Development of real-time sensitive chiral analysis technique using quartz crystal analyzer

Sensors and Actuators B 171–172 (2012) 478–485

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Development of real-time sensitive chiral analysis technique using quartz crystal analyzer Jong Min Kim a , Sang-Mok Chang a , Xin-Kuai He b , Woo-Sik Kim c,∗ a b c

Department of Chemical Engineering, Dong-A University, Busan 604-714, Republic of Korea School of Packaging and Materials Engineering, Hunan University of Technology, Zhuzhou 412007, China Department of Chemical Engineering, Kyung Hee University, Kyungki-do 446-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 4 December 2011 Received in revised form 1 May 2012 Accepted 4 May 2012 Available online 15 May 2012 Keywords: Chirality Chiral sensing Mandelic acid Mandelic acid derivative Quartz crystal analyzer Molecular recognition

a b s t r a c t A rapid and highly sensitive chiral sensing technique was developed for the chiral discrimination of mandelic acid (MA) using a quartz crystal microbalance (QCM) modified with an amide-type S-MA derivative. After the modification, the response difference when exposed to the target R-MA and S-MA was measured using a low concentration range from 10 ␮M to 3 mM combined with a novel analysis technique. For the given target concentrations, the chiral discrimination factor between S-MA and R-MA was 5.7 when using the normal frequency measurement. Remarkably, the novel sensing technique produced a chiral discrimination factor up to 9.4 based on an additional parameter at the same experimental condition. In addition, the interaction properties between the enantiomers and the chiral selector were investigated in detail using the F–R model. The main advantages of the novel sensing technique are that it is based on a real-time technique, requires a short detection time, and is highly sensitive within a 10–300 ␮M concentration range. In particular, the excellent sensitivity for a low target concentration was because the novel analysis method revealed the mechanical difference of the intermolecular binding properties between the target enantiomers and the selector. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Chiral recognition continues to attract research attention due to its importance in the fields of chemical, biomedical, and pharmaceutical engineering [1,2]. Various methods have already been developed for chiral sensing, analysis and separation, including capillary electrophoresis [3], high performance liquid chromatography [4], thin-layer chromatography [5], supercritical fluid chromatography [6], nuclear magnetic resonance [7], chiral ligand exchange chromatography [8], circular dichroism [9], diastereomeric crystallization [10], and molecular imprinting techniques [11]. Though these methods are efficient, they still require highcost equipment, sophisticated techniques, and a long process time. Plus, despite the established use of enantioselective synthesis, including asymmetric chemical synthesis and bio-synthesis, for obtaining chiral free molecules [12], developing a synthetic path to the desired enantiomer is still expensive and time-consuming, and the enantiomeric excesses obtained from an enantioselective procedure also require a further chiral separation step. Thus, chiral sensing and separation still remain important challenges in various fields of science and engineering [13–15].

∗ Corresponding author. Tel.: +82 31 201 2970; fax: +82 31 273 2971. E-mail address: [email protected] (W.-S. Kim). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.05.015

Recently, a quartz crystal microbalance (QCM) technique has been introduced for chiral recognition that offers several advantages over other techniques, including a low cost, simple equipment, and high sensitivity [16,17]. The QCM technique was essentially focused on the choice of the chiral selector. For example, Xu et al. [18] reported that ten chiral sensors immobilized with mercaptyl perfunctionalized-cyclodextrins exhibited enantioselectivity toward three pairs of enantiomers (R,S-methyl lactate, R,S-ethyl lactate, and R,S-2-octanol) in the gas phase, yet none of the discrimination factors was over 1.5. Meanwhile, Su et al. [19] used bovine serum albumin or human serum albumin as the selector to discriminate R,S-1-(3-methoxyphenyl)ethylamine, R,S-1-(4methoxyphenyl)ethylamine, R,S-tetrahydronaphthylamine, R,S-2octanol, and R,S-methyl lactate, yet none of their discrimination factors was over 1.6. In a previous study, the current authors developed an l-phenylalanine-modified QCM sensor for the highly sensitive recognition of chiral molecules. In this case, the discrimination factor between l-MA and d-MA was over 8 in a high concentration of 0.05 mol L−1 [20–22]. The proposed method was unfortunately based on an off-line technique and required a long detection time of over 6 days since the experiment was performed in the gas phase. Thus, gas and liquid phase experiments both have their own merits and weaknesses. While gas phase experiments can provide a high discrimination factor, they require a long detection time and samples with a high target concentration.

