Elegant Chemistry to Directly Anchor Intact Saccharides on Solid

Article pubs.acs.org/bc

Elegant Chemistry to Directly Anchor Intact Saccharides on Solid Surfaces Used for the Fabrication of Bioactivity-Conserved Saccharide Microarrays Kai Liang† and Yi Chen*,†,‡ †

Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡ Beijing National Laboratory for Molecular Science, Beijing 100190, China S Supporting Information *

ABSTRACT: An easy chemical strategy was proposed and used to establish a method for direct anchoring of intact saccharides on solid surfaces with well conserved bioaffinity. The anchoring was achieved by temperature-modulated stepwise reactions with cyanuric chloride as a key linker, and was successfully applied to the fabrication of saccharide chips. To demonstrate, 15 intact reducing and nonreducing saccharides with various molecular sizes were dotted on a cyanuric-chloride-modified chip (1.0 × 1.0 cm2) and made to react with lectins. As expected, the anchored saccharides were capable of recognizing their target lectins, and more exciting were the perfect conservation of the specific recognizing ability of the anchored monosaccharides such as mannose, glucose, and even fructose (interacting only weakly with concanavalin A). This conservation was ascribed to the maintenance of the original structure (especially the anomeric configuration) of saccharides after immobilization and to the allowance of the anchored saccharides to rotate with and/or on the scaffold of cyanuric chloride, which makes them easily adapt to the recognition-preferred spatial position. The expected linkage of saccharides on cyanuric chloride and the maintenance of their anomeric configuration were characterized by mass spectrometry and nuclear magnetic resonance, respectively. The new method can be highlighted not only by its conservation of saccharide bioaffinity and universal applicability but also by its merits of easy manipulation or facile control of the reactions and cost-effectiveness due to the use of extremely cheap cyanuric chloride.

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INTRODUCTION It is widely recognized that saccharides and their associated complexes can play crucial roles in a series of specific physiological processes,1 but they are also one class of the most complicated substances hard to detect and analyze, which has made glycomic research full of challenges.2−4 To attack the challenges, microarray method or chip technology has been tried and is drawing great attention5−9 because it offers a new high throughput and low consumption way to simultaneously observe the behaviors of various saccharides, even capable of simulating some functions of saccharides on cell surfaces.10 It has since been expected to be one of the most powerful potential tools for the future study of glycomics. However, a prerequisite to use such a chip is to anchor the saccharides on the chip with their bioaffinity conserved. This has activated correlative researchers to find the anchoring strategies.11−15 Physical adsorption was first tried for its ease of manipulation. This strategy is applicable to the fixing of polysaccharides on some absorptive surfaces such as nitrocellulose-16 or black polystyrene-coated17 glass slides but commonly not applicable to less or not adsorptive substances © 2012 American Chemical Society

such as mono- and oligo-saccharides unless their adsorbability is largely increased by some special ways such as the fluoroustargeting technique.18 It may also have the problem of gradual loss of the adsorbed polysaccharides during a continuous wash or reaction in solutions. Chemical reactions were then explored to stably immobilize the saccharides on solid surfaces by the formation of covalent bounds through Schiff’s base19,20 or amidation.21,22 This strategy is easily widened by the exploration of more reactions such as click chemistry,23 Staudinger ligation,24 Diels−Alder reaction,25 Michael addition,26 and even streptavidin- and biotin-based immunoreactions.27 In general, chemical strategy is flexible and applicable to the firm immobilization of saccharides on various surfaces,28−37 but at present, its manipulation is generally not as easy as the physical adsorption. Chemical strategy is in many aspects favorable, but it may not be applicable to the direct immobilization of intact or Received: March 20, 2012 Revised: April 29, 2012 Published: May 6, 2012 1300

