Screening Lectin-Binding Specificity of Bacterium by Lectin Microarray

Anal. Chem. 2010, 82, 9240–9247

Screening Lectin-Binding Specificity of Bacterium by Lectin Microarray with Gold Nanoparticle Probes Jingqing Gao,†,‡ Dianjun Liu,† and Zhenxin Wang†,* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China, 130022, and Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China To develop a novel high-throughput tool for monitoring specific affinity of microbes with lectins, a kind of lectin microarray has been fabricated by immobilizing lectins on epoxide-derivatized glass slides and used to capture microbes. The capturing events are marked by attachment of lectin-conjugated gold nanoparticles followed by silver deposition to enhance the resonance light scattering (RLS) of the particles. The interactions of 16 lectins with four bacteria and one fungus were profiled by this approach. We demonstrated that the gold-nanoparticle-labeled array was suitable for identifying the binding affinity of lectin with bacterium, as well as determining the bacterium with high sensitivity. More importantly, we found that the growth of microbial strains in different culture media resulted in significant changes in their binding affinities with lectins, which might be important to the pathogenesis of the organisms. The recent discovery of glycomics highlights our lack of knowledge about glycosylation in bacteria because their surfaces are fully decorated with carbohydrates, which exist as glycoconjugates, such as glycoproteins, glycolipids, glcosaminoglycans, and proteoglycans.1-3 Expression of these molecules, especially their carbohydrate structures, is frequently specific to bacterium or cell type.4-12 In particular, specific binding affinities between carbohydrates and proteins (such as lectins and antibodies) also initiate * To whom correspondence should be addressed. E-mail: [email protected]. Fax: (+86) 431-85262243. † Changchun Institute of Applied Chemistry. ‡ Graduate School of the Chinese Academy of Sciences. (1) Luzhetskyy, A.; Bechthold, A. Appl. Microbiol. Biot. 2008, 80, 945–952. (2) Griffith, B. R.; Langenhan, J. M.; Thorson, J. S. Curr. Opin. Biotechnol. 2005, 16, 622–630. (3) Schmidt, M. A.; Riley, L. W.; Benz, I. Trends Microbiol. 2003, 11, 554– 561. (4) Pilobello, K. T.; Mahal, L. K. Curr. Opin. Chem. Biol. 2007, 11, 300–305. (5) Tateno, H.; Uchiyama, N.; Kuno, A.; Togayachi, A.; Sato, T.; Narimatsu, H.; Hirabayashi, J. Glycobiology 2007, 17, 1138–1146. (6) Yildiz, F. H. Res. Microbiol. 2007, 158, 195–202. (7) Hsu, K.-L.; Pilobello, K. T.; Mahal, L. K. Nat. Chem. Biol. 2006, 2, 153– 157. (8) Disney, M. D.; Zheng, J.; Swager, T. M.; Seeberger, P. H. J. Am. Chem. Soc. 2004, 126, 13343–13346. (9) Joo, K. L.; Mao, S.; Sun, C.; Gao, C.; Blixt, O.; Arrues, S.; Hom, L. G.; Kaufmann, G. F.; Hofmann, T. Z.; Coyle, A. R.; Paulson, J.; FeldingHabermann, B.; Janda, K. D. J. Am. Chem. Soc. 2002, 124, 12439–12446. (10) Ertl, P.; Mikkelsen, S. R. Anal. Chem. 2001, 73, 4241–4248. (11) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357–2364.