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Fig. 1. Schematic presentation of fabrication process of S-MA derivative-modified QCM sensor: 4-ATP modification (a), S-MA coupling reaction (b), protecting group removal (c), and finally modified S-MA derivative surface (d).

Conversely, while liquid phase experiments can achieve a high speed analysis, a decrease in the discrimination factor is unavoidable in the QCM method. In addition, the previous QCM techniques only used the resonant frequency change for the chiral discrimination. Yet, a clear discrimination and related mechanism analysis were difficult to achieve when only measuring the resonant frequency change, especially when the frequency change was small. Thus, to obtain a rapid highly sensitive response, a new analytical method is required for liquid phase experiments. Chiral recognition is normally possible as one of the enantiomers is preferentially bound to the selector surface by a different 3-D structure. In this process, the functional groups of the selector form supramolecules with the target molecules through a non-covalent interaction, such as a dipole–dipole interaction, van der Waals force, electrostatic interaction, hydrophobic interaction, or hydrogen bonding [23,24]. Thus, setting an adequate criterion, which can clearly reveal the binding property difference between the enantiomers and selectors, is essentially required for highly sensitive chiral discrimination. The practical application of such a stereoselective analysis could lead to the formation of homochiral compounds from racemates in autocatalytic reactions [25]. Thus, sensing experiments could provide possible routes for chiral separation techniques. Accordingly, this study presents a new rapid chiral sensing and analysis technique for the highly sensitive chiral discrimination of MA based on an additional parameter measurement. Thus, a novel QCM method was used for the definitive distinction of a small difference in the affinity interaction between the target and the selector. 2. Experimental 2.1. Materials and reagents 4-Aminothiophenol (4-ATP), 2-ethoxy-a-(ethoxycarbonyl)-1,2dihydroquinoline (EEDQ), S-mandelic acid (S-MA) (99%, chiral purity), and R-mandelic acid (R-MA) (99%, chiral purity) were all obtained from Sigma–Aldrich (ACS grade, U.S.A.). All the other reagents were commercially available and of an analytical reagent grade.

The response of the quartz crystal sensors was monitored using an oscillation detector (QCA917, EG&G, Tokyo, Japan) at a measuring temperature of 20 ± 1 ◦ C. The detector measured both the resonant frequency and the resonant resistance simultaneously. The accuracy and principle of the instrument was previously reported [26,27]. Briefly, the instrument has the frequency and resistance accuracy of 0.1 Hz and 0.01  at a gate time of 1 s. Two QCM measuring setups were created: (i) a toluene media measuring system for monitoring the procedures of the selector immobilization and (ii) an aqueous media measuring system for the chiral sensing experiment. Each system consisted of a computer, oscillation detector, reaction cell, and quartz crystal sensor. In the first measuring system, both sides of the quartz crystal electrodes were directly exposed to the nonpolar measuring solution. In the case of the aqueous media measuring system, a liquid phase QCM protecting cell was applied so that only one electrode side was exposed to the liquid. The oscillation frequency and resistance of the QCM sensor were recorded after stabilizing the baseline of the resonant frequency to a fluctuation of ±1 Hz min−1 and gate time of 1 s. The mass change of the QCM sensor was calculated according to the Sauerbrey equation [28], and the QCM system had a mass sensitivity of 1.07 ng Hz−1 . The contact angles were measured to confirm the surface property change using a Phoenix 300 contact angle meter (Surface Electro Optics Co., Korea) at room temperature. 2.3. Immobilization process of chiral selector on quartz crystals The amide-type S-MA derivative-modified QCM sensors were prepared using a three-step assembly process, as illustrated in Fig. 1. First, a monolayer of 4-ATP was self-assembled on the electrode surface through Au S bonding. S-MA was then selfassembled on the 4-ATP-coated quartz crystal sensors through a coupling reaction between the carboxylic group (S-MA) and the amine group (4-ATP) with the addition of EEDQ [29–32]. Finally, the amide-type S-MA derivative was obtained by a hydrolytic reaction after removing the protecting group [29,32]. (The detailed immobilization process is described in supporting information.) 2.4. Data analysis using F–R diagram

2.2. Apparatus and QCM measurements A quartz crystal chip was a 9 MHz, AT-cut standard quartz crystal plate coated both sides with Au electrodes (area 19.635 mm2 ).

