Selective detection of Escherichia coli caused UTIs with surface imprinted plasmonic nanoscale sensor

Materials Science & Engineering C 104 (2019) 109869

Contents lists available at ScienceDirect

Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec

Selective detection of Escherichia coli caused UTIs with surface imprinted plasmonic nanoscale sensor

T

Sinem Diken Güra, Monireh Bakhshpourb, Adil Denizlib,

⁎

a b

Hacettepe University, Department of Biology, Ankara, Turkey Hacettepe University, Department of Chemistry, Ankara, Turkey

ARTICLE INFO

ABSTRACT

Keywords: E. coli Urinary tract infections Surface plasmon resonance Nanosensor Au nanoparticles Surface imprinting

The aim of the present study was developing a surface plasmon resonance (SPR) nanosensor to detect Escherichia coli (E. coli) for the diagnosis of urinary tract infections by using surface imprinted Au nanoparticles (AuNPs) as a recognition element. In order to realize imprinting, Cu(II) ions were used to provide interaction between E. coli cell wall and amine functionalized AuNPs forming cavities on the surface of nanosensor. E. coli surface imprinted AuNPs nanosensor was characterized by using ellipsometry, contact angle measurement, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The real time detection of E. coli was evaluated by using E. coli suspensions in the concentration range of 1 × 103–0.5 × 101 CFU/mL. Combination of the signal enhancing properties of AuNPs and surface imprinting technique provided ultrasensitive detection with a comparatively low limit of detection value (1 CFU/mL) to the SPR nanosensor system. Selectivity experiments were performed by using Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. The highest response was recorded for E. coli, as expected. Additionally, the recognition of E. coli even in a complex medium such as artificial urine sample was achieved by the developed nanosensing system. Also, this chip can be used repeatedly without seen signal reducing for four-time consecutive. In the view of these results, it was emphasized that this novel sensing system has a potency for the selective, very sensitive, rapid and real time detection of causative agent in order to diagnose E. coli caused infections.

1. Introduction The most frequently encountered bacterial infections which are painful and economically costly infections, are urinary tract infections (UTIs) impacting mostly women due to the anatomic differences. Also, the most common pathogen associated with community acquired UTIs is uropathogenic E. coli (UPEC), responsible for approximately 85% of infections [1]. After invading the host epithelial cells in the urinary tract, UPEC can permeate a dormant state. Intracellular settlement ability of these bacteria provides them resistance properties to frequently used antibiotics in the clinic. Early treatment in UTIs is required for reducing morbidity and risk of recurrence [2]. However early intervention is only possible with rapid detection of causative agent, used diagnosis methods in clinic such as conventional culturing is time consuming. In addition, molecular methods are known to be more sensitive and rapid than culturing, have some disadvantages like high cost and professional staff necessity. For these reasons, development of sensitive and fast detection tools such as biosensors is deficient [3–5]. Biosensors are one of the most important devices due to their useful

⁎

properties such as low limit of detection, real time response, high selectivity and low cost. In recent years different sensors developed with graphene and Au nanocomposites were used for ultrasensitive determination of several targets [6–9]. Among different biosensors, surface plasmon resonance sensors which can obtain measurements in real time are one of the important sensing tools. Also, this property allows the determination of binding kinetics and strength [10,11]. Surface plasmon resonance (SPR) which is a label free optic sensor, allows for the analysis of pathogenic bacteria at low concentrations by the determination of refractive index changes due to the complementary binding of the recognition elements such as bacterial components (DNA, RNA, enzymes and produced substances) or whole bacteria to the optical transducer surface [12,13]. With the use of SPR sensors, minute changes in the refractive index can even be determined sensitively. Additionally, SPR sensors which offer multiplexing capacity, allow effective and point of care diagnosis of pathogen bacteria. Furthermore, optical sensors can be integrated with smart phone based systems to provide fast and cost effective sensing [14]. The selectivity of the sensor is the most significant part of the sensor

Corresponding author. E-mail address: [email protected] (A. Denizli).

https://doi.org/10.1016/j.msec.2019.109869 Received 13 March 2019; Received in revised form 21 May 2019; Accepted 5 June 2019 Available online 06 June 2019 0928-4931/ © 2019 Published by Elsevier B.V.