dx.doi.org/10.1021/bc300142s | Bioconjugate Chem. 2012, 23, 1300−1308

Bioconjugate Chemistry

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natural saccharides without a loss of their bioactivity.29,36 For example, reducing saccharides can directly be immobilized on hydrazide-, aminooxy-33,34 or aminooxyacetyl-modified35 substrates, but their delicate structure is changed after reactions: the aminooxy-based reaction preferentially generated acyclic products,33,38 while the hydrazide-based reaction predominantly yields β-anomeric configuration adducts.33,34 The immobilized saccharides, especially the small saccharides, will change or adjust their bioaffinity accordingly. A further question is that many of the chemical approaches are not universally applicable or only applicable to the immobilization of reducing saccharides.33−35 In order to establish a universal method, we have explored a photochemical approach by use of perfluorophenyl azide (PFPA) as a critical linker.37 It was demonstrated that, on a PFPA-functioned chip surface, all intact reducing and nonreducing saccharides, and even sugar alcohols, could firmly be immobilized through a solid phase photochemical reaction. Nevertheless, the loss of the bioaffinity of the anchored saccharides remains a problem and an interspacing arm of polylysine was used to recover and enhance the recognition of small saccharides.37 The interspacing arm could make the small saccharides work collaboratively or in a way of cluster(s).37,39 This implies that the saccharides were seemingly anchored too firmly to adjust their spatial position to favor the recognition. A better immobilizing way should thus be capable of allowing the saccharides to turn around a bond on the surface, which must lie greatly in the finding of a better linker. Some new chemical reactions were thus tried, and cyanuric chloride (CC) was shown to be at the top of the selection. CC is a heterocyclic trifunctional compound, manufactured on the scale of several millions of tons per year and is among the reagents of lowest price.40 Its most known feature is that the three chlorines on the triazine ring can be replaced stepwise by hydroxyls and/or amino groups through the regulation of reaction temperature. The additional attracting feature for us is that, after immobilization on a solid surface, it has the potential to turn around its immobilization bond and thus endow some extra possibility to the molecules linked on it to adjust their spatial position. Surprisingly, although the stepwise substitution of the chlorines on CC with nucleophiles has been documented well41−43 and has been applied extensively to organic synthesis44 and material surface modifications,45−47 there were only limited examples focused on microarray fabrications.48−52 To the best of our knowledge, no attempt has been made to directly immobilize various intact saccharides on solid surfaces aiming at the preparation of saccharide chips. In this article, we will demonstrate that the CC-based chemistry is indeed favorable as a new, bioaffinity-conserved strategy to covalently immobilize intact saccharides on solid surfaces. A model reaction was designed to conjugate saccharides with an alkoxyl group through CC in solution, studied to simulate the reaction of saccharides with a CCfunctionalized surface, and characterized by liquid chromatography−mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. On the basis of the study, an easy method was established to fabricate saccharide chips. To demonstrate, 15 mono-, oligo-, and polysaccharides were successfully dotted on 1.0 × 1.0 cm2 glass slides with or without a gold film and characterized by various techniques such as X-ray photoelectron spectra (XPS), vapor condensation imaging (VCi), surface plasmon resonance sensing and imaging (SPR and SPRi), and laser-induced fluorescence imaging

(LIFi). The immobilized saccharides, especially the monosaccharides, could all display their inherent recognizing ability with their target lectins such as concanavalin A (Con A) and peanut agglutinin (PNA). The new method can thus be highlighted not only by its conservation of saccharide bioaffinity and universal applicability but also by its merits of easy manipulation or facile control of reaction, and cost-effectiveness due to the use of extremely cheap CC.

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EXPERIMENTAL PROCEDURES Materials and Reagents. Concanavalin A (Con A), peanut agglutinin (PNA), fluorescein isothiocyanate (FITC), 3-aminopropyltrimethoxysilane (APTMS), and 1-ethyl-3-(3dimethyllaminopropyl)carbodiimide (EDC) hydrochloride were purchased from Sigma (St. Louis, Mo, USA). Cyanuric chloride (CC) and 11-mercaptoundecanoic acid (MUA) were obtained from Aldrich (Milwaukee, WI, USA). N-Hydroxysuccinimide (NHS) was purchased from Acros (New Jersey, USA). N,N-Diisopropylethylamine (DIPEA) was purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China), and 4hydroxyphenylacetic acid (HPA) was from Fluka (Switzerland). NaOH, NaCl, CaCl2, MnCl2, absolute ethanol, ethanolamine (EOA), ethanediamine (EDA), and tris(hydroxylmethyl)aminomethane (Tris) were of analytical reagent grade from Beijing Chemical Works (Beijing, China). All saccharides were of biochemical reagent grade from Beijing Reagent Work (Beijing, China). All reagents and solvents were used as received. Triply distilled water was used for the preparation of all solutions and rinsing. Preparation of Hydroxyl-Terminated Gold Film. Glass slides (1.0 cm ×1.0 cm, Shitai Co. Ltd., China) were deposited with 50-nm-thick gold films by vapor-deposition in a JEE-4X vacuum evaporator (JEOL Ltd., Tokyo, Japan). The cleaned gold chips were immersed in a solution of MUA in ethanol (1 mM) for at least 8 h, rinsed with ethanol and distilled water sequentially, and were then immersed for 25 min in an aqueous solution of EDC (75 mM) and NHS (15 mM). The resulting chips were treated with an aqueous solution of EOA (1 M, adjusted with 5 M HCl to pH 8.6) for 1.5 h. Finally, the substrates were flushed thoroughly with water and dried under N2 stream. Preparation of Hydroxyl-Terminated Glass Slides. First, glass slides (1.0 cm ×1.0 cm, Shitai Co. Ltd., China) were exposed to oxygen plasma for 5 min (PDC-MG, Chengdu Mingheng S&T CO. Ltd., China). Then they were immediately immersed in a freshly prepared APTMS solution (3% in methanol) for 2.5 h. The silanized glass slides were sonicated in methanol three times and dried under N2 stream. The resulted chips were treated with a 1:1 mixed solution of HPA in ethanol (113 mM) and EDC in water (219 mM) for 3 h. At last, the substrates were flushed thoroughly with ethanol and dried under N2 stream. Preparation of Saccharide Microarray Chips. First, the hydroxyl-terminated substrates (on gold film or glass slide) were incubated with a mixed solution of CC (100 mM) and DIPEA (100 mM) in acetone at 4 °C for 6 h. Then the substrates were flushed thoroughly with acetone and dried under N2 stream. An aqueous saccharide solution (30 mM for small saccharides or 3 mg/mL for polysaccharides, adjusted to pH 9.0 with 1 M NaOH) was spotted on the CC-functionalized chips by a laboratory-fabricated microprinting system, which allows the delivery of 15 nL solution onto the substrate to form a spot of 250 μm in diameter. After all the samples were 1301



