CHIRALITY 26:129–131 (2014)

Short Communication

Enantioselective Surface Plasmon Resonance Sensor Based on C60

Fullerene-Glutathione Self-Assembled Monolayer (SAM)†

RALUCA-IOANA STEFAN-VAN STADEN*

Laboratory of Electrochemistry and PATLABBucharest, National Institute of Research for Electrochemistry and CondensedMatter, Bucharest, Romania

© 2014 Wiley Perio

ABSTRACT A novel enantioselective surface plasmon resonance (SPR) sensor based on aself-assembled monolayer of C60 fullerene as the chiral selector is proposed. A binding assay,apparent affinity constant, and apparent dissociation binding constant were used to analyze andstudy the enantioselectivity of C60 fullerene-glutathione film for L-histidine, which was chosen asthe model analyte. The apparent affinity constant for the complex formed by L-histidine with C60fullerene-glutathione film was 5.2 x 109M-1. The proposed SPR sensor can be used for the assayof L-histidine in the 10-10 – 10-7mol/L concentration range. Chirality 26:129–131, 2014. © 2014Wiley Periodicals, Inc.

KEY WORDS: L-histidine; C60 fullerene; enantioselective surface plasmon resonance sensor

*Correspondence to: Raluca-Ioana Stefan-van Staden, Laboratory of Electro-chemistry and PATLAB Bucharest, National Institute of Research for Electro-chemistry and Condensed Matter, 202 Splaiul Independentei Str., 060021,Bucharest, Romania. E-mail: iustinavanstaden@yahoo.comReceived for publication 13 October 2013; Accepted 16 November 2013DOI: 10.1002/chir.22281Published online 21 January 2014 in Wiley Online Library(wileyonlinelibrary.com).†This article was published online on [21 January 2014]. The article category hassince been corrected to “Short Communication”. This notice is included in theonline version to indicate that this has been corrected [17 Febuary 2014].

INTRODUCTIONChiral analysis has become extremely important in the last

years, especially in the clinical and pharmaceutical fields.1

Different enantiomers of a chiral biomolecule can be markersfor different diseases, while different enantiomers of a pharma-ceutical compound may have different pathways in the body.Accordingly, there is a need to develop reliable analytical

methods for enantiomers discrimination and quantificationas well as to find the most reliable chiral selectors for a givenenantiomer. Thin-layer chromatography and surface plasmonresonance (SPR) techniques are very good for the qualitativestep of the analysis, in both cases making it possible to identifythe presence of each of the enantiomers. Chromatographic tech-niques such as gas chromatography, high-performance liquidchromatography (HPLC), and capillary electrophoresis simulta-neously provide qualitative and semiquantitative information re-lated to the enantiomeric composition of the sample, withcapillary electrophoresis providing the most reliable analyticalinformation, but at the same time is not sensitive enough to beused in all types of mixtures.2 Amperometric electrodes, amper-ometric biosensors, and enantioselective, potentiometric mem-brane electrodes are reliably used for the enantioanalysis ofenantiomers without any prior separation from their samples.3,4

SPR is a powerful technique that can be successfully used forthe selection of the best chiral selector for enantioanalysis. Thiswas demonstrated for the first time by Kieser et al.5 when theyused modified cyclodextrins for the design of enantioselectiveSPR sensors.L-histidine (L-His) is an essential component of many pro-

teins. Its residue is often found at the active site of enzymes.6

L-histidine acts as a neurotransmitter or neuromodulator in themammalian central nervous system, including retina.7 Anenantioselective, potentiometric membrane electrode based onC60 fullerene was used for the enantioanalysis of L-histidine.8

This article describes the construction of an enantioselectiveC60 fullerene-glutathione self-assembled monolayer on a goldelectrode, and its utilization for the enantioselective binding ofL-histidine, which was used as model analyte.

dicals, Inc.

The spontaneous formation of the monolayer was first dem-onstrated by Zisman and colleagues.9 Since then, numerousadsorbate/substance combinations have been found to formself-assembled monolayers (SAMs). On the other hand, filmsof fullerenes have been prepared using different strategies,including gas-phase deposition,10 Langmuir-Blodgett,11 andself assembly12 techniques. To our knowledge, SAM basedon fullerene was not used until now for enantioanalysis.