Materials Science & Engineering C 104 (2019) 109869

S.D. Gür, et al.

which is generally determined by the used biological recognition element [11]. In different biosensor studies, antibodies used as a biorecognition element to bind their antigens with high specificity. However, antibodies exhibit high affinity towards their target, they have some disadvantages such as high cost and lack of stability to harsh conditions [15]. In order to overcome these problems imprinting of target molecule have started to be used in recent studies. For obtaining imprinted polymer or film with cavities and binding sides which are complementary to the original target in regard to shape and chemical properties, the monomer and target mixture is polymerized. Then solvent is used to remove target for opening binding sides specific to the target [16]. In contrast to antibodies, imprinted materials show important advantages including high stability, low cost, high specificity and re-usability capacity. While imprinting of large structures such as bacterial cell, limited access or remove problem can be prevented by using surface imprinting method with a thin layer film. Thus, specific recognition regions are formed near to the surface of polymer or film [17–19]. When the large template molecules are imprinted, the poorly accessible and deeply embedded cavities are consisted in the polymer matrix. For this reason, slow binding kinetics and low binding capacity are observed. To accomplish these challenges, nanoscale imprinted polymers such as nanoparticles (NPs) have been introduced [20]. One of the nanoparticles used for this purpose is AuNPs. The composites or films prepared with imprinted AuNPs have an ability to enhance sensitivity and selectivity of nanosensor [21,22]. AuNPs offer some significant properties to the biosensor with their high surface energy and large surface to volume ratio. As a consequence, the detection limit of biosensor can reach to a single cell with properties of AuNPs [20]. In literature there is no report about determination of uropathogenic E. coli by using surface imprinted AuNPs on SPR chip. In this study, in order to real time detect E. coli from urine samples, surface imprinted nanoscale SPR biosensor was developed. The characterization studies of E. coli surface imprinted AuNPs SPR (E. coliAuNPs-SPR) chip was carried out by using ellipsometry, contact angle measurement, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The selectivity studies of developed nanosensor were performed using different bacteria such as Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. The SPR chip was used four times for investigating reusability performance. Then, artificial urine sample that contains E. coli was prepared to perform real sample experiments.

were incubated at 37 °C for 18 h in an incubator-shaker. Then the bacteria were centrifuged at 3000 rpm for 15 min for obtaining pellet. After removing supernatant, the pellet resuspended with PBS buffer. The washing process of bacteria was repeated three times. In order to determine the bacteria concentration, plate counting method was used. For this purpose, serial 10-fold dilutions of bacteria in sterile PBS buffer were prepared and 100 μL sample was inoculated onto nutrient agar plates. After incubation at 37 °C for 24 h, the formed colonies were counted and the concentrations were expressed as colony forming units in milliliter (CFU/mL). 2.3. Preparation of E. coli-AuNPs-SPR chip The modification of gold SPR chip surface by allyl mercaptan was carried out according to previous our group works [23,24]. In order to make modification on the surface of SPR chip with using allyl mercaptan (CH2CHCH2SH), firstly the chip surface was washed with acidic piranha solution. Then 4 μL allyl mercaptan was added and spread to chip surface. In order to remove the unbounded allyl mercaptan molecules, the chip surface was washed with distilled water and ethanol, and then dried under the vacuum at 200 mmHg, 25 °C. For preparation of E. coli surface imprinted AuNPs SPR chip, 1 mL amine modified AuNPs and 2.4 mg Cu(II) were mixed for 30 min at room temperature in rotator with 200 rpm. Then, 1.5 × 107 CFU/mL E. coli suspension was added to amine modified AuNPs-Cu(II) solutions and mixed for 10 min at room temperature. Then, the initiator AIBN was added into this stock solution. After the preparation of AuNPs-Cu (II)-E. coli solutions, 10 μL solution was added on to the modified SPR chip. Then applied UV polymerization for 20 min. By this way amine groups of Au nanoparticles bonded to –SH groups (based on thiol modification) of SPR chip surface. Also, non-imprinted AuNPs SPR chip was perpetrated with the same method without E. coli as target molecule. The preparation of AuNPs-Cu(II)-E. coli was shown in the Fig. 1. For removing the template molecules (E. coli), the chip surface was washed with 1 mM lysozyme, 1 mM NaCl and 100 mM ethanol solutions, respectively. 2.4. Surface characterization of E. coli-AuNPs-SPR chip The characterization of E. coli surface imprinted and non-imprinted AuNPs-SPR chips were carried out with using ellipsometry, AFM, SEM and contact angle. The thickness properties of the chip surface were observed using Nanofilm EP3-Nulling Ellipsometry (Gottingen, Germany). The measurement of contact angle was realized by KRUSS DSA100 (Hamburg, Germany). In order to characterize the 3D topography of E. coli-AuNPs-SPR chip, AFM analysis was performed in METU Central Laboratory in tapping mode. SEM analysis was evaluated for obtaining information about the morphological properties of Au chip surface in the METU Central Laboratory. Firstly, chip was dried in the freeze drier (Christ Alpha LD 1–2 plus, UK) at −50 °C (Christ Alpha LD 1–2 plus, UK) after the chip surface was washed by ethanol and purified water, then, coated with Au.