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Scheme 1. Process to Immobilize Saccharides on a Gold Film and/or a Glass Slide and Their Interaction with Lectins

XPS Characterization of Surfaces. X-ray photoelectron spectra were obtained by AlKα radiation at 300 W and base pressure of about 3 × 10−9 mbar with an ESCALab220i-XL electron spectrometer from VG Scientific (VG Scientific Ltd., East Grinstead, Sussex, U.K.). The binding energies were calculated with the C1s line at 284.8 eV from adventitious carbon as a reference. In Figure S2 (Supporting Information), the hydroxyl surface (step 1) and the CC modified surface (step 2) were prepared based on the procedure introduced above. The chip of step 3 was prepared through immersing the chip of step 2 directly into the mannose solution (pH 9.0, 30 mM) for 10 h followed by washing carefully with distilled water and drying with a N2 stream. Vapor Condensation Imaging (VCi). VCi was conducted under a stereo microscope model XTL-500 from Guilin Optical Instrument Factory (Guilin, China) with a charge-coupled device (CCD) camera. Images were recorded after a gentle breath from the operator. The recorded images were subsequently saved and analyzed. Surface Plasmon Resonance Imaging (SPRi). Saccharide microarray chips prepared on gold slides were studied using our laboratory-built SPR imager, SPRi-TX7100. Briefly, a collimated beam of p-polarized light was directed, at a fixed angle, toward a prism on which a microarray chip was assembled via a thin layer of index matching fluid and sealed in a flow cell. The reflected light beam filtered through a narrow bandpass filter (λ 645 nm) was collected by a CCD camera (WAT-902B, Watec Co., Ltd., Japan), and the images were saved and analyzed with a laboratory-edited imaging workstation, v 1.0. The general process to monitor the immune reaction of lectins to the saccharides on the microarray using SPRi is presented next. A Tris-HCl buffer (pH 7.6, 25 mM) containing 1 mM CaCl2, 1 mM MnCl2, and 0.1% Tween 20 was pneumatically pumped through the flow cell (located in the SPRi instrument) at a flow rate of 60 μL/min until the SPR signal reached equilibrium. Then a Con A solution (50 μg/mL, in the aforementioned Tris-HCl buffer) was introduced into the

