Quartz crystal microbalance based histidine sensor

Abstract Herein, quartz crystal microbalance (QCM) biosensor is prepared for the detection of l-histidine by attachment of l-histidine imprinted poly(EGDMA-MAH/Cu(II)) nanoparticles on QCM electrode. The imprinted nanoparticles with the size of 86.43 nm were synthesized via miniemulsion polymerization reaction. Prepared QCM sensors were characterized with ellipsometer, contact angle measurements and FTIR. The thickness measurements demonstrated that the particle thin films were almost monolayer. l-histidine solutions with a concentration range between 6.44 μM and 225.6 μM were introduced to QCM system to determine the adsorption kinetics. Selectivity of the l-histidine imprinted nanoparticles were examined using d-histidine and l-tryptophan as competitor molecules. l-histidine imprinted QCM biosensors was also used for RNAase, lysozyme, cytochrome-C and BSA to investigate the competitive adsorption of surface histidine exposed proteins.


Introduction
Histidine, an essential bioactive molecule, is highly important for human growth and repair of tissues and behaves as a neurotransmitter in human muscular and the central nervous system [1][2][3][4][5]. Additionally, histidine regulates metal ions transmission in biological systems and constitutes the active site of many enzymes and natural proteins [6,7]. Hence, excessive or deficiency of histidine/histidine rich proteins would cause many diseases like Parkinson's disease [2], epilepsy [8], histidinemia [9], chronic kidney disease [5], the failure of normal erythropoiesis development [10], inflammation and oxidative stress [5,11]. Sensitive, specific and accurate detection of histidine in biological samples has great significance in clinical analysis. The low concentration and similar structural features of other amino acids bother the specific detection of histidine in many biological samples. Various analytical methods including capillary electrophoresis, HPLC, colorimetry, fluorometry, SERS, NMR and electrochemistry [4,[12][13][14][15][16][17] have been proposed to overcome this challange. Some of these analytical methods suffer from the complicated operating procedures, complicated instrumentation, sophisticated detection systems and expensive cost [3].
Among these methods, molecular imprinting (templateassisted synthesis) offering selective and spesific binding sites for template molecule in polymeric network, has been recognized as one of the most promising technique [18][19][20][21]. The formation of tailor-made recognition materials based on the molecular imprinting technique can design a durable recognition layer on the transducer surface such as mass-sensitive devices, optrodes, or electrochemical electrodes [22]. QCM is a mass-sensitive device due to the piezoelectric effect. The oscillation frequency of the electrode shifts if a mass adsorbed on the surface of QCM electrode, that enables to examine any interaction through the frequency shift of the electrode [23][24][25][26][27]. Combination of molecular imprinting technique with QCM presents some benefits such as high sensitivity, quick response time, label-free sensing in real time and calculating the kinetic constans of biomolecules [28]. QCM can be used in analytical applications regarding some drawbacks of laboratory-based approaches like high cost, complicated instrumentation and hardship of operating procedures.
Herein, advantages of QCM and molecular imprinting technique were combined to develop QCM biosensor. QCM biosensor was prepared by attachment of L-histidine imprinted nanoparticles (MIP) on the QCM electrodes. QCM electrodes were characterized with ellipsometer, FTIR and contact angle measurements. L-histidine solutions with a concentration range between 6.44 lM and 225.6 lM were introduced to QCM to calculate the absorption kinetics. Competitive adsorption of ethanol were supplied by Merck AG (Darmstadt, Germany). a,a'-azobisisobutyronitrile (AIBN), ethylene glycol dimethacrylate (EGDMA) were supplied by Fluka A.G. Sodium dodecyl sulfate (SDS), polyvinyl alcohol (PVA), sodium bicarbonate, sodium bisulfite, ammonium persulfate, and potassium bromide (KBr) were supplied from Sigma-Aldrich Chemical Co (St.Louis, USA).

Formation of functional monomer
The synthesis of N-methacryloyl-(L)-histidine methyl ester (MAH) monomer was conducted according the previous study [29] and characterized with H-NMR and FTIR analyses.

Synthesis of MAH/copper(II) metal-chelating monomer
For the synthesis of MAH/copper(II), firstly 20% (w/v) MAH solution was prepared in EtOH, then 2.5 mg copper (II) nitrate was added into 20% (w/v) MAH solution and continuously stirred for 2 h. The resulting clear blue colored complex was crystallized from the ethanol/acetonitrile mixture through evaporating the solvent in the rotary evaporator.

