Selective Recognition of Kanamycin via Molecularly Imprinted Nanosensor

Herein, the molecular recognition sites on the surface of the chip were created by the molecular imprinting method to produce the surface plasmon resonance (SPR) based nanosensor for the real-time kanamycin (KAN) detection. Firstly, kanamycin imprinted nanofilm, which has specific recognition cavities for kanamycin were synthesized by insitu radical polymerization. Fabricated nanofilm for the detection of kanamycin was characterized with FTIR, ellipsometer, and atomic force microscope by the means of structurally and morphologically. The mean thickness values were determined for the imprinted and non-imprinted nanofilms as 102.4±3.1 nm and 101.8±4.7, respectively. The sensitivity performance of imprinted nanosensor was investigated by using the KAN solutions at different concentrations (25-200 ng/mL). The refractive index and the KAN concentration were found to be in perfect agreement with a regression coefficient (R2, 0.992). The detection limit was calculated as 0.40±0.05 ng/mL by using the equation in the calibration curve. The response of imprinted and nonimprinted nanosensors towards the chemical analogs of KAN (NEO and SPM) were investigated to prove the selectivity of KAN imprinted nanosensors. The reusability performance of imprinted nanosensor was investigated by spiking 25 ng/mL KAN solution with three replicates. When the kinetic analyzes were examined, high sensitivity real-time kanamycin analysis was performed at very low concentrations with good reusability. INTRODUCTION

electrochemical [4,7,10], mass spectrophotometry (MS) [9], capillary electrophoresis [11,12]. While substantial contributions and recompensing results have been provided to the KAN detection, the majority of these methods are time-consuming, laborious, and complicated [2,6,7]. Furthermore, they often entail a significant amount of raw material, professional personnel, and costly equipment [2,6,10]. Therefore, rapid, simple, and low-cost methods for sensitive and selective KAN detection need to be developed. The molecular imprinting technique (MIT) is a powerful tool to create selective recognition regions on the surface of interest [7,[13][14][15][16]. There are many applications such as drug delivery [17], diagnostics [18], biosensors [19], forensic science [20], regenerative medicine [21], tissue engineering [22], etc. that is used effectively and successfully to obtain high sensitivity, specificity, and accuracy. Surface plasmon resonance (SPR) with undeniable advantages is a suitable platform to obtain a low detection limit for the sensing application of MIT [23][24][25]. The sensing system produced by the

A B S T R A C T
H erein, the molecular recognition sites on the surface of the chip were created by the molecular imprinting method to produce the surface plasmon resonance (SPR) based nanosensor for the real-time kanamycin (KAN) detection. Firstly, kanamycin imprinted nanofilm, which has specific recognition cavities for kanamycin were synthesized by insitu radical polymerization. Fabricated nanofilm for the detection of kanamycin was characterized with FTIR, ellipsometer, and atomic force microscope by the means of structurally and morphologically. The mean thickness values were determined for the imprinted and non-imprinted nanofilms as 102.4±3.1 nm and 101.8±4.7, respectively. The sensitivity performance of imprinted nanosensor was investigated by using the KAN solutions at different concentrations (25-200 ng/mL). The refractive index and the KAN concentration were found to be in perfect agreement with a regression coefficient (R2, 0.992). The detection limit was calculated as 0.40±0.05 ng/mL by using the equation in the calibration curve. The response of imprinted and nonimprinted nanosensors towards the chemical analogs of KAN (NEO and SPM) were investigated to prove the selectivity of KAN imprinted nanosensors. The reusability performance of imprinted nanosensor was investigated by spiking 25 ng/mL KAN solution with three replicates. When the kinetic analyzes were examined, high sensitivity real-time kanamycin analysis was performed at very low concentrations with good reusability. selective molecularly imprinted nanofilm was obtained from the polymerization of MAA-KAN pre-complex towards EGDMA as a crosslinker and HEMA as a cofunctional monomer. For the preparation of MAA-KAN pre-complex, 2 mg of KAN was complexed with 7.25 μL of MAA by providing a 1:5 (mmol) template molecule and functional monomer ratio. The pre-complex was dissolved by mixing 41 µL of toluene and 164 µL of ACN to obtain a homogeneous solution of the pre-complex. 13.4 µL of HEMA monomer and 27.7 µL of EGDMA crosslinker were added to the pre-complex solution. After adding AIBN (10 mg) as initiator, the final solution (5 µL) was dripped on the pre-vinylated chip. The prepared chip was placed under a UV lamp and polymerization was achieved by photopolymerization for 75 minutes. The unreacted monomer was removed with ethyl alcohol. The ratio of the crosslinker, monomer, and template molecule is 20:14:1, respectively. The percentage of monomer in solution was determined as 25 % (w/v). After the polymerization steps, the imprinted nanofilm-coated SPR chip was washed with methanol and acetonitrile solution (4:1, MeOH:MeCN) to extract the template. The KAN imprinted nanofilm was washed with this desorption solution at room temperature for one hour, renewing the desorption solution every ten minutes. This process was repeated until KAN could not be detected by UV spectrophotometer at 276 nm. To prove the selectivity of KAN imprinted nanofilm, the non-imprinted nanofilm was also fabricated in the same manner as described earlier without adding KAN.