2. Experimental 2.1. Materials E. coli ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 25923, Klebsiella pneumoniae (K. pneumoniae) ATCC 13883 and Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853 were purchased from American Type Culture Collection (ATCC). SPR chips were obtained from Nanoreg (Ankara, Turkey). Fluka (Buchs, Switzerland) provided α-α′azoisobutyronitrile (AIBN). Allyl mercaptan, lysozyme (from chicken egg white), phosphate buffer saline (PBS) and amine modified Au nanoparticles (15 nm) were supplied from Sigma Chem (catalog number: 765325). Luria Bertani broth and nutrient agar which were used for culturing of bacteria were obtained from Merck. Other chemicals used in this study were of analytical grade, also distilled water was used for preparing mediums and buffers.

2.5. Real time detection of E. coli The real time detection of E. coli was carried out using SPR imager II system (GWC Technologies, Madison, USA). Firstly, the SPR nanosensor was washed with distilled water (20 mL, 2 mL min−1 flow-rate) and equilibration buffer (pH: 7.4, PBS, 20 mL, 2 mL min−1 flow-rate). After obtaining the steady resonance frequency, E. coli solutions with different concentrations (1 × 103–0.5 × 101 CFU/mL) in 0.1 M PBS (pH 7.4) (5 mL) were injected to SPR system (2.0 mL min−1 of flowrate). The resonance frequency changes were monitored instantly until reached to plateau. In desorption step, to remove bacteria from the surface of nanosensor, 1 mM lysozyme solution (5 mL), 1 mM NaCl

2.2. Preparation of bacterial strains The strains used in experiments were E. coli, S. aureus, P. aeruginosa and K. pneumoniae. Luria Bertani broth was used as growth medium. After the bacterial strains were inoculated into 10 mL LB broth, samples 2

Materials Science & Engineering C 104 (2019) 109869

S.D. Gür, et al.

Fig. 1. Graphical abstract of E. coli-AuNPs-SPR chip preparation and real time detection of E. coli using SPR nanosensor.

solution (5 mL) and 100 mM ethanol solution (5 mL) at the flow rate of 2.0 mL min−1 were used, respectively. Then, SPR chip was cleaned with distilled water and equilibration buffer to prepare chip for new injections. Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa were used to examine specificity and selectivity of SPR nanosensor. Due to the similar cell wall structure, shape and size, Klebsiella pneumoniae and Pseudomonas aeruginosa were preferred. On the other hand, S. aureus was included due to the different cell wall structure, shape and size from E. coli. For real sample studies, artificial urine spiked with different concentrations of E. coli (1 × 102–1 × 101 CFU/mL) was applied to SPR nanosensor. The reusability of the nanosensor was examined monitoring reflectivity changes at the repeated experiments using same constant concentration (1 × 101 CFU/mL) of E. coli suspension. Also, SPR 1001 software was used to show the kinetic data.

due to the localized surface plasmon resonance property of AuNPs, they exhibit strong UV visible absorption band which makes them ideal choice for optical biosensor applications. In this regard AuNPs incorporation with SPR devices cause signal amplification and improvement of sensitivity by providing increase in SPR incident angle shift [10]. In the light of these data, E. coli surface imprinted nanocomposite film on SPR chip was fabricated by using amine modified AuNPs and Cu (II) ions bound to amine groups. As a result of the electrostatic interactions between Cu(II) ions and negative charged parts of E. coli cell wall, the interaction between nanocomposite film and E. coli were occurred. The one of the main contributing cause for the recognition of the bacterial cell is the cavity shape and size that is similar to E. coli, second is chemical matching between nanofilm surface and cell wall of E. coli.

3. Results and discussion

The surface characteristics of E. coli imprinted nanoscale film were determined by ellipsometry, SEM, AFM and contact angle. The water contact angle (WCA) value of bare SPR Au chip was found to be 90 ± 0.91°. Modification realized with allyl mercaptan decreased this value to 66.9 ± 1.9° due to the polar allyl groups of allyl mercaptan [23]. After E. coli surface imprinting, the WCA value changed as 81.2 ± 0.5° and WCA value of non-imprinted SPR chip was found 78.9 ± 1.3°. These results indicated that surface imprinting could be achieved successfully on the modified SPR chip surface. According to ellipsometer measurements, the thicknesses of allyl mercaptan modified surface, non-imprinted and surface imprinted AuNPs-SPR chips were found to be 11.5 ± 1.3, 30.5 ± 6.5 and 32.8 ± 7.1 nm, respectively (Fig. 2A–C). As seen in Fig. 2D1–2, SEM images indicated that the smooth surface of the bare gold chip turned into a rough surface after surface imprinting formation on the SPR chip. The size of Au nanoparticles was showed ~15 nm with SEM photographs. Also, AFM analyse showed that the thickness of the formed film structure on the chip surface was 30 nm. Differentiating surface structure is also seen in AFM image (Fig. 2E).

