Zwitterionic molecularly imprinted polymer-based solid-phase micro-extraction coupled with molecularly imprinted polymer sensor for ultra-trace sensing of L-histidine

Bhim Bali PrasadKhushaboo TiwariMeenakshi SinghPiyush S. SharmaAmit K. PatelShrinkhala Srivastava

Analytical Division, Departmentof Chemistry, Faculty of Science,Banaras Hindu University,Varanasi, India

Original Paper

Zwitterionic molecularly imprinted polymer-basedsolid-phase micro-extraction coupled withmolecularly imprinted polymer sensor for ultra-trace sensing of L-histidine

The proposed L-histidine sensing system composed of a molecularly imprinted solid-phase microextraction component combined with a molecularly imprinted poly-mer sensor was used to determine critical levels of test analyte in a complex matrixof highly diluted human blood serum without any non-specific sorption and false-positive contributions. The molecularly imprinted polymer was a zwitterionic poly-mer brush derived from the disodium salt of EDTA and chloranil, grafted to solid-phase microextraction material. The hyphenated approach was able to detect L-histi-dine quantitatively with a limit of detection as low as 0.0435 ng/mL (RSD = 0.2%,S/N = 3).

Keywords: L-Histidine / Human blood serum / Molecularly imprinted polymer brush / Molecularlyimprinted polymer-sensor / Solid-phase microextraction /

Received: October 22, 2008; revised: December 15, 2008; accepted: December 15, 2008

DOI 10.1002/jssc.200800595

1 Introduction

One of the main challenges facing the analytical chemistis the development of polymer brushes that respond tothe growing need to perform ultratrace analysis of bio-molecules in ,real-life’ samples without any false-positivecontributions. Surface properties such as specific affin-ities toward bulk materials with protein-resistance char-acteristics are highly desirable for this kind of tailoringof materials. One way of controlling the surface proper-ties of a material is to deposit a polymer brush thereon.Polymer brushes are created by attaching (grafting) poly-mers by one end of their chains to a surface at a suffi-ciently high density to ensure that the chains, in theirpreferred configuration, substantially stretch away fromthe surface in order to avoid overlapping. Whereas the

thickness of the brush is known to depend strongly onthe length of the polymer, the brush grafting density atthe material surface is less easy to control and dependsstrongly on the method used to form the polymerbrushes. Various approaches for end-grafting (tethering)polymer chains on the surface such as “grafting to”(chemisorption), “grafting from” (in situ polymerizationof monomers), physisorption, and Langmuir–Blodgetttechniques are increasingly being utilized for modifyingthe surface properties of materials. Of these, the “graft-ing to” procedure has proven to be experimentally muchmore challenging for production of high densitybrushes, since it is known to normally produce a lowgrafting density and overlapped brushes at the surface.

In the present investigation, we have focused on thepreparation of a polymer brush based on the “graftingto” principle which could result in high density brusheswith a narrow molecular weight distribution, and at thesame time could be applicable in the development of asolid-phase microextraction (SPME) system with appa-rently no impediment to mass transfer. Besides protein-resistance characteristics of such polymer brushes, highspecificity (selectivity) can be achieved on the basis ofmolecular imprinting. It might be mentioned thatmolecularly imprinted polymer (MIP) brushes have notyet received the attention they deserve, despite offeringthe advantages of wettability, reduced friction, and pro-tein resistant surfaces for better mass transfer and selec-tive recognition. Further, for ultratrace analysis, no sin-

Correspondence: Professor B. B. Prasad, Analytical Division, De-partment of Chemistry, Faculty of Science, Banaras Hindu Uni-versity, Varanasi – 221005, IndiaE-mail: [email protected]: +91 5422 368174

