9
Fullerene derived molecularly imprinted polymer for chemosensing of adenosine-5 0 -triphosphate (ATP) Piyush S. Sharma a , Marcin Dabrowski a , Krzysztof Noworyta a , Tan-Phat Huynh a , Chandra B. KC b , Janusz W. Sobczak a , Piotr Pieta a , Francis DSouza b, **, Wlodzimierz Kutner a, c, * a Department of Physical Chemistry of Supramolecular Complexes, Institute of Physical Chemistry, Polish Academy of Sciences (IPC PAS), Kasprzaka 44/52, 01- 224 Warsaw, Poland b Department of Chemistry, University of North Texas, 1155 Union Circle, #305070, Denton, TX 76203-5017, USA c Faculty of Mathematics and Natural Sciences, School of Science, Cardinal Stefan Wyszynski University in Warsaw, Wóycickiego 1/3, 01-815 Warsaw, Poland H I G H L I G H T S G R A P H I C A L A B S T R A C T Two MIP based chemosensors for ATP were devised and fabricated by using fullerene based functional monomers. The presence of the ionic liquid allows dissolving all components for electropolymerization. The imprinted cavities of MIP-ATP discriminated the ATP analyte from its common interferences. A R T I C L E I N F O Article history: Received 21 April 2014 Received in revised form 4 July 2014 Accepted 8 July 2014 Available online 11 July 2014 Keywords: Molecularly imprinted polymer Fullerene polymer Electroactive functional monomer Adenosine-5 0 -triphosphate Capacitive impedimetry Piezoelectric microgravimetry A B S T R A C T For molecular imprinting of oxidatively electroactive analytes by electropolymerization, we used herein reductively electroactive functional monomers. As a proof of concept, we applied C 60 fullerene adducts as such for the rst time. For that, we derivatized C 60 to bear either an uracil or an amide, or a carboxy addend for recognition of the adenosine-5 0 -triphosphate (ATP) oxidizable analyte with the ATP- templated molecularly imprinted polymer (MIP-ATP). Accordingly, the ATP complex with all of the functional monomers formed in solution was potentiodynamically electropolymerized to deposit an MIP-ATP lm either on an Au electrode of the quartz crystal resonator or on a Pt disk electrode for the piezoelectric microgravimetry (PM) or capacitive impedimetry (CI) determination of ATP, respectively, under the ow-injection analysis (FIA) conditions. The apparent imprinting factor for ATP was 4.0. After extraction of the ATP template, analytical performance of the resulting chemosensors, including detectability, sensitivity, and selectivity, was characterized. The limit of detection was 0.3 and 0.03 mM ATP for the PM and CI chemosensor, respectively. The MIP-ATP lm discriminated structural analogues of ATP quite well. The Langmuir, Freundlich, and LangmuirFreundlich isotherms were tted to the experimental data of the ATP sorption and sorption stability constants appeared to be nearly independent of the adopted sorption model. ã 2014 Elsevier B.V. All rights reserved. * Corresponding author at: Institute of Physical Chemistry, Polish Academy of Sciences, Department of Physical Chemistry of Supramolecular Complexes, Kasprzaka 44/52, 01-224 Warsaw, Poland. Tel.: +48 22 343 3217; fax: +48 22 343 3333. ** Corresponding author. Tel.: +940 369 8832; fax: +940 565 4318. E-mail addresses: [email protected] (F. DSouza), [email protected] (W. Kutner). http://dx.doi.org/10.1016/j.aca.2014.07.005 0003-2670/ ã 2014 Elsevier B.V. All rights reserved. Analytica Chimica Acta 844 (2014) 6169 Contents lists available at ScienceDirect Analytica Chimica Acta journa l home page : www.e lsevier.com/loca te/aca

Fullerene derived molecularly imprinted polymer for chemosensing of adenosine-5′-triphosphate (ATP)

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Page 1: Fullerene derived molecularly imprinted polymer for chemosensing of adenosine-5′-triphosphate (ATP)

Analytica Chimica Acta 844 (2014) 61–69

Fullerene derived molecularly imprinted polymer for chemosensing ofadenosine-50-triphosphate (ATP)

Piyush S. Sharma a, Marcin Dabrowski a, Krzysztof Noworyta a, Tan-Phat Huynh a,Chandra B. KC b, Janusz W. Sobczak a, Piotr Pieta a, Francis D’Souza b,**,Wlodzimierz Kutner a,c,*aDepartment of Physical Chemistry of Supramolecular Complexes, Institute of Physical Chemistry, Polish Academy of Sciences (IPC PAS), Kasprzaka 44/52, 01-224 Warsaw, PolandbDepartment of Chemistry, University of North Texas, 1155 Union Circle, #305070, Denton, TX 76203-5017, USAc Faculty of Mathematics and Natural Sciences, School of Science, Cardinal Stefan Wyszynski University in Warsaw, Wóycickiego 1/3, 01-815 Warsaw, Poland

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

� Two MIP based chemosensors forATP were devised and fabricated byusing fullerene based functionalmonomers.

� The presence of the ionic liquidallows dissolving all componentsfor electropolymerization.

� The imprinted cavities of MIP-ATPdiscriminated the ATP analyte fromits common interferences.

