Supramolecular scaffolds on glass slides as sugar based rewritable sensors for bacteria

Article abstract of DOI:10.1039/c5cc01019e

We describe here the sugar functionalized β-cyclodextrin-ferrocene glass slides as fully reversible bacterial biosensors under the influence of external adamantane carboxylic acid. The prototype d-mannose-E. coli ORN 178 and l-fucose-P. aeruginosa interactions serve as a model to illustrate the new approach.

Full text of DOI:10.1039/c5cc01019e

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Supramolecular scaffolds on glass slides as sugar
based rewritable sensors for bacteria†
Cite this: Chem. Commun., 2015,
1, 6346
5
a
b
a
c
Madhuri Gade, Ajay Paul, Catherine Alex, Devika Choudhury,
b
a
Received 3rd February 2015,
Accepted 3rd March 2015
Hirekodathakallu V. Thulasiram and Raghavendra Kikkeri*
DOI: 10.1039/c5cc01019e
www.rsc.org/chemcomm
We describe here the sugar functionalized b-cyclodextrin–ferrocene attach a multitude of sugar molecules in close proximity by
glass slides as fully reversible bacterial biosensors under the influence of attaching them to its skeleton. We have synthesized a b-CD
external adamantane carboxylic acid. The prototype D-mannose – scaffold which contains seven sugar molecules with increased
E. coli ORN 178 and L-fucose – P. aeruginosa interactions serve as a carbohydrate mediated interactions. This new sugar-modified
model to illustrate the new approach.
b-CD derivative was used for developing a sensor on glass sub-
strates. The attachment of this compound to the glass surface was
Food- and water-borne diseases cause over 3 million deaths every achieved by formation of host–guest b-CD–ferrocene complexes.
year, of which, according to WHO publication, about 90% are The specific advantage of ferrocene over the adamantane linker is
7
children younger than 5 years from poor families and communities the difference in the strength of the host–guest complexation,
1
in developing countries. The major reasons for the rapid increase which yields a facile method to regenerate the surface to develop a
in global pathogen infections are strain mutations and emergence protocol for removal of the bacteria and sugar after binding so that
2
of antibiotic resistance. However, appropriate medical treatment the same slide can be used continuously for similar or different
based on early diagnosis can decrease fatalities. Currently, pathogen–carbohydrate interactions.
detection of pathogenesis is done using DNA based PCR assay
and protein-based ELISA techniques but these methods require strains mannose-FimH of E. coli ORN 178 and fucose-PIM-2 of
trained personnel and are also influenced by the environment. A P. aeruginosa to establish the selectivity, sensitivity and reversibility
We used mannose and fucose as sugars and the specific bacterial
3
8
different approach for detection is based on carbohydrates that are of the detection platform.
used as biomarkers for pathogens and is based on the highly
Mannose-modified b-cyclodextrin (C-2) was synthesized as
9
selective recognition of bacterial lectins through specific oligo- described previously. C-3 was synthesized from peracetylated
4
saccharide epitopes. Carbohydrate–protein interactions are fucose 2 which was glycosylated with bromoethanol in the
5
weak but multivalent interactions amplify recognition. We presence of NIS and TfOH, converted to thiocyanate, followed
and others have harnessed these interactions as templates for by Zn/AcOH reduction to yield 3. This derivative was reacted
6
recognition of bacteria.
2 3
with peracetylated hepta iodo-b-CD 4 in the presence of Cs CO
In this communication we describe a technique to prepare a yielding 5 that was deacetylated in the presence of sodium
glass substrate covered with sugar dendrimers which can be methoxide to yield C-3 (Scheme 1).
used as a platform for detection of bacteria. Our approach
Ferrocene derivative L-1 was prepared by coupling mono-boc
utilizes the special high tendency of b-cyclodextrin (b-CD) to protected ethylene diamine and ferrocene monocarboxylic acid,
form host–guest complexes with small molecules, especially followed by deprotection using TFA in DCM (Scheme 1).
7
with adamantane and ferrocene molecules and the ability to
Robust ferrocene-based monolayers were formed by assembling
ferrocene derivative L-1 on silylepoxide-coated glass or silicon slides.
This was done by washing glass slides (approx. 1 Â 1 cm in size)
with piranha solution followed by dipping them immediately into a
solution of 3-glycidyloxypropyltrimethoxysilane (GOPTMS) in
toluene. The substrates were heated at 85 1C for 52 h in a pressure
tube, rinsed with toluene to remove excess GOPTMS and dipped in
a solution of L-1 (0.02 mM) in ethanol for 24 h. Finally the substrates
were rinsed with ethanol to remove the excess ferrocene derivative
and to remove residual epoxide groups (Fig. 1). The process yielded
a
Indian Institute of Science Education and Research, Dr. Homi Bhabha Road,
Pashan, Pune 411008, India. E-mail: rkikkeri@iiserpune.ac.in;
Fax: +91-20-25899790; Tel: +91-20-25908207
b
Chemical Biology Unit, Division of Organic chemistry, CSIR-National Chemical
Laboratory, Dr. Homi Bhabha Road, Pune-411008, India
c
Department of Energy Science and Engineering, IIT Bombay, Mumbai-400076,
India
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5cc01019e
6
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formed by complexation of the ferrocene skeleton in the
cyclodextrin cavity. The substrates were rinsed with deionized
water to remove the uncomplexed compound and the slides
were checked again by SIMS-TOF and XPS to reveal the
presence of sulphur atoms in addition to those that were found
on the slide before complexation, confirming the presence of
C-2 or C-3 on the surfaces (Fig. S2 and S3, ESI†). Finally, the