Abbreviations: Chl, chloranil; DPCSV, differential pulse, catho-dic stripping voltammetry; ECD, electrochemical detection;HMDE, hanging mercury drop electrode; IPN, interpenetratingnetwork; LH, L-histidine; MIP, molecularly imprinted polymer;MISPME, molecularly imprinted solid-phase microextraction;NIP, non-imprinted polymer; PMMA, poly(methyl methacry-late); SAM, self-assembled monolayer; TEOS, tetraethyl orthosili-cate

i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

1096 B. Bali Prasad et al. J. Sep. Sci. 2009, 32, 1096 – 1105

J. Sep. Sci. 2009, 32, 1096 –1105 Other Techniques 1097

gle technique, whether it is based on a direct sensingprobe (sensor) or SPME systems (commercial SPME fibers[1], silica fibers [2], MIP fibers [3, 4]), is able to detect com-pounds at stringent limits of detection, particularly inhighly diluted real samples. Alternatively, a combinedapproach employing both techniques has reportedlybeen beneficial against surface fouling in ultratrace sens-ing of biomolecules [5]. Here the surface modification ofSPME fibers with an interpenetrating network (IPN) oforganic– inorganic–organic hybrid was found advanta-geous for the selective separation and preconcentrationof target analyte. The sol–gel technique was introducedas a convenient way to produce a highly stable three-dimensional matrix as a sandwich to support an IPNhybrid over an SPME fiber.

Of the two isomers (D- and L-form) of histidine, only L-histidine (LH) is bioactive and can convert into hista-mine, which is a major neurotransmitter in the brainand throughout the nervous system. The chiral recogni-tion and quantitative assay of LH isomer are essential atboth elevated and deficient levels (normal level: 0.31–26.35 lg/mL [6], elevated levels: 29.5 lg/mL [7]). Such lev-els are indicative of LH metabolism disorders, namelyhistidinemia, Friedreich ataxia, rheumatoid arthritis,fibromyalgia syndrome, and schizophrenia. Manyhyphenated techniques like GC–MS [8], LC–MS [9],HPLC–electrochemical detection (ECD) [10], HPLC–elec-trospray tandem mass spectrometry [11], solid-phaseextraction with GC–FID [12], FIA with chemilumines-cence [13], in addition to other conventional methods,viz. chromatography [14], electrophoresis [15], spectro-scopy [16], ECD [17], biosensor [18], fluorometry [19], aswell as a number of MIPs [20–25] have been reported forLH analysis. Some of these techniques, however, encoun-tered various drawbacks such as labor-intensive samplepreparation, long analysis time, tedious synthetic proce-dure, and expensive instrumentation. To the best of ourknowledge, the SPME technique has not hitherto beenemployed for LH analysis. Bearing in mind the notoriouspassivation of electrochemical sensors under the ordi-nary aqueous conditions of an LH aqueous solution [26],a zwitterionic MIP-brush adopting charge-transfer com-plexation between EDTA and chloranil (chl) is utilized inthe present work to modify a hanging mercury drop elec-trode (HMDE). This MIP sensor was employed after per-forming a molecularly imprinted solid-phase micro-extraction (MISPME) experiment to achieve a highly spe-cific and sensitive detection as low as ng/mL. The intro-duction of a chl moiety, in analogy with the surfactantfor direct electron transfer within a zwitterionic

,gemini’ film [27], could help to develop an electrocon-ductive environment (see Section 3) at the surface of anMIP sensor for the direct electrochemical reduction of LHin the differential pulse, cathodic stripping voltammetry(DPCSV) mode [28]. The MIP plays, in the present

instance, a double role as a MISPME material for LH pre-concentration and as a recognition receptor in subse-quent DPCSV sensing with an MIP sensor.

2 Experimental

2.1 Materials and reagents

Poly(methyl methacrylate) (PMMA) optical fibers wereobtained from a fiber optic UFO lamp (New Bright Deco-rative Co., Foshan-City/Nanhai, China). Reagents EDTA(disodium salt), chl, tetraethyl orthosilicate (TEOS), testanalyte (LH), and all interferents used in the presentstudy were purchased from GlaxoSmithKline Pharma-ceuticals, India; Loba Chemie, India; Sigma–Aldrich,India; Sisco Research Laboratories, India and SD Fine,India. Solvents DMSO and water were used after distilla-tion. The real-world sample in this investigation washuman blood serum obtained from a local pathologycenter. All test samples were prepared from LH aqueousstock solution (500 lg/mL) by appropriate dilution, andthe solution pH was maintained at 4.0 with the aid of afew drops of 0.01 M HNO3.

