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Realisation and chemical characterisation of a model system forsaccharide-based biosensor
G. Leone ⁎, M. Consumi, A. Tognazzi, A. MagnaniDepartment of Pharmaceutical and Applied Chemistry, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy
Article history:Received 6 October 2009Received in revised form 21 July 2010Accepted 23 July 2010Available online 30 July 2010
Keywords:SaccharidesSelf-assembled monolayersReflection absorption infrared spectroscopyTime of flight secondary ion massspectroscopyClick chemistry
A procedure to immobilise saccharides on a gold substrate based on self-assembled monolayer (SAM)methodology associated to click chemistry is presented. Alkanethiol self-assembled on gold was obtained.The SAM was modified in order to introduce an alkyne-terminated arm which could react with azidomodified saccharides through click chemistry. The substrates were characterised at each modification step byreflection absorption infrared spectroscopy and time of flight secondary ion mass spectrometry. The stabilityof the organic arm immobilised on the gold substrate was also verified.
The detection of biological agents and in particular bacteria, is atopic of great interest. Specific identification of microorganismsinvolves complex and laborious microbiological methods requiringbetween 2 and 4 days, such as DNA-microarray combined withpolymerase chain reaction [1], culture and colony counting methods[2] and immunology-based methods [3]. In this context, rapid andsensitive detection of pathogenic bacteria is a key requirement forprevention and identification of problems related to health andenvironment [4]. A valid solution can be represented by the use ofbiosensors. A biosensor is defined by the National Research Council(part of the US National Academy of Sciences) as a detection devicethat incorporates a living organism or product derived from livingsystems (e.g., an enzyme or an antibody) and a transducer to providean indication, signal, or other form of recognition of the presence of aspecific substance in the environment [5].
In their infection strategy, microorganisms often use sugar-bindingproteins, that are lectins and adhesins, to recognise and bind to hostglycoconjugates where sialylated and fucosylated oligosaccharides arethemajor targets. The lectin/glyconjugate interactions are characterisedby high specificity [6], being the glycosylation specific for species,tissues, cell types anddevelopment. The variations in animal glycansas afunction of time and space are mirrored by a variety of strategies thatpathogens use to exploit the host surface and escape the defence [7].
Furthermore, adhesins represent the antigenic determinant present onthe bacterial cell and can represent the target of the specific antibodytowards that specific microorganism.
In recent years, a large number of lectins and adhesins werecharacterised, demonstrating that, in opposition to plant and animalthat produce lectins with low affinity to glycan, bacterial receptoroften present submicromolar affinity toward monosaccharides [8]. Itis well known that cellular recognition is based on protein–protein,carbohydrate–protein and carbohydrate–carbohydrate interactions[9]. All living organisms contain a huge amount of receptors torecognise and identify compounds that are necessary for eitherregulation, such as hormones and neurotransmitters, or immunolog-ical defence, where the body tracks external proteins and triggers offreactions to destroy them. These receptors are usually highly selectivetowards their respective substrates; this feature makes thempotentially very interesting for analytical purposes especially if fastresponses without the need of sample preparation are required.
In vivo interactions are extremely complex processes and conse-quently the identification and the analysis of each single process isextremely difficult. Consequently, it is of huge importance to have amodel system which can simplify the whole system and its analysis.The main advantage to get model systems consists in the possibility todesign specific “host” for specific “guest”. Models, which are used forthe study of molecular recognition processes, can have a simplemonovalent carbohydratic structure, or a complex polyvalent carbo-hydrate structure, up to models which mimic the entire bacterial cell.
Direct deposition of a natural product on a suitable transducer,moreover, is often limited. Biological receptors frequently consist of acluster of several protein molecules, and work properly solely under
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physiological conditions in respect to temperature, solvent and pH.Thus, the challenge is to create artificial materials with syntheticreceptors that combine biological recognition abilities with thethermal, chemical and physical stability of chemical compounds [10].
Several techniques to functionalise surfaces with saccharides,which are the target of microorganism lectins, have been developed[11–17].
However, these approaches require multistep modifications ofmonosaccharides or oligosaccharides moieties with chemical activegroups or ligands, which are tedious and laborious. Ideally, nomodification orminormodification of the carbohydratemoieties shouldbe required for the attachment, and the immobilised oligosaccharidesshould be organised in a regular and homogeneous environment so thatall immobilised sugars have equal activity towards the ligand [18].
