Mass spectrometry of carbohydrate-protein interactions on a glycan array conjugated to CVD graphene surfaces

In general terms, the functionalization of a graphene-based microarray was based on the formation of a hydrophobic bilayer via the combination of covalent and non-covalent modification of graphene (Scheme 1). Initially, the graphene surface was covalently functionalized with stearyl alkoxide-substituted aniline derivative 1 via a diazonium salt reaction [28] and further modified by hydrophobic interactions with the N-hydroxysuccinimide (NHS) activated bidentate 1,2-sn-dipalmitoyl glycerol 2. Onto this NHS activated hydrophobic bilayer, amine-containing ligands can be robotically printed and immobilized as highly stable carbamates. In addition, the terminal reactivity of the bidentate linker might be easily changed to azide, alkyne [29] or amine groups to accommodate other ligand types. Hereupon, p mol amounts of 5-amino-pentyl modified carbohydrates were spatially arrayed on the different surfaces, generating micrometer sized spots of the immobilized carbohydrates.

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2.1.1. Covalent modification of pristine graphene

In a proof of concept experiment, we studied the covalent modification of CVDG with the diazonium salt generated from aniline 1 on SiO₂ and characterized the resulting surface by Raman spectroscopy and AFM (see supporting information). Pristine CVDG on SiO₂ measured with a 532 nm excitation laser showed the characteristic G, D and 2D bands at 1350, 1580 and 2700 cm⁻¹, respectively (figures S5(a) and (b) (stacks.iop.org/TDM/7/024003/mmedia)). After treatment with the in situ generated diazonium salt from the aniline 1, the graphene surface showed a slight increment of the D band (Δ(I_D/I_G)  =  0.05, figure S5(b)). This increased intensity might be explained by the formation of structural sp³ defects in the graphene structure where the corresponding functional groups are covalently attached. This covalent modification, tends to produce a low number of defects (low I_D/I_G ratio) in order to preserve the intrinsic properties of CVDG [30, 31]. Interestingly, AFM showed significant changes in the surface morphology, probably due to the well-known oligomers derived from the generated phenyl radicals (figure S6) [32]. For improved clarity we depict the attachment of a 3,4-octadeciloxi-phenyl monomer on the graphene surface (Scheme 3) but, probably, oligomers derived from 3,4-octadeciloxi-phenyl radicals are obtained and immobilized (figure S7).

After confirming the covalent modification of CVDG on a generic substrate, the preparation of the different graphene-based microarray platforms were addressed. Consequently, CVDG on ITO-coated glass and uncoated glass slides were covalently functionalized and characterized by Raman spectroscopy, x-ray photoelectron spectroscopy (XPS) analysis, water contact angle measurements and AFM. Raman mapping studies of areas around 30  ×  20 µm² for graphene on ITO-coated glass showed an increment of D and D' band at 1350 and 1615 cm⁻¹, respectively (figures 2(a) and S5(c), (d)). In addition, XPS analysis of the chemical composition of the graphene surface was performed to provide evidence of the graphene functionalization. The XPS analysis showed an increase of atomic carbon concentration after functionalization (table S1). Focusing on the atomic carbon composition, pristine graphene substrates showed a C1s core level composed of graphenic C  =  C asymmetric component (284.37 eV) and oxygenated carbon groups such as C–O and C=O components (~286 and ~288 eV, respectively; table S2). However, after covalent functionalization, C  =  C/C–C component in the C1s core level significantly increased, probably because of the introduction of the dialkoxyphenyl moieties. In order to determine the influence of the functionalization on the surface hydrophobicity, the water contact angle was measured (figures S14(a)–(c)). The contact angle on functionalized graphene on ITO-coated glass was slightly increased with respect to the pristine material, probably, due to the introduction of alkane chains. In addition, the surface morphology was studied by AFM after functionalization and a thorough washing process. The AFM images of functionalized graphene on ITO-coated glass suggested the existence of an amorphous layer on the surface composed of a significant fraction of aryl oligomers (figures 2(b)–(d) and S8) [32].