2.2 Instrumentation

Extracts desorbed from MISPME fiber (mounted in theplunger of an insulin syringe (Hindustan Syringes andMedical Devices, India)) were analyzed using a PrincetonApplied Research (PAR) model 264A voltammetric ana-lyzer/stripping voltammeter (in conjunction with 303 Astatic mercury drop cell stand and an x-y recorder (PARmodel RE 0089)) following DPCSV technique. The work-ing electrode was an HMDE (surface area 0.0092 cm2).The reference and auxiliary electrodes were, respectively,a saturated Ag/AgCl electrode with porous Vycor frit anda platinum wire electrode. Elemental analyses were per-formed using an Exeter Analytical Inc. elemental ana-lyzer (Model CE-440), Mexico. Spectral characterizationand morphological evaluation of fiber were performedwith an FTIR spectrophotometer (Varian 3100 FT/IR, USA)and a scanning electron microscope (SEM model XL-20,Philips, The Netherlands), respectively.

2.3 Preparation of MISPME fibers

The fabrication details for MIP-coated SPME fibers arereported in our earlier work [5]. Insofar as MIP synthesisis concerned, normally in a single batch, EDTA (3.72 g/10 mL DMSO) and chl (2.46 g/10mL DMSO) solutionswere refluxed together for 1 h at ca. 1858C to yield a col-ored (crimson-red) polymer solution. To this preformedpolymer solution was added LH (1.55 g/10 mL DMSO) at alater stage and the mixture was again refluxed for 4 h.After complete evaporation of DMSO (porogen) at



ca 1858C, an MIP-LH adduct was obtained in melt condi-tion. The complete retrieval of LH from film was ensuredby soaking MIP-adduct modified PMMA fibers in 500 lLof 0.1 M HCl, under vortex stirring, until no currentresponse of LH was detectable in a DPCSV run. The aver-age thickness of MIP-coated fibers including non-imprinted polymer (NIP) fibers (10 fibers per batch) wasmeasured with the aid of Vernier Calipers (Mitutoyo,Japan). A reproducible average film thickness of131.5 l 0.8 lm was obtained for each fiber in each batch,which included a 10 lm sol–gel film and an MIP brushof 21.5 lm (RSD = 3.7%) thickness.

2.4 SPME–DPCSV procedure

The direct-immersion SPME method was followed in thepresent study as described earlier [5]. The modification ofa hanging mercury drop by an MIP–DMF casting solu-tion (Fig. 1C) and the voltammetric procedure adoptedhere were based on earlier work [29]. The operationalconditions for DPCSV measurement optimized in thepresent case were: MIP concentration, 250 lg/mL; MIPdeposition time, 90 s; accumulation potential of LH,+0.1 V (vs. Ag/AgCl); LH accumulation time, 90 s; pH, 4.0;scan rate, 10 mV/s; pulse amplitude, 25 mV. The reprodu-cible regeneration of modified SPME fiber and the preci-sion (fiber-to-fiber and sample-to-sample) of the proposedmethod have been examined. The use of HMDE (in lieu ofa solid electrode) has an environmental edge resultingfrom the distinct advantages of always being able toobtain a new mercury drop for modification with areproducible thin membrane film of MIP for highly sensi-tive measurement without the attendant problem of sur-face fouling, in contrast to solid electrodes and conven-tional SPME systems.

3 Results and discussion

3.1 SPME fiber coating and morphology

The method employed for the preparation of sol–gelimmobilized MIP coating comprises – in short – contact-ing in succession or simultaneously an activated PMMAsurface with sol–gel melt and a melt of brush-formingfunctionalized telomer chains in order to allow a bond-ing reaction to occur between the sol–gel surface andthe MIP. Upon completion of the nucleophilic attack ofSiO – on C5 (chl), the sol–gel forms a sandwich betweenthe PMMA fiber surface and the brush MIP chains. This isevident from Fig. 2, which shows two covalently bondedmicrophases: (i) between hydrolyzed PMMA (containingCOOH groups) and sol–gel and (ii) between sol–gel andterminally functionalized (chl) polymer chains via Si–O–R links [5]. A “melt” in the context of sol–gel and MIPas used in this work means a preferentially liquid poly-

mer composition wherein both matrices are at a criticaltemperature (above the characteristic glass transitiontemperature and below the degradation temperature) of808C. Above this temperature both are soft and flexible,and there is enough thermal energy available to allowtorsional angle changes and mobility of the molecule.Since the IPN primarily involved an organized sequencelayer of PMMA-sol–gel-MIP adduct, the binding sites inthe exterior layer of grafted MIP brushes were notdeformed (as was evident from 100% analyte uptake effi-ciency), as a consequence of the template removal, interms of size and affinity. The sol–gel, once attached tothe PMMA surface through covalent bonding, still has ahigh density of unreacted reactive groups (SiO – ) to whichbrush polymers can be attached with high grafting den-


Figure 1. (A) MIP-LH adduct, (B) MIP, (C) MIP-coatedHMDE sensor.


sity. The increased electrostatic repulsion between intra-and inter-chains of zwitterionic MIP allowed polymerbrushes to be stretched away from the fiber surfaceunder any conditions before and after the use of acid(0.1 M HCl) as template extractant [30].