The immobilisation of saccharides as self-assembling monolayer(SAM) is a major methodology [19]. The SAMs are spontaneouslyprepared with the precise structure and the saccharide-immobilisedsubstrates are easy to handle due to the covalent bond formationbetween the molecule and the substrate [20]. Nevertheless, a directdeposition of saccharides on metallic substrate, such as gold, cannotpermit a good interaction with the lectins and/or adhesins of cells. Infact, it is a fundamental aspect to achieve a good spatial structure of thesaccharide to permit the correct saccharide–cell interaction. Basing onthe work of Fukuda et al. who demonstrated that saccharidesimmobilised onto gold or silica SAMs by click chemistry maintainedtheir selective capability to interact with specific lectins [9,21], in thiswork a strategy to bind saccharides without modified them on asubstrate was realised by combining the SAM methodology [22] withthe click chemistry [23], with the aim of developing a simple modelsystem which can represent a starting point for the realisation ofbiosensor. In particular, the gold substrates were functionalised with11-mercaptoundecanoic acid which was conjugated to propargyla-mine. The terminal alkynic group was used to bind the azido modifiedsaccharide through click chemistry. The substrates were characterisedat each step of modification by reflection absorption infraredspectroscopy (RAIRS) and time of flight secondary ion mass spec-trometry (ToF-SIMS). The stability of the organic arm immobilised onthe gold substrate was also verified. The aim of the paper is limited tothe synthesis and chemical characterisation.
Scheme 1. Scheme of the functionalisation of g
2. Experimental details
2.1. Chemicals
11-Mercaptoundecanoic acid (MUA) (purum≥98%), N-hydroxy-succinimide (NHS) (purum,≥97.0%), 1-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (purum, ≥98.0%), N, N-diisopropylethylamine (DIEA) (purum≥98%), CuI (puriss≥99.5%),propargylamine (purum≥95%), and 1-azido-1deoxy-β-D-glucopyrano-side (purum≥98%),were purchased fromSigma-Aldrich (Switzerland).All solvents were reagent-grade. Reagents were used without anyfurther purification. Experimentswere carriedout at room temperature.
2.2. Realisation of the biosensor-model system
Si wafer (11 mm×11mm) coated with a 200 nm thick gold layer ofgold were purchased from Aldrich (Switzerland). The gold coatedsubstrates were washed using three different solvents: dichloro-methane, acetone and ethanol in ultrasonic bath for 15 min. Eachwashing step was repeated for three times.
Then, the substrates were immersed in a solution of MUA inethanol absolute (1×10−3 M) for 24 h and rinsed in ethanol and driedunder a N2 flow (step 1). Subsequently, the substrates were treatedwith a solution containing NHS (20 mM) and EDC (10 mM) inultrapure water for 2 h following by an immersion in a aqueoussolution of propargylamine (1 mM) for 24 h (step 2). The residualNHS esters were blocked with 1 M ethanolamine solution (pH 9 for20 min). After washing with ultrapure water, the substrates werefinally immersed in an ethanol solution of azide-terminated saccha-ride (0.5 mM) with DIEA (2.5 mM) and CuI (2.5 mM) for 24 h. Then,the substrates were sonicated with ethanol and MilliQ water for15 min and dried under N2 (step 3).
2.3. RAIRS measurements
The infrared characterisation was performed by a Nicolet 5700(Thermo) spectrometer equipped with a MCT detector.
Before recording the RAIR spectra the samples were washed with50% ethanol in an ultrasonic bath for 15 min in order to remove all the
Fig. 1. RAIRS spectra of: A: gold surface after immersion in a MUA solution; B: alkyne-terminated thiolate SAM; C: saccharidemodified substrate. The absorbance is expressedin arbitrary units (arb. units). The spectra were recorded at room temperature.
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no covalently bound molecules. The samples were then washed withwater, ethanol and dried under nitrogen flow.
RAIR spectra were recorded using a RAIR disposal (SMART SAGA,Thermo) under a N2 atmosphere. Typically, 512 scans at a resolutionof 2.0 cm−1 were averaged. The frequency scale was internallycalibrated with a helium–neon reference laser to an accuracy of0.01 cm−1.