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infection of host cells by bacteria and viruses, as most host receptors or coreceptors of microbes are glycocomplexes.1-15 Therefore, understanding of the molecular basis for carbohydrate-protein interactions not only provides valuable information on biological processes in living organisms but also aids the development of potent biomedical agents.13-15 Many biophysical and biochemical methods [such as chromatographic methods, mass spectrometry, and nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) spectroscopy] have been used to study the details of carbohydrate-protein interactions.16-22 Usually, these methods examine a single carbohydrate motif, which is a slow process that is limited by expertise, equipment, and time. In addition, many of these assays are also disadvantaged by requirements of extraction and purification steps for modifying the structures of these glycans. Compared with its counterparts, genomics and proteomics, glycomics has suffered from a lack of powerful microarray-based high-throughput screening tools that are readily available for studying nucleic acids and proteins.4,23-29 Microarray-based assays offer prominent advantages over common screening (12) Kitov, P. I.; Sadowska, J. M.; Mulvey, G.; Armstrong, G. D.; Ling, H.; Pannu, N. S.; Read, R. J.; Bundle, D. R. Nature 2000, 403, 669–672. (13) Disney, M. D.; Seeberger, P. H. Chem. Biol. 2004, 11, 1701–1707. (14) Sharon, N.; Lis, H. Glycobiology 2004, 14, 53R–62R. (15) Peters, T.; Scheffler, K.; Ernst, B.; Katopodis, A.; Magnani, J. L.; Wang, W. T.; Weisemann, R. Angew. Chem., Int. Ed. Engl. 1995, 34, 1841–1844. (16) Blixt, O.; Han, S.; Liao, L.; Zeng, Y.; Hoffmann, J.; Futakawa, S.; Paulson, J. C. J. Am. Chem. Soc. 2008, 130, 6680–6681. (17) Wakao, M.; Saito, A.; Ohishi, K.; Kishimoto, Y.; Nishimura, T.; Sobel, M.; Suda, Y. Bioorg. Med. Chem. Lett. 2008, 18, 2499–2504. (18) Ratner, D. M.; Adams, E. W.; Su, J.; O’Keefe, B. R.; Mrksich, M.; Seeberger, P. H. ChemBioChem 2004, 5, 379–383. (19) Dam, T. K.; Brewer, C. F. Chem. Rev. 2002, 102, 387–430. (20) Perou, C. M. Nature 2000, 406, 747–752. (21) Somers, W. S.; Tang, J.; Shaw, G. D.; Camphausen, R. T. Cell 2000, 103, 467–479. (22) Mann, D. A.; Kanai, M.; Maly, D. J.; Kiessling, L. L. J. Am. Chem. Soc. 1998, 120, 10575–10582. (23) Weinrich, D.; Jonkheijm, P.; Niemeyer, C. M.; Waldmann, H. Angew. Chem., Int. Ed. 2009, 48, 7744–7751. (24) Oyelaran, O.; McShane, L. M.; Dodd, L.; Gildersleeve, J. C. J. Proteome Res. 2009, 8, 4301–4310. (25) Danica, P. G.; David, Y. G. Nature 2007, 446, 1000–1007. (26) Wilson, R.; Cossins, A. R.; Spiller, D. G. Angew. Chem., Int. Ed. 2006, 45, 6104–6017. (27) Park, S.; Lee, M. R.; Pyo, S. J.; Shin, I. J. Am. Chem. Soc. 2004, 126, 4812– 4819. (28) Fukui, S.; Feizi, T.; Galustian, C.; Lawson, A. M.; Chai, W. G. Nat. Biotechnol. 2002, 20, 1011–1017. (29) Houseman, B. T.; Mrksich, M. Chem. Biol. 2002, 9, 443–454. 10.1021/ac1022309  2010 American Chemical Society Published on Web 10/21/2010

