Surface Imprints: Advantageous Application of Ready2use Materials

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Surface Imprints: Advantageous Application of Ready2use Materials for Bacterial QCM-Sensors Anna-Maria Poller, Eva Spieker, Peter Alexander Lieberzeit, and Claudia Preininger ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13888 • Publication Date (Web): 12 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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ACS Applied Materials & Interfaces

Surface Imprints: Advantageous Application of Ready2use Materials for Bacterial QCM-Sensors Anna-Maria Poller †, Eva Spieker ‡, Peter A. Lieberzeit ‡, Claudia Preininger *, † †

AIT - Austrian Institute of Technology, Konrad-Lorenz-Straße 24, 3430 Tulln, Austria

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University of Vienna, Faculty for Chemistry, Department of Physical Chemistry, Währinger

Straße 42, 1090 Wien, Austria keywords: imprinting, ready-to-use materials, surface MIP, bacterial sensor, QCM ABSTRACT Four different materials (two ab initio synthesized polyurethanes; ready-to-use: Epon1002F and PVA-SbQ) for the generation of E. coli surface imprints are compared in this work. The use of commercially available, ready-to-use materials instead of self synthesized polymers represents an innovative and convenient way of molecular imprint fabrication. This was herein investigated for large, biological templates. Fully synthesized imprint materials (polyurethanes) were developed and optimized regarding their OH-excess and the use of catalyst in the polymerization reaction. No to low OH-excess (0-10 %) and a non-catalyzed synthesis was determined to be superior for the imprinting of the Gram-negative bacteria. Imprints were characterized using atomic force microscopy, with Epon1002F yielding the most distinguished imprints, along with a smooth surface. The imprints were afterwards tested as plastic antibody coatings in a masssensitive QCM measurement. Dilutions of E. coli suspensions, down to a LOD of 1.4*10^7

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CFU/mL, were successfully measured. Best results were obtained with Epon1002F and self synthesized, stoichiometric polyurethane. Since ready-to-use Epon1002F was superior in terms of signal intensities and sensitivity, it can advantageously replace self synthesized polymers for the generation of imprinted sensor surfaces. Easy day-to-day reproducibility and further shortening of imprint fabrication time are other advantages of employing the ready-to-use material instead of conventionally synthesized polymers. 1 INTRODUCTION Bacteria are - besides yeast and molds - a main concern in food spoilage and can also lead to various food-borne illnesses 1,2. For instance, the Gram-negative, rod shaped Escherichia coli is a bacterium with a diameter of ~1 µm and a length between 2 to 6 µm that is ubiquitously present. E. coli functions as hygiene indicator bacteria in food and environment 3,4. There are non pathogenic and pathogenic E. coli strains. Latter one can cause a variety of pathological states, which can range from mild (traveler’s diarrhea) to very severe (hemolytic uremic syndrome) illnesses. There are well-established methods for enumerating E. coli, such as the most probable number technique, the solid medium method or membrane filtration methods. All these rely on growing the bacterium, resulting in testing times of 12 h up to 5 days. While often antibodies are immobilized as receptors on sensor surfaces 5,6, we herein propose to generate molecular surface imprints selective to E. coli to be used as receptors for fast bacterial detection. To do so, a template (analyte) is first deposited onto a stamp which is afterwards placed onto a thin film of polymer. During polymerization, the template is enclosed by a polymer network, from which it is afterwards removed, when the stamp is lifted off the surface. After a washing step, the surface is structured with cavities – called imprints – that match the template in terms of size, shape and polarity. This allows for non-covalent interactions