Characterization of nanofilms
The thickness measurements on the surfaces of the prepared sensors were characterized by Nanofilm EP3-Nulling Ellipsometer (Göttingen, Germany). The laser with the wavelength of 532 nm at an incidence angle of 62° was used to perform thickness measurements. The SPR sensor is positioned beneath the laser light source. The average of the kinetic measurement results repeated three times in six different regions is reported. The contact angles of nanofilms were measured using a KRUSS DSA100 (Hamburg, Germany) instrument. The contact angles were determined by pouring water on the SPR chip surfaces using the Sessile Drop technique. For each drop, five independent photographs were taken from different regions of the chip surface to calculate contact angle data. Five independent photographs were recorded from various regions of the surface of the chips, and the contact angle data were calculated for each drop. The structural characterization of the nanofilm surfaces was carried out using Fourier transform infrared spectrometer (Thermo Fisher Scientific, Nicolet iS10, Waltham, MA, USA). combination of SPR and MIT is widely used in the detection of cells [20], pathogens [21,26], biomolecules [27,28], drugs [29], antibiotics [30][31][32], etc.
Herein, the nanosensor fabricated via MIT was developed to detect KAN with sensitivity and selectivity by using SPR. The KAN imprinted nanofilm which has specific recognition cavities for KAN were synthesized by in-situ radical polymerization on the chip surface. Before kinetic measurement of SPR chip surfaces with KAN imprinted nanofilms, FTIR, Ellipsometer, and AFM were used for the characterization of nanofilms on the surface of chips. The responses of nanosensors against KAN and competitors were investigated for selectivity studies. The KAN detection using the assay was successful, with high selectivity and sensitivity.

Functionalization of the chip surface
The chip surface was functionalized with allyl mercaptan (CH2CHCH2SH). Before the modification, acidic piranha solution (3:1, H2SO4 / H2O2) was utilized to clean the gold surface. Then, ethyl alcohol was used as a washing solution for the cleaned chips, and the chips were placed to dry in a vacuum oven (200 mmHg, 40°C) for 3 hours. 10 μl of allyl mercaptan was applied to the cleaned SPR chip surface and allowed to stand for 12 hours. Following this, the final chips were washed with ethanol and dried in a nitrogen atmosphere. The modification resulted in the functionalization of the chip surface with allyl groups.

In situ synthesis of nanofilms
The following process was carried out for the fabrication of nanofilm on the surface of chips. The KAN