The spectra were subjected to baseline correction, smoothingfunction and depletion of humidity.
2.4. ToF-SIMS measurements
ToF-SIMS measurements were carried out on a TRIFT III spec-trometer (Physical Electronics, Chanhassen, MN, USA) equipped witha gold liquid–metal primary ion source.
Before acquiring positive and negative spectra the samples weremaintained overnight in a conditioning pre-chamber with a vacuumvalue of about 10−4 Pa and then moved to the analysing chamber in
Table 1Main wavenumbers observed in the RAIR spectra together with their assignments.
Sample
Step 1
Step 2
Step 3
which the vacuum value raised up to 10−8 Pa. Positive and negative ionspectra were acquired with a pulsed, bunched 22 keV Au+ primary ionbeam, by rastering the ion beam over a 100 μm×100 μm sample areaand maintaining static SIMS conditions (primary ion dose densi-tyb1012 ions/cm2). Positive ion spectra were calibrated with CH3
(25.008m/z), in the lowmass region and with I− (m/z=125.09) in thehigh mass region. A number of peaks of increasing mass were assignedand added to the calibration set for an accurate mass calibration. Themass resolution (m/Δm) was 6000 at 27m/z.
Chemical images were acquired with a pulsed, bunched 22 keV Au+
primary ion beam, by rastering the ion beam over a 250 μm×250 μmsample area and maintaining static SIMS conditions. Chemical imagingwas performed in high mass resolution mode (m/Δm)=8000 at m/z29).
2.5. Stability of the biosensor-model system
The thermal and chemical stability was determined by incubatingthe samples in different solutions (solution A: NaCl 0.9%; solution B:EtOH 70%; solution C: EtOH:NaCl 1:1) over a period of 30 days at bothroom temperature and 37 °C. After incubation RAIR spectra of thesamples were recorded using the procedure previously reported(Section 2.3).
The mechanical stability was determined by analysing the SAMsRAIR and SIMS spectra after ultrasonic bath and ultrahigh vacuumtreatments.
3. Results and discussion
The biosensor-model system based on the immobilisation of amonosaccharide onto gold substrate was realised by a “three-stepprocedure”which involves SAM and click chemistry methodologies as
Fig. 2. Negative ion ToF-SIMS spectra of MUA-SAM on gold in the characteristic regionof gold–sulphur clusters (from AuS− up to Au3S−): A: gold–sulphur clusters Au2S (m/z=426) and Au2SH (m/z=427); B: Au2S2 (m/z=458) and Au2S2H (m/z=459);C: Au3S (m/z=623).
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shown in Scheme 1. Alkenthiol-Au based SAM was chosen for its wellknown high chemical stability and ease of handling. The reactivity ofsilane functionalities towards hydroxyl groups makes not easy toprepare surfaces with hydroxyl or carboxylic acid termination. In fact,as reported by Leggett [24], rather than adsorb the ω-carboxylic acidsubstituted silane directly, it is necessary to first adsorb a silane with aterminal group that is unreactive towards the head group functionalityand then convert this terminal group to the desired functionality witha loss of efficiency due to the obtainment of a not purely monofunc-tional substrate.
The activation of carboxylate groups towards nucleophilic attack ofaminic moieties, once adsorbed onto solid substrates, required about2 h.
The immobilisation of saccharide units was achieved by applyingthe click chemistry to SAMmodified substrates basing on the works ofboth Miura who applied this technique to bind modified saccharide onsilicon substrates [9], and Fukuda who applied the methodology on Au[21]. In his work, Fukuda pointed out that the saccharides once boundontomodifiedgold surface through click chemistry aswell as onto silicasubstrates retained their capability to selectively recognise and bindbiological target, or lectins, demonstrating the ability of the saccharideto maintain a correct spatial distribution and the biorecognitionactivity.
At each step the sample was analysed by RAIR spectroscopy andToF-SIMS to validate the proposed strategy and to gain usefulstructural information on the realised system.
3.1. RAIRS analysis
The RAIR spectra relative to each of the three steps involved in thesynthesis of the biosensor-model system (Scheme 1) are reported inFig. 1. The main wavenumbers observed in the RAIR spectra aresummarised in Table 1 together with their assignments [25].