A R T I C L E I N F O

Article history:Received 21 April 2014Received in revised form 4 July 2014Accepted 8 July 2014Available online 11 July 2014

Keywords:Molecularly imprinted polymerFullerene polymerElectroactive functional monomerAdenosine-50-triphosphateCapacitive impedimetryPiezoelectric microgravimetry

A B S T R A C T

For molecular imprinting of oxidatively electroactive analytes by electropolymerization, we used hereinreductively electroactive functional monomers. As a proof of concept, we applied C60 fullerene adducts assuch for the first time. For that, we derivatized C60 to bear either an uracil or an amide, or a carboxyaddend for recognition of the adenosine-50-triphosphate (ATP) oxidizable analyte with the ATP-templated molecularly imprinted polymer (MIP-ATP). Accordingly, the ATP complex with all of thefunctional monomers formed in solution was potentiodynamically electropolymerized to deposit anMIP-ATP film either on an Au electrode of the quartz crystal resonator or on a Pt disk electrode for thepiezoelectric microgravimetry (PM) or capacitive impedimetry (CI) determination of ATP, respectively,under the flow-injection analysis (FIA) conditions. The apparent imprinting factor for ATP was �4.0. Afterextraction of the ATP template, analytical performance of the resulting chemosensors, includingdetectability, sensitivity, and selectivity, was characterized. The limit of detection was 0.3 and 0.03 mMATP for the PM and CI chemosensor, respectively. The MIP-ATP film discriminated structural analogues ofATP quite well. The Langmuir, Freundlich, and Langmuir–Freundlich isotherms were fitted to theexperimental data of the ATP sorption and sorption stability constants appeared to be nearly independentof the adopted sorption model.

ã 2014 Elsevier B.V. All rights reserved.

* Corresponding author at: Institute of Physical Chemistry, Polish Academy of Sciences, Department of Physical Chemistry of Supramolecular Complexes, Kasprzaka 44/52,

Contents lists available at ScienceDirect

Analytica Chimica Acta

journa l home page : www.e l sev ier .com/ loca te /aca

01-224 Warsaw, Poland. Tel.: +48 22 343 3217; fax: +48 22 343 3333.** Corresponding author. Tel.: +940 369 8832; fax: +940 565 4318.

E-mail addresses: [email protected] (F. D’Souza), [email protected] (W. Kutner).

http://dx.doi.org/10.1016/j.aca.2014.07.0050003-2670/ã 2014 Elsevier B.V. All rights reserved.

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62 P.S. Sharma et al. / Analytica Chimica Acta 844 (2014) 61–69

1. Introduction

Complementary receptor–analyte binding is a prerequisitefor building supramolecular recognition governed analyticaldevices. With this regard, the design and synthesis of functionalmonomers and then polymers capable of selective sensing hasrecently become an active research area [1–5]. Towards this,molecular imprinting is a frequently used procedure. Thisimprinting consists of impressing molecular cavities in apolymer matrix with template molecules. The cavity shapeand size as well as orientation of recognition sites generated inthese cavities correspond to binding sites of the templatemolecule. The analyte itself or its close analogue is selected as atemplate. Briefly, this procedure involves, first, pre-organizationof the template with functional monomers to form a pre-polymerization complex in solution followed by polymerizationof this complex in the presence of a cross-linking monomer.Subsequent removal of the template from the resulting polymermatrix leaves behind imprinted cavities complementary to thetemplate molecules.

Electroactive functional monomers are often used for prepara-tion of a conducting or non-conducting thin molecularly imprintedpolymer (MIP) film on a conducting transducer surface [6,7]. Forpolymerization of these monomers, electropolymerization is anappealing method of choice [6]. Moreover, molecular imprintingwith the use of an electroactive functional monomer is advanta-geous compared to that with electroinactive monomers because ofthe possibility of deposition of an MIP film directly on the electrodesurface for devising chemosensors. Generally, a selective chemo-sensor integrates a unit of chemical recognition of an analyte andthat of transduction of the recognition signal into a measurableanalytical signal [8]. An MIP film proved to serve as a reliablerecognition unit [6].

If an electroactive template (analyte) is to be imprinted byelectropolymerization, an apparent recognition difficulty mayarise from electroactivity of this template if it proceeds under theconditions of the electropolymerization. Then, products of anelectrode reaction of the template may be imprinted instead of thegenuine template itself. Moreover, these products may adsorb and,consequently, foul the electrode surface. Therefore, the electrodereaction of such a template should be eliminated. Toward that,several procedures have been developed. In one, an electroinactiveclose analogue can be used as the surrogate template. Unfortu-nately, not many electroactive analytes have their electroinactiveclose analogues. Another possibility involves self-barrier forma-tion, which uses a resistive MIP self-barrier preventing electrodeprocesses of redox templates [9]. One more appealing alternative isto deposit, first, a barrier underlayer of some conducting polymer,which precludes electrochemical transformation of the templateon the one hand and allows electrodeposition of an MIP film on topof it on the other [10,11]. However, the most elegant procedureseems to be using electroreducible functional and cross-linkingmonomers for imprinting of electro-oxidizable templates and viceversa. To our best knowledge, only electro-oxidizable monomersare so far used for electropolymerizational imprinting [6]. As aproof of concept, for the first time to our best knowledge, we useherein (C60) fullerene derivatives as reductively electroactivefunctional monomers for imprinting of an electro-oxidizabletemplate, adenosine-50-triphosphate (ATP) 1.

ATP plays a key role in the energy turnover in a living cell [12]. Ittransports metabolic energy and, therefore, can be considered as abiological “energy unit”. Hence, elevated ATP levels can just beused to indicate the presence of microorganisms and somatic cellsin environments where their presence would exert a harmful effect[13]. For instance, in the food and water safety as well as in thetoxicity and contamination analysis, the presence and growth of

microorganisms are widely analyzed by an ATP test. Therefore,determination of the ATP concentration is effective in analyzingmicrobial contamination of water, and that way, the health safety[14].