The morphology of IPN hybrid material coated onSPME fibers was studied by SEM (magnification 1486)after removal of the template. Although a compact andhighly dense coating was observed (Fig. 3), its rough sur-face exhibits several micropores (microvoids) with appa-rent apertures of different sizes for analyte rebinding.The nanometer scale morphology observed here mostprobably results from the heterogeneity of the brushlayer in terms of grafting density (r = 70.8 chains/nm2),as calculated from using Eq. (1) [31]

r = hqNa/Mn (1)

where h is the thickness of the dry polymer brushes, NAisAvogadro's number, Mn (3878 g/mol) is the MIP-LH adductnumber average molecular weight, and q (21.26103g/m3) is the density of anchored polymer. The average dis-tance between grafting points (D = 2(pr) – 1/2 = 0.19 nm) islower than the end-to-end distance (5.8 nm) of the poly-mer, confirming that the MIP grafter layer is in the brushregime. The dense morphology, as shown by the SEMimage, is a consequence of the higher grafting density(70.8 chains/nm2) of polymer brushes. The grafting den-

sity of a polymer brush is mainly governed by the brushpolymerization degree (N) and the polymer weight frac-tion in the grafting solution (b = 1, 100 wt%), as repre-sented by Eq. (2) [32]

m = (N – 1/2 b7/8)/a2 (2)

where, a is the segmental monomeric length (a =1.46 nm) of the MIP. Accordingly, the maximum graftingdensity (mmax) achievable via the “grafting to” approach is


Figure 2. Schematic representation of MIP-coatedSPME fiber preparation.

Figure 3. SEM image of MIP-coated SPME fiber at magnifi-cation 1486.


equal to 0.24 chains/nm2. Interestingly, in the presentcase, the experimental grafting density realized withcylindrical fibers is comparatively high. This may beattributed to the fact that the brush polymers are immo-bilized onto the pre-coated sol–gel surface in the melt(non-swollen) state, which allows for higher grafting den-sity. In contrast to the method in which polymer chainsof the brush are immobilized onto the surface in theswollen conformation because they are in a dissolvedstate and are contacted with the surface in solution, thebrush chains in the present case are immobilized in anessentially non-swollen state because they are contactedwith the surface in the form of a melt. Since the volumeof a single polymer is reduced in this approach, thepresent method allows for an abnormally high graftingdensity.

3.2 Structural characterization and recognitionmechanism

Since chain propagation during polymerization is onlyfeasible with EDTA terminal nitrogen (underivatized), LHdoping occurred at alternate nitrogen centers (Fig. 1A),particularly when the EDTA –chl–template molar ratiowas 1:1:1. It may be noted in this context that a stoichi-ometry of 1:1:2 would only lead to a monomeric adductin the absence of free terminal nitrogen of EDTA andhence chain propagation. Furthermore, the stability ofthe MIP (1:1:1) (mp A 3008C) is thermodynamicallyfavored in the absence of any steric compression. The N-terminus of LH need not be protected since this wasadded at a later stage in the matrix of the pre-formed pol-ymer chain. The probable structures of the MIP-LHadduct and the MIP, as shown in Fig. 1A, B, are based onthe following elemental analyses: (i) MIP–LH adduct(3n + 1) DMSO; n = 3, Mn 3878. Experimental (%):C = 31.90, H = 3.94, N = 6.97; calculated (%): C = 31.56,H = 3.38, N = 6.14. (ii) MIP nDMSO; n = 3, Mn 2754. Experi-mental (%): C = 30.90, H = 2.34, N = 4.30; calculated (%):C = 30.50, H = 2.69, N = 4.10.

The chloride ion estimates in MIP–LH adduct and MIP,as obtained conductometrically in DMSO–water (50%,v/v), were 1.51 and 2.83 mole Cl – ion/1000 g, respectively,which are in accord with the proposed structural formu-lations.