Step 1: In Fig. 1A the RAIR spectrum of the gold surface afterimmersion in a MUA solution is reported. Two typical bands appear inthe ν(C–H) region, at 2848 cm−1 and 2918 cm−1 corresponding to thesymmetric and asymmetric stretching vibrations of the CH2 chain,respectively. The wavenumber position of these bands suggests ahighly order layer. In fact as explained by Nuzzo et al. [26] andconfirmed by Briand et al. [22] the C–H stretching band at 2923 cm−1
is characteristic of densely packed layer suggesting a quasi-crystallinearrangement of the alkyl chains thiolates. When a disorder layer isobtained slightly higher wavenumbers are observed with a band shiftfrom 2923 cm−1 to 2930 cm−1. The band centred at 1736 cm−1 pointsout the presence of the carboxylic moieties in the protonated form.
Step 2: In Fig. 1B theRAIR spectrumof the alkyne-terminated thiolateSAM is reported. The spectrum shows the characteristic band of alkynicgroup. In fact, it is evident a broad band centred at 3287 cm−1which canbe related to the stretching of C–H of terminal alkyne and to thestretching of amidic NH. The alkyne-terminated SAM spectrum shows adecrease of the COOH band and the appearance of a sharp band centredat 1657 cm−1 (amidic C=O stretching) and a band at 1541 cm−1
(amidic NH bending), confirming that the second step occurred.Furthermore, this spectrum shows both the symmetric and asymmetricν(C–H) wavenumbers slightly higher than for MUA-SAM. This may berelated to a partial loss of order after the formation of the amidic bond.
Step 3: The RAIR spectrum reported in Fig. 1C shows the typicalbands of saccharide moieties: a broad band in the 3300–3500 cm−1
range, relative to the stretching of intramolecular and intermolecularbonded OH; two bands at 2925 and 2852 cm−1 relative to asymmetricand symmetric stretching of CH2 moieties respectively and the bandrelative to the carbohydrate unit. In particular, the broad band relatedto saccharide moiety can be evidenced three shoulders centred at1147 cm−1, 1090 cm−1 and 1042 cm−1. On the basis of the assign-ment of IR bands in Hyaluronan [27] the three shoulders can berelated to the antisymmetric stretching (C–O–C) of the glycoside unit,
the stretching of C–O–C ring mode and the stretching of C–OH,respectively. The sugar band is also accompanied by the appearance oftwo new sharp bands centred at 2117 cm−1 and 1265 cm−1 whichcan be related to the triazole ring deriving from the Huisgen [2+3]cycloaddition. The unambiguous assignment of the band centred at2117 cm−1 to the triazole ring and not to the azido group of the nativesaccharide is due to the fact that the sample, before recording the RAIRspectra, has been subjected to a 15 min washing in ultrasonic whichguaranteed a complete removal of unbounded molecules. Further-more, as reported in literature, the presence of the triazole ring isconfirmed by the appearance of the band centred at 1265 cm−1 whichcorresponds to C–N stretching of the 1,3-triazole ring [21].
3.2. ToF-SIMS analysis
The formation of the MUA-SAM on gold substrate and thesubsequent functionalisation steps leading to the surface immobilisa-tion of the syntheticmonosaccharide receptorwere further investigatedby ToF-SIMS, a surface-sensitive technique which provides uniquemolecular informationon the chemical structure and composition of thefirst surface monolayer. If molecular information is desired the primaryion dose density has to be lowered to less than 1012 ions/cm 2. Underthese so called static conditions atmaximum the 0.1% of the uppermostmonolayer is destroyed so that spectral information comes predomi-nantly from undisturbed areas.
ToF-SIMS positive and negative ions spectra were acquired at eachstep of Scheme 1.
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The positive spectra, (not shown), in particular in the high massregion, provided very few relevant information on the SAM formationand subsequent functionalisation, mostly due to the electronegativityof sulphur ion which favours negative ion formation [28]. Thus, onlythe negative ion spectra are reported and discussed. In fact, asreported by Legget in his overview on Static SIMS studies onalkanethiol-on-gold self-assembled monolayers [24] negative ionspectra exhibit a range of high m/z peaks that could be specifically
Fig. 4. Negative ion ToF-SIMS spectra of MUA-SAM on gold in the characteristic regionof fragments clustered with gold: A: C2H2SAu− and C2H3SAu−; B: C3H2SAu, C3H4SAuand C3H5SAu.
