Development of a highly sensitive MIP based-QCM nanosensor for selective determination of cholic acid level in body fluids

Development of a highly sensitive MIP based-QCM nanosensor for

selective determination of cholic acid level in body

fluids

Aytaç Gültekin

⁎

, Gamze Karan

_fil

, Sava

_{ş Sönmezoğlu}

, R

_{ıdvan Say}

Anadolu University, Faculty of Science, Department of Chemistry, Eskişehir 26470, Turkey

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 20 November 2013 Received in revised form 6 May 2014 Accepted 29 May 2014

Available online 4 June 2014 Keywords:

Cholic acid MIP QCM Nanosensor

Determination of cholic acid is very important and necessary in bodyfluids due to its both pharmaceutical and clinical significance. In this study, a quartz crystal microbalance (QCM) nanosensor, which is imprinted cholic acid, has been developed for the assignation of cholic acid. The cholic acid selective memories have been generated on QCM electrode surface by using molecularly imprinted polymer (MIP) based on methacryloylamidohistidine-copper (II) (MAH-Cu(II)) pre-organized monomer. The cholic acid imprinted nanosensor was characterized by atomic force microscopy (AFM) and then analytical performance of the cholic acid imprinted QCM nanosensor was studied. The detection limit was found to be 0.0065μM with linear range of 0.01–1000 μM. Moreover, the high value of Langmuir constant (b) (7.3 * 105_{) obtained by Langmuir graph}

showed that the cholic acid imprinted nanosensor had quite strong binding sites affinity. At the last step of this procedure, cholic acid levels in bodyfluids were determined by the prepared imprinted QCM nanosensor.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Bile acids, a main metabolite of cholesterol, are a mixture of steroids, and play a significant physiological role in removal of cholesterol from the body and in simplifying an absorption of dietary lipids and fat-soluble vitamins by forming micelles[1,2]. The concentration of bile acids in body is concerned with hepatitis, gallstone and other diseases in liver. Hence, the quantification of bile acids in body fluids (such as plasma and urine) is an important tool for the diagnosis of hepatobiliary diseases. Cholic acid as well as its derivatives is a major component of bile acids[3].

Several methods such as high performance liquid chromatography_– tandem mass spectrometry (LC–MS–MS)[4], ultraviolet absorption spectrophotometry [5], solid phase radioimmunoassay [6] and a cholate-selective liquid membrane electrode[7]have been reported in the literature for detection of cholic acid level in serum. Since these methods are relatively expensive and require pretreatment, the development of alternative analytical methods to determine cholic acid level in bodyfluids is very important.

A designing of highly selective, cost-efficient, stable, sensitive and surefire chemical sensors by using nanoparticles, quantum dots or various electrodes is an interesting research area[8–11]. Quartz crystal microbalance (QCM) is well feasible for selective and sensitive detection and portable in situ measurement. QCM is widely used in biochemistry,

environment, food, and clinical analysis[12–16] and also for the detection of viruses[17], bacteria[18], and DNA[19]due to its attractive performance such as low cost, easiness to use and promptness of detection. Although it has great properties, it has no specific selectivity. Consequently, various chemicals and biomaterials can be used to modify (physically or chemically) the QCM electrode surface in an effort to obtain selectivity[20].

Molecular imprinted polymer (MIP) is employed for QCM as a selective polymer layer. Molecular imprinting is a technology to generate recognition sites in a macromolecular matrix using a molecular template. In other words, both a shape image of the template and an alignment of the functional moieties are memorized in the macromolecular matrix for the recognition or separation of the template during formation of the polymeric materials to interact with the moieties in the template[21]. MIPs are easy to prepare, stable, inexpensive, has high selectivity for template molecules and capable of molecular recognition[22–28].

Recently, the combination of QCM and MIPs has been applied in se-lective sensing detection. A lot of applications of QCM/MIPs have been reported for the determination of different molecules such as detection of albumin[29], nucleobases which provided rapid methods for DNA/ RNA detection[30], folic acid[31], caffeic acid[32], 8OHdG[33]and nerve agent and paraoxon[34].

The qualitative and quantitative analysis of cholic acid that is one of the major bile acids in the body has both pharmaceutical and clinical importance. In this study, we report a new, cost-effective and sensitive method for the fabrication of molecularly imprinted QCM nanosensor

⁎ Corresponding author. Tel.: +90 338 226 5055; fax: +90 338 226 2116. E-mail address:aysari@yahoo.com(A. Gültekin).

http://dx.doi.org/10.1016/j.msec.2014.05.055

0928-4931/© 2014 Elsevier B.V. All rights reserved.