Preparation of the QCM biosensor
The synthesis of MIP was performed by two-phase mini-emulsion polymerization method [28]. Same synthesis approach was applied for the synthesis of non-imprinted nanoparticles (NIP) without using L-histidine. Prior to attaching of MIP onto the gold surface of QCM electrode, OCM electrode was immersed into the acidic piranha solution for 20 s then cleaned with deionized water and EtOH. Afterwards, 5 lL of MIP suspension was drop-casted on the QCM electrode and it was dried in a dust-free environment at 37 C for 6 h. MIP attached QCM electrode was cleaned with DI and pure EtOH. FTIR-ATR characterization measurement of MIP biosensor was conducted by using the FTIR-ATR spectrophotometer. An auto-nulling imaging ellipsometer was used for thickness measument. All measurements were done at a wavelength of 532 nm with an incidence angle of 62 . Contact angle measurements were performed with Kruss DSA100 with sessile drop method.

Analysis of QCM biosensor response
Binding events were performed with L-histidine solutions at different concentrations. A concentration range between 6.44 lM and 225.6 lM were prepared in phosphate buffer (10 mM, pH 7.4). MIP and NIP attached QCM biosensors were used in kinetic analysis studies. MIP QCM biosensor was equilibrated with phosphate buffer at a flow rate of 1.0 mL/min. L-histidine solutions in a concentration range between 6.44 lM and 225.6 lM were individually introduced into the QCM system. L-histidine was desorbed from the surface of MIP QCM biosensor with equilibrium buffer containing 1.0 M NaCl. Interaction between metal-chelating monomer and L-histidine is due to secondary forces. However, coordination bonds between MAH-Cu metal-chelating monomer and L-histidine are the basic interactions. Competitive adsorption of L-histidine, D-histidine and l-tryptophan was investigated to show the selectivity of MIP and NIP QCM biosensors. MIP QCM biosensors were also used for RNAase, lysozyme, cytochrome-C and BSA to investigate the competitive adsorption of surface histidine exposed proteins. The obtained kinetic data were analyzed by using RQCM (Maxtek) software.

Results and dissussion
Characterization studies When the FTIR spectrum in Figure S2 is examined, the characteristic carbonyl bands of the MAH structure are observed at 1653 and 1629 cm À1 , and the amide II stretching vibration band is observed at 1529 cm À1 . Enantiomeric purity of the synthesized MAH monomer was determined with HPLC using the Chirobiotic T column. The single peak observed at 2.12 min on the chromatogram indicates that MAH monomer has an enantiopurity of 99% ( Figure 1). Figure 2 indicates the results of zeta size analysis of MIP and NIP. The average size of L-histidine imprinted nanoparticles was found as 86.43 nm with PDI of 0.138. For MIP, these values were found to be 70.27 nm and 0.150, respectively. As a result of the polymerization processes, the average sizes of MIP and NIP were similar.
The FTIR-ATR spectrum of MIP attached QCM sensor surface was shown in Figure 3. Absorption bands belonging to MIP attached QCM sensor surface were marked on the spectrum. The hydroxyl band at 3409 cm À1 , aliphatic C-H band at 2995 cm À1 and carbonyl band at 1731 cm À1 of the methacrylate structure were observed. In addition, amide bands originating from MAH monomer were observed at 1644 cm À1 (C=O, amide I) and 1447 cm À1 (N-H bending, amide II). C=C-H asymmetric stretching band belongs to imidazole group were observed at 3025 cm À1 , C-C=C symmetrical stretching and C-C=C asymmetric stretching bands were observed at 1584 cm À1 1469 cm À1 , respectively.
Surface characterization of the unmodified QCM sensor, MIP attached QCM surface and NIP attached QCM surface was performed. DSA2 software was utilized to calculate   contact angle values. Table 1 summarizes the surface contact angles of the unmodified QCM sensor, MIP and NIP QCM sensors. The contact angle of the unmodified, MIP and NIP sensor surfaces was 68.7 , 59.8 , 62.2 , respectively. The decrease in the contact angle value remarks that the hydrophilic property increases owing to the hydrophilic character of MAH-Cu(II) metal chelating monomer to fabricate of both MIP and NIP. The MAH-Cu metal chelating monomer is an amino acid (histidine) based material with hydrophilic character. Thus, the attachment of a hydrophilic polymer on to the surface of QCM electrodes increased the surface hydrophilicity and reduced the contact angle value. Figure 4 indicates contact angle images of unmodified (a), MIP (b) and NIP (c) sensors. Surface depth obtained from ellipsometer of MIP attached QCM surface and NIP attached QCM surface was found as 54.9 and 52.0 nm. The thickness measurements showed that the nanoparticle distribution on the surface was almost homogeneous.