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On the other hand, the characterization of covalently modified CVDG on bare glass showed only a low increment in the D band (Δ(I_D/I_G)  =  0.01, figure S5(f)) by Raman spectroscopy. XPS analysis confirmed the expected increment in the C  =  C/C–C component (284.37 eV), due to the presence of phenyl oligomers (table S2). Nevertheless, the contact angle measurements and the morphology study by AFM for functionalized graphene on bare glass were not conclusive due to the high roughness of the glass substrate (figure S9). Overall, characterizing CVDG on bare glass was quite challenging, due to the poorer surface characteristics of bare glass as compared to ITO coatings.

2.1.2. Non-covalent modification of covalently modified graphene

After the covalent modification, the immobilization of N-hydroxysuccinimide (NHS) activated bidentate 1,2-sn-dipalmitoyl glycerol linker was carried out onto ITO/CVDG and glass/CVDG modified graphene substrates. To evaluate the adsorption of the lipidic bidentate linker MALDI-TOF MS after matrix deposition was carried out and the immobilization could be clearly confirmed on both CVDG-based substrates (figure S15). The activation of the surface with reactive NHS groups permits the immobilization of amine containing biomolecules. Then, as a proof of concept, solutions (15 nl) of 5-aminopentyl modified carbohydrates (figure S16(a)): N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-lactosamine (LacNAc), lactose (Lac) and Lewis^x trisaccharide (Le^x) were robotically dispensed on both NHS activated substrates. Each spot contains p mol amounts of each carbohydrate forming subarrays of four different carbohydrates each of them printed in five replicates (figure S16(b)). All hydrophobic bilayers showed high stability during aqueous washings and excess of buffers or reagents in the printing solutions could be removed by a simple washing procedure. Non-reacted NHS groups were quenched by the immersion of the substrates in ethanolamine solution.

The successful immobilization of the carbohydrates was monitored by MALDI-TOF MS after matrix deposition. Under laser irradiation, the hydrophobic bilayer is disrupted allowing the detection of carbohydrates coupled to bidentate linker on individual spots. For an accurate comparison of the different CVDG substrate performance, all spectra were recorded under identical laser settings and power conditions and a sum of 500 shots was acquired in each spectrum. Both ITO/CVDG and glass/CVDG showed similar performance in the analysis of immobilized carbohydrates (table 1). Clean, mass spectra with high ion intensity and good signal to noise ratio were obtained from individual spots (figures 3(a) and (b)). The performance of CVDG-glass microarrays was compared with previously described hydrophobic coated ITO based microarrays [27]. CVDG coated glass slides showed a similar performance as the C-18 alkane coated ITO microarrays (figure S17 and table S3) Therefore, CVDG-glass could become an alternative to the use of ITO-coated glass in carbohydrate microarray fabrication.

Table 1. MS detection of different immobilized carbohydrates using CVDG-based substrates by MALDI-TOF.

Carbohydrate	m/z [M+Na]+	ITO/CVDG		Bare glass/CVDG
		Peak intensity	S/N	Peak intensity	S/N
GlcNAc	923.65	3525	292.1	5292	736.4
LacNAc	1085.71	3281	743.5	2016	385.3
Lactose	1044.68	2587	365.0	1577	276.2
Lex	1231.76	2783	271.2	2789	417.1

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In addition, carbohydrates immobilized on glass/CVDG microarrays were efficiently detected under matrix-free conditions (figure 3(c)). Thus, the role of graphene as a SALDI platform was confirmed. As graphene surfaces show high hydrophobicity (figures S14(b) and (e)) and their interaction with phospholipids has been previously described [33], we evaluated the assay performance for arrays with the bidentate activated linker 2 directly immobilized on the non-derivatized CVDG surface. MS spectra recorded under identical conditions showed that the direct neoglycolipid immobilization on graphene surface produced spectra with far lower S/N and spectral quality than spectra obtained from graphene, which had been hydrophobically derivatised with the dialkoxy aniline ligand 1 (figure S18 and table S4).

This experimental evidence suggests that the covalent hydrophobic modification of the graphene layer, although does not affect substantially the hydrophobic properties of the material, can lead to a higher loading of the bidentate linker 2 and consequently to an increase in the loading of immobilized carbohydrates.