Contents lists available atScienceDirect

Materials Science and Engineering C

to detect cholic acid based on the polymerization of metal-chelating monomer in the presence of template molecule (cholic acid). Cholic acid level in body_{fluids is determined by using the prepared cholic} acid imprinted QCM nanosensor.

2. Experimental 2.1. Chemicals

Cholic acid, ethanol (absolute), azobisisobutyronitrile (AIBN) and allyl mercaptan were supplied from Aldrich Chem. Co. (Milwaukee, WI, USA). EDMA was obtained from Fluka A.G. (Buchs, Switzerland), distilled under reduced pressure in the presence of hydroguanin inhib-itor and stored at 4 °C until use. All other chemicals were reagent grade and purchased from Merck AG (Darmstadt, Germany). All water used in experiments was obtained from Zeneer Power II water purification system.

2.2. Instrumentation

Fourier transform infrared (FTIR) spectrum was recorded on Perkin-Elmer Spectrum 100 between the range of 4000 and 400 cm−1.

An AT-cut, gold/Cr polished, 5-MHz quartz crystals and a quartz crystal analyzer (SRS Stanford Research Systems, Model QCM200 Quartz Crystal Microbalance Digital Controller) were used to perform microgravimetric measurements. Sauerbrey's equation has been established for an AT-cut shear mode QCM:

ΔF ¼ −2F 2 0Δm A pqμq

 1=2 ð1Þ

whereΔF is the measured frequency shift due to the added mass in hertz, F0is the fundamental oscillation frequency of the dry crystal,

Δm is the surface mass loading in grams, pqis the density of quartz

(2.65 g cm−3),μqis the shear modulus (2.95 × 1011 dyn cm−2), and

A is the electrode area (0.19 ± 0.01 cm2). For the 5 MHz quartz crystals used in this work, Eq.(1)estimated that a frequency change of 1 Hz corresponds to a mass increase of 1.03 ng cm−2on the electrode[29].

The topographic characteristics of QCM electrode surface for every stage were studied by atomic force microscopy (AFM) (hpAFM, Nanomagnetics Instruments, Oxford, UK).

2.3. Preparation of metal-chelate monomers and cholic acid having pre-organized complexes

Histidine-functional monomer, methacryloylamidohistidine (MAH), was synthesized according to the published procedure[35]. The FTIR spectra of MAH had characteristic carbonyl bands at 1653 and 1629 cm−1and amide II absorption band at 1529 cm−1. In the same manner, metal-chelate monomer, methacryloylamidohistidine-copper (II) [MAH-Cu(II)], was synthesized with respect to the published procedure[36]. Cu–N vibration bands at 498 cm−1_{in FTIR spectrum}

showed that Cu(II) was incorporated into MAH structure. Ligand exchange monomer, methacryloylamidohistidine-copper (II)-cholic acid [MAH-Cu(II)-cholic acid] was synthesized using MAH-Cu(II) and template cholic acid. MAH-Cu(II) (0.01 mmol) and cholic acid (0.01 mmol) were dissolved in ethanol and the two solutions were added each other, thenfinal solution was stirred 24 h. When cholic acid has been attached into the monomer system, existence of alkyl C–H stretching vibration at 2950 cm−1_{and also more strong carbonyl}

stretching absorption (C = O, –COOH) at 1782 cm−1_{in the FTIR}

spectrum demonstrated the interaction between MAH-Cu(II) and cholic acid.

2.4. Preparation of cholic acid imprinted QCM nanosensor

Before coating, gold surfaces were cleaned with piranha solu-tion (1:3 30% H2O2/concentrated H2SO4). The cleaned surfaces

were immersed into allyl mercaptan solution (2-propene-1-thiol) (0.30 mmol) for 24 h in order to insert thiol groups onto the gold surface of the QCM electrode. The thiol group of allyl mercaptan directed the interaction between the QCM gold surface and imprinted polymer and the allyl group procured the polymeriza-tion of metal–chelate monomer from this group. The electrode was then washed with ethanol and deionized water to remove the excess of thiols.

For the polymerization, the reaction mixture containing the metal– chelate (MAH-Cu(II)-cholic acid) pre-organized monomer, EDMA crosslinking monomer and initiator (AIBN) in ethanol was prepared. Then, a small amount of reaction mixture was dripped onto the allyl ac-tivated QCM electrode. Polymerization was carried out at room temper-ature under UV light irradiation for 4 h (Fig. 1). As a reference, the non-imprinted polymer-coated QCM sensors (NIP) were also prepared in a similar manner as with MAH-Cu(II).