The ATP concentration decreases upon cell necrosis orapoptosis. Deficiency in ATP results in ischemia [15], Parkinson’sdisease [16], and hypertension [17].

Moreover, the ATP assay can be used to determine the metabolicrate before and after administration of a tested drug, thus helpingto determine the susceptibility of a patient to this drug [18].Adoption of this assay may reduce the number of unsuccessfuldrug administrations. Thus, selective recognition of ATP inbiological aqueous media has emerged as an important researcharea of the microbiological and biochemical analysis.

Several MIP-based synthetic receptors for selective recognitionof ATP have been reported [18–25]. However, they mostly report onthe use of MIPs as the solid-phase extraction materials. Moreover,selectivity of these MIPs is not high �1.5 times [24] and the kineticsof binding is slow [19]. Therefore, further development of selectiveATP sensing is desired.

One of the easiest ways of fullerene electropolymerizationinvolves preparation of two-component polymers of sandwichedfullerene and some metal complex [26–31]. These polymers aredeposited as thin films on an electrode surface by reductiveelectropolymerization. Both redox conductivity and specificcapacity of these films are high [32]. By optimum adjustment ofpolymerization conditions, such as the fullerene-to-Pd mole ratio[26], one can prepare – (Pd–C60)n – polymers of either short orhighly cross-linked chains.

The use of fullerenes in sensing and biosensing is well known[33], however, utilization of fullerene in developing MIPs is yet tobe explored. So far, only one report described the use of C60

fullerene as an imprinting base to prepare chiro-selective bindingsites for sugars. The chiro-selectivity reached with this MIP wasvery high (�44–82%) [34].

In the present work, we investigate feasibility of fullerenederivatives to electropolymerize reductively for preparation of anelectroactive MIP film as a recognition unit of a chemosensor forATP. Accordingly, the ATP template was imprinted by the Pd(II)acetate dimer, Pd(ac)2, assisted co-polymerization of4-(fulleropyrollidine)-amidophenylbenzene 2, 4-(fulleropyrolli-dine)-benzoic acid 3 and 4-(fulleropyrollidine)-uracil 4 [35–39].These three fullerene derivatives served as functional monomersfor preparation of an MIP-ATP based recognition unit of twochemosensors for selective ATP recognition. For this fullerenederivatization, Prato’s fulleropyrrolidine synthetic procedure wasused [40]. Initially, the amide, carboxylic acid, and uracil adduct offullerene were allowed to complex, in solution, the phosphate,ribose, and adenine moiety of ATP, respectively. Then, the self-assembled complex was potentiodynamically electropolymerized.The resulting MIP-ATP film was deposited either on a Pt diskelectrode or on an Au-film electrode of a quartz crystal resonator(Au-QCR) for the capacitive impedimetry (CI) or piezoelectricmicrogravimetry (PM), respectively, determination of the ATPanalyte under the FIA conditions. Before this determination,however, the ATP template was extracted from the ATP-templatedMIP-ATP film, thus leaving the vacant impressed molecularcavities.

2. Experimental

2.1. Chemicals

Acetonitrile (ACN), 2-propanol, and toluene for syntheses werepurchased from Sigma–Aldrich and tetra-n-butylammoniumperchlorate [(TBA)ClO4] was supplied by Fluka. Potassium fluoride

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P.S. Sharma et al. / Analytica Chimica Acta 844 (2014) 61–69 63

(KF), and sodium fluoride (NaF) were from CHEMPUR. Pd(ac)2 wasfrom Alfa-Aesar. 1-Butyl-3-methylimidazolium trifluoro-metha-nesulfonate ionic liquid (IL) and ATP were purchased fromSigma–Aldrich. The functional monomer 4 was prepared asdescribed previously [39]. The functional monomers 2 and 3 wereprepared according to the herein developed procedures, describedin detail in Supporting Information.

2.2. Instruments and procedures

An AUTOLAB computerized electrochemistry system, equippedwith expansion cards of the PGSTAT12 potentiostat and the FRA2frequency response analyzer, and controlled by the GPES 4.9software, both of Eco Chemie, were used for the potentiodynamic,cyclic voltammetric (CV), and electrochemical impedance spec-troscopic (EIS) measurements of CI.

Atomic force microscopy (AFM) imaging was performed withthe use of a Multimode 8 microscope under control of theNanoscope V controller, of Bruker. For this imaging, the films weredeposited on the 7 �4 mm2 strips of indium-tin oxide (ITO)electrodes of Image Optics Components, Ltd.

For measurement of the film thickness, some part of the filmwas carefully removed from the electrode surface, i.e., scratchedwith a small piece of Teflon, under optical microscope. That way,only one part of the electrode remained coated with the film. WithAFM, the height of the resulting step was measured in severalsurface points along the step edge (sufficiently far from its partiallydetached front) for determination of the average value of thisheight.

The X-ray photoelectron spectroscopy (XPS) spectra wererecorded on a PHI 5000 VersaProbe (ULVAC-PHI) scanning ESCAmicroprobe using monochromatic Al Ka radiation (hn = 1486.6 eV).The Casa XPS software was used to evaluate the XPS data.Background was subtracted using the Shirley method, and peakswere fitted with the (Gaussian–Lorentzian)-shaped profiles. Thebinding energy (BE) scale was referenced to the C1s electron peakwith BE = 284.6 eV.