Fig. 3. Negative ion ToF-SIMS spectra of MUA-SAM on gold in the characteristic regionof: A: the deprotonated intact thiol (M–H)− molecular ion; B: the deprotonated intactmolecular thiol fragment clustered with Au; C: the 1:2:1 triplet formed by (M-3H)Au2
−
(m/z=608), (M–2H)Au2− (m/z=609), and (M–H)Au2− (m/z=610) ions; D: TOF-SIMS
image of (M–H)Au2− (m/z=610) with a resolution of 256×256 pixels over an area of
250×250 μm2. The image was normalised to total counts.
associated with ions containing entire adsorbate molecules. Thedetection of (M–H)−, AuM−, AuMS−, Au2(M–H)−, and Au(M–H)2−
fragments, where M indicates the entire molecule, demonstrates thepresence of the covalently bound organic arm.
3.2.1. Step 1: MUA-SAM on gold substrateThe most characteristic regions of the negative ion ToF-SIMS
spectrum of the MUA-SAM on gold are reported in Figs. 2–4. All the
Table 2Characteristic peaks relative to step 1 with their chemical composition and iondescription.
1° Step
M=HS–(CH2)10–COOH
Characteristic peak Chemical composition Ion description
197 Au Substrate217 C11H21O2S Deprotonated M229 AuS –
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peak characteristics of the modified gold substrate at step 1 arereported in Table 2 together with their relative assignments.
Following the chemisorption of MUA on gold, several peaks arestrongly enhanced and new characteristic peaks appear in comparisonwith native gold substrate. In the low mass region of the negative ionspectrum, the small fragments S− and SH− at m/z=32 and 33respectively, resulting from the fragmentation of the surface mole-cules, are particularly enhanced. At higher mass, the strong gold peakis still observed at m/z=197. Several gold–sulphur clusters Au2S,Au2SH, Au2S2, Au2S2H and Au3S are also observed, evidencing the Au–S bond at the gold–thiol interface (Fig. 2A,B,C).
However, the most characteristic peaks are those resulting from adirect desorption of intact thiol molecules, which are observed in thehigh mass region. In particular, the deprotonated intact thiol (M–H)−
molecular ion is detected at m/z=217 and the deprotonated intactmolecular thiol fragment clustered with Au is observed at m/z=413,with M denoting the MUA molecule (C11H21O2S) (Fig. 3A, B).Furthermore, the presence of thiolate bound to gold is also provedby the 1:2:1 triplet formed by the (M–H)Au2− ions in the negative ionspectrum (Fig. 3C) [29]. SIMS imaging has been used, in addition toToF-SIMS spectrometry, to gain information on the spatial distributionof MUA on the sample surface. The chemical map of the tripletfragment, reported in Fig. 3D, reveals a homogeneous distribution ofthese peaks in the investigated surface, without the presence of anyaccumulation area.
A series of fragments clustered with gold are also detected, theseare C2H2SAu, C2H3SAu at m/z=255 and m/z=256 respectively, and
Fig. 5. Negative ion ToF-SIMS spectra of the propargylamine conjugated to the MUA-SAM on gold (step 2) in the region of: A: the depronated thiol (M–H)−; B: thedepronated thiol clustered with Au (M–H)Au− and the intact thiol clustered with Au(MAu−); C: the depronated thiol clustered with Au (M–H)Au2
−.
C3H2SAu C3H4SAu, and C3H5SAu at m/z=267 m/z=269 and m/z=270, respectively (Fig. 4A,B).
The presence of the peak at m/z=261 due to AuSO2 fragment,resulting from oxygen attack of the Au–S bonds [28] is observedalthough with low but constant intensity, in all the analysed samplesrelative to three steps, suggesting that a partial oxidation of thesurface monolayer occurred.
3.2.2. Step 2: conjugation of propargylamine to the MUA-SAMThe chemi-adsorbed MUA was conjugated with propargylamine
after carboxylic group activation with NHS/EDC.
Fig. 6. A: Negative ion ToF-SIMS spectrum of propargylamine conjugated to MUA-SAMon gold in the 41–43 m/z region showing the peak at m/z=42, relative to the CNO−
fragment; B: ToF-SIMS chemical map of CNO− fragment. The rastered area is250×250 μm2 with a resolution of 256×256 pixels. The map has been normalised tototal ions.