Fig. 2shows AFM images of pure, allyl modified, MIP coated, MIP coated (after template removing) and NIP coated gold electrodes. As seen inFig. 2, the roughness of the surface is well distributed through all surface of the electrode. This result shows that the homogen and nanometer-size of about 150 nm cholic acid imprinting on the QCM electrode has been successfully performed. This specialty is one of the significant parameters to control the selectivity and recognition rate of the sensor.

2.5. Monitoring of the response of cholic acid imprinted QCM nanosensor response

Cholic acid imprinted QCM nanosensor was used for real time detection of cholic acid. The cholic acid was dissolved in ethanol and the frequency of the nanosensor was monitored until it became stable. The frequency shift for each concentration (0.01–1000 μM) of cholic acid was calculated using the equationΔF = F0–F1and

the evaluation was performed in triplicate. After each analysis, the desorption process was applied using 2 M sodium hydroxide/ tetrahydrofuran (NaOH/THF) (3:1, v/v)[3]. After desorption step, the cholic acid imprinted QCM nanosensor was washed with deion-ized water. This washing process was repeated until the frequency of the nanosensor recovered to the F0value. For each cholic acid

sample application, adsorption–desorption–cleaning steps were repeated.

2.6. Selectivity of MIP coated QCM nanosensor

The selectivity property is one of the important advantages of MIPs. The selectivity of the prepared nanosensor for cholic acid was estimated using chenodeoxycholic acid which has very simi-lar molecusimi-lar structure with cholic acid. The concentration of competitive molecule obtained 100 μM in ethanol. The QCM nanosensor was treated with this competitive molecule. After the equilibrium, the frequency shift of chenodeoxycholic acid mea-sured by designed cholic acid imprinted QCM nanosensor and Δm and Q (nmol) values were calculated with respect to Eq.(1). The selectivity coefficient for the binding of cholic acid in the oc-currence of opponent species can be acquired from equilibrium binding data consistent with k = Qtemplate molecule/Qopponent species.

The relative selectivity coefficient (k′ = kimprinted/knon-imprinted) con-sists of the comparison of the k values of the imprinted polymer with non-imprinted polymers for the prediction of the effect of imprinting on selectivity.

3. Results and discussion

3.1. Measurement of binding interaction of molecularly imprinted QCM nanosensor via ligand interaction

The binding of cholic acid (CA) to the cholic acid imprinted metal-chelate polymer [MAH-Cu(II)] on a gold quartz crystal causes a mass change,Δm, that was observed in the crystal frequency. The relationship between Δm and the frequency shift (ΔF0) was expressed by

Sauerbrey's equation[29]. The QCM electrodes were washed with deionized water and then dried. The frequency (F0) was monitored

after drying and then cholic acid solution was instilled on a confined type detector cell. The frequency of the nanosensor (F1) decreased

after adding cholic acid solution, then achieved the constant value in 30 min (Fig. 3).Fig. 3shows that the reaction reached an equilibrium swiftly. Moreover, these frequency changes strongly indicated that the cholic acid molecules were bound to the imprinted polymer on the quartz crystal. When non-imprinted polymer was used, less binding of cholic acid molecules to non-imprinted polymer was observed.

The binding interaction and equilibrium information between imprinted polymer and cholic acid template can be obtained by the Langmuir adsorption isotherm. This analysis can be acquired using the following equation: 1 Q¼ 1 Qmax:b ½ x 1 C   þ _Q1 max ½  ð2Þ

where Q is the amount of cholic acid bound to polymer, as calculated by the mass frequency alteration upon addition of analyte, and C is the con-centration of free cholic acid. Qmaxexpresses the apparent maximum

number of binding sites, and b is the Langmuir constant.

The results obtained from linearized form of the Langmuir isotherm by plotting 1/Q as a function of 1/C is (Fig. 4):

1 Q¼ 0:0474x 1 C   þ 0:0347 ð3Þ

Therefore, Langmuir constant, b, and the apparent maximum number of recognition sites, Qmax, values for the specific interaction

between the template imprinted polymer and the template itself

were 7.3 * 105_{and 28.8, respectively. The high value of Langmuir}

constant (b) represented that affinity of the binding sites was very strong.

