Whole cell imprinting in sol-gel thin films for bacterial recognition in liquids: macromolecular fingerprinting.

Int. J. Mol. Sci. 2010, 11, 1236-1252; doi:10.3390/ijms11041236
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Article
Whole Cell Imprinting in Sol-Gel Thin Films for Bacterial
Recognition in Liquids: Macromolecular Fingerprinting
Tally Cohen, Jeanna Starosvetsky, Uta Cheruti and Robert Armon *
Faculty of Civil & Environmental Engineering, Division of Environmental, Water and Agricultural
Engineering, Technion, Haifa 32000, Israel; E-Mails: tallyco@tx.technion.ac.il (T.C.);
starjean@tx.technion.ac.il (J.S.); uta@tx.technion.ac.il (U.C.)
* Author to whom correspondence should be addressed; E-Mail: cvrrobi@tx.technion.ac.il;
Tel.: +972-4-829-2-77; Fax: +972-4-829-3309.
Received: 28 January 2010; in revised form: 26 February 2010 / Accepted: 22 March 2010 /
Published: 24 March 2010

Abstract: Thin films of organically modified silica (ORMOSILS) produced by a sol-gel
method were imprinted with whole cells of a variety of microorganisms in order to develop
an easy and specific probe to concentrate and specifically identify these microorganisms in
liquids (e.g., water). Microorganisms with various morphology and outer surface
components were imprinted into thin sol-gel films. Adsorption of target microorganism
onto imprinted films was facilitated by these macromolecular fingerprints as revealed by
various microscopical examinations (SEM, AFM, HSEM and CLSM). The imprinted films
showed high selectivity toward each of test microorganisms with high adsorption affinity
making them excellent candidates for rapid detection of microorganisms from liquids.
Keywords: sol-gel; bacteria; protozoan parasite; whole cell imprinting; biosensors;
sol-gel films

1. Introduction
In the last decades sol-gel (SG) chemistry has played a major role in the evolution of hybrid
materials through bridging organic and inorganic chemistry. It is considered one of the fastest growing
fields of contemporary chemistry [1–5]. Organically modified silicate materials (ORMOSILS)
involved in sol-gel processes under ambient conditions, are a highly attractive research area due to
their simplicity and versatility. Sol-gel technology combining composition and microstructure control
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at a molecular level yields the ability to shape these materials in various configurations: bulk, thin
films, fibers, monoliths and powders. Reagents/reactants can be readily incorporated in a stable host
matrix by simply adding it to a sol precursor prior to its gelation at room temperature. In some cases,
the matrix may even stabilize the entrapped reagent from photodegradation and other extreme
environmental conditions [6,7]. Therefore, SG materials are chemically, photochemically and
electrochemically stable [4]. The attractiveness of these materials can be attributed to three main
factors: (1) the ability to generate an almost infinite number of such hybrid materials that demonstrate
both the mechanical stability of an inorganic framework and the particular reactivity (e.g., selective
recognition, optical properties, electrochemical activity, etc.) of the organic components;
(2) SG-derived materials can be used to encapsulate bio-molecules (e.g., enzymes, antibodies and
other proteins) in a functional state without structural damage; (3) discovery of the supra-molecular
template approach which can generate ordered microstructures over long length scales [5].
At present SG technology is practically applied in all known branches of modern industry:
advanced medical technologies, electronics, analytical chemistry, biochemistry, electrochemistry,
catalysis, optics, development of new composite materials, imprinting, etc. [2,5–7].
Among the many materials produced by the SG technique, sensing devices are a major area [7–9].
High sensitivity, good selectivity, long term durability (which also implies good reproducibility and
reversibility) ease of modification and flexible processing are key parameters for successful
applications of this methodology in biosensor construction and development [10–14]. The introduction
of molecularly imprinted polymers (MIP) in sensor applications opened a most promising field with
industrial implementations [15–17]. At present molecular imprinting (MI) is a well established method
for designing highly selective sensors [18]. According to this technique, a polymeric network is
assembled and molded around a suitable template molecule, which upon removal, yields micro-
cavities with a specific size, shape, and/or chemical functionality in a highly cross-linked matrix. Such
molecularly designed cavities show affinity for the imprinted molecule over other structurally and
chemically related compounds. Preparation of thin films by this method is very attractive for chemical
and biological sensing applications, since the reduced response time for the reagent due to significant
diffusional path length shortening. Another feature that may improve diffusional penetration into the
polymeric matrix is its porosity. SG materials inherently combine these two requirements, namely the
ability to form very thin films and variable matrix porosity. In addition, the ease of SG fabrication,
mild reaction conditions, commercial availability of a wide variety of functional monomers, physical
rigidity of the matrix, chemical inertness, and resistance to thermal and solvent stresses, had all made
the SG methodology attractive for molecular imprinting of thin films and a perfect basis for designing
various biological applications [4–7].
During the last decades, the scope of analytical chemistry has shifted from simple molecules to
increasingly complex organic and biological systems (e.g., proteins, carbohydrates, lipids and nucleic
acids) and even more towards entire biological species, such as eukaryotic, bacteria and viruses cells
[10–13,19–21]. For these analytes (in the present study, bacterial cells) an increasing demand for fast,
high value and on-line analysis has developed. Development of such detection methods is essential in
many areas like medicine (infectious diseases), food industry, water supply and environmental
microbiology. The most common chemical and biological methods relay on sample inoculation of a
specific nutrient media (solid or liquid) followed by incubation at a certain temperature allowing