In this paper, we use the concept of resonant resistance as well as the resonant frequency. The resonant resistance of the quartz crystal is the motional resistance for the resonant oscillation. For the

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quartz crystal in contact with liquids, the equation for the resonant resistance has been derived as follows [26]: R1 =

(2FL )1/2 A k2

where F, L , , and k are the resonant frequency, the density of the contacted liquid, the viscosity of the contact liquid and electro-mechano coupling factor, respectively. More detailed understanding is possible through the original paper [26]. The resonant resistance value is the degree of vibrational energy loss due to a mass change on the QCM surface. Thus, the mechanical energy loss is larger when a viscoelastic film is loaded on the QCM surface than when an elastic film is loaded. In the present study, the measured QCM data were analyzed using the F–R model [33–36]. For easier understanding, the F–R diagram model is briefly depicted in Fig. 2, where the dots show quantitative patterns that represent the changes in the viscoelasticity of the viscoelastic film based on comparing the resonant resistance with the resonant frequency shift. Here, the path B → C → D → E shows an increase in the viscosity of the liquid film. In this pattern change, the viscous penetration depth of the film increases as the viscosity increases, the resonant frequency decreases as the oscillation decay layer increases, and the resonant resistance increases due to the increased shear stress of the film. Meanwhile, the path B → F → G → H shows an elasticity increase in the liquid film coated electrode. In this pattern change, the resonant frequency decreases as the mass increases, yet the resonant resistance is unchanged, as there is no vibration energy loss from the film. This path is ideal for using the Sauerbrey equation [28,33]. The path I → H → E shows a viscosity increase in the elastic film without mass loading. As a result, the F–R diagram model is also able to provide information on the structural changes (including rigidity, density, and thickness) occurring during the formation of the adsorbed layer based on comparing the slopes of the F and R curves as well as providing the reliability of the real mass change [35,36]. A more theoretical explanation is available in the original reports [33–36]. 3. Results and discussion 3.1. Characterization of S-MA derivative modification procedure 3.1.1. QCM detection A typical QCM response for the 4-ATP self-assembly process is presented in Fig. 3(a) as a function of the reaction time. As shown, the resonant frequency decreased immediately, along with a continuous increase in the resonant resistance after the injection of the 4-ATP (the region ‘A’), indicating a rapid self-assembly process of 4-ATP on the bare Au electrode. Meanwhile, a slower assembly process occurred in the region ‘B’, where the resonant frequency shift remained almost unchanged, while the resonant resistance continued to increase even after 7 h of reaction time (the region ‘C’). The vibration energy loss could be estimated based on the F–R relation, as illustrated in Fig. 2, where the change in the resonant resistance (R) was plotted as a function of the resonant frequency change (F) for the assembly process. In order to show the reliability of the result, original resonant frequency and resistance values of the bare sensor chip in air are also plotted in Fig. 3(b). In Fig. 3(b), the dotted line is correspondent to the A → B → C → D → E path in Fig. 2, and the left side of the dotted line is possible area of the F–R change [36]. The film deposition data were shown in the left side of the line, but these could not reveal clearly because of the graphic scale. Thus, the deposition area ‘A’ is reproduced in Fig. 3(c) using the shift value at the starting point of the film deposition. Fig. 3(c) provides valuable information on the vibration energy change due to the mechanical resistance changes occurring with the

Fig. 2. Explanation of quantitative patterns in F–R diagram model.