techniques such as enzyme-linked immunosorbent assays (ELISAs), because several thousand binding events can be screened on a single glass slide and only minuscule amounts of analytes and ligands are required.4,23-29 Recently, lectin-microarray-based techniques have been developed for analysis of the dynamic bacterial glycome and rapid evaluation of microbial cellsurface carbohydrates because lectins have been used as a profiling tool to identify and differentiate bacterial strains, typically through agglutination assays.7,30,31 Meanwhile, successful pathogen detection is important for public health because the threat of infectious disease is increasing dramatically as bacteria develop new drug resistances, bioterrorism looms, and contaminated food reaches the public. Current standards for pathogen detection rely on culture plating and microscopy technologies. However, these methods are very timeconsuming, as organisms have to be isolated and grown, and usually a series of biochemical tests (such as ELISAs) must be completed for identification.32 Techniques such as the polymerase chain reaction (PCR) are also used for the amplification of pathogen-specific DNA sequences and identifying different types of bacteria with high sensitivity. Because impurities contained within the sample can inhibit the PCR, the PCR system falls short in its ability to analyze environmental samples; thus, a degree of sample preparation is necessary prior to the analysis.33 Because of their potential specificity and sensitivity (governed by the recognition element, e.g., an antibody), biosensors are particularly attractive as a means to detect and identify potential pathogenic microbes in a timely manner.34,35 In particular, microarray-based sensors can offer high-throughput assays for bacterial gene expression, toxin detection, and determination of the bioavailability of bacterial growing matrixes.36-40 Currently, microarray techniques mainly rely on the use of fluorescent molecular dye labels that have several potential drawbacks, such as low sensitivity and photoinstability of the dyes employed.41,42 Inorganic nanoparticles, such as gold and silver nanoparticles, can offer an unique set of physical properties that can be exploited in biological detection assays as an alternative (30) Garnett, J. A.; Liu, Y.; Leon, E.; Allman, S. A.; Friedrich, N.; Saouros, S.; Curry, S.; Soldati-Favre, D.; Davis, B. G.; Feizi, T.; Matthews, S. Protein Sci. 2009, 18, 1935–1947. (31) Hsu, K.-L.; Gildersleeve, J. C.; Mahal, L. K. Mol. BioSyst. 2008, 4, 654– 662. (32) Murray, P. R.; Baron, E. J.; Landry, M. L.; Jorgensen, J. H.; Pfaller, M. A. Manual of Clinical Microbiology, 9th ed., American Society of Microbiology: Washington, DC, 2007. (33) Fagan, P. K.; Hornitzky, M. A.; Bettelheim, K. A.; Djordjevic, S. P. Appl. Environ. Microbiol. 1999, 65, 868–872. (34) Jongerius-Gortemaker, B. G. M.; Goverde, R. L. J.; van Knapen, F.; Bergwerff, A. A. J. Immunol. Methods 2002, 266, 33–44. (35) Hartley, H. A.; Baeumner, A. J. Anal. Bioanal. Chem. 2003, 376, 319–327. (36) Van Dyk, T. K.; DeRose, E. J.; Gonye, G. E. J. Bacteriol. 2001, 183, 5496– 5505. (37) Mbeunkui, F.; Richaud, C.; Etienne, A. L.; Schmid, R. D.; Bachmann, T. T. Appl. Microbiol. Biotechnol. 2002, 60, 306–312. (38) Brogan, K. L.; Walt, D. R. Curr. Opin. Chem. Biol. 2005, 9, 494–500. (39) Lee, J. H.; Mitchell, R. J.; Kim, B. C.; Cullen, D. C.; Gu, M. B. Biosens.Bioelectron. 2005, 21, 500–507. (40) Suo, Z. Y.; Yang, X. H.; Avci, R.; Deliorman, M.; Rugheimer, P.; Pascual, D. W.; Idzerda, Y. Anal. Chem. 2009, 81, 7571–7578. (41) Schweitzer, B.; Wiltshire, S.; Lambert, J.; O’Malley, S.; Kukanskis, K.; Zhu, Z.; Kingsmore, S. F.; Lizardi, P. M.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10113–10119. (42) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757–1760.