spotted, the chips were put in a sealed humid bottle (humidified by a paper filter wetted with 1 μL water per milliliter volume) at room temperature for 10 h to allow the completion of the coupling reaction. They were then washed by sonication in water at least three times to remove the unbound saccharides and dried under N2 stream. These saccharide chips were used directly or stored at −20 °C before use. For biological tests, the chips were further blocked in 1 M EOA aqueous solution (adjusted to pH 9.0 with HCl) for 3 h. Synthesis of 2,4-Dichloro-6-methoxy-triazine (MeO− CC). A 3-mL solution of 1.7 mmol CC (310 mg in acetone) was mixed with 5 mL of methanol and 0.593 mL of DIPEA (3.4 mmol). After 30 min of stirring in an ice bath, the mixed solution was concentrated by vacuum rotary evaporation, and the residue was purified through silica column chromatography (homemade with silica gel of 45−75 μm) eluted with petroleum ether/acetone (v/v = 25:1). The first eluted peak was collected and dried by vacuum rotary evaporation to give the targeted compound (MeO−CC) as white powder, with data of 1H NMR (400 MHz, DMSO, δ) at 3.9 (s, 3H, −O− CH3) and EI-MS at m/z 179 for both of calcd [C3H3N3Cl2O]− and found. Synthesis of MeO−CC-mannose. A 74-mM mannose aqueous solution (80 mg in 6 mL) was mixed with 26.4 mg of solid NaOH (equal to 0.66 mmol), 80 mg of MeO−CC (0.45 mmol), and 1 mL of acetone. After stirring at 25 °C for 8 h, the mixed solution was freeze-dried, and the crude product was purified by preparative thin layer chromatography (HP-TLC from Merck KGaA, Darmstadt, Germany) developed by 12:1:0.4 (v/v/v) isopropanal/methanol/water to remove the unreacted MeO−CC and free mannose. The product was collected from the original points, and after isolation with methanol and drying by vacuum rotary evaporation, the resulting white powder was characterized by ESI-MS, giving m/z at 306 ([M+H]+) for MeO−CC-mannose. Some MeO− CC-(mannose)2 was also found at m/z 468 ([M+H]+). This procedure is applicable to other saccharides. 1302



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cell and incubated for 10−20 min. After that, the Tris-HCl buffer was pumped into the cell again to wash the unbound ConA molecules away. The experiments were carried out at 25 °C, and if needed, an aqueous solution of phosphoric acid (0.1 M) could be introduced into the cell to remove the ConA molecules bound on the saccharide microarray, and then the Tris-HCl buffer was utilized to regenerate the chip surface. LC-MS of MeO−CC-Saccharide. All LC-MS characterizations were conducted on the LC-MS-2010EV from Shimadzu (Hong Kong) LTD., mounted with a Waters Sunfire C18 column (particle diameter 3.5 μm; length 50 mm, internal diameter 2.1 mm). The separation was conducted by elution, at a flow rate of 0.2 mL/min, with 0.1% formic acid in water (solution A) and acetonitrile (solution B) at a volume ratio of A/B changed continuously from 95/5 to 0/100 in 1.2 min. The eluted peaks were detected by a UV detector and evaporative light-scattering detector. Preparation of FITC-Labeled Con A. FITC-labeled Con A was synthesized according to a previously reported procedure.37 Briefly, 1.12 mL of Con A solution (5 mg/mL) in Tris-HCl buffer (pH 9.5) was mixed with 56 μL of FITC solution in acetone (1 mg/mL) at 20 °C and stirred for 20 h. The resulting product was dialyzed against Tris-HCl buffer (0.1 M, pH 7.0) 3 times (8 h for each). The dialyzed ConA solution was diluted with the Tris-HCl buffer (25 mM, pH 7.6, containing 1 mM CaCl2 and 1 mM MnCl2) to 0.05 mg/mL and used immediately for the subsequent experiments with microarray chips. Laser-Induced Fluorescence Imaging (LIFi). A saccharide microarray chip prepared on a glass slide was allowed to react with a solution of FITC-labeled Con A in pH 7.6 TrisHCl buffer (20 μg/mL) for 1 h. Fluorescent images were recorded using a Typhoon Trio Variable Mode Imager (Amersham, Biosciences/GE Healthcare) at a pixel size of 25 μm. The fluorescence was excited by a 488 nm laser and collected through a 520 nm filter (40 nm band-pass).

Figure 1. pH value of spotting solutions (A) controls the reaction of saccharides and even OH− from NaOH (the first line in A) with the CC-terminated surface as viewed by vapor condensed imaging (VCi) (B) and in turn their recognition abilities with Con A (C) and PNA (D) as viewed by SPRi (C and D, with pseudocolor). The spots of NaOH at pH 9.0 were used as blanks.

