molecular imprinting studies for developing qcm sensors for bacillus cereus

Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference doi: 10.1016/j.proeng.2016.11.525 ScienceDirect 30th Eurosensors Conference, EUROSENSORS

Procedia Engineering 168 ( 2016 ) 561 – 564

1877-7058 © 2016 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license

( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference

doi: 10.1016/j.proeng.2016.11.525

ScienceDirect

30th Eurosensors Conference, EUROSENSORS 2016 Molecular imprinting studies for developing QCM-sensors for

Bacillus cereus

Eva Spiekera, Peter A Lieberzeita*

a University of Vienna, Department of Physical Chemistry,Währinger Straße 42, 1090 Vienna, Austria

Abstract

Herein we report the design of a novel molecularly imprinted polymer (MIP) for B cereus In a first step, we screened different polymers for their respective affinities towards B cereus with polymer coated quartz crystal microbalance (QCM), namely

polystyrene (PS), polyacrylate (PA), polyvinylpyrrolidone (PVP), polyacrylamide (PAA) and polyurethane (PU) Of these, PU

and PAA revealed the highest inherent affinity to B cereus Light microscopy studies on polymer-coated glass plates

demonstrated that PU is more promising than PAA, because the PU film contain more than three times as many bacteria compared to PAA These experiments also showed that stamp imprinting is preferable to sedimentation imprinting MIP was successfully transferred to QCM: Cavities generated are suitable to re-incorporate the bacteria Both this and washing can be monitored in real-time The sensor response time in distilled water is less than 10 min

© 2016 The Authors Published by Elsevier Ltd

Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference

Keywords: Molecularly Imprinted Polymers; MIP; QCM; Bacillus cereus; bacteria detection

1 Introduction

Public and environmental health protection requires safe drinking water Among others, this generates the need to monitor pathogenic microorganisms Standard microbiological methods require a few days up to a few weeks to determine bacteria in drinking water and PCR strategies are still rather costly for that purpose [1] Hence it is highly desirable to design a sensor to detect such contamination online in water, which is one of the aims of the EU-FP7

* Corresponding author Tel.: +43-1-4277-52341; fax: +43-1-4277-9523

E-mail address: peter.lieberzeit@univie.ac.at

© 2016 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license

( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference

project 312330 “ISIS”: To address such needs, the project among others develops MIP sensors to detect B cereus

[2]

Molecular Imprinting is a technique to design artificial, biomimetic receptors for a given analyte [3] The resulting Molecularly Imprinted Polymers (MIP) are usually highly selective and can be applied for example as receptor system for proteins [4, 5], bacteria [6] or viruses [7, 8] Purification and separation [9, 10], catalysis [11] or drug delivery [12, 13] are further application areas MIPs are robust recognition elements that mimic natural recognition entities [14] Depending on the size of the analyte different strategies (bulk or surface imprinting) for creating recognition cavities are to be applied In the case of large analyte species, such as bacteria, it is best to

generate surface MIP Within this work, we developed two different methods leading to B cereus MIP, namely

sedimentation and stamp imprinting, as summarized in Fig 1 During sedimentation imprinting the sensor transducer surface is spin coated with a pre-polymer solution After this, a small amount of analyte suspension is dropped onto the polymer (typically a few µl) followed by covering it with a PDMS stamp and polymer hardening During stamp imprinting the analyte suspension is drop coated onto a PDMS slide, which is used as a stamp to pattern the spin coated prepolymer solution on the device of interest Both methods require a washing step to remove bacteria from the generated cavities

2 Results and Discussion

The first step of sensor development comprised of screening different polymers for their natural affinity towards

B cereus (ATCC® 11778TM), because it is known that optimal MIP can be achieved in that case [15]

Fig 1 Overview of imprinting strategies Left: Sedimentation imprinting; Right: Stamp imprinting

For that purpose we utilized dual-electrode quartz crystal microbalance (QCM) sensors One electrode was coated with polystyrene (PS), polyacrylate (PA), polyvinylpyrrolidone (PVP), polyacrylamide (PAA) and polyurethane (PU), respectively The other one remained uncoated as a reference Then, the sensors were exposed to solutions

containing the same concentration of B cereus and the corresponding frequency shifts against distilled water were

recorded The exact bacteria concentration of the solution was unknown, because for screening purposes it is enough

to know that all sensors are exposed to the same amount of bacteria Fig 2 summarizes the results: Of all polymers tested, PAA and PU revealed highest affinity and thus are the most suitable candidates for imprinting tests

Preliminary imprinting experiments on glass substrates were carried out for both polymers including both stamp and sedimentation imprinting Fig 3 shows the corresponding light microscope images of bacteria after imprinting and polyurethane hardening: For better visibility, bacteria were stained with crystal violet prior to imprinting Evidently, the image of the sedimentation-imprinted polyurethane in Fig 3a) reveals fewer bacteria on the polymer surface than Fig 3b) on the right-hand side, which resulted from stamp imprinting Furthermore, bacteria are also more evenly distributed over the polymer in the case of stamp MIP, which is hence superior to sedimentation imprinting Bacteria could be removed from the polymer by an aqueous solution of 40 mM sodium hydroxide containing 0.2 % sodium dodecyl sulfate