to the use of fluorescent dyes.43-48 As a pioneering effort, Mirkin and co-workers developed a DNA-modified gold-nanoparticlebased assays for highly sensitive and selective detection of DNAs and proteins on microarrays.42,49-51 In these assays, DNA-gold nanoparticle conjugates are used as labels to detect specific analytes, namely, DNA or proteins, followed by a resonant light scattering (RLS) enhancement step based on the electroless deposition of silver onto the gold particles. Based on the same detection principle, a microarray format for detection of carbohydrate-lectin interactions and kinase functionality/inhibition by gold nanoparticle probes has also been developed by Wang et al.52,53 Compared with the conventional fluorescence-based microarray format, the RLS-based microarray format can be read by a white-light source scanner, thereby providing significant cost savings in terms of instrumentation. As reported in this article, we developed a lectin-microarraybased RLS assay for the examination of microbial glycans. In this assay, microbes are captured by the lectin arrays and marked by attachment of GS II- (Griffonia simplicifolia II-) conjugated gold nanoparticles (GS II@GNPs) followed by silver deposition for signal enhancement. To test the feasibility of the assay, the interactions of 16 lectins (details of these lectins are listed in Table 1 and in Table S1 of the Supporting Information) with four bacteria and one fungus (E. coli, E. cloacae, B. subtilis, S. aureus, and S. cerevisiae) were examined. Specific binding patterns of microbes with the immobilized lectins were observed, which provides a simple means to fingerprint microbes based on their surface glycans. EXPERIMENTAL SECTION Materials and Reagents. Escherichia coli DH5R (E. coli) bacterial strain was purchased from Dingguo Ltd. (Beijing, China). Staphyloccocus aureus Rosenbach (S. aureus) was obtained from China-Japan Union Hospital of Jilin University (Jilin, China). Enterobacter cloacae (E. cloacae), Bacillus subtilis (B. subtilis), and Saccharomyces cerevisiae (S. cerevisiae) microbial strains were purchased from China Center for Virus Culture Collection, Institute of Microbiology, Chinese Academy of Sciences (Beijing, China). Sixteen lectins [Ricinus communis agglutinin I (RCA 120), Maackia amurensis lectin I (MAA I), Maackia amurensis lectin II (MAA II), Ulex europaeus agglutinin I (UEA), soybean agglutinin (SBA), Erythrina cristagalli lectin (ECA), Sambucus nigra lectin (SNA), Griffonia simplicifolia I (GS I), Griffonia simplicifolia II (GS II), wheat germ agglutinin (WGA), concanavalin A isolated from Canavalia ensiformis seeds (Con A), Aleuria aurantia lectin (43) Yguerabide, J.; Yguerabide, E. E. J. Cell. Biochem. 2001, 37 (Suppl), 71– 81. (44) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631–635. (45) Alivisatos, A. P. Nat. Biotechnol. 2004, 22, 47–52. (46) You, C.-C.; De, M.; Rotello, V. M. Curr. Opin. Chem. Biol. 2005, 9, 639– 646. (47) Leggett, R.; Lee-Smith, E. E.; Jickells, S. M.; Russell, D. A. Angew. Chem., Int. Ed. 2007, 46, 4100–4103. (48) Medley, C. D.; Smith, J. E.; Tang, Z.; Wu, Y.; Bamrungsap, S.; Tan, W. Anal. Chem. 2008, 80, 1067–1072. (49) Cao, Y. C.; Jin, R.; Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 14676–14677. (50) Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884–1886. (51) Kim, D.; Daniel, W. L.; Mirkin, C. A. Anal. Chem. 2009, 81, 9183–9187. (52) Gao, J. Q.; Liu, D. J.; Wang, Z. X. Anal. Chem. 2008, 80, 8822–8827. (53) Sun, L. L.; Liu, D. J.; Wang, Z. X. Anal. Chem. 2007, 79, 773–777.