Since LH exists as zwitterion (side chain) in the pHrange between 4 to 8 [33], three molecules of LH caneasily be paired via electrostatic interaction with threezwitterionic (–N+ –CH2 –COO – ) pendant arms of the MIP,releasing three moles of HCl (to waste). The protrudingimidazole rings could be accommodated in cavities cre-ated by amino acetic (N-carbobetaine) pendant arms in aspatial arrangement with proximate chl molecules,involving hydrophobically induced hydrogen bondingbetween both coplanar hydrophobic imidazole amine

and chl –C=O, Fig. 1A. Such hydrogen bonding is sup-ported by downward IR peak shifting of –C=O of chlfrom 1654 cm – 1 to 1632 cm – 1 and upward peak shiftingof –NH bending of imidazole from 1411 cm – 1 to1470 cm – 1 (Fig. 4a). Interestingly, these shifted peaksreassumed their original positions upon removal of LHby 0.1 M HCl. Other characteristic IR peaks (Fig. 4b) indi-cate major functionalities of MIP (after templateremoval) as described below for fiber–sol-gel–MIP (mmax,cm – 1): 3440 (–COO – Na+ stretching), 2990 (–COOHstretching/ –CH stretching), 2880 (stretching of quater-nary amine salt), 1720 (–C=O stretching of free carb-oxylic acid), 1654 (–C=O stretching for ionized carboxy-late/–C=O of chl), 1411 (–CH bending), 1220 (–CNstretching), 1080 (–C–O stretching), 720 (–C–Cl stretch-ing). The IR peaks (Si–O–Si, Si–O–C at ca. 1085, Si–OHat ca. 960 and SiO – vibrations at ca. 795 and ca. 460) asobserved in Fig. 4b correspond to the sol–gel sand-wiched between PMMA fiber and MIP, as discussed ear-lier [5].

The present MIP system could be regarded as a polycar-bobetaine (a special class of polyampholytes having poly-electrolyte nature), in which each monomer unit carriesboth a positive and negative charge. Although the recog-nition mechanism on such zwitterionic stationaryphases is still ambiguous, a combination of two majorcomponents may be considered responsible for the ana-lyte sorption. The first is electrostatic attraction of theanalyte to the stationary phase and the second involveshydrophobic attraction and water structure inducedpairing of ionic groups. Furthermore, it is highly prob-able that the very small perturbation effect of the zwit-terionic polymer on the structure of water is crucial for


Figure 4. Infrared (KBr pellet) spectra: (a) fiber–sol–gel–MIP–LH adduct, (b) fiber–sol–gel–MIP.


the absence of non-specific adsorption of proteins to thepolymers resulting in excellent biocompatibility of thepolymers [34]. In the present case, polymer surface modi-fications were significantly thicker (21.5 lm) than thoseof self-assembled monolayers (SAMs; typically 3–5 nmthick) [35], thus creating an increased density of non-foul-ing zwitterionic groups and interactions on the surface.With a higher number of non-fouling interactions thepolymer brush surface exhibits an increase in the surfacehydration layer and, as a result, the hydration force usedto resist protein adsorption from biological fluids ismuch greater on the polymer surface.

3.3 Binding performance

The sorbed LH molecules in the exterior film of MIPbrushes were first desorbed and again loaded for re-enrichment into the same MIP directly coated over theHMDE sensor. The HMDE revealed a film adherence ofthe MIP film through coulombic interactions betweenpositively charged HMDE (+0.1 V vs. Ag/AgCl) and elec-tron-rich chl carbonyls; the quaternary nitrogensremained electroneutral in the polymer chains (Fig. 1C).The presence of chl may also induce a charge-transfercomplexation between LH and chl, creating a ,p-type’semiconductor property in LH, thus permitting elec-tronic communication (heterogeneous electron transferdirectly from electrode to the LH redox centers) in thezwitterionic MIP film [36]. Accordingly, under such con-dition, the firmly entrapped ,electro-inactive’ LH mole-cules were now set to behave as ,electro-active’ centersand responded with a broader cathodic DPCSV peak at