formation of the 4-ATP self-assembled layer. Three separate regions can be distinguished in Fig. 3(c). In the case of the region ‘A’, which extends to the frequency change of −110 Hz, the resonant resistance gradually increased with a rapid decrease of F, and the value of R/F was about −0.1045  Hz−1 (11.5 /−110 Hz). This step is similar to path B → C → D → E in Fig. 2, indicating that the 4ATP was initially adsorbed in a flat conformation based on forming many contacts with the gold surface through Au S bonding, and the surface coverage gradually increased until most of the electrode area was covered by the adsorbed 4-ATP molecules. In the case of the region ‘B’, which extends to the frequency change of −130 Hz, R did not show any distinct change in spite of a significant change in the F value. This step is similar to the path B → F → G → H in Fig. 2, indicating the formation of an elastic film on the QCM surface. In this study, the formation of a highly dense film may explain the increased elasticity, as full surface coverage has already been found to change the slip condition of the adsorbed molecules, where the dense attachment of the adsorbed molecules causes the increased elasticity [35]. Finally, the region ‘C’ exhibited the most rapid increase in R with only a slight change in the resonant frequency (R/F was about −1.375  Hz−1 ). This step is similar to the path I → H → E in Fig. 2, indicating an increased viscosity without much mass binding. This step is also important for understanding the response change of the QCM related to the self-assembly mechanism, representing minimal additional mass loading. Thus, the increase in the resonant resistance was due to an interfacial viscoelastic property change between the adsorbed molecules and the solvent due to a so-called coupling reaction [37]. As discussed above, 139.1 ng of 4-ATP was assumed to be firmly attached to the sensor surface, corresponding to the frequency shift of 130 Hz, while 8.6 ng of 4-ATP was weakly adsorbed, corresponding to the frequency shift of 8 Hz. A typical response of the 4-ATP-modified sensor exposed to a 5 g L−1 S-MA toluene solution containing 10 g L−1 EEDQ was also measured using the QCM detection (see supporting information, Fig. S1). The changes in the sensor response during the overall S-MA self-assembly process were similar to those of the overall 4ATP self-assembly process. The result was not surprising because S-MA forms a strong chemical complex with 4-ATP to produce a highly dense complex film [29]. The surface coverage of the S-MA derivative was calculated with relative to the 4-ATP coverage. The surface coverage of self-assembled small alkane thiol has already been well-documented by Love et al. [38], and is

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Fig. 3. Typical response of QCM sensor exposed to 1 mM 4-ATP/toluene solution (a) total F–R change compared with the condition in the bare (b) and an detailed view for the film deposition area (c).

normally treated as a highly dense film without a severe void area. As such, a full coverage of 4-ATP could be assumed. Therefore, for the S-MA derivative, in the case of full coverage, the decreasing frequency ratio between the 4-ATP and the S-MA derivative was expected to assume the same ratio as the molecular weight ratio, since the molecular binding was based on a 1:1 molecular ratio. In this study, the molecular weight of 4-ATP was 125 g mol−1 , while that of the additionally attached S-MA derivative (see the step ‘C’ in Fig. 1) was 207 g mol−1 . Consequently, the molecular weight ratio between the S-MA derivative and the 4-ATP was about 1.656, while the frequency ratio was about 1.217 (168 Hz/138 Hz). As a result, the surface coverage of S-MA when compared with that of 4-ATP was assumed to be over 73%. 3.1.2. Contact angle The change of surface property was additionally evaluated by the surface wet ability measurement after every modification process. After cleaning with a Piranha solution, the base sensor surface exhibited a relatively high hydrophilicity with a contact angle of 44.2◦ (Fig. 4(a)). This contact angle was then increased to 69.2◦ after the 4-ATP modification (Fig. 4(b)), indicating an increased surface hydrophobicity due to the hydrophobic property of the benzoic ring in 4-ATP. Plus, the further modification with the S-MA derivative produced a slight change in the hydrophobicity of the surface, and the contact angle became 71.4◦ (Fig. 4(c)). This last change was assumed to result from the increased number of hydrophobic