In the next step we coated QCM with such stamp-imprinted PU In order to make sure that sensor effects in fact result from imprinting, both electrodes were coated with the same polymer but only one of them – the future MIP

channel - were imprinted with B cereus The other electrode acted as a reference containing the non-imprinted

polymer (NIP)

Fig 4 shows the outcome of such a sensor measurement in distilled water with four different concentrations of

Fig 3 Light microscope images (1000x) of B cereus in PU with sedimentation imprinting (a) left) and stamp imprinting (b) right).

B cereus: After adding the analyte, it binds to the film which leads to mass loading and thus to a change in

frequency After flushing the system with water the analyte is washed out and the frequency rises back to baseline revealing reversibility At the same time, the NIP leads to no change in frequency The peaks around the injection points are caused by flushing the system, which changes the properties inside the measuring cell for some seconds This shows successful imprinting Furthermore the sensor signals shown in Fig 4 clearly depend on bacteria concentration thus showing that the sensor is inherently useful for quantification These first experiments indicate linear relation between sensor signal and concentration with a sensor response time of around 10 min Next steps will comprise of selectivity studies and evaluating sensor effects in real-life samples, i.e in drinking water

Acknowledgements

The research leading to these results has received funding from the European Union’s Seventh Framework Programme FP7/2007-2013 under grant agreement n°312330

References

[1] S Park, H Kim, J Kim, T Kim, Simultaneous detection and identification of Bacillus cereus group bacteria using multiplex PCR.

Journal of microbiology and biotechnology 17 (2007) 1177

[2] A Rompré, P Servais, J Baudart, M.-R de-Roubin, P Laurent, Detection and enumeration of coliforms in drinking water: current

methods and emerging approaches Journal of Microbiological Methods 49 (2002) 31-54

[3] M Hussain, J Wackerlig, P.A Lieberzeit, Biomimetic strategies for sensing biological species Biosensors 3 (2013) 89-107

[4] A Bossi, F Bonini, A.P.F Turner, S.A Piletsky, Molecularly imprinted polymers for the recognition of proteins: The state of the art.

Biosensors and Bioelectronics 22 (2007) 1131-1137.

[5] S Chunta, R Suedee, P.A Lieberzeit, Low-Density Lipoprotein Sensor Based on Molecularly Imprinted Polymer Analytical

chemistry 88 (2015) 1419-1425

[6] R Samardzic, H.F Sussitz, N Jongkon, P.A Lieberzeit, Quartz crystal microbalance in-line sensing of escherichia coli in a bioreactor

using molecularly imprinted polymers Sensor Letters 12 (2014) 1152-1155

[7] F Dickert, O Hayden, R Bindeus, K.-J Mann, D Blaas, E Waigmann, Bioimprinted QCM sensors for virus detection—screening of

plant sap Analytical and Bioanalytical Chemistry 378 (2004) 1929-1934

[8] G.M Birnbaumer, P.A Lieberzeit, L Richter, R Schirhagl, M Milnera, F.L Dickert, A Bailey, P Ertl, Detection of viruses with

molecularly imprinted polymers integrated on a microfluidic biochip using contact-less dielectric microsensors Lab on a Chip 9

(2009) 3549-3556

[9] B Sellergren, Imprinted chiral stationary phases in high-performance liquid chromatography Journal of Chromatography A 906

(2001) 227-252

[10] L.I Andersson, Molecular imprinting: developments and applications in the analytical chemistry field Journal of Chromatography B:

Biomedical Sciences and Applications 745 (2000) 3-13

[11] O Ramström, K Mosbach, Synthesis and catalysis by molecularly imprinted materials Current Opinion in Chemical Biology 3

(1999) 759-764

[12] B Sellergren, C.J Allender, Molecularly imprinted polymers: A bridge to advanced drug delivery Advanced Drug Delivery Reviews

57 (2005) 1733-1741

[13] J.Z Hilt, M.E Byrne, Configurational biomimesis in drug delivery: molecular imprinting of biologically significant molecules.

Advanced Drug Delivery Reviews 56 (2004) 1599-1620

[14] G Vasapollo, R.D Sole, L Mergola, M.R Lazzoi, A Scardino, S Scorrano, G Mele, Molecularly imprinted polymers: present and

future prospective International journal of molecular sciences 12 (2011) 5908-5945

[15] C Baggiani, C Giovannoli, L Anfossi, C Passini, P Baravalle, G Giraudi, A connection between the binding properties of

imprinted and nonimprinted polymers: a change of perspective in molecular imprinting Journal of the American Chemical Society

134 (2012) 1513-1518