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microorganisms to grow and form colonies. The newly developed colonies are counted and identified
based on biochemical tests or genotypically by polymerase chain reaction (PCR) and electrophoresis
[22]. These methods are suitable for this task and yield the desired detection limits with high
specificity, however they are still too long in time terms for the rapid determinations required by
several areas such as medicine and security. For example, bacterial cultivations on specific media may
require many hours or even days. Recently, a large effort has been made to overcome this problem
when dealing with environmental samples that beside complex composition harbor few bacteria
therefore requiring a concentration step. Many sophisticated methods for detection and identification
of specific microorganisms were developed, however little effort had been devoted to develop methods
for selective capture and concentration of microorganisms at species level from the environment. Such
methods would play a critical topmost role as a capture/concentrator/identifier for biological warfare
agents or medical/environmental pathogens of concern. Various sensors for microorganisms detection,
were proposed based on different approaches using various bioactive materials like antibodies,
proteins, cell's membrane components, etc. [8–12,22]. Although microorganism's sensors provide
critical screening capability as early warning systems, they generally lack specificity toward specific
microorganisms. An obvious requirement exists for a rapid, selective seizure and detection of
microorganisms from highly complex environments.
The new emerging approach for selective organism capture based on sol-gel bio-imprinting was
firstly demonstrated by Dickert et al. [23,24] that developed a soft, lithographic technique used to
produce an imprinted surface on a quartz crystal microbalance (QCM) sensor. The imprinted layer was
capable of selective capture of different yeast genera. Cunliffe et al. [25], through a complex multi-
step organic synthesis, prepared bacterially imprinted polymeric surfaces favoring attachment of
affinity ligands solely onto an imprinted site. These unique imprinted materials are capable of
microorganism trapping by combined size-shape discrimination and affinity recognition. Perez et al.
[26] demonstrated selective binding of rod-shaped (Listeria monocytogenes) and coccoidal
(Staphylococcus aureus) bacteria from a mixture of both on beads imprinted with one or the other.
Harvey et al. [27] used affinity augmented beads imprinted with Bacillus thuringiensis and Bacillus
anthracis as a semi-selective matrix to capture and concentrate their specific spores based on
same principles.
In our laboratory, previous investigations revealed a variety of successful applications of ormosil
sol-gel processes in environmental biotechnology such as: entrapped Thiobacillus thiooxidans free
cells extract in a SG matrix effectively oxidized H2S in water [28]; epifluorescent microscopy
detection of E. coli esterase/lipase activity by thin SG films doped with fluorescein diacetate [29]; SG
entrapment of Pseudomonas spp. parathion hydrolase catalyzing hydrolysis of organophosphates [30],
effective reduction of nitrate/nitrite in solution by up-flow sludge blanket denitrifiers whole cell
entrapped in SG matrix [31] immobilization of humic acids in SG matrix and investigation of
adsorption kinetics of hydrophobic contaminants to soil [32] and tissue culture growth on sol-gel thin
layers for rapid detection of viral plaques [33,34].
The aim of the present study was to prepare and evaluate SG imprinted surfaces (ormosils-based)
with various microorganisms to efficiently discriminate between different mixed microorganisms in
liquid environment (e.g., water) based on their morphology or outer layer components regardless of
their similar morphology. The basic idea was to perform whole cell imprinting in SG precursor