A model EQCM 5610 [41] and EQCM 5710 quartz crystalmicrobalance [42], driven by the EQCM 5710-S2 software, all of IPCPAS, were used to conduct the PM measurements under the FIAand batch analysis conditions, respectively. The resonant frequen-cy change was measured with 1-Hz resolution using 14-mmdiameter, AT-cut, plano-plano, Au-QCRs of 10-MHz resonantfrequency with 5-mm diameter Au disk film electrodes. Prior tothe polymer film electrodeposition, Au-QCRs were cleaned with a“piranha” solution for 10 s (H2O2–H2SO4, 1:3, v:v; caution: thissolution is dangerous in contact with skin and eye as it violentlyreacts with most organic compounds). A coiled Pt wire and an AgClfilm coated Ag wire were used as the counter and pseudo-referenceelectrode, respectively. For electropolymerization, the EP-20potentiostat of IPC PAS was interfaced with the EQCM 5710microbalance whose Au-QCR holder was mounted horizontally,with the Au-QCR facing upward, in order to use as low volume ofthe sample solution as 100 mL in the Au-QCR well of the holder.

The ATP-extracted MIP-ATP films deposited on Au-QCRs wereused for ATP determination by PM under the FIA conditions [43].The 0.1 M NaF carrier solution was pumped at the 20 mL/min flowrate through the EQCM 5610 holder with a model NE-500 syringepump of New Era Pump Systems. The 200-mL samples of the ATPtest solutions were injected with a model 7725i rotary six-portvalve of Rheodyne. Composition of the ATP test solutions was thesame as that of the carrier solution.

A large-volume radial-flow thin-layer electrochemical cell [44]was used for the CI determination of ATP, under FIA conditions,controlled by the AUTOLAB system. In the cell, 1-mm diameter Ptdisk working electrode was axially mounted, opposite to the inlet

capillary tip, at the distance of 300 mm. A Pt wire and an Ag/AgClelectrode served as the auxiliary and reference electrode,respectively. The applied frequency and potential was hold atf = 20 Hz and Eappl = 0.20 V (vs. Ag/AgCl), respectively. No faradaicprocess occurred at this potential. The ATP samples were preparedwith the solution of the same composition as that of the carriersolution, i.e., 0.1 M KF. Sample injection was the same as that for PMdetermination of ATP described above. The carrier solution flowrate was 35 mL/min. This electrochemical cell was completely filled(�35 mL) with the carrier solution before starting the measure-ments.

3. Results and discussion

The use of fullerenes bearing recognizing addends as functionalmonomers is much appealing in the sensing field. This is becausethe presence of these addends in the fullerene MIP film improveselectrochemical characteristics of the film by decreasing itsresistivity [45,46]. Toward that, we have utilized three differentderivatives of C60, viz., 2, 3, and 4 (Scheme 1) to serve as thesefunctional monomers.

3.1. Preparation and characterization of the MIP-ATP film

The amide derivatized synthetic receptors are widely used forselective recognition of phosphate in solution [47,48]. Accordingly,we have synthesized an amide derivative of C60 2 for complexationof the phosphate moieties of ATP.

The presence of the ribose vicinal diol in ATP encouraged us touse carboxy adduct of C60 3 as one more functional monomer forimprinting.

The functional monomer 4 is capable of hydrogen bonding viacomplementary nucleobase pairing of adenine, adenosine, and ATP[39]. Notably, this pairing plays decisive role in biologicalmolecular recognition, such as replication of nucleic acids,maintenance of tertiary structure of proteins, and enzyme-substrate binding. The hydrogen bond involved in this pairing isa directional secondary valence force compared to other non-covalent bonds, such as the electrostatic, van der Waals, andhydrophobic interactions. Because of its capability of nucleobasepairing of ATP, 4 prompted us to consider it as a promisingfunctional monomer for the MIP-ATP film preparation.

In accordance with the desired stoichiometry of the pre-polymerization complex, a solution of 1, 2, 3, 4, and Pd(ac)2 in themole ratio of 1:3:1:1:80, respectively, was prepared for electro-polymerization. For dissolution of all the components, a mixedsolvent solution of toluene, ACN, IL, and 2-propanol was optimized.However, toluene and the ACN-IL solution are immiscible.Therefore, 2-propanol had to be added to dissolve completely allthe components. Scheme 1 proposes one of possible bindingswithin the pre-polymerization complex formed in solution.

The C60–Pd(ac)2 polymer film was grown on either the Pt-diskelectrode or on the Au-QCR electrode for the CI or PMdetermination of ATP, respectively. For that, the 0.1 M (TBA)ClO4

mixed solvent solution of toluene-ACN-IL-(2-propanol) (8:1:1:1, v:v:v:v) containing 2, 3, 4, and Pd(ac)2, in the presence of ATP, as thefunctional monomers, cross-linker, and template, respectively, wasprepared. Before electropolymerization, the solution was deaer-ated with the argon purge. The imposed potentiodynamicconditions involved 12 potential cycles between 0 and �1.30 Vvs. Ag/AgCl at the sweep rate of 0.05 V/s (Fig. 1). The cathodiccurrent decrease with the increase of the cycle number and theaccompanying growth of a black solid on the electrode indicateddeposition of an electrochemically active film. The non-imprinted(NIP) control film was prepared under the same conditions exceptof the absence of the ATP template (data not shown). After

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Scheme 1. The proposed simplified structural formula of the pre-polymerization complex of 1 with functional monomers 2, 3, and 4 as well as with the Pd(ac)2 cross-linker.