Fig. 8. Negative ion ToF-SIMS spectrum of immobilised glucose functionalised goldsubstrate showing the peaks relative to the saccharidemolecular ion C6H11O6 (m/z=179)and the deprotonated one C6H10O6 (m/z=178).
Fig. 7. Negative ion ToF-SIMS spectrum of immobilised glucose functionalised goldsubstrate showing the peak relative to the molecular ion (AuM–H) (m/z=655) and(AuM-2H) (m/z=654) (with M denoting the entire molecule (C20H36N4O6S)). Thesignal/noise ratio relative to the molecular peak with respect to noise is 4.9.
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The negative ion ToF-SIMS spectra are reported in Fig. 5. All thepeak characteristics of the substrate as modified at step 2 are reportedin Table 3 together with their relative assignments.
Fig. 9. Negative ion ToF-SIMS spectrum of immobilised glucose functionalised goldsubstrate showing the: A: peaks typical of saccharide fragmentation: C3H5O3 (m/z=89) and C5H6O2 (m/z=98); B: peaks relative to the gold clustered alkyne-terminated thiolate+triazole ring (AuSC14N4H24=492 m/z) and to the deprotonatedfragment (AuSC14N4H23=491 m/z). The signal/noise ratio relative to the 492 m/z peakwith respect to noise is 3.8.
In the high mass region the most characteristic peaks of thissample are the depronated thiol (M–H)− at m/z=254, intact orclustered with Au (M–H)Au− atm/z=451; (M–2H)Au− atm/z=450and (M–H)Au2− at m/z=648, with M denoting the entire molecule(SC14H25NO) (Fig. 5A,B,C).
In the lowmass region the fragment CNO− atm/z=42 is detected,too. This peak, resulting from the amidic bond formed between theamine moiety of propargylamine and the carboxylic group of MUA-SAM, is used to confirm the conjugation reaction. In fact, it is absent inthe ToF-SIMS spectrum recorded for step 1 (Fig. 6A). The chemicalmap of the CNO− fragment, reported in Fig. 6B, clearly evidences thehomogeneous distribution of this peak over the investigated surface,and as occurred for 610m/z peak no accumulation areas are detected.
3.2.3. Step 3: immobilisation of the monosaccharideThe most characteristic regions of the negative ion ToF-SIMS
spectrum of the biosensor-model system are reported in Figs. 7–10.All the characteristic peaks of this sample are summarised in Table 4together with their relative assignments.
Fig. 10. Negative ion ToF-SIMS spectra of immobilised glucose functionalised goldsubstrate showing: A: saccharide fragmentation C2H3O2 (m/z=59), C3H3O3 (m/z=71),peaks of triazole (m/z=67) and triazole+C2H2 fragment (m/z=93); B: peaks of thetriazole+saccharide fragments: triazole+C4H4N3O (m/z=110) and triazole+C4H5N3O (m/z=111); C: ToF-SIMS chemical map of fragment C4H4N3O (m/z=110)with a resolution of 256×256 pixels. The rastered area is 250×250 μm2. The map hasbeen normalised to total ions.
Fig. 11. A: RAIR spectra of the biosensor-model system sample after incubation in NaCl0.9% solution, 70% EtOH and EtOH–NaCl 1:1 at 37 °C over a period of 30 days. TheAbsorbance is reported in arbitrary units (arb. units); B: RAIR spectra of the biosensor-model system sample before (black curve) and after incubation (grey curve) in 70%EtOH at 37 °C over a period of 30 days.
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Saccharidic moieties are generally unstable and rapidly degrade.Thus, it is quite difficult tofind thepeak relative to the intact synthesisedmolecule.
Anyway, the SIMS spectrum in the 653–675 m/z range shows thepresence of a peak even with very low intensity relative to themolecular ion clustered with Au (C20N4O6H35SAu m/z=655) (Fig. 7).