3.2. Characterization of E. coli-AuNPs-SPR chip

3.1. Preparation of E. coli-AuNPs-SPR chip Surface plasmon resonance biosensing that is a versatile method for bacteria detection to monitor refractive index changes that occur as a result of recognition. For a recognition element, antibody can be used and antigen-antibody reaction occurred on the chip surface can be determined by SPR sensor which is a label free method [25]. Additionally, template specific interaction cavities/sites can be fabricated to mimic the naturally occurring biorecognition elements using molecular imprinting method. Among the recognition systems used in the literature, molecularly imprinted recognition sides have stimulated a wide range of interest over these last years due to the capabilities that offer such as cost effective, easy synthesis, stability under harsh conditions, selectively recognizing, binding template molecules with high affinity and reusability [20]. However, the molecular imprinting applications of small molecules (nucleotides, amino acids, peptides etc.) is well established, imprinting templates with large molecular size such as protein, bacteria, cell is still a challenging because of the binding sites are contained within the bulk, tend to make mass transfer slow. In order to achieve this condition, surface imprinting method can be used and artificial recognition sides for template fabricated near the surface of formed film. When combined with imprinting technology, nanotechnology based imprinted nanomaterials show enhanced sensitivity and selectivity, which may lead to the development of more proper film for SPR chip to detect bacteria with a low limit of detection [26]. For this purpose, noble metal nanoparticles such as Au and Ag are widely used for obtaining nanoscale biosensor. When compared, AuNPs have some advantages of chemical stability and biocompatibility [16]. Also

3.3. Real time detection of E. coli After equilibration step, different E. coli concentrations (1 × 103–0.5 × 101 CFU/mL) were applied to SPR nanosensor for instant detection of E. coli. After injection, reflectivity index change occurs due to the interaction between the recognition cavities and E. coli. Fig. 3A showed the response of SPR nanosensor for different concentration of E. coli after applying to the E. coli surface imprinted AuNPs-SPR chip. As seen in Fig. 3B, when 5 × 101 concentration of E. coli was injected to both imprinting and non-imprinted AuNPs-SPR 3

Materials Science & Engineering C 104 (2019) 109869

S.D. Gür, et al.

Fig. 2. Characterization of SPR nanosensor surface. A–C, Ellipsometry results of allyl mercaptan modified, non-imprinted, and surface imprinted AuNPs-SPR chips; D1–2, SEM images of surface imprinted AuNPs; E, AFM image of E. coli AuNPs-SPR chips.

chip, the response with the determination of E. coli was recorded with imprinted chip. Furthermore, linear relationship between % Resonance frequency change (%ΔR) and the increased concentrations of E. coli was monitored and the validity percentage of assay was found to be 87%

(Fig. 4A). The interaction model between E. coli strains and surface imprinted AuNPs-SPR chip was examined using Langmuir/Freundlich, Langmuir and Freundlich isotherms. The most conforming isotherm model was found as Langmuir/Freundlich with a R2: 0.9925 coefficient (Fig. 4B). Calculated values of the isotherm models given in Table 1.

Fig. 3. Real time responses of A, E. coli surface imprinted AuNPs SPR nanosensor at different E. coli concentrations and B, E. coli surface imprinted and non-imprinted AuNPs SPR nanosensor. 4

Materials Science & Engineering C 104 (2019) 109869

S.D. Gür, et al.

Fig. 4. A, Calibration curve for E. coli detection using AuNPs SPR nanosensor under optimum experimental conditions (flow rate: 2 mL min−1; equilibration buffer: 10 mM phosphate buffer saline, pH 7.4; desorption buffer: 1 mM lysozyme solution (5 mL), 1 mM NaCl solution (5 mL) and 100 mM ethanol solution (5 ml); T: 25 °C). B1, B2, B3, Langmuir/Freundlich, Langmuir and Freundlich isotherms of SPR nanosensor.

confirmed as 4 CFU/mL and 1 CFU/mL, respectively according to Eqs. (5) and (6) [25].