64 P.S. Sharma et al. / Analytica Chimica Acta 844 (2014) 61–69

deposition, the films were rinsed with the ACN-water (1:1, v:v)solution to remove the supporting electrolyte.

Fig. 2a shows the AFM image of the ATP-templated MIP filmdeposited by electropolymerization on a gilded glass slide. Thisfilm was composed of 20(�5)-nm diameter grains, aggregated toform clusters. Thickness and roughness of the film was358 � 22 nm and 44 � 22 nm, respectively. This roughness madethe film permeable enabling diffusion of the ATP analyte, thusfacilitating its access to the emptied imprinted cavities (Fig. S1).Interestingly, extraction of the ATP template from the MIP-ATP filmdid not cause much change in the film topography (Fig. 2b). That is,

Fig. 1. Potentiodynamic curves for 0.1 mM 1, 0.3 mM 2, 0.1 mM 3, 0.1 mM 4, and8 mM Pd(ac)2 in 0.1 M (TBA)ClO4, in the toluene-ACN-IL-(2-propanol) (8:1:1:1, v:v:v:v) solution recorded at the 1-mm diameter Pt disk electrode for 12 potential cyclesin the 0 to �1.30 V vs. Ag/AgCl potential range. The potential scan rate was 50 mV/s.

grains visible in Fig. 2a are also present in Fig. 2b but they formsmaller clusters than those present in the ATP-templated MIP film.ATP extraction slightly increased the film thickness to 429 � 26 nmbut did not change the film roughness. Apparently, the film swellsupon template extraction. Presumably, the presence of vacatingmolecular cavities promoted the MIP-ATP film swelling [25]. Fordeposition of the MIP-ATP film on Au-QCR, curves of the current,resonant frequency change, and dynamic resistance change vs.potential were simultaneously recorded during the electropoly-merization (Fig. 3). The resulting decrease of resonant frequency(Fig. 3b) corresponded to the increase of the Au-QCR mass due todeposition of the MIP-ATP film. For rigid films, the mass of thedeposited film is calculated from Sauerbrey’s equation [49], Eq. (1)

Df ¼ � 2f 20Dm

Aac mqrq

� �0:5 (1)

where f0 is the fundamental frequency of oscillations of Au-QCR(10 MHz), Aac is the acoustically active area of Au-QCR (0.1963 cm2),mq is the shear modulus of quartz (2.947 � 1011 g s�2 cm�1), and rq

is the quartz density (2.648 g cm�3). A small (�150 V) change indynamic resistance (Fig. 3c) accompanying the MIP-ATP filmdeposition evidenced that the film was rigid and changes of itsviscosity and density only negligibly influenced the total frequencychange.

After the electropolymerization, the ATP template wasextracted from the MIP-ATP film with 0.1 M HCl for 2 h at 50 �C.Template removal was confirmed by measuring the current limitedby diffusion of the K4Fe(CN)6 redox probe through the MIP-ATPfilm to the electrode surface by using differential pulse voltam-metry (DPV) (Fig. S2). As a result, the DPV peak for the K4Fe(CN)6electro-oxidation was practically absent for the MIP-ATP film fullyloaded with the ATP template (curve 1 in Fig. S2) meaning thatdiffusion of the probe was effectively prevented because the

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Fig. 2. The AFM images of (a) ATP templated MIP-ATP film and (b) ATP-extracted MIP-ATP film.

P.S. Sharma et al. / Analytica Chimica Acta 844 (2014) 61–69 65

imprinted molecular cavities were occupied by the ATP molecules[6,7]. However, template extraction for different time intervalsresulted in well-developed peaks (curves 3 and 4 in Fig. S2).Apparently, the K4Fe(CN)6 electrode reaction was facilitated in thiscase, because the K4Fe(CN)6 diffusion through the film, populatedby vacated imprinted cavities, was enabled. There was no change inthe DPV peak height for the redox probe when the same

Fig. 3. Simultaneously recorded curves of potential dependence of (a) current aswell as the change of (b) resonant frequency and (c) dynamic resistance fordeposition of the MIP-ATP film by potentiodynamic electropolymerization on theAu-film electrode of the 10-MHz quartz crystal resonator. The solution compositionwas the same as that described in caption to Fig. 1.

experiment was performed using an NIP film modified electrode(curve 2 in Fig. S2).

The XPS measurements provided additional proof of removal byextraction of the ATP template from MIP-ATP. That is, highresolution XPS spectra for nitrogen, phosphorous, and carbon ofC60 revealed that the P-to-(C of C60) and N-to-(C of C60) ratio afterextraction decreased from 0.08 to 0.01 and from 0.51 to 0.26,respectively.

3.2. ATP determination using the capacitive impedimetry (CI)chemosensor under FIA conditions

In the CI determination of ATP, the change of the capacity of theelectrical double layer, Cdl, with the change of the ATP concentra-tion in solution was measured. At constant and sufficiently lowfrequency (20 Hz) this capacity can readily be determined from themeasured imaginary component of impedance, Z0 0, by using Eq. (2)[50].