4 in Figure 1). The improper pH values will also influence later recognition experiments (Figure 1 C and D), which will be discussed in the next section. By basic conditions, the anchoring of a saccharide could be achieved through temperaturemodulated stepwise replacement of the three chlorines on CC. Although this stepwise replacement is mild, applicable to both of hydroxyl or amino groups,41−43 and has been explored to derivatize and/or synthesize various molecules,44−47 the direct reaction of CC with saccharide’s hydroxyls has not yet been studied in depth. To the best of our knowledge, this is the first attempt, especially with respect to the direct construction of saccharide microarray chips.55 By using CC as a linker, hydroxyl surfaces were shown to be better than amino ones. We have tried to use an aminoterminated surface to anchor intact saccharides via CC, but the spot quality and signals were poor or even undetectable (Figure S1, Supporting Information). Only when a hydroxyl surface was used and reacted with the CC through replacement of its first chlorine could the remaining chlorines keep high reactivity with the saccharide’s hydroxyls, which also agrees with the literature.42 Hydroxyls on a solid surface are also preferred to suppress nonspecific adsorptions, which is in generel a big issue in surface-related recognition reactions. To exclude possible nonchemical adsorptions, saccharides were purposely spotted on a hydroxyl-terminated surface (before CC modification) (Figure 2B and E) and on a NaOH (pH 9.0)-hydrolyzed CC-surface (Figure 2C and F). No recognizable or only negligible signals were verified by VCi. These results effectively foreclosed the possibility of physical or nonspecific adsorption from the designed strategy. The stepwise reaction was verified by XPS measurement using mannose as a testing sample. The immobilization of CC on a hydroxyl-terminated surface (step 1 in Figure S2, Supporting Information) was found to cause an increase of nitrogen peak at ca. 400 eV and an appearance of a new binding energy peak at 200.8 eV corresponding to chlorine (step 2 in Figure S2, Supporting Information). The chlorine signal disappeared (step 3 in Figure S2, Supporting Information) after the CC-immobilized surface was further treated with a mannose solution in basic conditions (required to make the reaction happen and to hydrolyze all the unreplaced chlorine). An evident decrease of the nitrogen signal was also observed

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RESULTS AND DISCUSSION Strategy and Mechanism to Anchor Intact Saccharides via CC Chemistry. A universal strategy was designed to immobilize any an intact saccharide on a solid surface by CCbased chemistry as illustrated in Scheme 1. The chemistry includes three key steps: formation of hydroxyl-terminated surfaces; modification of the hydroxyl terminal with CC; and spotting of saccharide solution on the CC-terminated surfaces. The hydroxyl-terminated surfaces can be prepared by different reactions dependent on the nature of solid materials used. For example, a glass slide can be hydroxyl-terminated by silanization with a reagent containing hydroxyl groups. In this article, it was achieved by treating the glass slides first with APTMS and then with EDC/HPA to have biocompatibility. The hydroxyl groups can be introduced onto a gold surface by direct treatment with a hydroxyl alkyl mercaptan or by more steps of reactions, such as, treatment first with MUA and then with EDC/NHS/EOA. The latter one needs more reaction steps but is also more costeffective and was adopted in this work. On the hydroxyl-terminated surfaces, a layer of CC was easily formed, followed a layer of saccharides, in basic conditions. Figure 1 shows that neutral and acidic conditions (lines 2 and 5 in Figure 1) cannot produce visible images by the vaporcondensed imaging (VCi) method37,53,54 (useful to quickly reveal the hydrophilic position from the hydrophobic). Only at pH above 8.0 can the immobilization be successful (lines 3 and 1303



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Figure 2. VCi images of saccharide microarray prepared on (A) gold/ hydroxyl/CC surface, (B) gold/hydroxyl surface (free of CC), (C) gold/hydroxyl/hydrolyzed CC surface (treated by NaOH beforehand), (D) glass/hydroxyl/CC surface, (E) glass/hydroxyl surface (free of CC), and (F) glass/hydroxyl/hydrolyzed CC surface (treated by NaOH beforehand).

Figure 3. Comparison of 1H NMR spectra between MeO−CCmannose (A), free mannose (B), and free MeO−CC (C).