Nevertheless, many other peaks relative to fragments resultingfrom the saccharide immobilisation can be evidenced demonstratingthat the reaction between glucose and the functionalised SAMoccurred. In particular, the peaks relative to the intact saccharide atm/z=178 and m/z=179 (C6H10O6 and C6H11O6) (Fig. 8) and typicalsaccharide fragments like C2H3O2, C3H3O3, C3H5O3 and C5H6O2 at m/z=59m/z=71m/z=89 andm/z=98, respectively are observed [30](Figs. 9A and 10A). The triazole and triazole+C2H2 fragment areevident at m/z=67 and m/z=93, respectively. Finally, the unequiv-ocal presence of the peaks at m/z=110 and m/z=111 relative totriazole with a part of saccharide C4H4N3O and C4H5N3O confirms thebinding of the glucose to the functionalised gold surface (Fig. 10B). Thechemical map of the C4H4N3O fragment, reported in Fig. 10C, under-lines the presence of these peaks homogeneously distributed all overthe investigated area. Furthermore, the evidence that the reactionbetween the azido saccharide and the alkyne-terminated thiolateoccurred at step 2 was provided by the presence of a peak centred at492 m/z. This peak corresponds to the gold clustered molecular ion ofthe step 2+ triazole ring derived by the Huisgen [2+3] cycloaddition.Finally, the detection of this fragment as a gold cluster points out thecovalent bond of the organic arm to the support.
3.3. Stability of the saccharide-based monolayer
The chemical and thermal stability of SAMs in EtOH and NaClsolutions was confirmed by the RAIR spectra reported in Fig. 11A.
Sample incubation in EtOH or NaCl solutions over a period of30 days does not affect the stability of the monolayer neither at roomtemperature (≈25 °C) nor at 37 °C. In fact, all the spectra in Fig. 11Ashow the characteristic absorption bands of the functional groups ofthe immobilised molecule. In particular, the bands relative to the
triazole ring centred at 1265 cm−1 and 2117 cm−1 are still presentafter sample incubation in EtOH or NaCl solutions as well as those ofsaccharide moiety (1147, 1091, and 1039 cm−1 relative to the O–C–Ohemiacetal vibrations of carbohydrates).
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Furthermore, by comparing the three spectra no significantdifferences are present between them. In Fig. 11B a comparisonbetween the IR spectrumof themonolayer before and after the 30 daysimmersion in 70% EtOH solution at 37 °C is reported. No significantdecrease in the intensity of the band relative to saccharide moiety isdetected. Thus, all the three solvents can be used as conserving mediafor the synthesised monolayer.
Temperatures higher than 37 °C were not tested because of therisk of saccharide decomposition. Furthermore, the foreseen applica-tion (as biosensor) does not suppose themaintenance of the sample athigh temperature having it to interact with cells or bacteria.
The stability of the monolayer to ultrasonic waves and highvacuum environment was deduced from the RAIR spectra recordedafter sample washing for 15 min in an ultrasonic bath and the ToF-SIMS spectra recorded after maintaining overnight the sample in avacuum chamber at 10−4 Pa, respectively.
4. Conclusion
We presented here the covalent immobilisation of single mole-cules of glucose on gold substrate using a combination of SAMmethodology and click chemistry.
RAIR spectroscopy and ToF-SIMS spectrometry have been appliedto validate the preparation procedure.
The proposed procedure appears to be useful for the realisation ofhomogeneous surfaces containing the glucose receptor covalentlybound to the substrate without extensive modification. Previousworks demonstrated that the association between SAM and clickchemistry permitted to obtain a correct spatial arrangement ofsaccharide guaranteeing their interacting capability. Consequently,the realised systems, due to the glucose moiety accessibility can beproposed for the preparation of model systems of biosensors. Thevalidation of this methodology will allow us to modify our model intoa more specific system changing the glucose residue with a morespecific saccharide or oligosaccharide moiety directly involved inspecific interactions with specific cells or bacteria. Detailed work onthe optimization of biorecognition properties with respect to bacterialor cell interaction with more specific saccharidic moieties such asSialic acid residues will appear later. In fact, Sialic acid residues, suchas Lewis a (Lea) Lewis X (Lex) formed by differently arranged Fucose,N-acetylglucosamine and Galactose residues as well as Sialyl Lewis X(LSex) constituted by Sialic acid, Fucose, N-acetylglucosamine andGalactose residues often constitute the termini of glycan chains ofmany cell surface glycoproteins or glycolipids [6]. They are a major
target for numerous pathogens and therefore are crucial determinantsnot only for cell-recognition and attachment but also for hostspecificity.
Acknowledgment
The authors would like to thank “Fondazione Monte dei Paschi diSiena” for financial support.
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