Z00 ¼ 12pf CdlA

(2)

The Cdl value determined herein was higher the higher was theconcentration of ATP in the consecutively injected samplesolutions (Fig. 4). The measured Cdl increase most likely originatedfrom binding ATP by imprinted molecular cavities, thus changingboth dielectric properties and structure of the film [51]. Moreover,no contribution of capacity of the diffuse-layer part of the doublelayer to the total capacity is expected at a relatively highconcentration of the supporting electrolyte of non-specificallyadsorbing ions (0.1 M KF) used. Therefore, the Helmholtz model ofthe compact layer was adopted, in which the double-layer capacitysolely depends on electric permittivity, e, and thickness, d, of thecompact layer, according to Eq. (3)

Cdl ¼ee0d

(3)

where e0 is the electric permittivity of free space. Apparently, thepermittivity increase due to the ATP ingress to the MIP-ATP filmwas mainly responsible for the measured capacity increase (Fig. 4).Subsequent ATP elution with excess of the carrier solution causedthe capacity decrease to the baseline. The linear dynamicconcentration range extended from 0.06 to 1 mM ATP with thecalibration plot described by the linear regression equation of Cdl/(mF cm�2) = 0.069(�0.003) cATP/mM – 0.002(�0.002) with thecorrelation coefficient of 0.98; the detectability at the signal-to-noise ratio (S/N) of 3 and sensitivity were 31 mM and 0.069(�0.003) mF cm�2mM�1, respectively (curve 1 in Fig. 4, inset).

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Fig. 4. The time dependence of the capacity change due to consecutive injections ofATP solutions of concentrations indicated at peaks, recorded at the ATP-extractedMIP-ATP film coated 1-mm diameter Pt disk electrode, at 0.20 V vs. Ag/AgCl and20 Hz, except of (2) in inset. Inset: calibration plots for (1) ATP, (2) ATP at NIP, (3) GTP,(4) TTP, (5) ADP, (6) AMP, (7) guanosine, (8) adenine, (9) phosphate, and (10) CTP.

Fig. 5. The time dependence of the resonant frequency change due to consecutiveinjections of ATP solutions of concentrations indicated at peaks recorded at the ATP-extracted MIP-ATP coated Au electrode of 10-MHz QCR, except of 2. Inset:calibration plots for (1) ATP, (2) ATP at NIP, (3) GTP, (4) TTP, (5) ADP, and (6) CTP.

66 P.S. Sharma et al. / Analytica Chimica Acta 844 (2014) 61–69

In order to confirm the imprinting, an NIP film was also used forthe ATP determination in a control experiment. Apparently, theATP binding by NIP was much lower than that by the ATP-extractedMIP-ATP film (curve 2 in Fig. 4, inset). That is, Cdl for the former wasproportional to cATP between 0.06 and 1 mM obeying the linearregression equation of Cdl/(mF cm�2) = 0.0169(�0.004)cATP/mM – 0.002(�0.002) (curve 2 in inset in Fig. 4). The NIPfilm sensitivity to ATP was rather lower amounting to 0.0169(�0.004) mF cm�2mM�1.

Selectivity of the MIP-ATP film with respect to structuralanalogues of ATP was quite pronounced (Fig. 4, inset). That is,sensitivity to ATP was nearly nine times that to adenosinemonophosphate, (AMP) 0.0078 (�0.001) (curve 6 in inset inFig. 4), four times that to thymidine triphosphate (TTP) 0.016(�0.002) mF cm�2mM�1 (curve 4 in inset in Fig. 4), and guanosinetriphosphate (GTP) 0.016(�0.002) mF cm�2mM�1 (curve 3 in insetin Fig. 4) and nearly two-and-half times that to adenosinediphosphate, (ADP) 0.028(�0.004) mF cm�2mM�1 (curve 5 ininset in Fig. 4). However, it was merely twice that to cytidinetriphosphate (CTP) 0.033(�0.003) mF cm�2mM�1 (curve 10 ininset in Fig. 4). But the latter is not much surprising in view ofstructural similarity of CTP and ATP with all the H-bond forminggroups of ATP also present in CTP. Moreover, sensitivity to adeninewas approximately fifteen times lower equaling 0.0048 (�0.0019).Advantageously, phosphate and guanosine did not interfere at all.

3.3. ATP determination using the piezoelectric microgravimetry (PM)chemosensor under FIA conditions

An Au-QCR, coated with the ATP-extracted MIP-ATP film, wasmounted in the EQCM 5610 holder for the ATP determinationunder FIA conditions.

After each injection of the ATP solution of different concentra-tion, Df decreased (Fig. 5) because mass of the MIP-ATP filmincreased as ATP entered it. Then, an excess of the carrier solutioneluted the ATP from the film and Df returned to the baseline level.The maximum value of Df for each injection was proportional tothe ATP concentration in solution at least in the range of 0.6–10 mM, according to the linear regression equation ofDf/Hz = �9.05(�1.10) cATP/mM – 2.91(�5.7) (curve 1 in Fig. 5,inset). The sensitivity and correlation coefficient was �9.05(�1.10) Hz mM�1 and 0.95, respectively. The LOD at S/N = 3 was

0.31 mM. The calibration plot for the NIP film was described by thefollowing linear regression equation Df/Hz = �2.4(�0.70) cATP/mM– 7.85(�3.64) (curve 2 in Fig. 5, inset). From the ratio of thesensitivity of MIP-ATP (curve 1 in Fig. 5, inset) and that of NIP(curve 2 in Fig. 5, inset) to ATP, a quite appreciable apparentimprinting factor of �4.0 was determined.

Similarly as for the CI chemosensor, the selectivity of the PMchemosensor was determined (Fig. 5, inset). Advantageously, thesensitivity to ATP of �9.05(�1.10) Hz mM�1 was nearly five-and-half times that to GTP �1.63(�0.20) Hz mM�1 (curve 3 in Fig. 5,inset), three-and-half times that to TTP �2.57(�0.20) Hz mM�1

(curve 4 in Fig. 5, inset), nearly three times that to ADP �3.30(�1.33) Hz mM�1 (curve 5 in Fig. 5, inset), and merely one-and-halftimes that to CTP 6.60(�0.48) Hz mM�1 (curve 6 in Fig. 5, inset).Apparently, the selectivity trends were quite similar for bothchemosensors.