–0.3 V vs. Ag/AgCl at 10 mV/s scan rate and pH 4.0. InSPME, the LH recovery was found to gradually increase,attaining a limiting plateau beyond 20 min on magneticstirring (600 rpm). The desorbing process (0.1M HCl/15 min, under dynamic condition) required an optimumvolume of 500 lL, as ascertained on the basis of a normal-ized elution profile (amount of analyte detected/amountof analyte taken versus eluent volume; Figure not shown)of analyte eluted in SPME experiments. The MIP-coatedSPME fiber showed a maximum uptake of LH at pH 4.0due to the fact that a tautomerizable nitrogen-boundhydrogen in the imidazole cation is readily available forformation of a charge-transfer (LH –chl) complexthrough hydrogen bonding at this pH. This is not favoredat pH >4.0, and in basic media, the basic requirement ofLH in zwitterionic form for formation of such a complexis no longer fulfilled. In spite of the protein and non-spe-cific resistant zwitterionic MIP brush, non-specific bind-ing of LH molecules still occurred to the extent of 28% inthe present study. Nevertheless, this could easily bewashed off by multiple water-washings and analyte mol-ecules could then diffuse specifically toward bindingsites. The observed imprinting factor (MIP response/NIPresponse) for LH-imprinted polymer was found to be 5.0after single washing (160.5 mL water) of MIP- and NIP-coated SPME fibers. As suggested by Fig. 5A, a maximumof three water-washings (360.5 mL) of NIP-coated SPMEfibers were required to completely mitigate non-specificinteractions. This is also recommended for MIP-coatedSPME fibers.

As shown in Fig. 6, when used alone, the MIP sensoreither afforded non-detectable signals (DPCSV curve ,c’)


Figure 5. (A) Recoveries of LH (20.0 ng/mL) from aqueous solutions by MIP-coated (9) and NIP-coated (0) fibers by 0.5 mL HCl(0.1 M), the fibers were washed prior to recovery with 0.5 mL portion of water in each washing. (B) Effect of dilution of bloodserum on recovery of LH from MIP-coated (9) and NIP-coated (0) fibers by 0.5 mL HCl (0.1 M).


or a less quantifiable (DPCSV curves ,a’, ,e’ and ,g’)response. However, a combined MISPME-MIP sensor tech-nique responded with at least three-fold enhanced mag-nitude of current for LH (DPCSV curves ,b’, ,d’, ,f'’, and ,h’)under optimized operating conditions. Consequently,the dual preconcentration of LH, first on the SPME fiberand secondly on the MIP sensor, enhanced sensitivity byas much as a factor of about three compared to the MIP-modified HMDE sensor (LOD = 0.128 ng/mL). Analyticalresults for LH analysis obtained through a combined MIS-PME-MIP-sensor method are summarized in Table 1; how-ever, associated enrichment factors only increased up to10-fold, recoveries were always quantitative with perfectlinearity between [analyte]taken and [analyte]desorbed. Thecalibration equation between peak current (Ipc, lA) andanalyte concentration (C, ng/mL) for LH and respectivelimit of detection (LOD) computed as 3r by standard pro-cedure) [37] were: For the concentration range of 0.15–90.00 ng/mL), Ipc = (0.0897 l 0.0003)C + (0.0166 l 0.0107),m = 0.99, n = 11. LOD = 0.0435 ng/mL (3r, RSD = 0.2%).

The stability and performance of the MISPME fiber waschecked for the same fiber regenerated every alternateday, and also in terms of fiber-to-fiber variability fromthree batches of MISPME fibers. The regeneration (tem-

plate removal) of an MISPME fiber, after each measure-ment, gave multiple runs (Fig. 6f) confirming its reprodu-cible behavior. As is also evident from Table 1, the MIS-PME fiber was found to be reusable, after regeneration(template removal), for as many as 33 consecutive extrac-tions giving reproducible results, after which the graftedlayer was found to gradually deteriorate, giving a dimin-ishing peak. The reproducibility among three differentMISPME fibers taken from three different batches for LHconcentration 7.0 ng/mL (Table 1) yielded a high degreeof precision (RSD 0.1%, n = 3), and this confirmed the val-idity of the proposed method. The ruggedness describingthe reproducibility of the method in any DPCSV measure-ment, after SPME extraction on a single fiber, was furtherconfirmed by multiple DPCSV runs (Fig. 6h) of threedifferent MISPME extracts obtained from three identicalaqueous samples (LH concentration 1.50 ng/mL).