MATERIALS AND METHODSL- and D-histidine were purchased from Sigma-Aldrich (St. Louis, MO).

C60 was purchased from Fluka (Buchs, Switzerland). Glutathione was pur-chased from Aldrich (Milwaukee, WI). Phosphate buffer (pH= 6.89) waspurchased from Merck (Darmstadt, Germany). Ethanol, acetone andbenzene were purchased from Fluka. Gold disks were supplied byEcoChemie (Utrecht, The Netherlands). Gold discs were coupled to thecuvette and adhered to the prism via a matching oil with the same refrac-tive index (n = 1.52). The index-matching oil was purchased from CargilleLaboratories (Cedar Grove, NJ). All reagents were of analytical grade.Deionizedwater was obtained using aModulab System (ContinentalWater

Systems, San Antonio, TX). Solutions of L- and D-histidine were preparedby serial dilution from stock solutions of L- and D-histidine (0.1mol/L) andbuffered 1:1 deionized water:buffer solution at pH=6.89. Glutathione andC60 solutions were prepared in deionized water and ethanol, respectively.

Preparation of SAM on the Gold DiskA gold disk electrode (99.99% ~0.8mm diameter) sealed in a special

kind of soft glass was prepared by manual polishing with graded alumina(Buehler, Lake Bluff, IL) from 1.0 to 0.05m with a soft polishing cloth(Buehler); it was sonicated in absolute ethanol and then in distilled water.

STEFAN-VAN STADEN130

The covalent attachment of SAM of C60 fullerene-glutathione wastargeted to the well-defined substrate. The approach involves activationof the gold sensor surface with glutathione molecules that expose theC60. The gold disk was first immersed in the glutathione aqueous solution(0.001mol/L) for 16 h, then it was rinsed with water to remove thenonchemisorbed material and dried. After drying, the disk was immersedin the C60 fullerene solution (0.001mol/L) for 24 h and then again dried.Before each experiment, the surface of this sensor was washed withexcess volumes of ethanol and phosphate-buffered saline (PBS).

ApparatusSPRmeasurementswere performedwithESPRIT instrument (EcoChemie),

using the Kretschmann configuration and a monochrome laser with a 635nmincidentwavelength. TheSPR cuvettewas equippedwith a single channelflow.

Recommended ProcedureSurface plasmon excited themetallic/dielectric interface, upon total inter-

nal reflection of polarization light from an He laser. The optical processes atthe gold surface were detected at a fixed incident angle by in situ monitor-ing reflectivity changes as a function of time. The solutions were injectedinto the cuvette using a plastic needle. The steps of each measurementconsisted of sample injection, mixing of solution, and washing. All experi-ments were performed at room temperature. All bottles were thoroughlyrinsed with deionized water and absolute ethanol and dried before use.

RESULTS AND DISCUSSIONMechanism of Enantiorecognition

An enantioselective SPR sensor based on C60 fullerene-glutathione SAM was used for the enantiorecognition ofL-histidine. The results obtained using this SPR sensor are com-parable to those obtained using the enantioselective, potentiomet-ric membrane electrode based on C60 fullerene.

8 Establishing themechanism of enantiorecognition is essential at this point.C60 fullerene is composed only of carbon atoms, which

make it hydrophobic and nonpolar. It is also known that C60fullerene is an electron acceptor, and is capable of formingdonor-acceptor types of bonds. In contrast, the tripeptide thiolglutathione (GSH) has facile electron-donating capacity,linked to its sulfhydryl (�SH) group. Also, glutathione actsas a bifunctional building block. Gold shows maximum affinityfor the thiol group; accordingly, the –SH group of glutathionewill be covalently attached to the gold. The building of theC60-GSH monolayer on gold is shown in Fig. 1. As shown in