functional groups in the benzoic ring in S-MA and additional alkyl chain. In the final step, after the immersion in a 0.01 M HCl aqueous solution, the contact angle decreased to 64.6◦ (Fig. 4(d)), indicating an increase in the hydrophilicity of the surface due to the removal of the hydrophobic alkyl chain through a hydrolytic reaction. Thus, all the QCM detection results and contact angle measurements confirmed the successful modification of the amide-type S-MA derivative on the QCM sensor surface. 3.2. Response of S-MA derivative-modified sensor to chiral mandelic acid 3.2.1. Novel QCM techniques for chiral analysis The chiral recognition of R-MA and S-MA by the selectormodified QCM sensor was performed in an aqueous solution. After obtaining a stable base resonant frequency in the reaction cell containing distilled water, 1 ml of an R-MA or S-MA aqueous solution diluted at various concentrations was injected into the reaction cell for the chiral discrimination. In our experiment, we have repeated 5 chiral recognition measurements at a same concentration in the given target concentration range from 0.01 mM to 3 mM to obtain a statistical error scale. The typical changes in the sensor response during the chiral recognition reaction are shown in Fig. 5 for the resonant frequency and resistance changes. Although 5 data sets were collected for different MA concentrations, only one set was shown for easier

Fig. 4. Contact angles for QCM sensor surface obtained with deionized water: bare QCM sensor after cleaning with Piranha solution (a), 4-ATP self-assembled QCM sensor (b), S-MA-modified QCM sensor before immersion in 0.01 M HCl aqueous solution (c), and S-MA-modified QCM sensor after immersion in 0.01 M HCl aqueous solution (d).

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Fig. 5. Typical frequency (a) and resistance (b) responses of S-MA derivative-modified QCM sensor exposed to aqueous 0.05 mM R-MA or S-MA solution, and the F–R representation based on (a) and (b).

explanation. A summary of the maximum resonant frequency and resistance changes corresponding to all 5 data sets for the different concentrations is shown in Fig. 6. As shown in Fig. 5(a), the resonant frequency exposed to 0.05 mM R-MA decreased to −4, −15, and −17 Hz, whereas the resonant frequency exposed to 0.05 mM SMA decreased to −38, −62, and −97 Hz after 1, 5, and 9 h of reaction time, respectively. Thus, the reaction speed between the selector and the target S-MA was faster than that between the selector and the target R-MA. In addition, the results also demonstrated the feasibility of discriminating the molecules after only 1 h of reaction time. The chiral discrimination factor between R-MA and S-MA, ˛S-MA/R-MA = FS-MAX /FR-MAX , as normally used in a QCM analysis, was found to be 5.7. Remarkably, the change in the resonant resistance was different to the tendency of the resonant frequency change, as shown in Fig. 5(b). In the case of the R-MA, the resonant resistance increased to 1.13 , which was bigger change than that

of the S-MA (0.74 ). In the initial step up to the reaction time of 2 h, the resonant resistance of the S-MA exhibited a significant increase due to a large mass loading, as shown in Fig. 5(a) and (b), yet thereafter increased slowly when compared to the resonant resistance of the R-MA. As described above, the resonant resistance values indicated the mechanical resistance of the coating films, which was small when the film was attached firmly to the surface of the quartz crystal. As such, the attachment of the R-MA was not firm in spite of the small mass gain. To compare this result, Fig. 5(c) and (d) was then reproduced using Fig. 5(a) and (b). In Fig. 5(c), it could confirm the both the chiral sensing responses were performed with an elastic mass loading mechanism similar to the path B → F → G → H in Fig. 2, but detailed understanding was impossible only with the large graphic scale. Thus, we additionally produced an enlarged graphic scale shown in Fig. 5(d) using the same operational technique as described in Fig. 3. As shown, the slope values (R/F) for

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Fig. 6. Summarized resonant frequency and resistance changes according to the function of the applied target concentration for R-MA (a) and S-MA (b).