The choice and amount of functional monomers used for MIPpreparation was based on the number and nature of binding sitesavailable on the ATP template molecule. To study the effect of thechange of the ratio of the monomers on the ATP binding behavior,the PM response to ATP of the film of MIP that did not containfunctional monomer 2 was examined. The PM response of this filmwas nearly four times lower than that for the film of MIP, whichcontained functional monomer 2 (Fig. S3). Apparently, thecomposition of the MIP film is important to devise a MIP cavitysensitive to ATP.

As a stability test, chemosensor binding to ATP was examinedafter keeping the MIP-ATP film coated Au-QCR in the carriersolution for 48 h. Even after this long time storage, the chemo-sensor response was quite similar to that of the freshly preparedMIP film (Fig. S4). Apparently, the MIP-ATP film behavior is quiteinvariable to flow analytical conditions.

3.4. Characterization of ATP sorption by the MIP-ATP film

For characterization of the ATP sorption by the MIP-ATP film,stability constants of the complex of ATP and its complementarilyimprinted cavity, i.e., sorption equilibrium constants, weredetermined under both the steady-state and flow-injectionsolution conditions. Three most typical isotherms were testedfor the former conditions while the rate constants of complexformation and dissociation were determined and used for

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P.S. Sharma et al. / Analytica Chimica Acta 844 (2014) 61–69 67

calculation of the stability constant under the latter conditions[52–54].

For the former determinations, the batch-solution ATP bindingexperiment was performed using the ATP-extracted MIP-ATP filmcoated Au-QCR, mounted in the EQCM 5710 holder. After eachaddition of the ATP solution of different concentration to theworking solution, Df stepwise decreased because of the ATPbinding by the MIP-ATP film (Fig. S5a). The PM response reachedplateau at �4.5 mM ATP in the solution indicating saturation of theMIP-ATP film. The Langmuir, Freundlich, and Langmuir–Freundlichadsorption isotherms were then fitted to the experimentalsorption data acquired (Fig. S5) and isotherm parametersdetermined [53,54].

Application of the Langmuir isotherm assumes, among others,that molecules are sorbed at predefined homogeneous bindingsites within the sorbent with only one molecule occupying one siteand the immobilized molecules do not interact mutually. Theconcentration of the bound analyte, B, is expressed by Eq. (4)

B ¼ NKscATP1 þ KscATP

(4)

where CATP is the concentration of ATP in solution, N represents thedensity of imprinted cavities in the ATP-extracted MIP-ATP filmaccessible to the ATP analyte or, in other words, the monomolecu-lar saturation of imprinted cavities, and Ks is the sorptionequilibrium constant, related to the Gibbs free energy changedue to sorption, DGs = �RT ln Ks.

The Langmuir isotherm can easily be linearized for the linearregression analysis with graphical determination of its adjustableparameters. By rearranging Eq. (4), one obtains Eq. (5).

cATPB

¼ 1NKs

þ 1NcATP (5)

With the use of Eq. (5), the N and Ks constants were determinedby plotting cATP/B vs. cATP (Fig. S5b).

The Freundlich isotherm describes B as a power function of CATP,according to Eq. (6)

B ¼ acmATP (6)

where a is the Freundlich parameter related to the binding affinityand m is the index of heterogenity of the sorbent. This index variesin the range 0 � m � 1; m approaches 1 as heterogenity decreasesand equals 1 for a homogeneous system (Fig. S5c).

The Freundlich isotherm, Eq. (6), can be rearranged to its linearform of Eq. (7).

logB ¼ mlogcATP þ loga (7)

Plotting log B vs. log cATP allows for determination of a and m by thelinear regression analysis. These two, a and m, fitting parametersyield a measure of binding. The pre-exponential factor a is themeasure of N and Ks. However, the individual contributions of Nand Ks to this factor cannot directly be obtained without additionalexperiments performed or assumptions adopted. Moreover, theinability of the Freundlich isotherm to model the saturationbehavior limits the types of binding parameters that can becalculated from it.

Table 1Isotherm fitting parameters calculated for the MIP-ATP film.

Isotherm N (mmol/g) Isotherm fitting param

a

Langmuir 17.85 � 0.32 –

Freundlich – 5.67 � 0.16

Langmuir–Freundlich 18.53 � 1.32 –

N – density of imprinted cavities accessible to ATP, Ks – sorption equilibrium constant,

Accurate determination of the N and Ks values for heteroge-neous systems and the heterogenity index requires adoption of ahybrid model accommodating both the saturation and sub-saturation regions of the isotherm, such as that of theLangmuir–Freundlich isotherm. This isotherm describes equilibri-um between B and cATP, as follows.

B ¼ NKms cATPm

1 þ Kms cATPm

(8)

The fitting parameter, m, is identical to the heterogenity index inthe Freundlich isotherm (Fig. S5d).

As evident from Table 1 summarizing the sorption results,values of the sorption equilibrium constants determined for theATP sorbed by the MIP-ATP film are quite similar, independentlywhich sorption model is adopted. Moreover, the determined valuesof the molecular cavity density are similar (Table 1). The Freundlichisotherm deviates from the experimental data points for high ATPconcentrations (Fig. S5c) being incapable of modeling thesaturation behavior. Therefore, the heterogeneity index deter-mined from the Freundlich isotherm was quite different from thatdetermined from the Langmuir–Freundlich isotherm (Table 1).