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(hydrogen bonds, dipole-dipole, Van der Waals, electrostatic, ionic, or hydrophobic) of the polymer with the template similar to antibody-antigen interactions. When addressing larger templates, such as bacteria, surface imprinting is preferred over other techniques (e.g. bulk imprinting), since macromolecules and their assemblies hardly penetrate into polymer pores and could be trapped during polymerization 7. Aside from using a suitable imprinting technique, choosing a suitable polymer is crucial, because it strongly influences sensor characteristics: for instance, molecularly imprinted polymers (MIP) based on methacrylate copolymers containing vinyl pyrrolidone proved five times more sensitive in detecting folic acid, than the pure methacrylate 8. Conventionally, MIPs are synthesized “from scratch”; i.e. starting with a defined set of monomers. For that purpose, most papers report using carboxylic acids (acrylic acid, methacrylic acid, vinylbenzoic acid), sulphonic acids (2-acrylamido-2-methylpropane sulphonic acid) and heteroaromatic bases (vinylpyridine, vinylimidazole). Especially methacrylic acid (MAA) has been widely utilized 9. Regarding crosslinkers, trimethylolpropane trimethacrylate (TRIM) and ethylene glycol dimethacrylate (EGDMA) are most frequently employed 10,11. EGDMA is reported to additionally increase stability of specific binding-areas of template molecules 12. In the case of large, biological templates, such as bacteria, also polyurethanes have been reported 13,14. However, to achieve reproducible, appreciable binding behavior, one has to consider a variety of experimental parameters, such as different functional monomers, their molar ratios, the amount of crosslinker, and others. During actual imprinting, stamp inking and polymerization process are also subject to variation. Therefore it is important that the polymer material itself remains a constant factor. Using ab initio synthesis protocols, it is possible to exactly reproduce polymers on a day-to-day basis, but the approach is laborious and time consuming. Thus we investigated


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the application of commercially available, ready-to-use materials to generate MIP. Originating from techniques such as nanoimprint lithography (NIL), a wide range of polymers and resins are commercially available 15. They differ in e.g. epoxy functionalities or molecular weight. Their properties (viscosity, crosslinking, rigidity) vary and can hence be chosen according to the respective needs 16. Ready-to-use materials are less prone to batch-to-batch variations and represent an innovative and convenient way to manufacture surface imprints. In this work, on one hand conventional imprinting protocols for E. coli in polyurethanes synthesized from scratch were developed. Additionally, we tested imprinting of these large bio-templates in ready-to-use materials (Epon1002F, PVA-SbQ). Bacteria imprints were assessed via bright field and atomic force microscopy to gain information on surface properties and imprint success. Afterwards the sensing properties of ab initio synthesized materials were compared to the ready-to-use materials in a mass sensitive, label-free quartz-crystal microbalance (QCM) experiment. Special attention was given to applicability and assay sensitivity to evaluate, if ready-to-use materials are able to replace fully synthesized imprint materials, and to thus save time and efforts during synthesis. 2 EXPERIMENTAL SECTION 2.1. Materials and Reagents Epon 1002F was from Miller Stephenson Chemical, Co. (USA). Cyclopentanone, tetrahydrofuran (THF) and safranin solution were from Sigma Aldrich (USA). Bisphenol A (BPA), phloroglucinol (PG), glucose, tryptic soy broth (TSB) growth medium, 1,4diazabicyclo[2.2.2]octane (DABCO) and 4,4'-diphenylmethane diisocyanate (DPDI) were purchased from VWR (Germany) in analytical or highest available synthetic grade. Poly(vinyl alcohol)/N-methyl-4(4’-formylstyryl)pyridinium methosulfate acetal (PVA-SbQ) was from


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Polysciences Inc. (USA). Polydimethylsiloxane (PDMS, Sylgard 184 silicone elastomer) and PDMS-curing agent (Sylgard 184 silicone agent) were from Dow Corning Corporation (USA). Microscopic glass slides were purchased from Schott (Germany), while the gold paste for electrode fabrication was from Heraeus (GGP 2093). Escherichia coli (DH5alpha) was obtained from DSMZ (Germany). Bacteria were spread onto TSB plates and incubated at 36°C overnight. The next day TSB liquid growth medium was inoculated with CFUs and incubated at 36°C overnight. Bacterial cells were harvested by centrifugation at 4500 rpm for 10 min, washed with sterile 0.9 % NaCl, pelleted again and resuspended in sterile 0.9 % NaCl. Cell counting was performed with an improved Neubauer counting chamber. 2.2. MIP synthesis To generate polyurethane polymers, Bisphenol A (517 µmol), phloroglucinol (246 µmol) and 4,4'-Diphenylmethane diisocyanate (772 µmol) were dissolved in 200 µL tetrahydrofuran (THF) and pre-polymerized at 70°C for 30 min. Afterwards the pre-polymerized stock was diluted 1:20 in THF to result in polymer coating stock. Additionally we compared polyurethanes synthesized with and without catalyst, respectively. For catalyzed PU, we further diluted a 2% solution of 1,4-diazabicyclo[2.2.2]octane (DABCO) in THF with THF (1:45). Then, the pre-polymerized stock was diluted 1:10 with the catalyst solution. After 5 min incubation at 70°C, the catalyzed pre-polymerization stock was diluted 1:5 with THF to give the catalyzed polymer coating stock. Beside this general synthesis protocols, we tested various proportions of the phenolic components (BPA, PG) was tested. This is reported to impact the properties of the resulting polymer by improving wettability as well as enhance wash-out efficiency 17.