Table 1 Calculated values of the isotherm models. Equilibrium analysis Scatchard ∆Rmax (CFU/ cm2) KA (CFU/mL)

Langmuir 6.31 0.6

KD (mL/CFU)

1.6

R2

0.97

∆Rmax (CFU/cm2) KA (CFU/ mL) KD (mL/ CFU) R2

Freundlich 4.97 0.46 2.16

∆Rmax (CFU/cm2) 1/n R

2

LangmuirFreundlich 2.81 1.41 0.90

0.96

∆Rmax (CFU/cm2) KA (CFU/ mL) KD (mL/ CFU) R2

R eq)]}

0.98 1.02 0.99

(1)

R = { Rmax [C]/KD + [C]}

(2)

R = { Rmax [C]1/n }

(3)

R = { Rmax [C]1/n /KD + [C]1/n }

(4)

(5)

LOQ = 10S/m

(6)

where S is the standard deviation of the intercept and m is the slope of the regression line. Using Au NPs which have large surface to volume ratio and high surface energy as an immobilization platform for bacteria in SPR nanosensor was provided more binding side and signal enhancement. Changes in refractive index occurring at the gold chip surface due to the recognition of target molecule were determined SPR sensor [27]. In this study, for amplifying the SPR changes the surface plasmon wave of gold chip surface was coupled with the localized plasmon of Au NPs. In different studies Au NPs were found to be effective for enhancing the SPR signal [28–30]. The different sensor technologies used in recent years for E. coli determination were compared in Table 2. As seen in the table, the lowest LOD value was obtained in this study. In this regard this data was indicated that synthetic recognition sides are effective tools for detecting template molecules inside the suspensions. Also, the usage of AuNPs to form nanofilm structure offered some advantages to the applied biosensing technology with the properties that they have owned such as advanced thermal and chemical stability, large surface area, high number of recognition sites, easy preparation with low cost and application potential to a wide range of analytics [20]. It has been suggested that the improvement of the detection sensitivity and reducing LOD value depends on the two important reasons. One is the increasing accessible recognition sides for binding template due to the nanoscale heterogeneous surface structure and the other one is the synergetic effect of the surface plasmon oscillations belongs to Au

11.1

The applied linear model of Scatchard, Langmuir, Freundlich, and Langmuir-Freundlich isotherms can be determined using the Eqs. (1), (2), (3), and (4) respectively.

R ex /[C] = {KA ( Rmax

LOD = 3. 3S/m

where ΔR is the response measured by binding; C is concentration of E. coli (CFU/mL); 1/n refers Freundlich exponent; KA (CFU/mL) and KD (mL/CFU) are also the forward and reverse equilibrium constants; subscripts ex, max, and eq indicate experimental, maximum, and equilibrium, respectively. This model indicates multilayer on heterogeneous surface. The limit of quantification (LOQ) and the limit of detection (LOD) values were Table 2 Summary of recent sensor studies for E. coli detection. Method Ferrocene-antimicrobial peptide modified biosensor Aptamer mediated strand displacement amplification Micro contact whole cell imprinting Micro contact whole cell imprinting Micro contact whole cell imprinting Antibody modifies magnetic Au NPs Mesoporous silica of Au NRs@SiO2 Surface imprinting with Au NPs

Sensor type

LOD

Electrochemical impedance spectroscopy Lateral flow biosensor SPR QCM Capacitive biosensor Capacitive biosensor LSPR SPR

5

3

10 CFU/mL 10 CFU/mL 3.72 × 105 CFU/mL 1.54 × 106 CFU/mL 70 CFU/mL 10 CFU/mL 10 CFU/mL 1 CFU/mL

Reference [31] [32] [23] [23] [33] [34] [35] This study

Materials Science & Engineering C 104 (2019) 109869

S.D. Gür, et al.

Fig. 5. A) Selectivity of E. coli-AuNPs-SPR chip nanosensor against different bacterial strains; E. coli, P. aeruginosa, K. pneumoniae and S. aureus (flow rate: 2 mL min−1; equilibration buffer: 10 mM phosphate buffer saline, pH 7.4; desorption buffer: 1 mM lysozyme solution (5 mL), 1 mM NaCl solution (5 ml) and 100 mM ethanol solution (5 ml); T: 25 °C). B) Real time responses of E. coli surface imprinted SPR chip with thin film prepared using Au NPs against artificial urine spiked with different concentrations of E. coli (1 × 102–1 × 101 CFU/mL).