3.2. Results of selectivity studies of imprinted polymer on QCM nanosensor The adsorption of chenodeoxycholic acid, which is similar in chemical structure with cholic acid (Fig. 5), on the cholic acid imprinted nanosensor was studied for a better understanding of in-teractions between the binding sites of MIP-QCM nanosensor and its template molecules, and the ability of this imprinted polymer to define the cholic acid molecules.

According to experimental studies, Q values of cholic acid and chenodeoxycholic acid were determined as 8.92 and 0.60 nmol for the MIP nanosensor and 0.65 and 0.30 nmol for the NIP nanosensor. Selec-tivity coefficients (k) and relative selectivity coefficients (k′) values were shown inTable 1. As seen fromTable 1, MIP nanosensor was 15 times more selective for cholic acid than for chenodeoxycholic acid. Moreover, the adsorption capacity of the cholic acid imprinted polymers for adsorption of cholic acid plus related molecule chenodeoxycholic acid was utilized (Fig. 6).

3.3. Analytical performances

Fig. 7shows the calibration curve was obtained by plotting differ-ent concdiffer-entrations of cholic acid (0.01–1000 μM) imprinted quartz crystal versus frequency shifts. The detection limit, defined as the concentration of analyte giving frequency shift equivalent to three standard deviation of the blank, plus the net blank frequency shift, was 0.0065 ± 0.0004μM for the MAH-Cu(II) based nanosensor. The linear range was established between 0.0100 and 1000μM with coefficient (R2_{) of 0.9894. The experiments were performed in}

replicates of three and the samples were analyzed in replicates of three as well. In the literature, the lowest detection limits were re-ported as 4.27μM for cholic acid by HPLC[37]and 38.3μM[38]for deoxycholic acid by electrokinetic chromotography. Furthermore, in our previous works, the detection limit of the cholic acid imprinted gold nanoparticles nanosensors and the cholic acid imprinted gold-silver nanocluster nanosensors were 0.1μM[21]

H2C O O O O O CH3 CH3 O Cl O O O O H3C H3C O H3C _O O O O H3C H3C O H3C S Au QCM electrode HN O CH H2 C C O O N NH Cu2+ O OH2 OH H H HO H H HO OH H2C O O O O O CH3 CH3 O Cl O O O O H3C H3C O H3C _O O O O H3C H3C O H3C S Au QCM electrode HN O CH H2 C C O O N NH Cu2+ OH2 Cholic acid template Template removal

Fig. 2. AFM images of (a) pure and (b) allyl modified, (c) MIP coated, (d) MIP coated (after template removing), and (e) NIP coated QCM electrode. Scanning mode: dynamic; scanning area: 5.0 × 5.0μm.

and 0.05μM[28], respectively. It can be clearly said for this study that the low detection limit of new cholic acid imprinted QCM nanosensor was obtained by comparison with previous works.

3.4. Determination of cholic acid content in human serum and urine by pre-pared QCM nanosensor

The cholic acid imprinted QCM nanosensor was tested against blood serum and urine. Before the measurement, some pre-treatment steps were applied to the blood plasmas which belong to diabetic patient and healthy person. One milliliter of plasma was taken and plasma pro-tein was precipitated by the addition of an equal volume of acetonitrile. Precipitated protein was centrifuged at 4000 rpm for 15 min. The super-natant was transferred to a new tube and mixed with four volumes of water. The eluent, which was obtained after the pre-treatment step, was dripped onto the prepared cholic acid imprinted nanosensor. Sim-ilarly, urine sample was squeezed onto the cholic acid imprinted QCM

-100 -90 -80 -70 -60 -50 -40 -30 -20 -10 00 5 10 15 20 25 30 35 40 45 50 55 60 Frequency Shift(Hz) Time (Minute)

Cholic acid imprinted sensor

non-imprinted sensor

Fig. 3. QCM responses of the cholic acid imprinted and non-imprinted nanosensors (CCA= 0.1μM). y = 0,0474x + 0,0347 R² = 0,9861 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,001 0,01 0,1 1 10 100 1/Q 1/C 0 200 400 600 Fr e que nc y ( H z)

Concentration of cholic acid (µM)

Fig. 4. Langmuir isotherm of cholic acid imprinted nanosensor.

OH O H HO H OH H OH OH O H HO H OH H

a)

b)

Fig. 5. Structure of (a) cholic acid and (b) chenodeoxycholic acid.

