto ferrocene–ferrocenium couple of FcCB. The oxidation ferrocenylgroup in FcCB under similar conditions can be attributed to theelectron withdrawing effect of carbonyl group attached at thecyclopentadienyl ring that causes the electron removal fromferrocene difficult. The 2nd anodic peak at a high positive potentialcan be related to the oxidation of cyclopentene part of FcCB.

3.1.2. Square wave voltammetrySquare wave voltammetry (SWV) is a fast electroanalytical

technique associated with minimal chances of adsorption. Thistechnique is especially used for getting evidence about thereversible or irreversible nature of the reaction [32,33]. Thecurrent in square wave voltammetry is sampled in both positiveand negative pulses, so that oxidation and/or reduction peaks ofelectroactive species can be registered at the electrode surface inthe same experiment.

Square wave voltammograms of 1mM FcCB recorded at pH 7.0(Fig. 3) showed results quite similar to that of CV. Two anodic peaksat 0.548V and 1.096V can be seen in the net current of the firstscan of SWV (solid line in Fig. 3A). The opposite direction offorward and reverse current components corresponding to peak 1a(dash and dot lines in Fig. 3A) confirmed the reversibility of theferrocenyl group. At the same time, peak 2a demonstrated theirreversible nature of cyclopentenyl oxidation.

Multiple successive scans of square wave voltammograms of1.0mM FcCB obtained at pH 7.0 have been depicted in Fig. 3B. Nonew peak(s) appear on the successive scans, indicating the electro-

pH; plots of Epa (B) and Ipa (C) for peaks of FcCB vs. pH.

[(Scheme_2)TD$FIG]

Scheme 2. Proposed oxidation mechanism of FcCB.

C. Wu et al. / Analytica Chimica Acta 857 (2015) 39–45 43

inactivity of oxidative products of FcCB. The inset in Fig. 3B showsgradual decrease of peak currents with consecutive scans. It wasdue to adsorption of FcCB and oxidation products at the electrodesurface. Peak currents decreased sharply in the first three scans,and almost stayed stable after the third scan. Another observationwas that the current of peak 2a decreased more quickly than peak1a. At the third scan, 50.62% and 74.11% decrease in the intensitywas noticed for peak 2a and peak 1a, respectively. This peculiarvoltammetric feature can be related to the adsorption of theoxidative products of FcCB at the electrode surface that will reducethe sensing area of the working electrode [34].

3.1.3. Differential pulse voltammetryDifferential pulse voltammetry (DPV) of FcCB was performed

for comparison of their ease of oxidation and determining thenumber of electrons involved in anodic processes. The DPV

[(Fig._5)TD$FIG]

Fig. 5. (A) The time dependence of current percentage for the FcCB after incubatingwith cof currents for the FcCB after incubating with different concentration of cells. The li800 cellsmL�1. The inlet showsDPV of FcCBwith different cell concentrations. (C) DPV of F5min. (D) box plot of peak potentials for FcCB before and after incubating with leukem

demonstrated in Fig. S1 were obtained at pH 10.0 at 5mVs�1. Thepotential difference at half peak height, DE1/2 corresponded to theinvolvement of 1e� in each step of FcCB. However, both anodicsignals of Fc2CB are broad and high. It was due to the possiblemerging of two sub peaks. Thesewide oxidationwaves of Fc2CB aresuggestive of two electrons loss. The reason of this remarkablydifferent behavior from FcCB is the presence of two ferrocenylgroups in Fc2CB.

3.2. Influence of pH values and redox mechanism

The voltammetric response of FcCB was examined in support-ing electrolytes of different pH values, ranging from 3.0 to 11.0(Fig. 4). Potentials of peaks 1a and 2a increased linearly with the pHvalues of the medium (Fig. 4A and B). As shown in Fig. 4B, the plotswere fitted as Ep1a (V) = 0.05381�pH+0.18571 (R2 = 0.97657) andEp2a (V) = 0.5257�pH+0.69516 (R2 = 0. 99707). The slopes of fittinglines are near to 59mV per pH unit at the pH interval 3.0–8.0,indicating the oxidation of FcCB to occur with the transfer of thesame number of electron and proton. However, above pH 8.0, thepotentials of peaks 1a and 2awere not influenced by the increase inpH, suggesting the oxidation to exhibit the loss of only electronswithout the involvement of protons [35]. Hence, pH 8.0 is the pKa

of FcCB at which chemical protonation–deprotonation takes place.The pH dependent redox behavior may be due to the pH sensitivefunctionality and conjugated structure of FcCB, as shown inScheme 1. The maximum peak currents of peak 1a at pH 7.0 and 2aat pH 5.0 can be attributed to the close accessibility of theircorresponding electrophores to the electrode surface under theseconditions (Fig. 4C).

ells. The inlet showsDPV of FcCB at different timewith cells. (B) The logarithmic plotnear range is from 1.0�103 to 3.0�104 cellsmL�1 and the limit of detection atcCB before (—) and after incubatingwith leukemia cells (� � �) and leucocytes (- - -) foria cells, leucocytes and HepG2 cells. *P<0.05.