surface and the localized surface plasmon oscillations of the AuNPs. Additionally, the diffusion limitations that occur when binding to recognition cavities due to the huge size of bacteria have been arranged by the use of surface imprinting method [10,36]. As a consequence, when analytes immobilize to the cavities formed on the AuNPs film surface, signal amplification enhanced with surface plasmon, causing the reduce of detection limit to the single bacterial colony level [37]. Demonstrating the selectivity of E. coli surface imprinted AuNPs nanofilm that coated SPR chip, S. aureus, K. pneumoniae and P. aeruginosa were applied to SPR nanosensor. Besides S. aureus that has Gram positive cell wall and the coccus morphology, K. pneumoniae and P. aeruginosa were selected due to the similar shape and cell wall structure with E. coli. The responses of SPR nanosensor system for applied bacteria suspension are given in Fig. 5A. While the highest ΔR value was evaluated for E. coli, the lowest responses were monitored for S. aureus which has a different size, shape and cell wall properties than E. coli. The reason of the recorded highest response against E. coli may depend on complementary binding sides to E. coli formed on the surface of AuNPs nanofilm. The selectivity efficiency of SPR nanosensor for E. coli was found to be 47–50% times higher than the bacteria that have similarities with E. coli (P. aeruginosa and K. pneumoniae). The values given in Table 3 presented that specific binding sides for E. coli cell wall were successfully established by using metal ion mediated imprinting application. Metal ion interactions utilized in surface imprinting of E. coli is effective to obtain highly specific and stabile recognition sides. For this reason, the stoichiometric amount of the metal ion Cu(II) needed to consist specific interaction between the template and the amine modified AuNPs to form cavities complementary to the template [38]. According to these results AuNPs based bacteria surface imprinted SPR nanosensor can be utilized in different applications in order to detect E. coli and to make quantitative analysis. Real sample studies were evaluated by using artificial urine sample spiked with E. coli strains in the different concentrations range because the contamination in urinary tract could be determined obtaining urine samples from patients. According to these experiments, increasing signal response was seen depending on increasing E. coli concentration (Fig. 5B). Thereby developing nanosensor system has the capability to determine E. coli even in a complex medium. The reusability of nanosensor was examined by repeating

Fig. 6. Reusability of E. coli-AuNPs-SPR chip at 1 × 101 CFU/mL concentration of E. coli.

equilibration-adsorption-regeneration cycles four times. An artificial urine sample spiked with E. coli suspension in the 1 × 101 CFU/mL concentration was used in order to obtain SPR sensorgram based on refractive index change. The results indicated that the developed new sensor system could be used for repeated assays (Fig. 6). It has been suggested that the use of lysozyme-NaCl-ethanol suspensions for the subsequent removal of E. coli from the nanosensor is a proper method before starting new cycle [39]. 4. Conclusions In the present study, we developed novel E. coli surface imprinted amine modified AuNPs based SPR nanosensor for fast and sensitive E. coli detection from urine sample. Whole cell surface imprinting strategy offers the opportunity of being able to provide detection of analyte without using recognition elements that obtaining costly and hardly. Also, this nanoscale sensing system demonstrated a high affinity against E. coli as a result of the consisting of metal ion preorganization between bacterial cell wall and Cu(II) ions bounded AuNPs. Occurring refractive index changes and accompanying SPR shifts due to the formations of the metal ion interactions between the Cu(II) ions and negative charged cell wall partitions, amplified because of the localized plasmon of the AuNPs and the surface plasmon wave coupling. This provided ultrasensitive E. coli determination with a very low LOD value (1 CFU/mL). While organic or inorganic monomers were used in the previous imprinting studies that focused on forming small bead sized polymer matrices [40,41], our imprinting technique may be considered as nanoscale imprinting due to the use of the modified AuNPs as monomer units. As a consequence, this study clearly indicates the potential of AuNPs to enhance the signal and sensitivity of SPR system.

Table 3 Selectivity coefficients of E. coli-AuNPs-SPR and non-imprinted SPR nanosensor (ΔR: SPR response, k: selectivity coefficient for E. coli versus other bacterial strains, k′: relative selectivity coefficient for E. coli-AuNPs-SPR versus non-imprinted SPR). Bacterial strains

E. coli P. aeruginosa K. pneumoniae S. aureus

MIP

NIP

k’

∆R

k

∆R

k

5.07 0.11 0.17 0.09

– 47.47 50.8 72.57

0.15 0.11 0.1 0.1

– 1.36 1.5 1.5

Acknowledgements

– 34.81 33.86 48.38

The present study was supported by Hacettepe University Scientific Research Projects Coordination Unit (Project number: FHD-201817102). 6

S.D. Gür, et al.