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As ready-to-use materials Epon1002F and PVA-SbQ were tested. To generate working stocks, a 10% solution of Epon1002F was prepared in cyclopentanone, while PVA-SbQ was diluted 1:3 with H2O and centrifuged for 2 min at 3000 rpm to get rid of air bubbles. 2.3. Stamp Inking PDMS stamp material was polymerized by mixing Sylgard 184 silicone elastomer and curing agent 1:10. Afterwards the PDMS was cut into small pieces. For inking, stamps were incubated with aqueous E. coli suspension (20 µL of 2.8*10^7 cells/mL per stamp) for 45 min. After bacteria sedimentation onto the stamp surface, excess suspension was removed by spin-off (2 s at 1800 rpm). To cover stamps with glucose overlayer, 20 µL glucose solution (100 mM) was incubated (20 min) on the regular sedimentation stamps, and again spun off (2 s at 1800 rpm). For comparison, non-imprinted stamps were generated the same way as the bacterial stamps, but PDMS was inked with 20 µL H2O. 2.4. Spin Coating and Imprint Fabrication MIPs were coated onto microscopy glass slides and QCM, respectively. The former served as platform for testing washing steps and polyurethane formulations, while the latter served in QCM sensor assay. The glass slides were cut into 10x10 mm squares and coated with the respective material. QCM discs were fabricated as described previously by Dickert et al. 2002 18. First 10 MHz AT-cut quartz discs of 13.8 mm diameter were screen-printed with Au-paste (12%), to form dual electrodes geometries. These electrodes where subsequently coated with imprint material. 10 µL of the respective imprinting material was spin coated on glass slides or QCM disks. Polyurethanes and PVA-SbQ were spun at 2000 rpm for 3 s, while Epon1002F was coated at 3000 rpm for 30 s. This resulted in films of 350 – 550 nm thickness. Afterwards, the


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prepared stamps were placed onto the coated films and fixed with a clamp (see figure 1). Polyurethane films fully polymerized overnight, while PVA-SbQ imprints were cured with UV (365 nm) for 30 min. The Epon 1002F films were baked for curing at 95°C for 10 min. Consecutively the stamps were removed and potentially remaining bacteria were removed from imprint cavities in a washing step. 2.5. Surface and Imprint Characterization 2.5.1. Bright Field Microscopy Bright field microscopy was performed on a Nikon Eclipse LV-2 microscope. Cut glass slides with E. coli surface imprints were examined. Imprinted E. coli were stained with safranin solution to clearly identify Gram-negative bacteria. To assess numbers of imprinted bacteria and removal after wash-out, image data from bright field microscopy was analyzed and quantified by using the particle analysis function of ImageJ. For that purpose, images were converted to 8-bit greyscale. Then brightness and contrast were adjusted to maximize the visibility of the bacteria. Afterwards threshold was adjusted to reduce and exclude background signal. If bacteria were clustered together, they were split up using ImageJ’s watershed segmentation. After these steps bacteria were quantified by performing particle analysis. All results shown here are mean values from triplicate measurements. 2.5.2. AFM – Atomic Force Microscopy AFM measurements were performed on a Nanoscope VIII (Bruker Metrology, USA). Images were acquired in air, operating in contact mode. A NP-S oxide-sharpened silicon nitride probe (Veeco Probes) was used. Image procession was done with the AFM control software