44 C. Wu et al. / Analytica Chimica Acta 857 (2015) 39–45

According to electrochemical behaviors and UV–vis absorp-tion spectra (see Supplementary materials Fig. S2), we proposedthese redox mechanisms of FcCB in comparison to Fc2CB. FcCBis proposed to oxidize in two 1e� and 1H+ reversible and quasi-reversible steps from the appearance of two anodic peaks withDE1/2 values corresponding to the transfer of one electron peroxidation step, ratio of forward and backward components of thetotal current, and shift of peak potential with rise in pH values(Scheme 2). The irreversibility of the 2nd step can be related tothe dimerization or adsorption of the product at the electrodesurface.

3.3. Specific recognition of cancer cells

Here we have explored a new electrochemical strategy in adroplet system to recognize cancer cells from normal cells ofclinical samples. Leukemia cells and leucocytes were taken asan example to explore the potential analytical and clinicalapplications in specific recognition of normal cells and cancercells. The limit of detection of FcCB for peak 1a was 2.32mMand it showed nice linearity in the concentration range of 50–1000mM (shown in Supplementary materials Fig. S3 andTable S1). The low detection limit and board linearity detectionrange of peak 1a for FcCB allowed the potential application insensitive bioanalysis of specific recognition of cancer cells(Fig. 5). The in vitro cytotoxicity was explored by using MTTassay. IC50 value of FcCB to K562 cells was about 38.2mM asshown in Supplementary materials.

The time dependence of the efficiency of incubation time wasalso investigated (shown in Fig. 5A). The corresponding currents ofpeak 1a of FcCBwere decreasedwith an increasing incubation time.After 5min, peak current decreased to about 82% and then almostreached to a constant value. Therefore, the optimal incubation timewas set to 5min for subsequent experiments. As seen from Fig. 5B,peak current values decreased logarithmically with the increasingconcentration of cancer cells (from 1.0�103 to 3.0�104 cellsmL�1,that the equation was fitted as Ipa (mA) =0.4016� lgC (cellsmL�1)�3.65877 (R2 = 0.99867). The limit of detection of cancer cells was800 cellsmL�1. Meanwhile, the peak potential was almost stable.The cell concentration was not the decisive factor of theexperiment. Besides, the cell concentration in this study wasmuch less than that in clinical samples, where there was between4500 and 11,000 white blood cells per cubic millimeter of blood[36], suggesting promising application of this strategy for therelevant clinic diagnostics.

Fig. 5C and D illustrates the electrochemical study of FcCBbefore and after incubatedwith different type of cells.We observedthat potential of peak 1a for normal samples negatively shifted to0.536V and for leukemia samples positively shifted to 0.553V. It isevident that there were significant difference of peak potentials ofFcCB after incubated with leukemia cells and leucocytes due to theresults of statistical analysis (P<0.05, Fig. 5D). There were alsosignificant differences in peak potential of FcCB between normalcells (leucocytes) and HepG2 cells. Our previous observationsdemonstrated that the carborane derivatives had a potent selectiveinhibition effect on the proliferation of the cancer cell lines and noeffect on normal cell lines [37]. It was also observed that carboranederivatives can lead to specific binding modalities with macro-molecules, such asmyoglobin [38], hemoglobin [39], nicotinamidephosphoribosyltranferase [40], low-density lipoprotein [41], rele-vant carborane ligands had been considered as effective pharma-cophores as non-BNCT anticancer agents [42–44]. Based on theseconsiderations, it is possible to readily utilize the novel carboranederivatives to target and distinguish different kinds of cells for thespecific cancer diagnostics and treatment.

4. Conclusions

The electrochemical behavior of novel ferrocenyl basedcarborane was thoroughly examined in a droplet electrochemicalsystem. Both compounds were found to have robust anodic signalsat a glassy carbon electrode. Cyclic voltammetry, square wavevoltammetry and differential pulse voltammetry were successfullyused in awide pH range for the establishment of electrode reactionmechanism. The results revealed that ferrocenyl group gotoxidized by the abstraction of one electron and one proton in areversible manner. The oxidationwas found to depend strongly onthe pH of the medium up to pH 8.0. Potential-pH plots revealedthat in strongly alkaline conditions the analytes oxidize only by theloss of electrons. Squarewave voltammetry revealed the reversiblenature of the first anodic peak and quasi-reversible characteristicof the second oxidationwave. Limit of detection (LOD) and limit ofquantification (LOQ) were evaluated in order to determine thesensing ability of glassy carbon electrode for ferrocenyl carboranes.The established ferrocenyl carboranes can be utilized as sensitiveelectrochemical probes in specific recognition of normal cells andcancer cells like leukemia cells. This electrochemical bioanalysis ina droplet electrochemical system based on ferrocenyl substitutedcarboranes has a great potential application for relevant diag-nostics of clinical samples.

Acknowledgments

This work is supported by the National Science Foundation forDistinguished Young Scholars of China (81325011), National HighTechnology Research and Development Program of China(2012AA022703), National Natural Science Foundation of China(21175020,21327902 and 21271102) and the National BasicResearch Program of China(2010CB732404, 2013CB922101) aswell as Suzhou Science & Technology Major Project (ZXY2012028).Dr. Afzal Shah thanks the support of Quaid-i-Azam University andHigher Education Commission Islamabad, Pakistan.

Appendix A. Supplementary data

Supplementary data associatedwith this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.aca.2014.12.019.

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