The proposed method is compared with the adsorptivecathodic stripping voltammetric method [38]. Whereasboth methods are precise and accurate on the basis ofStudent's t-test (tcal a ttab, confidence level 95%, m = 0.99),Table 1, the sensitivity of the present method is manytimes higher than that of the adsorptive cathodic strip-ping voltammetric method (adsorptive cathodic strip-


Table 1. Analytical results of DPCSV measurements of LH in an aqueous environment by combined MISPME-MIP-sensormethod.

[LH]takena) (ng/mL) Sample

Volume(mL)

[LH]desorbedb)

(ng/mL)Volumeeluted(mL)

Efc) Recoveryd)

(%)RSDe) (%)(n = 3)

mf) tg)

0.15(nd) 5.0 1.498 l 0.036 0.5 9.9 99.9 2.40.50 (0.48, nd) 5.0 5.058 l 0.036 0.5 10.1 101.2 0.71.50 (1.49, nd) 5.0 14.742 l 0.054 0.5 9.8 98.3

(98.0,99.0,100.0)0.4

2.50 (2.49, nd) 5.0 25.240 l 0.054 0.5 10.1 100.9 2.17.00 (6.95, nd) 5.0 71.118 l 0.072 0.5 10.2 101.7 {100 (i),

98 (ii), 90 (iii),80 (iv)}

0.1 (0.1)

15.00 (14.77, nd) 5.0 149.184 l 0.084 0.5 9.9 99.5 0.123.00 (22.96, 22.98) 5.0 230.76 l 2.322 0.5 10.0 100.3 1.039.50 (39.46, 39.48) 5.0 394.182 l 1.898 0.5 9.9 100.2 0.5 tcal = 1.666.00 (65.80, 65.98)78.00 (77.54, 77.98)90.00 (89.42, 81.11)

5.05.05.0

666.288 l 3.204783.342 l 2.970900.00 l 3.168

0.50.50.5

10.110.010.0

100.9100.4100.0

0.50.40.4

0.99 ttab = 2.8

a) Values in parenthesis denote LH concentration determined by MIP-modified HMDE sensor and adsorptive stripping voltam-metry [37], respectively. nd, not detectable.

b) [LH]desorbed denotes the concentration of desorbed amount (i. e. LH amount eluted by the fiber/optimized volume of thedesorbing solvent).

c) Enrichment factor, [LH]desorbed/[LH]taken.d) Recovery (amount of desorbed LH)/(amount of LH in sample), values in parentheses indicate recoveries from three identical

samples. Values in curled brackets indicate recovery after (i) 33, (ii) 34, (iii) 35, and (iv) 36 consecutive extractions by thesame MISPME fiber regenerated on every alternate day.

e) Relative standard deviation, value in parentheses indicates precision among three different MISPME fibers taken from threedifferent batches of MISPME fibers.

f) m, Correlation coefficient.g) Student's t-test for comparison of two methods at confidence level 95%.

Hffjffh


ping voltammetric method; LOD = 12.412 ng/mL; MIS-PME-MIP-sensor method, LOD = 0.0435 ng/mL).

3.4 Interference studies

Interferents examined for cross-reactivity on MIP- or NIP-coated fibers either had common functionalities orexclusively zwitterionic (amino acid, creatine) struc-tures. These were tryptophan (Trp), tyrosine (Tyr), phenyl-alanine (Ph), urea, glycine (Gly), creatine (Crea), D-histi-dine (DH), imidazole (Imz), dopamine (DA), glucose (Glu),uric acid (UA), and ascorbic acid (AA). The MISPME fibershowed a quantitative (100%) recovery of the target ana-lyte (LH). On the other hand, it was totally non-responsiveto any of the interferents studied (Fig. 7). In the case ofNIP-modified SPME fibers, LH recovery was quite signifi-cant (20.1%), while all interferents showed sorption withsubstantial recoveries between 12.5% and 38.5% after thefirst wash (160.5 mL water). However, these non-specifi-cally sorbed molecules were completely washed awayfrom the surface of NIP-coated fibers using two morewashings with 260.5 mL water. Insofar as binary andmultiple mixtures (containing both test analyte andinterferents in clinically relevant concentration ratios)(Fig. 8) are concerned, LH recoveries (10.8–32.9%) weredrastically reduced after the first wash and completelyremoved in the final wash with NIP-coated SPME fiber.