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Nanoscope, provided by the instrument’s manufacturer. AFM scans yield images that describe the exact topography (z-axis) of a sample surface and factors such as regularity, topography or roughness of a surface can be determined. Molecularly imprinted as well as non-imprinted samples were analyzed. When creating sensor layers, these properties are important quality criteria. 2.6. QCM – Measurement The QCM measurement setup was as described by Dickert et al. 2002 18: The QCM was mounted into a custom-made measuring cell. Its design ensures that only one face of the QCM is exposed to the liquid sample. The cell is tightly closed with a PDMS lid which holds an inlet and outlet for sample feed. This setup ensures tight contact of the cell and quartz electrodes as well as it excludes interferences from air bubbles in the sample volume. To operate the QCM at its resonance frequency, dual channel oscillator circuits are employed. These self-designed circuits are complimentary to the dual electrode quartz design and make the sensor application even under highly damped conditions possible. For measurement, the cell was filled with 200 µL sample solution (E. coli suspensions: 7.3, 3.6, 1.8, 0.9, 0.7, 0.4 *10^7 CFU/mL). After each sample the cell was flushed with H2O to remove bacteria. To guarantee that decreasing sensor responses are no artifacts from consecutive dilution of the highest bacteria concentration (7.3*10^7 CFU/mL) it was re-measured after experiment’s completion. On all surfaces this resulted in – again – highest sensor response, also indicating the renewability of the sensor. The change in frequency (corresponding to the change in mass on the sensor surface) was detected through an HP 53131A frequency counter. The measurement is carried out in stopped flow. Afterwards the frequency data is analyzed as a function of time. To obtain limit of detection (LOD) values, the mean value and standard deviation were calculated from 50 measurement


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points after the signal reached euqilibrium. 3x standard deviation was added to the mean value thus leading to the calculated LOD. 3 RESULTS AND DISCUSSION To assess the possibility of replacing fully synthesized polymers (polyurethanes) with readyto-use materials (Epon1002F, PVA-SbQ) these two groups of materials were compared for imprinting bacteria (E. coli). Polyurethanes have been reported to adhere well to QCM surfaces and have been described previously in successful QCM measurements of large biospecies 14,17–19. The ready-to-use materials were chosen based on their relative rigidity, which is essential for mass sensitive measurements. Additionally Epon1002F and PVA-SbQ require different curing mechanisms. While Epon1002F requires heat-curing, PVA-SbQ is hardened by UV irradiation. 3.1. Polyurethane Synthesis Polyurethane-based MIP was previously described for generating surface imprints of E. coli 14. In contrast to this previous work, we utilized BPA instead of Poly-(4-vinylphenol) PVP as a monomer, different molar ratios of monomers (Samardizic et al.: DPDI:PVP:PG = 7:12:1; herein we use: DPDI:BPA:PG = 3:2:1), and assessed catalysis with DABCO. This section deals with optimizing the functional ratio of monomers (excess of OH groups) in the MIP and investigating the influence of the catalyst. Eight different polyurethane synthesis protocols were tested. By varying the amounts of phenolic monomers (BPA = 517 – 789 µmol; PG = 246 – 365 µmol), we produced MIP containing varying excess of OH-functionalities, namely 0%, 10%, 25% and 50%, all with and without the use of catalyst, respectively. The imprints were produced on glass support using a plain PDMS sedimentation stamp. Any bacteria remaining in the polymer after