concentration of mannose on the slide was quantified by
phenol–sulfuric acid assay.
Glass slides thus modified were used for binding different
bacterial strains via interaction with the sugar molecules found
on the slides. Three bacterial strains, differing in their sugar
recognition properties, were used to assess whether the sugar
functionalized glass slides could recognize the specific bacterial
strain. We used E. coli ORN 178 having a mannose receptor
(FimH), its mutant strain E. coli ORN 208 and P. aeruginosa
having a fucose binding PIM-2 receptor. First, bacteria were
grown at 37 1C and a standard growth curve was recorded at
OD600. Glass slides coated with L-1, C-1/L-1, C-2/L-1 and C-3/L-1
Scheme 1 Synthetic procedures used to prepare the compounds used were dipped in a solution with a concentration of approximately
8
in this work: (a) DIC/HOBT/DCM/BocNH–(CH
2
)
2
–NH
O; (d) bromoethanol/NIS/TfOH; KSCN/DMF; AcOH/Zn;
3
/DMF; (f) NaOMe/MeOH.
2
; (b) TFA/DCM;
1
0 bacteria for 30 minutes. The slides were then washed several
(
c) PhSH/BF
e) Cs CO
3
ÁEt
2
times with distilled water to remove unbound or weakly bound
bacteria and finally, bound bacteria were treated with DAPI or
FITC or rhodamine and imaged microscopically. Glass slides
coated with L-1, C-1 or C-3 were not found to bind bacteria of
E. coli strain. However, C-2 functionalized glass slides revealed a
strong cluster of ORN 178, which could not be removed even
upon vigorous rinsing with water. On the other hand, all glass
slides with ORN-208 did not show binding. These results
demonstrate selective and specific carbohydrate–protein inter-
actions in bacterial recognition. Similar experiments were
carried out using a P. aeruginosa strain. As expected, specific fucose
mediated aggregation of bacteria was clearly observed (Fig. 2) which
corroborates the selectivity of the binding assay.
(
2
glass slides covered with monolayers of the ferrocene derivative
1
0
L-1 which were analyzed by SIMS-TOF and XPS. Presence of
L-1 on the glass slides was confirmed by the relative abundance
of carbon, oxygen and iron atoms on the chips (Fig. S1 and S3,
ESI†). In the final step, the freshly prepared ferrocene mono-
layers were immersed in solutions of the b-CD derivatives, C-2
or C-3 (10 mM), for 30 min at RT, and sugar coated surfaces were
After assessing the selectivity of the platforms in bacterial
recognition, we determined the sensitivity of the detection platform.
Glass slides with C-2/L-1 modified surfaces were immersed and
incubated in serially diluted solutions of mannose-binding E. coli
ORN 178 for various periods of time, and surface-bound bacteria
5
were visualized by DAPI staining. We found that as little as 10
bacterial cells bound to the surfaces could be observed by
1
1
fluorescent staining of the clusters (Fig. 3 and Fig. S4, ESI†).
It is interesting to note that this is also the minimum number
6
of bacterial cells needed for formation of each bacterial colony.
In order to assess the feasibility of the system to serve as a
biosensor, it was crucial to demonstrate the reversibility of the
system, i.e., to prove that the slides could be used for more than
one binding event and regeneration–generation cycles. This
was done by utilizing the high binding constant of the b-CD–
4
À1
adamantane complex (5.7 Â 10 M ) as compared to that of
3
À1
7
ferrocene (9.9 Â 10 M ). When glass substrates coated with
C-2 sugar bound to ORN 178 bacteria were incubated in a
solution of adamantane carboxylic acid (0.1 mM) for 5 minutes,
the sugar was repelled from the slide. The adamantane skeleton
replaced ferrocene in the cavity of the b-cyclodextrin skeleton
and detached it from the glass surface. After washing the glass
Fig. 1 Molecular structures and schematic representation of different
steps to immobilize sugar-modified b-CD supramolecular scaffolds onto
glass slides.
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Fig. 2 Representative images of bacterial adhesion on different sugar substrates coated onto glass slides.
Fig. 3 Detection limit for the staining of mannose binding E. coli on the
C-2/L-1 surface. The number of bacteria used in the incubation mixture is
shown above the images.
Scheme 2 Schematic describing the regeneration process of sugar
coated glass slides and subsequent interaction with E. coli (ORN 178)
and P. aeruginosa – regeneration–generation cycles.
substrate with PBS, fresh ferrocene modified surfaces were
obtained. Visualizing through fluorescence microscopy revealed
no bacterial aggregation and corroborated the regeneration high sensitivity. The interactions that control the binding are
process. When the same slide was incubated again in solutions carbohydrate–protein interactions and the sensitivity is determined
of C-2 or C-3 for 30 minutes, sugar respective bacterial aggrega- by the sugar moieties placed on the slide. The assay is reversible;
tions were observed on the glass slides (Scheme 2, Fig. 4). Several and regeneration of the glass substrate has been demonstrated.
cycles (5) of degeneration–generation processes were performed The platform described here can be used for continuous bacterial
and the reproducibility was found to be excellent.
sensing and could potentially be used as a detection method of
We have demonstrated that CD-based glycodendrimers choice in point-of-care testing. The possibility of producing
placed on glass substrates by formation of complexes, controlled microarrays with multitude types of carbohydrate on a single
by host–guest interactions, produced a platform for detection of glass substrate for high-throughput detection of several bacteria on
specific types of bacteria, such as E. coli and P. aeruginosa, with a single platform is currently under study.
6
348 | Chem. Commun., 2015, 51, 6346--6349
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