Evaluation of QCM biosensor response
L-histidine solutions in a concentration range between 6.44 lM and 225.6 lM were introduced to QCM system to examine the effect of concentration on QCM respond. Concentration interval was determined in respect with the probability of L-histidine levels in human plasma. The solutions were interacted with the QCM sensors via a peristaltic pump.
Data obtained with RQCM (Maxtek) software were monitored simultaneously and kinetic data were collected. As seen from Figure 5, the frequency change (Df) and the mass change (Dm) increased with the increase of L-histidine concentration. An increase in the concentration enhanced also the driving force.
LOD and LOQ were calculated according to previous study [30]. The LOD and LOQ were found as 1.06 mM and 3.53 mM, respectively.

Selectivity and reproducibility studies
Distribution and selectivity coefficients for L-histidine, D-histidine and l-tryptophan molecules were determined according to Eq. 1.
K d defines distribution coefficient (1/mM); C i and C f , initial and final concentrations of analytes (mM); V, volume of solution (mL) and m is the weight of the polymer (g). Primary reason for this approach is the absence of meaningful difference between initial and final concentrations. The polymer mass can not be precisely determined and the concentration is linearly related to Dm [31]. So, selectivity coefficient becomes, K ¼ Dm template =Dm competitor (2) and relative imprinting selectivity obtained, The competitive adsorption of L-histidine, D-histidine, ltryptophan and binary mixtures of these analytes on MIP QCM biosensor was depicted in Figure 6. The mass changes  of MIP QCM biosensor against L-histidine, D-histidine and ltryptophan solutions at the concentration of 96.6 mM were measured as 0.35, 0.06 and 0.16, respectively. MIP QCM biosensor was 5.83 and 2.19 times more selective for L-histidine against to D-histidine and l-tryptophan, respectively. Therefore, the molecular imprinted polymers also allow the chiral recognition. The mass changes of NIP QCM biosensor against L-histidine, D-histidine and l-tryptophan solutions at the concentration of 96.6 mM was also determined to examine the imprinting effect as 0.224, 0.235 and 0.22, respectively (Figure 7). k and k' of MIP QCM biosensor were summarized in Table 2. k' of MIP QCM biosensor for L-histine are 6.12 and 2.15 according to D-histidine and L-tryptophan. The imprinted binding sides generated in MIP preferentially sense L-histidine better than D-histidine and L-tryptophan.
MIP QCM biosensor was also utilized for RNAase, lysozyme, citochrom-c and BSA to investigate the competitive adsorption of surface histidine exposed proteins as shown in Figure 8.
The reproducibility of a process is always considered not only from the economic point aspect, it is also important if the product controlled by a regulatory authority. To examine MIP QCM biosensors reusability, adsorption-equilibriumregeneration cycles was repeated three times using the L-histidine solution of 96.6 mM. Dm values were depicted in Figure  9. Dm values of MIP QCM biosensor against L-histidine were found as 0.210, 0.209 and 0.181. Fabricated biosensor responses are stable and could be used many times without significiant loss in L-histidine adsorption (Figure 9). In this regard, prepared biosensor is capable of L-histidine detection.

Equilibrium analysis
Maximum analyte adsorption on the sensor surface is defined as the sum of L-histidine binding sites, and concentration values can be defined as the QCM signals.
(dDm/dt, the rate of change of Dm (mM/cm 2 ); Dm and Dm max , binding signal and maximum signal; k a (mM/s), the binding rate constant; k d (1/s), the dissociation rate constant)   At equilibrium dm/dt =0; The binding constant K A (k a /k d ) was obtained from the graph of Dm equilibrium /C vs Dm equilibrium and the dissociation constant K D was obtained from 1/K A and given in Table 3 [31].

Mathematical approaches for MIP biosensor response
Interaction model between L-histidine and MIP QCM biosensor was evaluated by using Langmuir, Freundlich and Langmuir-Freundlich (LF) adsorption isotherm models ( Figure  S3-S7) ( Table 4). LF has higher correlation coefficient, R 2 of 0.98 compared to both Langmuir and Freundlich. LF facilitates to describe molecularly imprinted polymers of both homogeneous and heterogeneous distributions at both high and low concentrations. LF is applicable to define the corresponding binding parameters. LF fitting coefficients enable to measure the sum of binding sites, mean association constant and heterogeneity, so the binding behavior of molecularly imprinted polymers can be described by the heterogeneous LF isotherm [32].

Conclusions
L-histidine is an essential amino acid for biological functions. Excessive amounts or deficiency of histidine/histidine rich proteins can cause the life-threating diseases such as Parkinson and kidney disease. So, detection of L-histidine with high selectivity and sensitivity can facilitate the monitoring of mentioned diseases. For this purpose, molecularly imprinted QCM biosensor was designed for the detection of L-histidine. Molecularly imprinted nanoparticles with the size of 86.43 nm were synthesized via mini-emulsion polymerization. The LOD and LOQ were found as 1.06 mM and 3.53 mM, respectively.

Disclosure statement
No potential conflict of interest was reported by the authors.