Next, the detection of carbohydrate-lectin interactions on CVDG-based microarrays was explored (figure 4(a)). Lectins are proteins that recognize specific structural elements of carbohydrates with high selectivity, acting as translators of the information encoded by carbohydrates on the cell surface or in extracellular proteins. Lectins are widespread in all living organisms, from bacteria to mammals and display numerous biological functions, in cell–cell communication, fertilization and immune response, e.g. through the recognition of foreign carbohydrates presented in colonizing pathogens [34]. Carbohydrate microarrays have been extensively employed as high throughput screening tools for the characterization of these proteins [35, 36]. Commonly, carbohydrate-lectin interactions on microarrays are quantified via the emitted fluorescence of the adhered labelled lectins after a washing step to remove non-binders. It is well documented that graphene can quench fluorescent dyes via fluorescence resonant energy transfer (FRET) with high efficiency. In fact, bioassays based on the quenching and subsequent recovery of the fluorescence on graphene has been developed to study carbohydrate-lectin interactions [37, 38].

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We hypothesized that the hydrophobic bilayer constructed on CVDG based microarrays could provide enough distance to avoid FRET based quenching of the dyes allowing fluorescence measurements on these surfaces. To verify this hypothesis, we incubated CVDG carbohydrate microarrays with fluorescently labelled lectin Aleuria aurantia (AAL). AAL is isolated from orange peel fungus and recognizes L-fucose, with a broad specificity towards different linkages (α-1,2; α-1,3; α-1,4; α-1,6-). This lectin is commonly employed as a tool in the isolation and characterization of fucosylated glycoproteins which are often overexpressed in different types of cancers [39]. After incubation with AAL-555, the glass/CVDG carbohydrate microarray was washed and scanned in a microarray scanner (figure 4(b)). Fluorescence was only observed for spots corresponding to the immobilized Le^x carbohydrate, which was the only fucosylated structure on the array. Additionally, the label-free detection of carbohydrate-lectin interactions by MS was studied [40]. After incubation of array prepared on glass/CVDG with AAL solution, we could detect by MALDI-TOF MS a peak at m/z  = 33 691 Da [AAL]⁺ [41] corresponding to the monomeric lectin exclusively on spots of immobilized Le^x carbohydrate (figure 4(c)).

Chemicals were acquired from Merck and were used as received. Raman spectra were collected on a Renishaw Invia Raman microspectrometer comprised of a laser source at 532 nm wavelength, a high-resolution diffraction grafting of 1800 g mm⁻¹ and a Peltier-cooled front-illuminated CCD camera. XPS spectra were obtained using a SPECS Sage HR 100 spectrometer with a nonmonochromatic x-ray source of Aluminium with a Kα line (1486.6 eV, 300 W). XPS data was analyzed with CasaXPS software. Surface topologies of slides were characterized by AFM (JPK NanoWizard II) in intermittent contact tapping mode. A tapping etched silicon probe (TESPA-V2) with a 0.01–0.025 Ω/cm antimony (n) doped Si was employed, equipped with a rectangular cantilever: (3.8 µm thickness, nominal resonant frequency of 320 kHz, spring constant of 42 N/m, length of 123 µm, and width of 40 µm). The AFM-images were analyzed employing WSxM 5.0 Develop 7.0 software. For mass analysis, the spectra were obtained on a Bruker Ultraflextreme III TOF mass spectrometer and the data was acquired using the FlexControl 3.3 software (Bruker Daltonics, Bremen, Germany). The spectrophotometer was equipped with a pulsed Nd:YAG laser (λ  =  355 nm). 5000 shots per spectrum were acquired in positive reflector or positive linear ion mode (pulse duration of 50 ns, a 40 frequency of 1000 Hz, a laser fluence of 30%, laser focus settings: offset  =  0%, range  =  100%, and value  =  9.5%). The m/z range was chosen according to the mass of the analyte. The spectra were processed with FlexAnalysis v3.3 software. For calibration of mass spectra INTAVIS peptide calibration standard mixture II was employed.

Microarrays were prepared with a SciFLEXARRAYER piezoelectric spotter S11 (Scienion, Berlin, Germany). Fluorescence images were obtained on a microarray scanner system (Agilent G265BA, Agilent Technologies, Santa Clara, USA).