Materials Science & Engineering C 104 (2019) 109869

References

interactions, J. Am. Chem. Soc. 130 (2008) 9726–9733. [22] M. Riskin, Y. Ben-Amram, R. Tel-Vered, V. Chegel, J. Almog, I. Willner, Molecularly imprinted Au nanoparticles composites on au surfaces for the surface plasmon resonance detection of pentaerythritol tetranitrate, nitroglycerin, and ethylene glycol dinitrate, Anal. Chem. 83 (2011) 3082–3088. [23] E. Yilmaz, D. Majidi, E. Ozgur, A. Denizli, Whole cell imprinting based Escherichia coli sensors: a study for SPR and QCM, Sensors Actuators B Chem. 209 (2015) 714–721. [24] M. Bakhshpour, E. Özgür, N. Bereli, A. Denizli, Microcontact imprinted quartz crystal microbalance nanosensor for protein C recognition, Colloids. Surf. B Biointerfaces 151 (2017) 264–270. [25] M. Frasconi, R. Tel-Vered, M. Riskin, I. Willner, Surface plasmon resonance analysis of antibiotics using imprinted boronic acid-functionalized Au nanoparticle composites, Anal. Chem. 82 (2010) 2512–2519. [26] M. Irshad, N. Iqbal, A. Mujahid, A. Afzal, T. Hussain, A. Sharif, E. Ahmad, M.M. Athar, Molecularly imprinted nanomaterials for sensor applications, Nanomaterials 3 (2013) 615–637. [27] R. Ahmad, N. Griffete, A. Lamouri, N. Felidj, M.M. Chehimi, C. Mangeney, Nanocomposites of gold nanoparticles@molecularly imprinted polymers: chemistry, processing, and applications in sensor, Chem. Mater. 27 (2015) 5464–5478. [28] R. Liu, Q. Wang, Q. Li, X. Yang, K. Wang, W. Nie, Surface plasmon resonance biosensor for sensitive detection of microRNA and cancer cell using multiple signal amplification strategy, Biosens. Bioelectron. 87 (2017) 433–438. [29] K. Zeng, H. Li, Y. Peng, Gold nanoparticle enhanced surface plasmon resonance imaging of microRNA-155 using a functional nucleic acid-based amplification machine, Microchim. Acta 184 (2017) 2637–2644. [30] H. Yuan, W. Ji, S. Chu, S. Qian, F. Wang, J. Masson, X. Han, W. Peng, Fiber-optic surface plasmon resonance glucose sensor enhanced with phenylboronic acid modified Au nanoparticles, Biosens. Bioelectron. 117 (2018) 637–643. [31] Y. Li, R. Afrasiabi, F. Fathi, N. Wang, C. Xiang, R. Love, Z. She, H.B. Kraatz, Impedance based detection of pathogenic E. coli O157:H7 using a ferrocene-antimicrobial peptide modified biosensor, Biosens. Bioelectron. 58 (2014) 193–199. [32] W. Wu, S. Zhao, Y. Mao, Z. Fang, X. Lu, L. Zeng, A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification, Anal. Chem. Acta 861 (2015) 62–68. [33] N. Idil, M. Hedström, A. Denizli, B. Mattiasson, Whole cell based microcontact imprinted capacitive biosensor for the detection of Escherichia coli, Biosens. Bioelectron. 87 (2017) 807–815. [34] Y. Wang, C.E. Alocilja, Gold nanoparticle-labeled biosensor for rapid and sensitive detection of bacterial pathogens, J. Biol. Eng. 9 (2015) 16–23. [35] L. Song, L. Zhang, Y. Huang, L. Chen, G. Zhang, Z. Shen, J. Zhang, Z. Xiao, T. Chen, Amplifying the signal of localized surface plasmon resonance sensing for the sensitive detection of Escherichia coli O157:H7, Sci. Rep. 7 (2017) 3288–3296. [36] A.D. Taylor, Q. Yu, S. Chen, J. Mola, S. Jiang, Comparison of E. coli O157:H7 preparation methods used for detection with surface plasmon resonance sensor, Sensors Actuators B Chem. 107 (2005) 202–208. [37] K. Saha, S.S. Agasti, C. Kim, X.N. Li, V.M. Rotello, Gold nanoparticles in chemical and biological sensing, Chem. Rev. 112 (2012) 2739–2779. [38] A.G. Sarıkaya, B. Osman, T. Çam, A. Denizli, Molecularly imprinted surface plasmon resonance (SPR) sensor for uric acid determination, Sensors Actuators B Chem. 251 (2017) 763–772. [39] I. Perçin, N. Idil, M. Bakhshpour, E. Yılmaz, B. Mattiasson, A. Denizli, Microcontact imprinted plasmonic nanosensors: powerful tools in the detection of Salmonella paratyphi, Sensors 17 (2017) 1375–1388. [40] M. Golabi, F. Kuralay, E.W.H. Jager, V. Beni, A.P.F. Turnera, Electrochemical bacterial detection using poly(3-aminophenylboronic acid)-based imprinted polymer, Biosens. Bioelectron. 93 (2017) 87–93. [41] A.A. Lahcen, F. Arduini, F. Lista, A. Amine, Label-free electrochemical sensor based on spore-imprinted polymer for Bacillus cereus spore detection, Sensors Actuators B Chem. 276 (2018) 114–120.