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materials (tetraethyl orthosilicate-TEOS, tetramethyl orthosilicate-TMOS, etc.) in order to obtain
bacterially imprinted thin films (1–2 m thick) that after immersion for a short time in a liquid volume
will selectively adsorb planktonic cells into already formed micro-cavities.
2. Results and Discussion
2.1. Imprinting of SG Films with Whole Cell Bacteria
The extremely radioresistant Gram positive bacterium Deinococcus radiodurans strain UWO 288
was selected as template for sol-gel imprinting due to its coccoidal cells organized in pairs and tetrads
[35]. Beside radioresistance, D. radiodurans has another interesting feature relevant to the imprinting
process, namely its outer membrane structure containing a regular surface protein array (RS) or
so-called hexagonally packed intermediate layer (HPI) [42,44]. According to Thompson and Murray
[43] blebs from the outer membrane of this bacterium are shed as large vesicles from approximately
5% of the cell population during growth. Elution of entrapped D. radiodurans cells into thin SG films
(~1 m), casted on microscope slides, revealed paired/tetrad like cavities similar to D. radiodurans
cells morphology (Figure 1). Figure 1 (A, B and C) shows the morphology of D. radiodurans cells
entrapped into sol-gel film observed with different microscopes. Interestingly, it was found that sole
Gram staining procedure is enough to cause elution of the imprinted cells (at the moment under study
to comprehend this phenomenon) leaving clear paired or tetrad cavities as observed with SEM
(Figure 1D).
Figure 1. Images of imprinted D. radiodurans: entrapped in SG-film (SEM) (A), AFM
(B), HSEM (C) and SG-film cavities left after removal of entrapped bacterial cells (SEM) (D).

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Sol-gel imprinted films were also subjected to acridine orange (AO) staining to be used with
epifluorescent microscopy (Figure 2). AO staining of cavities formed following elution of imprinted
D. radiodurans revealed staining of residual components entrapped in a sol-gel layer (Figure 2A). As
already mentioned earlier, these components are apparently membrane blebs shed by this bacterium
during the imprinting process. A similar phenomenon was observed with other bacteria such as
Escherichia coli CN13, Sphaerotilus natans and Bacillus subtilis (data not shown). In order to test the
affinity of D. radiodurans planktonic cells in suspension towards imprinted SG film, an imprinted
probe was immersed into a fresh D. radiodurans suspension (Figure 2B) against a non-imprinted
sol-gel film as control (Figure 2C). The imprinted SG film was selectively covered with adsorbed D.
radiodurans only onto imprinted sites compared with control that showed only few sparse non-specific
adsorbed cells (Figure 2C).
Figure 2. D. radiodurans specific adsorption onto SG imprinted films as detected by
acridine orange staining by epifluorescent microscopy (×1,000). (A) D. radiodurans
imprinted SG film(Gram staining and AO); (B) imprinted SG film with D. radiodurans
exposed to new planktonic suspension of D. radiodurans (AO staining); (C) Control, non-
imprinted sol-gel film exposed to same D. radiodurans suspension (AO staining).



The imprinting specificity for D. radiodurans cells was further substantiated by SEM micrographs
(Figure 3). Imprinted SG film exposed to fresh D. radiodurans suspension clearly revealed adsorption
and location of these bacteria pairs/tetrads solely in formed cavities.