SPR measurement
Kinetic measurements by the KAN imprinted nanosensors were carried out by surface plasmon resonance. The kanamycin solutions at different concentrations (25-200 ng/mL) were interacted with SPR nanosensors by a peristaltic pump and kinetic data were obtained using SPRview software.
The following steps were applied for the real-time SPR analysis: first equilibrium buffer (Phosphate buffer, 10 mM, pH 7.5) then KAN solution was passed through the system until the system reached equilibrium again, and the desorption solution (2.0 M NaOH) was used in the last step. In all measurements, the equilibrium buffer waited for 5 minutes and kanamycin solution was waited for 10 minutes for the system to reach equilibrium. Desorption and regeneration processes were carried out in about 15 minutes. The isotherm models can be applied to examine the interaction between the imprinted nanosensor and KAN in the graphs. The response of imprinted and nonimprinted nanosensors towards the chemical analogs of KAN (NEO and SPM) were investigated to prove the selectivity of KAN imprinted nanosensors. The molecular structures of KAN, NEO and SPM were given in Figure 1. The reusability performance of imprinted nanosensor was investigated by spiking 25 ng/mL KAN solution with three cycles.

Characterization
The average surface thickness of the nanofilm was investigated through spectroscopic ellipsometry. As seen in Figure 2, the mean thickness values were determined for the imprinted and non-imprinted nanofilms as 102.4±3.1 nm and 101.8±4.7, respectively.
The surface hydrophobicity of the SPR chip surface was investigated by taking the contact angle measurements. The contact angles of imprinted, nonimprinted, and unmodified SPR chips were measured as 60.5°, 62.2°, and 76.4°, respectively ( Figure 3). The decreasing of contact angle values indicates an increment in wettability as a result of the decrease in hydrophobicity. The imprinted and nonimprinted nanofilms synthesized on the chip surface have a hydrophilic character originating from the monomers (HEMA, EGD-MA, and MAA) used, therefore, as expected, the contact angles are lower than the unmodified SPR chip.
FTIR-ATR spectroscopy analysis was carried out for the structural characterization of nanofilms. The similarities in the chemical structures of nanofilms caused by crosslinker and functional monomers are clearly visible in FTIR-ATR spectra (Figure 4). The most prominent bands in the spectra, as shown in Figure 4, were O-H stretching bands, which correspond to the hydroxyl group of HEMA at about 3200-3300 cm -1 ; aliphatic C-H stretching bands, which correspond to the methyl group of MAA and EGD-MA at about 2900-3000 cm -1 ; and C=O stretching bands,

Kinetic and Equilibrium Analysis
To evaluate the relationship between SPR signal and kanamycin concentration, kanamycin solutions at various concentrations (25-200 ng/mL) were analyzed by the KAN imprinted nanosensor ( Figure 5). The changes in the refractive index versus time were given in Figure 4a by applying the KAN solutions at the different concentrations to the imprinted nanosensors. As can be seen from the figures, the % refraction value increases with the application of kanamycin to the imprinted SPR nanosensor. The reason for this can be shown as the increase in the concentration difference, which is the driving force between the kanamycin solution and the surface. As seen in Figure 5, the % refraction value increases with the application of kanamycin to the surface. In a standard measurement; equilibrium solution was passed through the system first, then kanamycin solution until the system reached equilibrium again, and desorption solution was used in the last step. In all measurements, approximately 20 minutes were waited for the system to reach equilibrium. Desorption and regeneration processes were carried out in about 15 minutes. It was observed that the % refraction value increased greater with increasing the concentration. The reason for this can be explained as the increase in the concentration difference, which is the driving force between the KAN solution and the surface of imprinted nanofilm. The amount of KAN can be determined by using the calibration plot giving the relationship between SPR signal and KAN concentration in Figure 5b. The refractive index and the KAN concentra-tion were found to be in perfect agreement (R2, 0.992). The detection limit was calculated as 0.40±0.05 ng/mL by using the calibration curve. When the kinetic analyzes were examined, high sensitivity real-time kanamycin analysis was performed at very low concentrations. Considering the importance of detecting residues of kanamycin for human health even at low concentrations, this method is thought to be a very successful technique for kanamycin analysis compared to literature (Table 1).
It is required to determine the binding parameters from the measurements of the process to quantify binding features. For the determination of constants, the models, whose equations are given below, are applied to the data [37][38][39].
where dR/dt is the change of the refractive index in unit time, R and R max are experimental sensor responses measured during analyte molecule binding (Reflectivity %/s) and theoretical maximum sensor response, C is the concentration (M), k a is the association rate constant (L/mol.s), k d is the dissociation rate constant (1/s), and 1/n is the Freundlich heterogeneity index. The association constant, K a , can be computed as K a = k a /k d (L/mol), while the dissociation constant, K d (mol/L), is equal to 1/K a .
The rate and equilibrium parameters reveal the intensity of association and dissociation tendency for the interaction of surface and molecule. As shown in Table 2, the association rate constant and s for (k a ) and the dissociation rate constant (k d ) were calculated as 2.2x102 M.s -1 and 2.8x10 -3 s -1 , respectively, while the association (K a ) and dissociation (K d )  constants were 7.7 x 105 M -1 and 1.3 x 10 -6 M. As can be seen, the interactions between the nanosensor and KAN have a high affinity according to the association (K a ) and dissociation (K d ) constants.
Adsorption isotherm models were utilized to examine the surface heterogeneity and binding behaviours of imprinted nanosensors. The binding characteristics were investigated by using the Langmuir and Scatchard models for the imprinted nanosensor ( Table 3). The Langmuir model supposes that the surface has homogeneous binding sites with the same binding affinity coefficient. The Freundlich model describes the heterogeneous binding behavior of surfaces. This model is useful for estimating adsorption on the heterogeneous nature of surfaces and thus avoiding the limitation of higher concentration related to the Freundlich model. Therefore, this model has been explained by Freundlich Isotherm at low concentration and the Langmuir model at high concentration.
The Freundlich model was used to match the experimental binding isotherms based on correlation coefficient and linearity data, as shown in Table 3. According to the Freundlich model, Rmax and the Freundlich constant, 1/n, were calculated to be 1.87 and 0.947, respectively. Although the binding sites on the kanamycin imprinted nanosensor are diverse, the population of high-affinity binding sites is greater. Consequently, the Freundlich model is best suited to elucidate the binding behaviors of imprinted polymers.