[1] M.S. Conover, M. Hadjifrangiskou, J.J. Palermo, M.E. Hibbing, K.W. Dodson, S.J. Hultgren, Metabolic requirements of Escherichia coli in intracellular bacterial communities during urinary tract infection pathogenesis, mBio 7 (2016) 104–116. [2] A.E. Barber, J.P. Norton, A.M. Spivak, M.A. Mulvey, Urinary tract infections: current and emerging management strategies, Clin. Infect. Dis. 57 (2013) 719–724. [3] R. Singh, M.D. Mukherjee, G. Sumana, R.K. Gupta, S. Sood, B.D. Malhotra, Biosensors for pathogen detection: a smart approach towards clinical diagnosis, Sensors Actuatos B Chem. 197 (2014) 385–404. [4] Z. Iskierko, P.S. Sharma, K. Bartold, A. Pietrzyk-Le, K. Noworyta, W. Kutner, Molecularly imprinted polymers for separating and sensing of macromolecular compounds and microorganisms, Biotechnol. Adv. 34 (2016) 30–46. [5] S. Krishnan, T. Chinnasamy, S. Veerappan, K. Senthilkumar, D. Kannaiyan, Dual labeled Ag@SiO2 core–shell nanoparticle based optical immunosensor for sensitive detection of E. coli, Mater. Sci. Eng. C 45 (2014) 337–342. [6] S. Chen, Z. Wang, X. Cui, L. Jiang, Y. Zhi, X. Ding, Z. Nie, P. Zhou, D. Cui, Microfluidic device directly fabricated on screen-printed electrodes for ultrasensitive electrochemical sensing of PSA, Nanoscale Res. Lett. 14 (2019) 71–77. [7] Y. Huang, S. Zhong, Y. Shen, Y. Yu, D. Cui, Terahertz phase jumps for ultra-sensitive graphene plasmon sensing, Nanoscale 10 (2018) 22466–22473. [8] A. Zhang, W. Guo, H. Ke, X. Zhang, H. Zhang, C. Huang, D. Yang, N. Jia, D. Cui, Sandwich-format ECL immunosensor based on Au star@BSA-luminol nanocomposites for determination of human chorionic gonadotropin, Biosens. Bioelectron. 101 (2018) 219–226. [9] Y. Huang, S. Zhong, Y. Shen, L. Yao, Y. Yu, D. Cui, Graphene/insulator stack based ultrasensitive terahertz sensor with surface plasmon resonance, IEEE Photon. J. 9 (2017) 1–11. [10] E.E. Bedford, J. Spadavecchia, C.M. Pradier, F.X. Gu, Surface plasmon resonance biosensors incorporating gold nanoparticles, Macromol. Biosci. 12 (2012) 724–739. [11] S. Chen, Y.F. Cheng, Biosensors for bacterial detection, Int. J. Biosens. Bioelectron 2 (2017) 00048-31. [12] Y. Wang, W. Knoll, J. Dostalek, Bacterial pathogen surface plasmon resonance biosensor advanced by long range surface plasmons and magnetic nanoparticle assays, Anal. Chem. 84 (2012) 8345–8350. [13] O. Erdem, Y. Saylan, N. Cihangir, A. Denizli, Molecularly imprinted nanoparticles based plasmonic sensors for real-time Enterococcus faecalis detection, Biosens. Bioelectron. 126 (2019) 608–614. [14] S.M. Yoo, S.Y. Lee, Optical biosensors for the detection of pathogenic microorganisms, Trends Biotechnol. 34 (2016) 7–25. [15] G. Ertürk, R. Lood, Bacteriophages as biorecognition elements in capacitive biosensors: phage and host bacteria detection, Sensors Actuators B Chem. 258 (2018) 535–543. [16] A.J. Sposito, A. Kurdekar, J. Zhao, I. Hewlett, Application of nanotechnology in biosensors for enhancing pathogen detection WIREs, Nanomed. Nanobiotechnol. 10 (2018) 1512–1534. [17] Y. Li, H.H. Yang, Q.H. You, Z.X. Zhuang, X.R. Wang, Protein recognition via surface molecularly imprinted polymer nanowires, Anal. Chem. 78 (2006) 317–320. [18] Y. Lv, T. Tan, F. Svec, Molecular imprinting of proteins in polymers attached to the surface of nanomaterials for selective recognition of biomacromolecules, Biotechnol. Adv. 31 (2013) 1172–1186. [19] M. Bakhshpour, E. Tamahkar, M. Andac, A. Denizli, Surface imprinted bacterial cellulose nanofibers for hemoglobin purification, Colloids. Surf. B Biointerfaces 158 (2017) 453–459. [20] R. Ahmad, N. Griffete, A. Lamouri, N. Felidj, M.M. Chehimi, C. Mangeney, Nanocomposites of gold nanoparticles@molecularly imprinted polymers: chemistry, processing, and applications in sensor, Chem. Mater. 27 (2015) 5464–5478. [21] M. Riskin, R. Tel-Vered, T. Bourenko, E. Granot, I. Willner, Imprinting of molecular recognition sites through electropolymerization of functionalized Au nanoparticles: development of an electrochemical TNT sensor based on π-donor-acceptor

7