Except for the maintenance of the anomeric configuration, the CC did not actually selectively link a special site on the saccharide molecules. The linking reaction happened stochastically along all the hydroxyl groups on a saccharide. Figure 3A shows that, after the mannose reacted with the MeO−CC, a series of new peaks appeared at 4.4−5.5 ppm and 6.0−6.2 ppm, accompanied by a large reduction of peaks at 3.4−4.1 ppm for C2−C6 and 4.9−5.2 for C1. Clearly, the former peaks could be assigned to the protons on C2−C6 of the reacted mannose and the latter to the proton on C1.57 They shifted for about 1 ppm away the original position due to the attachment of CC. Unfortunately, calculation of an exact ratio on each reacted 1Hsite was not possible because the resonant 1H peaks overlapped each other seriously and with other signals. Fortunately, these overlapped peaks are enough to deduce that MeO−CC has roughly a similar reactivity toward the saccharide’s hydroxyls. This was further validated in the reaction of MeO−CC with fructose (Figure S5, Supporting Information). The proton signals on C1−C5 moved from 3.5−4.2 ppm for free fructose (Figure S5B, Supporting Information) to 4.2−4.5 ppm and 5.1−5.4 ppm, for MeO−CC-fructose (Figure S5A, Supporting Information), indicating again the nonselective linking of CC with the hydroxyls on fructose. Considering that the recognition ability of the immobilized saccharides can be well conserved (to be discussed in the next section), it can interestingly be concluded that which site is linked on a saccharide is not as important as what we expected if the original cyclic structure and configuration of a saccharide can be well conserved. Preparation of Bioaffinity-Conserved Saccharide Microarrays. On the basis of the proposed chemical strategy (Scheme 1), 15 different saccharides, including mono-, oligo-, and polysaccharides, were successfully immobilized on the same hydroxyl-terminated chip by spotting them in an addressable way (Figure 4A and B). Different from most of the currently used immobilizing strategies which may require complicated synthetic steps, intensive modification of saccharides, or expensive coupling reagents, this CC-based chemistry needs just to drop or spot the basic aqueous saccharide solutions on the chip surface and then leave it at room temperature to react for a certain time. Both reducing and nonreducing (such as sucrose and trehalose, spot (4,iii) and (3,i) in Figure 4B) saccharides can directly be spotted on a CC-terminated surface. Further modification of the saccharides is allowed but not

due to the embedment of its atoms under the newly immobilized mannose. These clear evidences revealed not only the ascertained route of step-by-step reactions during the deposition of mannose but also the substitution sites on CC. However, the XPS data revealed no information about the linking site(s) on a mannose molecule, which contains at least 5 different hydroxyl reaction positions. More importantly, we need to know if the anomeric configuration can be conserved after the reaction, which is critical to determine the binding affinity toward lectins.56 To have insight into the issues, the mannose-CC-deposited slides were inspected by solid state 1H NMR but failed; the sensitivity was too poor to yield any useable information. Instead, mannose-CC-gold nanoparticles were synthesized and subjected to 1H NMR, and the signals remained under recognition (Figure S3, Supporting Information). In order to increase the detection sensitivity, soluble methoxy-CC-sugars were then synthesized by making the CC react first with methanol (MeOH, Scheme S1A, Supporting Information), instead of the solid slides or particles, to give MeO−CC, and then with target saccharides (Scheme S1B, Supporting Information). By liquid chromatography−mass spectrometry (LC-MS) (Figure S4, Supporting Information), MeO−CC was confirmed to be capable of reaction with both of aldoses (Figure S4A, Supporting Information) and ketoses (Figure S4B, Supporting Information), as in the case of solid surfaceCC, while its third chlorine could be replaced by either a second saccharide or −OH (Figure S4A, Supporting Information). These MeO−CC-saccharides were thus considered to be capable of simulating the case of saccharides immobilized on CC-modified slides and subjected to 1H NMR. As expected and also to our surprise, the most preferred αanomer was well maintained after the saccharides reacted with MeO−CC. For example, the ratio of α-/β-anomers was 2.0:1 for MeO−CC-mannose measured by peak areas at 6.2/6.0 (Figure 3A), similar to 2.1:1 measured at 5.2/4.9 ppm for free mannose (Figure 1B). The immobilization reaction did not change the critical ratio or did not damage the original cyclic structure of saccharides. This foretells that the new method can well conserve the recognition function. 1304



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Figure 4. VCi (B) and SPRi (C and D) images of 15 intact saccharides (A) spotted on a CC-modified gold surface. VCi was conducted just after immobilization (B) and the pseudocolored SPRi after incubation with PNA (C) or ConA (D).