Under the PM-FIA conditions, kinetic parameters characterizingthe ATP binding by the ATP-extracted MIP-ATP film weredetermined. Formation of the complex of the molecule of theATP analyte with the MIP-ATP cavity is characterized by theassociation, ka, and dissociation, kd, rate constants. The equilibriumof formation of this complex can be written, as follows

MIP-ATP þ ATP ?ka

kdMIP-ATPð Þ-ATP (9)

for which Ks takes the form

Ks ¼ MIP-ATPð Þ-ATP½ MIP-ATPð Þ½ ATP½ (10)

the dependence of the (MIP-ATP)-ATP concentration, [(MIP-ATP)-ATP], on time is expressed as [55]

d½ðMIP-ATPÞ-ATPdt

¼ �Mdfdt

� �¼ kacATP fmax � fð Þ � kdf (11)

where M = �[Aac (mqrq)1/2/2 MwV f 20] is the proportionality factorobtained from Eq. (1). Mw is the molecular weight of the(MIP-ATP)-ATP complex, V is the volume of the MIP film, andfmax is the maximum change of the resonant frequency for aparticular ATP concentration. The Ks is defined by Eq. (12).

Ks ¼ kakd

(12)

Integration of Eq. (11) against time and substitution of Eq. (13)

f eq ¼ kacATPfmax

kacATP þ kdð Þ (13)

result in Eq. (14)

f eq ¼ fmax 1 � exp �kobstð Þ½ (14)

where

eter Ks (M�1) Correlation coefficient

m

– 530 � 13 0.9910.56 � 0.02 – 0.9901.00 � 0.06 476 � 57 0.997

a – Freundlich parameter, m – heterogenity index.

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68 P.S. Sharma et al. / Analytica Chimica Acta 844 (2014) 61–69

kobs ¼ kacATP þ kd (15)

Here, feq is the equilibrium frequency at time t and kobs is theapparent association rate constant. Values of kobs were derivedfrom Eq. (13) by best fit to the experimental data. Values of ka weredetermined from slopes and those of kd from intercepts of thelinear regression plots of kobs vs. cATP, (Fig. S6) according to Eq. (15).The mass transport effects were accounted for by performing acontrol experiment with the K4Fe(CN)6 redox probe, as describedpreviously [10]. The determined Ks value for the MIP-ATP complexwith ATP was, Ks = 645 �196 M�1. Importantly, this Ks value is closeto those determined under steady-state solution conditions byadoption of different sorption models (Table 1).

4. Conclusions

As a proof of concept, we have successfully demonstratedmolecular imprinting of the ATP oxidizable template in an MIP-ATPfilm prepared by reductive electropolymerization. For thatpurpose, three different fullerene adducts were utilized asfunctional monomers. The presence of IL in the solution for theelectropolymerization allowed us to dissolve all components,including the ionic ATP template. After polymerization, the ATPtemplate presence in the MIP-ATP film and its subsequent absenceafter extraction proved formation of the pre-polymerizationcomplex in solution, and then deposition of the polymerizedmolecular imprint. This fullerene based MIP-ATP film wasintegrated with two different transducers, vis., the Au-QCR andthe Pt electrode, for fabrication of the PM and CI chemosensor,respectively.

The FIA peak-like detection signals confirmed reversibility ofthe ATP binding by the imprinted cavities of MIP-ATP. That is, theinjected ATP analyte was, first, accumulated in the MIP-ATP film bynon-covalent binding to its molecular cavities, and then fullyreleased with the excess of the carrier solution. The lowest LODvalue was reached by the CI chemosensor being by an order ofmagnitude lower than that of the PM chemosensor. Thisdetectability difference can be ascribed to inherent properties ofthe transduction techniques used. Moreover, both these techni-ques showed that, except of CTP, the imprinted cavities of the MIP-ATP film successfully discriminated the ATP analyte from itscommon interferences, like AMP, ADP, GTP, and TTP. The hereindetermined selectivity with respect to ADP is higher than thosereported earlier [23,24]. Besides, advantageously for the ATPdetermination, its cell concentration is thousand-fold higher thanthat of ADP [56].

The Langmuir, Freundlich, and Langmuir–Freundlich isothermswere fitted to the experimental data of the ATP sorption by theMIP-ATP film to characterize the sorption phenomenon. Thedetermined density of imprinted cavities accessible for ATP and theATP sorption equilibrium constants appeared to be similarindependent of the sorption model adopted. Moreover, the latterwas nearly the same, determined independently either under thesteady-state or flow-injection solution conditions.

Good isotherm fit to the experimental ATP sorption data mayindicate fulfillment of the assumptions of the sorption models.That is, the imprinted cavities were homogenous and each ATPmolecule occupied only one imprinted cavity. Moreover, the ATPmolecules occupying these cavities did not interact one with theother.

Acknowledgements

The present research was financially supported by the EuropeanRegional Development Fund through Project ERDF (POIG.01.01.02-00-008/08 2007-2013) to W.K., K.N., P.S.S. and J.W.S., the PolishNationalCentre for Science(NCN, 2011/03/D/ST4/02596) toP.S.S., the

Foundation for Polish Science (MPD/2009/1/styp19) to T.P.H., in partby the European Union 7.FP (Grant No. REGPOT-CT-2011-285949-NOBLESSE), and the US National Science Foundation through GrantNo. 1110942 to F.D. Access to AFM was funded by the Foundation forPolish Science under the FOCUS Grant No. FG 3/2010.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.aca.2014.07.005.

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