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stamping were washed out with water followed by recording bright field microscopic images and processing them as described in section 2.5.1). Catalyzed polyurethanes generally resulted in smoother surfaces compared to non-catalyzed polymers. Nevertheless fewer bacteria are imprinted, than in non-catalyzed polyurethanes. Figure 2 compares bacteria present, directly after imprinting and after washing, in the respective polymer (mean values of three MIPs each). Data shows that generally, in uncatalyzed polyurethane MIP about two times more bacteria are imprinted (see figure 2a: e.g. mean 10% OH-excess = 210 E. coli / MIP), than in catalyzed polymer (see figure 2b: e.g. mean 10% OHexcess = 90 E. coli / MIP). Additionally bacteria appear to get irreversibly bound, since none of the tested washing protocols (H2O; HAc; SDS/NaOH) was successful for any of the four catalyzed polymers. This may be due to the elevated polymerization process that traps bacteria within the film layer. When employing harsher washing conditions, such as 10% acetic acid or 0.04% SDS+0.05M NaOH (results not displayed), bacteria were not washed out, but the polymer detached from the glass support. In all cases (catalyzed and non-catalyzed polyurethanes), highest immobilization rates were achieved using no (0%) to little (10%) excess of OH groups. Overall, template removal was much more effective in non-catalyzed systems, even though only very mild washing agent (H2O, 2 h) was employed. The superior washout effect in polyurethanes with 0 - 10% OH- excess - in comparison to higher excess (25-50%) polyurethanes - can be attributed to the hydrophilic nature of E. coli’s surface 20. Microbial adhesion to a surface is dependent on non-covalent interactions (electrostatic, van der Waals, hydrophobic). The hydrophilic E. coli surface is likely to promote elevated hydrogen bonding with polymers with excess OH groups, hindering the bacteria’s washout. Thus stoichiometric (0%) polyurethanes or polyurethanes with little (10%) excess of OH groups are rated most suitable for the imprinting of


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E. coli. For these polymers, the use of a plain PDMS stamp for imprinting and a mild wash (H2O) also proved feasible. 3.2. Washing Protocols Choosing an appropriate washing protocol for the respective imprint material is a crucial factor for generating successful bacteria imprints. As described in section 3.1, polyurethanes can be washed at very mild (H2O) conditions (see figure 3). For Epon1002F and PVA-SbQ a mild wash (H2O, 2 h) as well as a stronger detergent (0.04% SDS + 0.05M NaOH, 1 h) were tested. Epon 1002F was originally used as a photoresist layer in coatings, but is also applied in nanoimprint lithography (NIL) or microelectromechanical systems (MEMS) 21. The epoxy polyether resin consists of bisphenol A diglycidyl ether. PVA-SbQ is obtained by mixing polyvinyl alcohol (PVA) with quaternized stilbazole (styrylpyridinium, SbQ) groups 16. Commercially it is used for manufacturing screen-printing emulsions, but due to its excellent ability to form films and its adhesive properties, it is also used for biomedical coatings or controlled drug release 22. Also it has been reported as a coating during QCM measurements 23. SDS is a well-known detergent for lysing cells, which should enhance the efficiency of bacteria removal. Previous research also employed (more concentrated) SDS/NaOH mixture to remove lipopolysaccharide and lipoteichoic acid from imprints in Epon1002F 24. Depicted in figure 4, are microscopic images of the washing protocols tested for the ready-touse materials. Examining the images it is obvious that the SDS/NaOH washing solution appears to remove bacteria more efficiently than H2O. While for H2O washing a removal of about 3% of imprinted E. coli was detected, SDS/NaOH solution removed substantially larger amount of bacteria (~ 50 %) in both materials (Epon1002F, PVA-SbQ). Furthermore, the safranin solution


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seems to stain not only bacteria, but the entire MIP, especially after the H2O wash. One reason for this may be swelling and dye absorption during washing. Overall SDS/NaOH hence led to the best results for clearing up binding cavities of MIPs based on Epon1002F and PVA-SbQ, respectively. 3.3. Glucose as Blocker on Imprint-Stamps Template bacteria should not stick too firmly to the MIP to ensure that they can be efficiently removed after hardening. Therefore it is fundamentally important to avoid covalent binding between template and forming polymer. In the case of polyurethanes, it is sometimes necessary to use a blocker or sacrificial layer for that purpose due to the reactivity of the isocyanate groups. Glucose is able to form such a protective layer that avoids irreversible binding of bacteria to MIP surfaces 25,26. Since it is not possible to tune monomer composition in ready-to-use, glucose covered imprint-stamps represent a convenient opportunity to influence imprint properties. For that purpose we covered sedimentation stamps with 100 mM glucose solution. The results of this approach were assessed based on light microscopy, as before. In the case of Epon1002F bacteria are not visible on the surface after imprinting with the glucose-covered stamp (see figure 5, top). This may be due to the fact that colored template bacteria are almost fully removed during stamp removal when coating them with glucose. After washing, the imprint cavities become clearly recognizable. In comparison to the non-glucose covered stamp tested before (see figure 4, top) improvement is apparent. Bacteria are removed very effectively (~ 90%) from imprint cavities, leaving the surface clearly structured but otherwise smooth (see figure 5, bottom). Neither PVA-SbQ, nor polyurethanes revealed the same