Fig. 1. Formation of C60-glutathione self-assembled monolay

Chirality DOI 10.1002/chir

Fig. 1, the –NH2 group of the GSH molecule is involved inthe bond-forming reaction with C60 fullerene.The chiral recognition mechanism depends on affinity and

enantioselectivity, which are characterized by weak noncovalentforces such as hydrophobic interactions, hydrogen bonding,and electrostatic bonds. The interaction between C60 fullereneand L-histidine is based on hydrogen bonding interaction. theexperimental results obtained using the enantioselective SPRsensors and the enantioselective, potentiometric membraneelectrode based on C60 fullerene8 proved that only L-histidineis bound with C60 fullerene. No bond was observed in either ex-periment betweenC60 fullerene andD-histidine (Fig. 2). Accord-ingly, the proposed electrode ca be used for enantiorecognitionof L-histidine.

Enantioselective Binding AnalysisThere are many ways to calculate the affinity constant from

the response obtained using the SPR at equilibrium (Req). Inthis study we used the apparent affinity constant, KA andapparent dissociation constant, KC for binding analysis.Usually, the experimental data are presented in a Scatchard

plot (Req/[L-Histidine] vs. Req) to obtain a straight line. How-ever, we prefer nonlinear regression using the Langmuirbinding isotherm, as large errors can occur in Scatchardplots.13 To understand the binding strength between the C60fullerene based SAM and L-histidine, the dissociating bindingconstant was determined using the following equation:

Req ¼ CC þ KC

� �Rmax

where Rmax is the maximum binding capacity of C60 withL-histidine, KC is the apparent dissociation constant for theinteraction at the gold sensor disc, and C is the concentrationof L-histidine. Different concentrations of L-histidine were usedto determine the dissociation constant; the values obtainedfor the dissociation constants were 3.0(±0.3)x10-10mol/L, 9.0(±0.2)x10-10mol/L, and 3.0(±0.2)x10-9mol/L. During the SPRmeasurements, maximum binding was recorded for a concen-tration of L-histidine of 10-7mol/L. Binding sensograms ofL- and D-histidine at 10-7mol/L are shown in Fig. 2.

er on gold surface, and its interaction with L-histidine.

Fig. 2. Binding sensograms of I – L-histidine and II – D-histidine.

SPR SENSORS BASED ON C60 FULLERENE 131

The following derived equation was used in order to deter-mine the apparent affinity constant, KA:

14

1Req

¼ 1RmaxKA

x1Cþ 1Rmax

where Req is the SPR response to the injection of L-histidine atequilibrium, C is the concentration of L-histidine, and Rmax isthe equilibrium SPR electrode response when the value of theconcentration of L-histidine is infinity. The apparent affinityconstant for the reaction between L-histidine and C60 fullerene,determined from the graph, is 5.2(±0.4) x 109M-1. Also, theregression coefficient obtained for the plot was 0.9995, provingthat it is possible to perform quantitative enantioanalysis ofL-histidine. The linear concentration range in which L-histidinecan be determined is 10-10 – 10-7mol/L. The limit of determina-tion was 10-10mol/L, and the limit of detection 2.0(±0.2)x10-11.The recovery of L-histidine in pharmaceutical products was97.62 ± 0.85%, when 10 tablets containing L-histidine as theactive substance were analyzed. Therefore, the SPR sensingtechnique can be used for enantioanalysis of L-histidine inreal samples.

CONCLUSIONA novel enantioselective surface plasmon resonance sensor

based on a self-assembled monolayer of C60 fulleren-glutathi-one is proposed for enantiorecognition and enantioanalysis ofL-histidine. The design of the self-assembled monolayer issimple and reliable. The value obtained for the affinityconstant demonstrated that L-histidine is interacting withC60 fullerene, the bond formed being the hydrogen type.The results obtained in this study correlate well with thoseobtained using an enantioselective, potentiometric membraneelectrode based on the same chiral selector: C60 fullerene.The main feature of the method is also to identify the best

chiral selector for a given enantiomer, based on the bindingevent recorded in the sensogram as well as on the association/dissociation constants determined in the experiment.

ACKNOWLEDGMENT

This work was supported by UEFISCDI PNII Program Ideas2011–2014, Contract no. 123/05.10.2011.

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Chirality DOI 10.1002/chir