Figure 6. DPCSV response of LH (concentration ng/mL): (a)3.0 in serum, MIP sensor; (b) 3.0 in serum, MISPME–MIPsensor; (c) 0.15, (e) 0.50, (g) 1.50 in water, MIP sensor; (d)0.15, (f) 0.50, (h) 1.50 in water, MISPME–MIP sensor.

Figure 7. Extraction yields of LH and interferents with MIP-and NIP-coated fibers at 12.0 ng/mL level.


Interestingly, MIP-coated fibers gave quantitative recov-eries of test analyte (LH) in either of the mixture solu-tions studied and maintained recoveries unchangedafter water washings. Nevertheless, in view of the equaleffect of non-specific sorptions on both MIP and NIP, it isimperative to wash MIP-coated SPME fibers (like the NIP-coated fiber) prior to desorption and the detection via acomplementary MIP sensor. Despite being zwitterionic,some interferents (DH, Trp, Tyr, Gly, Crea, and Ph) werenot oriented to pair with zwitterions of MIP brushes, likethe test analyte in the present instance. This revealed anexcellent imprinting effect and enantioselectivity of theproposed MISPME system.

3.5 Application to real samples

The proposed MISPME-MIP-sensor method was examinedfor LH analysis in human blood serum without samplepretreatment. However, the serum sample was dilutedby a factor of 1000 to mitigate non-specific sorptions dueto complex matrices as observed with NIP-coated SPMEfibers (Fig. 5B). The need for dilution is to obtain a betterDPCSV response with quantitative recovery by MIP-coated SPME fibers (Fig. 5B and Fig. 6 (curve ,b’)). The dilu-tion factor of blood serum in the present investigation iscomparatively lower than in previous work [5], simply

because zwitterionic MIP brushes are known to haveexcellent low-fouling (protein resistant) properties [34,35]. Furthermore, dilution of the serum sample approxi-mated its behavior somewhat closer to that of the aque-ous samples in terms of the calibration equation andLOD value: Human blood serum, concentration range(0.20 –89.50 ng/mL); Ipc = (0.0893 l 0.0001)C + (0.0054 l0.0024), m = 1.00, n = 6. LOD = 0.0448 ng/mL (3r, RSD =0.2%).

Insofar as sample-to-sample validation is concerned,three different blood samples collected from three volun-teers and their respective spiked sera containing a totalof 3.0 ng/mL of LH revealed identical DPCSV peaks(Fig. 6b) after MISPME, and for each serum sample, a cali-bration equation similar to the one mentioned above isobtained.

4 Concluding remarks

The present investigation demonstrates the developmentof a zwitterionic MIP network for grafting to the surfaceof SPME fiber in brush regime and for use as a sensing ele-ment in the complementary MIP sensor. The proposedmethod may be considered as a unique hyphenated tool(MISPME-MIP sensor) for clinical investigations to


Figure 8. Recoveries of LH from MIP-coatedfibers in the presence of interferents in the aque-ous solutions. Concentrations (ng/mL) of LH andits interferents in the binary as well as multiplemixtures are: (A) 12.0 LH+12.0 Trp, (B) 12.0LH+12.00 Tyr, (C) 12.0 LH+120.0 Ph, (D) 12.0LH+10 800.0 Urea, (E) 12.0 LH+240.0 Gly, (F)12.0 LH+12.0 Crea, (G) 12.0 LH +12.0 DH, (H)12.0 LH+12.0 Imz, (I) 12.0 LH +120.0 DA, (J)12.0 LH+240.0 Glu, (K) 12.0 LH+600.0 UA, lL)12.0 LH+600.0 AA, (M) 12.0 LH+120.0 (each)mixtures of all interferents. Recoveries from NIP-coated fibers are shown after first wash (0.5 mL)and final wash (260.5 mL) with water.


enhance the detection sensitivity as low as the ng/mLlevel by repeat preconcentration and subsequent DPCSVdetection. Choice of the zwittterionic format of the MIPaffords a biocompatible SPME system and secures accu-rate and precise results without any false positives owingto protein and/or other non-specific sorptions in biologi-cal fluids.

Support of this work by the Department of Science and Technol-ogy, SR/S1/IC-18/2006, is gratefully acknowledged.

The authors declared no conflict of interest.

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