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Table 1. Lectins and Their Specially Binding Saccharide Group

a

binding specificitya

number

lectin

abbreviation

1 2 3 4 5 6 7 8 9 10 11

RCA 120 MAA I MAA II UEA SBA ECA SNA GS I GS II WGA ConA

Galβ(1,4)GlcNAcβ1 Galβ(1,4)GlcNAc R-2,3 sialic acid R-fucose terminal GalNAc Gal β-1,4GlcNAc R-2,6 sialic acid R-galactose terminal GlcNAc β-GlcNAc, sialic acid, GalNAc branched and terminal mannose, terminal GlcNAc

12

Ricinus communis agglutinin I Maackia amurensis lectin I Maackia amurensis lectin II Ulex europaeus agglutinin I soybean agglutinin Erythrina cristagalli lectin Sambucus nigra lectin Griffonia simplicifolia I Griffonia simplicifolia II wheat germ agglutinin concanavalin A isolated from Canavalia ensiformis seeds Aleuria aurantia lectin

AAL

13 14 15 16

Narcissus pseudonarcissus lectin Datura stramonium lectin Lens culinaris agglutinin Phaseolus vulgaris agglutinin

NPL DSL LcH PHA

R-fucose, fucose-(R-1,6) N-acetylglucosamine, or (R-1,3) N-acetyllactosamine R-mannose GlcNAc β-1,4GlcNAc oligomers complex (Man/GlcNAc core with R-1,6 fucose) D-GalNAc

The binding specificity is obtained from manufacturer’s introduction.

(AAL), Narcissus pseudonarcissus lectin (NPL), Datura stramonium lectin (DSL), Lens culinaris agglutinin (LcH), and Phaseolus vulgaris agglutinin (PHA); see Table 1 and Table S1 (Supporting Information) for more details] and biotinylated Griffonia simplicifolia II (GS II biotin) were products of Vector Laboratory Ltd. (Burlingame, CA). CALNN and CALNNGK(biotin)G were obtained from Scilight Biotechnology Ltd. (Beijing, China). Bovine serum albumin (BSA), hydrogen tetrachloroaurate trihydrate (HAuCl4 · 3H2O), and sliver enhancer were purchased from Sigma Corp. (St. Louis, MO). Epoxide-modified glass microscope slides were obtained from CapitalBio Ltd. (Beijing, China). All other chemicals were analytical grade and were used as received. Milli-Q water (18.2 MΩ cm-1) was used in all experiments. Preparation of Gold Nanoparticles and Lectin-Conjugated Nanoparticles. Peptide-stabilized nanoparticles (13 nm) were prepared by a previously reported peptide capping procedure.54 Generally, an aqueous solution of peptide mixture [with a molar ratio of CALNN to CALNNGK(biotin)G of 9:1] was added to the solution of 13-nm citrate-stabilized gold nanoparticles (5 nM) to give a final concentration of total peptide of 1.5 mM. After a 1-h incubation, excess peptides were removed by repeated centrifugation (∼16100g, three times) using an Eppendorf centrifuge (Eppendorf, Hamburg, Germany). Then, 1 mL of peptide-stabilized particles (5 nM) was subsequently incubated with 70 µL of avidin (1 mg/mL) for 1 h at room temperature, and the mixture was purified by centrifugation to obtain avidin-functional gold nanoparticles (avidin@GNPs). For the preparation of GS II-functionalized gold nanoparticles (GS II@GNPs), 1 mL of avidin@GNPs (5 nM) was reacted with 80 µL of GS II biotin (2 mg/mL) for 1 h at room temperature and then purified by centrifugation (∼9908g, three times). After purification, all of the materials were redispersed in D-Hanks buffer (137 mM NaCl, 5.4 mM KCl, 0.37 mM Na2HPO4, 0.44 mM KH2PO4, and 4.2 mM NaHCO3, pH 7.2-7.4, sterilized) and stored at 4 °C. Lectin Microarrays. Epoxide-modified glass microscope slides were used to fabricate lectin microarrays by the standard (54) Levy, R.; Nguyen, T. K.; Thanh, R.; Doty, C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem. Soc. 2004, 126, 10076–10084.