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behavior (results not shown). Therefore we only applied such protective layer in Epon1002F MIP from this point on. 3.4. Surface Characterization Figure 6 shows AFM images and height sections of the non-imprinted materials (polyurethanes, Epon1002F, PVA-SbQ). Comparing the height sections, Epon1002F and PVASbQ display lower roughness profiles and more flat surfaces, than ab initio synthesized polyurethanes (Epon1102F Ra = 11.6 nm; PVA-SbQ Ra = 7.0 nm; 0% OH excess Ra = 16.7 nm; 10% OH-excess Ra = 15.0 nm). Polyurethane with 10% OH-excess reveals a slightly smoother surface, than stoichiometric polyurethane. Molecularly imprinted materials, as depicted in figure 7, differ in their surface properties from the non-imprinted samples. In all cases, surface roughness is elevated in the imprinted materials (0% OH excess Ra = 18.3 nm; 10% OH-excess Ra = 44.4 nm; Epon1002F Ra = 27.3 nm; PVASbQ Ra = 23.8 nm). This can be accounted to surface structuring during imprinting. Cavity-like structures suggest successful imprinting of E. coli in all materials but PVA-SbQ. There – although overall surface roughness was elevated in comparison to the non-imprinted surface – no pores of suitable size could be found. It is likely that only small amounts of the bacteria from the stamp were actually able to form a stable imprint. In both polyurethanes imprinted cavities could be identified. When using 10% excess of OH, cavities are larger and deeper, than in the stoichiometric MIP. This clearly suggests imprinting efficiency is improved by slightly increasing hydrophilic properties of the polymer surface. Epon1002F revealed very distinct imprints on a smooth surface. Height sections show that cavity dimensions correspond well to those of E. coli.


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3.5. Quartz Crystal Microbalance Measurements 3.5.1. Sensing of E. coli The application of polyurethanes, PVA-SbQ, and Epon1002F was tested and compared in QCM experiment using 0.4 to 7.3*10^7 CFU/mL E. coli suspensions. QCM yields label-free, mass-sensitive detection based on shape and function recognition elements (MIP), in contrast to other label-free techniques such as voltammetry relying on oxidation processes 9,27,28. Figure 8 shows that all MIP resulted in frequency changes corresponding to the change in mass through binding of E. coli. Sensor response to different bacteria concentrations was very prompt, within 1 - 5 minutes, for all materials. The respective NIP gave rise to only minor effects: in the case of stoichiometric polyurethane and Epon1002F, it reaches less than 12 % of the signal on the respective MIP, while on polyurethane with 10% OH-excess and PVA-SbQ a rather intense sensor response (> 30 %) was visible also on NIPs. 3.5.2. Comparing of different MIP Surfaces Assessing the different response behaviors of the MIP does not only help to identify the most suitable material for E. coli MIP, but also provides information on advantages and disadvantages of ready-to-use formulations compared to polymers synthesized from scratch. Incubation with aqueous E. coli suspension (7.3*10^7 CFU/mL) resulted in an immediate response of all four imprinted sensor surfaces with frequency shifts of different extent (see figure 9). The highest decrease was observed on Epon1002F, while with stoichiometric polyurethane the signal was two times smaller. Polyurethane with 10% OH-excess showed a response similar to the one of the stoichiometric polymer. Ready-to-use PVA-SbQ resulted in a comparatively


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very small sensor response. Lower sensor responses on polyurethane with 10% OH-excess and PVA-SbQ can be attributed to the stronger response generated on the non-imprinted references (NIPs) of the two materials (see figure 8). Deducting from this, non-specific adsorption of the analyte (E. coli) to PVA-SbQ and 10% OH-excess polyurethane surfaces is by far higher compared to the other tested surfaces. Further differences in measurements can arise from differences in the materials’ electrostatic properties. Polyurethanes and Epon 1002F have overall negative material net charge, while PVA-SbQ on the other hand is positively charged. E. coli in aqueous suspension are overall negatively charged and are hence surrounded by a cationic layer 29,30