Figure 5. LIFi (A) and SPRi (B, pseudo colored) images of saccharide microarrays measured after reaction with FITC-labeled ConA (A) or intact ConA (B). The chip for LIFi was prepared on a glass slide and for SPRi on gold film. The linear profiles represent the relative signal intensities across the dashed line on the images.

required, suggesting that the new strategy is universally applicable. Anchoring becomes now much simple and easy to manipulate. The experimental cost is greatly reduced due to the use of very cheap CC. The bioaffinity of saccharides spotted this way was shown to be well preserved. To demonstrate, the chip with 15 different saccharides was made to react with two lectins, concanavalin A (ConA) and peanut agglutinin (PNA). According to the literature,56 ConA will specifically recognize terminal α-Dglucose and α-D-mannose in an affinity order of mannose > glucose, and PNA specifically recognizes terminal β-D-galactose. The expected results were observed by surface plasmon resonance imaging (SPRi): Figure 4 shows clearly that the galβ1-structured saccharides, such as lactose and galactose, exhibit recognition signals with PNA (Figure 4C); all the gluα1- or manα1-structured saccharides, including glucose, mannose, trehalose, maltose, sucrose, malhextose, and dextran, have strong binding signals with ConA (Figure 4D); and all the nonlectin targets do not show any visible signal. Unexpectedly, fructose was measured to have a weak but visible interaction with ConA in our measurement (spots (1,i) in Figure 4D). After some search of the literature, this weak affinity was found to have been documented,58−60 but it is generally too weak to be visible by many existing methods. The visibility of this weak recognition further highlights the merit of our new method. These SPRi results were experimentally compared with laserinduced fluorescence imaging (LIFi) by using FITC-labled Con A, which is frequently used in bioassays. Figure 5 shows a parallel illustration of both LIFi and SPRi images. The recognition behaviors of the immobilized saccharides agree well with each other, demonstrating that the newly proposed strategy is reliable and applicable to also LIFi assays if labeled samples are available. It is until now satisfactory that all the tried saccharides, small and large or reducing and nonreducing, can be well immobilized by the new method and can play their expected recognition roles with the selected lectins. A special highlight is that the new strategy can well conserve the recognition ability of small saccharides, including even monosaccharides. According to the above-elucidated chemical mechanism, there are two reasons responsible for the conservation of bioaffinity of the immobilized saccharides: first, the CC-based coupling reaction

is mild and gentle. Different from the direct coupling of intact saccharides by reductive amination of the aldehyde groups, the CC-reaction does not directly touch the atoms of the cyclic scaffold of saccharides but reacts only at room temperature with the far and nonring atoms on hydroxyls having the original cyclic structure and with the anomeric configuration easily maintained (Figure 3). Second, CC offers three rotation axes with a theoretical angle of 120°. It can rotate on a surface even after anchoring, and similarly, the saccharides anchored on the CC ring can also revolve freely round the linking bond, making them more easily adjust or adapt their orientation to favor their collaborative recognition for a better “cluster effect”39 (steps 3 and 4 in Scheme 1). In recognition, the hydroxyl groups should be important because they contribute to the formation of the hydrogen bond,56 but in this test, the use of one hydroxyl for saccharide immobilization (Figure 3 and Figure S4, Supporting Information) did not show detectable impact on the recognition behavior of saccharides. This can also be explained by the free rotation offered by CC. In short, the CC-based chemistry opens an ideal way to prepare bioaffinity-preserved saccharide chips through chemical immobilization, which coincides with our original intention to design this strategy. Recognition Features of the Immobilized Saccharides. By the chip in combination with imaging techniques, the recognition behaviors were inspected to validate the real applicability of the proposed method. Both LIFi (Figure 5A) and SPRi (Figures 5B and 4C,D) showed that the recognition signals were dependent on the type of saccharides immobilized and lectins used, which agrees with the theoretical expectation. By closer inspection of Figure 5A or B, it could be found that the signals increased also with the molecular weight or residue number of saccharides. Figure 4D shows that the larger dextran (spots (5, iii) in Figure 4D) gives stronger signals than the smaller one (spots (5, i) or (5, ii) in Figure 4D). This is reasonable because a molecule with more binding sites must link more targets and produce stronger signals than a molecule with less binding sites. There are also differences in the recognition dynamics as revealed by the SPR observation shown in Figure 6. Mannose recognized Con A as the fastest, glucose as the second, and dextran as the slowest. This implies that the large molecules need a longer time than small ones to 1305



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binding−regenerating cycles. On shelf life, more experimental checks were conducted, and it was found that the fabricated microarray chips could maintain their binding performance after they were stored at −20 °C for at least 2 months. These results demonstrate that the immobilization strategy is robust and can benefit its practical application in the future.