Table 1

Selectivity of cholic acid imprinted QCM nanosensor. Q (nmol) (imprinted) Q (nmol) (non-imprinted) k (imprinted) k (non-imprinted) k′ Cholic acid 8.92 0.65 Chenodeoxycholic acid 0.60 0.30 14.87 2.16 6.88 -250 -200 -150 -100 -50 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Frequency Shift (Hz) Time(Minute) Cholic acid Chenodeoxycholic acid

Fig. 6. QCM responses of the cholic acid imprinted nanosensors for cholic acid and chenodeoxycholic acid (all concentrations are 100μM).

y = 0,1707 x + 101,617 R² = 0,9894 0 50 100 150 200 250 300 350 0 0.01 0.1 1 10 100 1000 Frequency Shift (Hz)

Concentration of cholic acid (µM)

Cholic acid imprinted sensor

Non-imprinted sensor

nanosensor. Cholic acid level in these samples was determined by the calibration curve (Fig. 7) using the frequency values.

High pressure liquid chromatography (HPLC, Shimadzu UV–Vis de-tector) was used for comparison of the analytical performances of the prepared cholic acid imprinted QCM electrode. For this purpose, Perkin Elmer C18 (5μm, 150 × 4.6 mm) column was used. The amount of cholic acid in bodyfluids using both MAH-Cu based QCM nanosensor and HPLC were given inTable 2. The nanosensor results were justified by comparative chromatographic measurements. The amount of cholic acid in the blood of a healthy person is about 6μM[39]. At the same time, the results confirmed that the cholic acid level in diabetic patient's bodyfluids were significantly higher than that in healthy person. Conse-quently, the ability of cholic acid level determination in bodyfluids of the cholic acid imprinted QCM nanosensor was determined by these results.

4. Conclusion

In the present study, we have developed MIP-based QCM nanosensor with the aim of specific determination of cholic acid level in bodyfluids. QCM nanosensor was prepared by modifying the gold surface of the quartz crystal with the cholic acid imprinted polymer. All experimental results indicated that cholic acid imprinted nanosensor with good reproducibility, short response time (30 min), wide linear range (0.01–1000 μM), low detection limit (0.0065 ± 0.0004 μM) and high selectivity (14.87 for MAH–Cu(II)–cholic acid complex with re-spect to chenodeoxycholic acid having similar structure with cholic acid) has been developed for the determination of cholic acid level by using the cholic acid MIP/QCM detection system as a recognition mate-rial. Moreover, in order to understand the suitability of the prepared nanosensor in real samples, blood serum and urine samples were inves-tigated with the cholic acid imprinted nanosensor and cholic acid level was found to be 9.77 ± 0.008 and 7.20 ± 0.004μM for diabetic person and 6.30 ± 0.006 and 3.54 ± 0.009μM for healthy person, respectively. In conclusion, the developed cholic acid imprinted QCM nanosensor has measured up for the selective determination of cholic acid level in body fluids.

Acknowledgments

Thefinancial support from the Commission of Scientific Research Projects of Karamanoğlu Mehmetbey University (Project No. 49-M-12) is gratefully acknowledged.

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Table 2

Quantities of cholic acid in bodyfluids.

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Aytaç Gültekin has received his PhD degree in January 2009 at Trakya University and has been working as an Assistant Professor at the Department of Energy Systems Engineering of the Karamanoğlu Mehmetbey University, Karaman, Turkey. His current research inter-ests are synthesis of nanomaterials, molecularly imprinted polymers, sensor systems for biomolecules,flourimetry, QCM and SPR.

Gamze Karanfil received her MS degree from Selçuk University, Konya, Turkey, in 2013. She is a PhD student in Mechanical Engineering. Her major research interests include syn-thesis of nanomaterials, molecularly imprinted polymers, sensor systems for biomolecules and QCM.

Savaş Sönmezoğlu is an Assoc. Prof. at the Department of Materials Science and Engineer-ing of the Karamanoğlu Mehmetbey University, Karaman, Turkey. He is the head of the Sonmezoglu Research Group. His research mainly focuses on the fabrication, design and characterization of micro/opto-electronic devices. Moreover, he has also studied the syn-thesis of organic, inorganic and biological based nano-materials. Dr. Sönmezoğlu has authored or co-authored more than 30 peer-reviewed scientific publications.

Rıdvan Say has received his PhD degree in June 1999 at Hacettepe University and has been working as a Professor at Anadolu University. His current research interests are synthetic receptors, molecularly imprinted polymers, purification of proteins by chromatographic methods, biosorption of fungus, biotechnology and nano systems.