Selectivity of nanosensor
The response of imprinted and nonimprinted nanosensors towards the chemical analogs of KAN (NEO and SPM) were investigated to prove the selectivity of KAN imprinted nanosensors. The assay for the detection of KAN was separately applied to the chemical analogs solutions onto the imprinted and nonimprinted nanosensor and the sensograms are shown in Figure 6. As seen in Figure 6b, the nonimprinted nanosensor is non-selective and has a poor sensitivity to detect KAN. However, the imprinted nanosensor produces a strong signal with good selectivity for the detection of KAN (Figure 6a). The selectivity results prove that the imprinted nanofilm has specific cavities to recognize the KAN with high sensitivity and selectivity.

Reusability of nanosensor
The reusability performance of imprinted nanosensor was investigated by spiking 25 ng/mL KAN solution with three cycles. As seen in Figure 7, the change in the response of nanosensor after a three-cycle is not significant to detect kanamycin. Consequently, the developed nanosensor system indicates good repeatability to recognize kanamycin in an aqueous solution.

CONCLUSION
In this research, the KAN imprinted nanofilm for the SPR sensor was constructed and successfully synthesized by in-situ radical polymerization. Various methodologies were used to characterize the constructed system, and the refractive index changes were analyzed depending on the relation between the KAN concentrations and the signal enhancements. the refractive index changes depending on the relationship between kanamycin concentrations and signal enhancements were analyzed. The responses of imprinted nanosensors were found to be effective for detecting analytes of interest, whereas non-imprinted nanosensor responses were ineffective. For the detection of kanamycin, numerous analytical approaches have been used, including solid-phase extraction (SPE), spectrophotometry, electrochemical, high-performance liquid chromatography (HPLC), mass spectrophotometry (MS), cantilever array sensor, and capillary electrophoresis [3][4][5][6][7][8][9][10][11][12]. Such systems, in contrast to the suggested sensing system, are arduous, expensive, time-consuming, and difficult. Based on these findings, the imprinted nanosensors are capable of selectively capturing KAN from aqueous media. The imprinted nanofilms appear to be a promising tool for sensing applications in this area. Based on this successful approach and ease of extension to the analysis of additional analytes, the SPR system in combination with the MIP method is envisioned as a potential technique for detecting residues.