. Thus negatively charged MIP, such as polyurethanes and Epon1002F, will result in higher

electrostatic attraction of bacteria than PVA-SbQ. This explains why PVA-SbQ is comparably insensitive. This is further corroborated when regarding the sensor characteristics over the whole detection range (0.4-7.3*10^7 CFU/mL)(see figure 9): again Epon1002F showed highest sensitivity, followed by the polyurethanes and PVA-SbQ. A steep slope of the calibration line indicates good sensitivity, thus capability of clear distinction between similar analyte concentrations. Imprints in Epon1002F showed the steepest slope of all materials tested. In comparison, slopes obtained with polyurethane imprints were less than half as steep. The calibration line obtained for PVA-SbQ shows little slope and could only distinguish four of the six tested E. coli concentrations. Sensitivity of a sensor strongly depends on the recognition element used 31. Antibodies are often used for rapid detection of bacteria and viruses. These usually yield more sensitive LODs than chemical receptors due to their very specific interactions with the analyte. Farka et al. 2015 6

pursued recognition with E.coli antibodies, immobilized via glutaraldehyde on the sensor

surface, resulting in a LOD of 9*10^5 CFU/mL for DH5alpha E.coli. For regeneration of the


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sensor citrate buffer (pH 4.0) was used. Wang et al. 2008 32 and Li et al. 2011 33 reported detection limits as low as ~ 2*10^2 CFU/mL E. coli using antibodies immobilized on selfassembled monolayers of 16-mercaptohexadecanoic acid or 3-mercaptopropionic acid. Nevertheless all antibody-based techniques have to deal with the inherent fragility of these biorecognition elements. If not stored or used under proper conditions, activity loss and thus sensor failure is the consequence 31. Therefore other non-biological recognition elements such as surface imprints that are more robust and do not require special conditions or handling are an attractive alternative. Samardzic et al. 2014 14 developed polyurethane-based E.coli sensors using drop imprinting and glass as stamping material. In stopped-flow measurement of E.coli in nutrient solution, the LOD obtained was 1.6*10^8 CFU/mL. Regeneration of the sensor was possible using water. In comparison to these works, analysis of six different concentrations of E. coli using Epon1002F as surface imprinted sensor material, resulted in frequency shifts between 2.8 – 0.3 kHz and a calculated LOD of 1.4*10^7 CFU/mL. Sensors were regenerated through washing with water. For the polyurethanes smaller frequency shifts were detected (stoichiometric polyurethane: 1.3 – 0.07 kHz; polyurethane 10% OH-excess: 1 – 0.02 kHz). Elevated noise in the measurement of polyurethane with 10% OH-excess resulted in a higher calculated LOD (2.9*10^7 CFU/mL), in comparison to stoichiometric polyurethane (1.5*10^7 CFU/mL). Imprints in PVA-SbQ resulted in inferior frequency shift (0.4 – 0.02 kHz) and the highest calculated LOD (8*10^7 CFU/mL), in comparison to the other tested materials. Generation of E. coli imprints was successful using ready-to-use materials as well as ab initio synthesized polyurethanes. Both were capable to reliably detect E. coli. Nevertheless, molecularly imprinted ready-to-use Epon1002F showed superior sensing properties in terms of signal intensity and sensitivity.


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4 CONCLUSION For the generation of E. coli surface imprints, commercially available materials (Epon1002F, PVA-SbQ) were compared to ab initio synthesized polyurethanes. Using ready-to-use materials for molecular imprinting of large, biological templates is an innovative and convenient way of molecular imprint fabrication. In contrast to lengthy culturing methods (12 h – 5 d), the masssensitive QCM generates a sensor response to different bacteria concentrations within minutes. Best results were obtained with Epon1002F and self synthesized, stoichiometric polyurethane. Epon1002F displayed superior sensitivity (LOD = 1.4*10^7 CFU/mL) and signal intensities (2.8 – 0.3 kHz), in comparison to the fully synthesized material. Easy day-to-day reproducibility and further shortening of imprint fabrication time are additional advantages of the ready-to-use epoxy resin. Therefore we propose the use of Epon1002F instead of conventionally synthesized polymer for the generation of imprinted sensor surfaces. Herein the advantageous use of the commercial product was proven for the imprint generation and detection of large, biological analytes (E. coli). To help MIPs become more widely available and commercialized in the future, development towards more reproducible imprint fabrication processes (e.g. stamp optimization) and further selectivity assessments are needed. LIST OF ABBREVIATIONS BPA