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CONCLUSIONS A new strategy was proposed to chemically immobilize various intact saccharides on a solid surface using CC as a key linking agent. An easy, cost-effective, and universal method was then established for the preparation of saccharide-dotted chips with well-preserved bioaffinity and enough stability. The key innovation present was the use of CC as a linker, which is very economical, abundant, and easily available. It is due to the use of CC that all the intact saccharides could directly be immobilized on hydroxyl-terminated solid surfaces which are biocompatible and can largely suppress the nonspecific adsorption effect. The highly attractive features are that the recognition affinity of the covalently anchored small (monosaccharides) and large (polysaccharides) saccharides could be well conserved as was proved by the recognition with lectins. This conservation was ascribed to the rotable structure of CC that offers three rotary axes, allowing the anchored saccharides to adapt their spatial position to favor their recognition in a cluster format, and to the mild and gentle reaction of CC with the nonring-determining atoms of hydroxyls, allowing maintenance of the original structure and configuration of the saccharides after immobilization. The CC was shown to react stochastically with each hydroxyl on a saccharide. This phenomenon suggests that replacement of one of the hydroxyl protons on a saccharides may not necessarily impact its bioaffinity if its ring structure is not disturbed, which opens a new trace to anchor saccharides. Furthermore, the CC-based chemistry is in theory extendable to the immobilization of other biological molecules having hydroxyl group(s) or amino groups (better on amino-terminated surfaces). In short, the new strategy offers a favorable toolbox with various means for exploration of better methods, not only the discussed one.

Figure 6. SPR sensorgram of ConA binding to the different saccharides immobilized by CC-based chemistry. The dynamic experiment includes 2 steps: (a) incubation of the saccharide chip with ConA solution (50 μg/mL, in the pH 7.6 Tris-HCl buffer); and (b) washing of the chip surface with Tris-HCl buffer at pH 7.6.

adjust their structure and/or position for recognition or that the dextran has less affinity than mannose toward ConA. Nevertheless, the recognition process took several minutes, not surpassing 15 min. These are fast recognition reactions. In contrast, the large dextran showed the fastest washing-off dynamic among the tested saccharides (Figure 6), which agrees with the suspicion that its binding affinity is weaker than that of mannose with ConA. This is also highly consistent with the reported results.37,56 It can thus be concluded that the CCbased chemistry can preserve not only the recognition ability of the immobilized saccharides but also a way to study the relative strength of the binding affinity. Stability of the Microarrayed Saccharide Chips. By stability, we mean two events here: the first is with respect to their shelf life or storage time; and the second is concerning their reusable times in a continuous recognition experiment. The reusable times were checked by SPR sensing of the ConAbased recognition−regeneration cycle. In a typical experiment, a chip could be reused for more than 10 cycles or for 48 h in acidic washing conditions without an evident loss of SPR sensitivity. Figure 7 shows an example where the highly reproducible SPR signals were measured during 4 successive

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ASSOCIATED CONTENT

S Supporting Information *

VCi images of saccharide microarray spotted on CC-aminofunctioned surfaces; the XPS N 1s and Cl 2p spectra of the surface in different preparing steps; 1H NMR spectra of gold nanoparticle modified with mannose through CC-chemistry; LC-MS characterization of products from the reaction of MeO−CC with saccharides; comparison of 1H NMR spectra among MeO−CC-fructose, free fructose, and free MeO−CC; and scheme of model reaction of CC with saccharides. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun North First Street 2, 100190 Beijing, PR China. Tel: +86-10-62618240. Fax: +86-10-62559373. E-mail: [email protected].

Figure 7. SPR sensorgram of successive binding-regenerating cycles measured from the recognitions of ConA with maltose and mannose immobilized on the same CC surface. Each cycle includes 4 steps: (a) incubation of the saccharide chip with ConA solution; (b) wash of the chip surface with Tris-HCl buffer at pH 7.6; (c) removal of the ConA molecules by flashing the chip surface with 0.1 M phosphoric acid; and (d) regeneration of the chip surface with Tris-HCl buffer at pH 7.6.

Notes

The authors declare no competing financial interest. 1306



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ACKNOWLEDGMENTS We acknowledge the financial support from NSFC (No. 21027003 & 91117010), MOST, and CAS.

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ABBREVIATIONS

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REFERENCES

Con A, concanavalin A; PNA, peanut agglutinin; FITC, fluorescein isothiocyanate; APTMS, 3-aminopropyltrimethoxysilane; EDC, 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide; CC, cyanuric chloride; MUA, 11-mercaptoundecanoic acid; NHS, N-hydroxysuccinimide; DIPEA, N,Ndiisopropylethylamine; HPA, 4-hydroxyphenylacetic acid; EOA, ethanolamine; EDA, ethanediamine

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Elegant Chemistry to Directly Anchor Intact Saccharides on Solid

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