All slides employed have the dimensions of 75 mm  ×  25 mm. ITO coated slides (ITO thickness of 130 nm) were purchased from Hudson Surface Technology, Inc. (Fort Lee, NJ), with a nominal transmittance of  >78%. Glass microscope slides (thickness 0.96–1.06 mm) were acquired from Corning Incorporated (Corning, NY).

Both ITO and bare glass slides were cleaned by immersion in piranha solution (H₂O:H₂O₂:NH₃, 40:15:15 ml) for 1 h at 60 °C before CVDG coating.

The high quality monolayer graphene was grown in a cold wall CVD reactor (4'' Aixtron BM). Then, CVD monolayer graphene was transferred to ITO-coated glass or bare glass substrates by using poly(methyl methacrylate) (PMMA) assisted wet transfer process. 100 nm of PMMA were spin-coated on top of graphene/copper samples followed by chemical etching of the graphene bottom layer. Afterwards the copper catalyst was etched away with ferric chloride for 90 min and cleaned with distilled water using a semi-automatic system. Finally, the supported polymer was removed by dipping in acetone and isopropanol for 30 min each bath and dried in a hotplate at 150 °C.

1,2-Dipalmitoyl-sn-glycerol was treated with N,N'-disuccinimidyl carbonate to produce 2, as previously reported [27]. Aleuria aurantia lectin was provided by Vector laboratories (Burlingame, CA, USA) and labelled with Alexa Fluor^™ 555 NHS succinimidyl ester from Thermo Fischer following manufacturer instructions.

3,4-bis(octadecyloxy)aniline (1, 403 mg, 0.64 mmol) was placed in a flask previously purged with argon and dissolved in dry DMF (80 ml) under sonication. Then, the graphene substrate was introduced in the flask and 3-methylbuthylnitrite (129 µl, 0.96 mmol) was slowly added dropwise. The resulting mixture was heated to 60 °C and left without stirring for 2 h. The substrate was removed from the reaction mixture and cleaned by immersion in toluene for 12 h and in EtOH for 30'. The modified graphene substrate was dried over a stream of N₂.

Modified graphene slide was incubated overnight at r.t. with (S)-3-((2,5-dioxocyclopentyloxy)carbonyloxy)propane-1,2-diyl dipalmitate (2, 1 mM solution in CHCl₃). The slide was left to dry under ambient conditions and stored at −20 °C or under vacuum.

Solutions (100 µM) of C5-amino linked carbohydrates (GlcNAc, LacNAc, Lac and Le^x) in phosphate buffer (300 mM, pH 8.5) were robotically printed (50 droplets, ≈  15 nl, ≈  1.7 pmol) onto slides functionalized with activated bidentate linker at a pitch of 750 µm in both x and y -directions, and left to react overnight at r.t. and controlled humidity of 90%. The slides were quenched by immersion in ethanolamine solution (50 mM) in sodium borate buffer (50 mM, pH  =  9.0).

For matrix assisted MS analysis, 2,5- dihydroxibenzoic acid (DHB) matrix (15 nl of 4 mg/ml in water:acetonitrile, 90:10 containing 0.1% trifluoroacetic acid (TFA) and 0.005% of NaCl) was spotted on top of the carbohydrates and then crystallized prior to MALDI-TOF measurements.

Subarrays were compartmentalized using 8-well ProPlate^® incubation chambers from GraceBiolabs and treated with Aleuria aurantia lectin (AAL). A solution containing AAL-555 or unconjugated AAL (50 µg/ml) in Hepes buffer (50 mM, pH  =  8.0) was incubated at room temperature for one hour. Before removing the incubation chamber the subarrays were washed sequentially with Hepes buffer and water. The complete slides were washed by immersion in water and dried under a stream of argon. For fluorescence measurements the slide was scanned in Agilent G265BA microarray scanner.

For MALDI-TOF mass detection of AAL a solution (15 nl) containing a mixture 1:1 of DHB solution and α-cyano-4-hydroxycinnamic acid (α-CHCA) matrix solution (4 mg/ml in water:acetonitrile 70:30 containing 5% formic acid) was spotted on top of the printed carbohydrates allowed to crystallize prior to MALDI-TOF measurements.