Bisphenol A

CFU

colony forming units

DABCO

1,4-diazabicyclo[2.2.2]octane

DPDI

4,4'-diphenylmethane diisocyanate

EGDMA

ethylene glycol dimethacrylate

H2O

water


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HAc

acetic acid

LOD

limit of detection

MAA

methacrylic acid

MIP

molecularly imprinted polymer

NaCl

sodium chloride

NaOH

sodium hydroxide

NIP

non-imprinted polymer

PG

phloroglucinol

PVA-SbQ

Poly(vinyl alcohol)/N-methyl-4(4’-formylstyryl)pyridinium methosulfate acetal

PDMS

Polydimethylsiloxane

PVP

Poly-(4-vinylphenol)

QCM

quartz crystal microbalance

SDS

sodium dodecyl sulfate

THF

tetrahydrofuran

TRIM

trimethylolpropane trimethacrylate

TSB

tryptic soy broth

AUTHOR INFORMATION Corresponding Author * (Claudia Preininger) E-Mail: [email protected] Tel.: +43 50550 3527.


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ACKNOWLEDGMENT The authors would like to thank Dr. Tanja Kostic from the AIT Austrian Institute of Technology for the preparation of spread plates and her support with culturing and handling of bacteria used in this work.


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FIGURE CAPTIONS Figure 1. Surface imprinting method. I: stamp inked with template is pressed into polymer film. II: enclosing of template during polymerization. III: stamp removal and generation of selective cavities. Figure 2 Comparison of polyurethanes for the imprinting of E. coli. The suitability of the material was rated by two factors 1) amount of imprinted E. coli (blue bar) and 2) number of remaining bacteria in material after a washing step with H2O (red bar). Polyurethanes with various OH-excess and synthesized without (a) or with (b) catalyst were tested. Data was obtained and analyzed (see section 3.5.1) from bright field microscopy images of three individual MIPs, for each polymer respectively. Figure 3 H2O washing of imprints in polyurethane with 0 % or 10% OH-excess. E. coli are stained with safranin solution to increase visibility. Exemplarily, single E. coli (~2 µm) are marked in the representative imprint image by a red circle. Figure 4 Comparison of H2O vs SDS/NaOH wash for the ready-to-use materials Epon1002F and PVA-SbQ. E. coli are stained with safranin solution to increase visibility. Exemplarily, single E. coli (~2µm) are marked in the representative imprint image with a red circle. Figure 5 Microscopy images of E. coli imprints in Epon1002F. Images were acquired after imprinting using glucose-layered bacteria on a PDMS-stamp (top) and after a washing (SDS/NaOH) step (bottom). For images of the non-glucose covered stamp imprints see figure 4. Figure 6 AFM images (top) and height sections (bottom) of the NIP (non-imprinted polymer) surfaces.


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Figure 7 AFM images and height cross-sections of the imprinted polymers. Imprints were generated using bacteria on PDMS stamps, for Epon1002F bacteria were additionally glucosecovered. Images were acquired after washing (polyurethanes: H2O; Epon1002F, PVA-SbQ: SDS/NaOH) and complete drying of the samples. Figure 8 Representative sensor curves for the measurement of E. coli obtained with different imprint materials. Sensor responses (MIP = red; NIP = green) of descending concentrations of aqueous E. coli suspensions (7.3, 3.6, 1.8, 0.9, 0.7, 0.4 *10^7 CFU / mL) are depicted. Figure 9 Representative QCM sensors characteristics of MIPs in different materials (▲Epon1002F, ♦ polyurethane 0% OH-excess, ■ polyurethane 10% OH-excess, x PVA-SbQ), incubated with various E. coli concentrations (7.3 - 0.4 *10^7 CFU/mL).


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Abstract graphic 83x35mm (300 x 300 DPI)



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Figure 1 34x51mm (300 x 300 DPI)


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Figure 2 85x102mm (300 x 300 DPI)



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Surface Imprints: Advantageous Application of Ready2use Materials

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