the R-MA and S-MA attached to the modified quartz crystal were different, with a larger value of about 0.066 /Hz for the R-MA, yet only about 0.007 /Hz for the S-MA. The R/F value indicates the change in the mechanical properties with the addition of a unit mass of approximately 1 ng. Thus, since this value includes information on various binding properties, such as the binding strength and interface phenomena during the binding process [33–36], it can be a more correct criterion as a discrimination factor, as it reflects the chemical or physical properties related to the binding interaction change. Therefore, ˛R-MA/S-MA = (R/F)R-MAX /(R/F)S-MAX was used as a novel discrimination factor. In the case of Fig. 5(c), the calculated discrimination factor was about 9.42, which was a larger value than when applying the normal discrimination factor (˛S-MA/R-MA = FS-MAX /FR-MAX = 5.7). Fig. 6 summarizes the resonant response changes based on a 9-h reaction time for the given concentration range. The standard deviation for the five repeated measurements was denoted as the error bars. Even though chiral discrimination was possible after a 1-h reaction time, as shown in Fig. 5, a 9-h reaction time was used to provide a comparison based on the full surface coverage of the target MA molecules. In Fig. 6(a), when increasing the R-MA concentrations to 0.01, 0.05, 0.2, 1.5, and 3.0 mM, the resonant frequency decreased to −15.5, −17.0, −22.0, −24.0, and −25.0 Hz, respectively, while the resonant resistance increased to 1.23, 1.13, 1.04, 0.89, and 0.52 , respectively. Meanwhile, in Fig. 6(b), when increasing the S-MA concentrations to 0.01, 0.05, 0.2, 1.5, and 3.0 mM, the resonant frequency decreased to −55.0, −97.0, −100.0, −110.0, and −134.0 Hz, respectively, while the resonant resistance increased to 0.47, 0.74, 0.70, 0.98, and 0.15 , respectively. When using the measured frequency values as the criteria, the chiral discrimination factors between R-MA and S-MA, ˛S , were evaluated to be about 3.5, 5.7, 4.5, 4.6, and 5.7 for concentrations of 0.01, 0.05, 0.2, 1.5, and 3.0 mM, respectively. The value was calculated by the deviation from the mean value of the five measurements. The standard deviation error does not exceed the possible measurement error. Thus, the reproducibility of the results was confirmed within an experimental error range. That is, normal replacement of the medium liquid could produce the resonant frequency change of 10 Hz and the resonant resistance change of 0.05  in our experiment. The difference in the response was not

clearly obtained within applied temperature range from 20 ◦ C to 30 ◦ C. Please see supporting Fig. S2 for the result at 30 ◦ C. For further clarification, the change in the R/F values based on Fig. 6(a) and (b) is represented in Fig. 7 as a function of the target concentration. The different R/F values between the two measuring systems revealed a different interaction behavior during the respective recognition reactions. The R/F values for the R-MA system were all larger than those for the S-MA system at the same concentration. The result was not surprising because the R-MA molecules were probably weakly adsorbed on the S-MA derivative-modified sensor surface based on bonding with a “oneor two-point interaction site” [23]. Conversely, the target S-MA molecules were probably strongly combined to form a more firmly

Fig. 7. R/F value change according to function of applied target concentration.

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adsorbed layer based on bonding with a “three-point interaction site” [23]. There are two common attractive interactions between the diastereoisomeric complexes of the S-MA derivative/target RMA and the S-MA derivative/target S-MA: a double hydrogen bond between the acylamide group of the S-MA derivative and the carboxyl group of the target S-MA or R-MA, and ␲–␲ interactions between the benzoic rings of the S-MA derivative and the target R-MA or S-MA. As such, the difference between the diastereoisomeric complexes of the S-MA derivative/target R-MA and the S-MA derivative/target S-MA was caused by the hydrogen bond between the hydroxyl group of the S-MA derivative and the optically active or inactive hydroxyl group of the target R-MA or S-MA, and it was this difference that facilitated the chiral recognition of the SMA derivative-modified sensor. Furthermore, as seen in Fig. 7, the R/F values for the target S-MA system only changed slightly when increasing the target concentration, indicating that the conformation of the adsorbed S-MA layer was only slightly changed with the different concentrations due to a strong host–guest interaction with a low concentration [39]. Conversely, the R/F values for the R-MA system decreased when increasing the target concentration, indicating that the conformation of the adsorbed R-MA layer was clearly changed with the different concentrations. This can be explained by the so-called bulk effect in literature [40]. Both the resonant frequency and the resistance are affected if the viscosity of the surrounding solution is changed due to a change in the solute concentration [41]. Importantly, at a low concentration, the R-MA molecules produced a much larger energy loss per added molecule (large R/F value) than at a high concentration, as shown in Fig. 7, implying a weaker interaction force between the selector and the target R-MA than that between the selector and the target S-MA. Thus, the discrimination factors based on the values of R/F, ˛R , were 10, 9.4, 6.7, 4.2, and 1.9. Importantly, a high discrimination factor was obtained when applying the novel discrimination technique, especially when the target concentration was low. In the further experiments, we have already confirmed the possible chiral analysis for the racemic mandelic acid solution applying the current conclusion. For proving this result, we had developed a technique that did not permit the interaction between R-MA and the modified sensor surface. This portion of the research will be reported elsewhere in near future. In addition, the applied chiral selector seems to have the various recognition capabilities to resolve different optical isomer possessing carboxyl ending group, and this result will be also reported in near future.

3.2.2. Contact angle measurement For further confirmation of the selective binding of the target R/S-MA on the S-MA derivative-modified surface, contact angle measurements were also performed. After a 9-h recognition reaction with the target S-MA molecules, the contact angle of the S-MA-modified sensor surface became 72.7◦ (see supporting information, Fig. S3(a)), which was 8.1◦ larger than the contact angle measured just after the S-MA derivative immobilization (Fig. 4(d)), indicating an increased surface hydrophobicity, most probably due to the binding interaction of the target SMA to the S-MA derivative-modified surface. Meanwhile, in the case of the R-MA (Fig. S3(b)), the contact angle of the S-MAmodified sensor surface did not show any distinct change after the 9-h reaction, indicating no noticeable binding interaction between the target R-MA and the QCM sensor. This was consistent with the QCM measurement results. Thus, the contact angle measurements also confirmed a specific binding interaction between the sensor-immobilized S-MA derivative and the target S-MA.

4. Conclusions A chiral recognition system for MA enantiomers was successfully constructed by immobilizing an amide-type S-MA derivative with an optically active hydroxyl group on a QCM surface. When using just the frequency measurement, the chiral discrimination factor between the target R- and S-MA was about 5.7. However, when adding the resonant resistance as a novel criterion, this enhanced the value of the chiral discrimination factor to about 10, which meant both the chiral selectivity and sensitivity were equally increased. The high speed sensing was also possible with the current method compared with our previous method. Importantly, the novel criterion provides a high discrimination factor with a low target concentration due to different binding property between the enantiomers and the selector.

Acknowledgment This work was financially supported by NRF (Korea, 20100017993).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2012.05.015.

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Biographies Jong Min Kim received his BS, MS and PhD degrees in chemical engineering from Dong-A University, Korea in 1994, 1996 and 1999, respectively. He has written over 50 research articles for electrochemistry, sensor, biochemistry, polymer rheology, quartz crystal technology, photochemistry and probe microscopy. His main interest is application development of the scanning probe microscopy, and he is currently interested in the nano scale separation techniques. Sang Mok Chang obtained BA degree from the department of chemical engineering, Seoul University in 1982, Master degree from Korea Advanced Institute of Science and Technology (KAIST), Korea, and doctorate degree from Tokyo Institute of Technology, Japan. He has experience as visiting researcher at RCAST, University of Tokyo, Japan as researcher, NAIR, Tokyo, Japan. Now he is a professor in Chemical Engineering at Dong-A University, Korea. His present research interests are self-assembly films, biosensor as monitoring system of the environment. Xin-Kuai He received his PhD in metallurgical physics and chemistry from Central South University in 2006, His research interests are in electrochemistry, quartz crystal technology, and chiral separation technology and biochemical sensor. Woo-Sik Kim received his PhD in Chemical Engineering at the Pennsylvania State University in 1992. He joined the KyungHee University in 1994 and is now full professor in Department of Chemical Engineering. His research interests are in crystallization technology, bio-materials separation and chiral separation.