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procedure with a SmartArrayer 48 system (Capitalbio Ltd., Beijing, China).55 Lectins were dissolved in 15 µL of spotting buffer [pH 8.0, 10 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), 0.15 M NaCl, 10 mM monosaccharide supplemented with 15% (v/v) glycerol] at a concentration of 1 mg/mL and spotted on the glass microscope slides by noncontact spraying. After an overnight reaction at 30 °C in a vacuum environment, the slides were rinsed with 50 mL of washing buffer [pH 8.0, 50 mM phosphate buffer (PB), 0.15 M NaCl supplemented with 0.1% (v/v) polyoxyethylene (20) sorbitan monooleate (Tween-20); three times] and then immersed in 10 mL of blocking buffer [pH 8.0, 50 mM PB, 0.15 M NaCl supplemented with 0.5% (w/w) BSA and 0.1 M ethanolamine] at 30 °C for 1 h to remove remaining free epoxide groups. Microbe Culturing. All microbial strains were cultivated following manufactures’ culturing guidelines. Typically, E. coli and S. aureus strains were cultured in Luria-Bertani (LB) culture medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl), B. subtilis and E. cloacae strains were cultured in nutrient medium (10 g/L tryptone, 3 g/L beef extract, 5 g/L NaCl), and S. cerevisiae strain was cultured in yeast nutrient medium (20 g/L tryptone, 10 g/L yeast extract, 20 g/L maltose). For the environmental effect studies, (1) E. coli and S. aureus strains were cultured in LB culture medium supplemented with a certain amount of milk for 16 h, (2) E. coli and S. aureus were cultured in nutrient medium or yeast nutrient medium for 16 h, (3) B. subtilis and E. cloacae strains were cultured in LB culture medium or yeast nutrient medium for 16 h, and (4) S. cerevisiae strain was cultured in LB culture medium and nutrient medium for 16 h. After culturing, all of these microbial strains were centrifuged at 5000g for 8 min and then resuspended in D-Hanks buffer at the desired concentrations. In all experiments, the concentrations of microbes were determined by optical density at 600 nm (OD600). Microbe Detection. For microbe detection, the lectin arrays were incubated with microbial strains that were diluted to the desired concentration with 50 µL of D-Hanks buffer. Following a 1-h incubation at room temperature with gentle shaking (130 rpm), the slides were rinsed with D-Hanks buffer (50 mL, three times) (55) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760–1763.

Figure 1. Schematic representation of (a) one spot on the microarray showing, from left to right, the binding of microbe with spotted lectins and subsequently labeled with GS II@GNPs, the silver enhancement step, and reading by detection of resonant light scattering (RLS) to obtain the binding patterns. (b) Details of binding microbe with lectin microarray and GS II@GNPs. The illustration is not drawn to scale.

and Milli-Q water (50 mL). Then, the slides were dried by centrifugation (200g for 5 min). After cell adhesion, the slides were incubated with 30 µL of GS II@GNPs (2 nM) in D-Hanks buffer for another 1 h at 30 °C. Then, the slides were subjected to a series of washing steps, namely, (1) 50 mL of washing buffer for 3 min (three times), (2) 50 mL of phosphate-buffered saline (PBS) buffer for 3 min (three times), and (3) 50 mL of Milli-Q water for 3 min (three times), and then dried by centrifugation (200g for 5 min). For fluorescence detection, after cell adhesion, the slides were incubated with 30 µL of FITC (0.01 mg/mL) in D-Hanks buffer for 30 min at 30 °C. Then, the slides were subjected to a series of washing steps, namely, (1) 50 mL of washing buffer for 3 min (three times), (2) 50 mL of PBS buffer for 3 min (three times), and (3) 50 mL of Milli-Q water for 3 min (three times), and then dried by centrifugation (200g for 5 min). Silver Enhancement and Detection. After being treated with gold nanoparticles, silver enhancer [1:1 mixed (1 mL total volume) solutions A (AgNO3) and B (hydroquinone) (SigmaAldrich)] was applied to each microarray and washed with water (three times). As previously reported, in this study, all results shown were obtained under optimum conditions (6-min exposure time) because the detection sensitivity and dynamic range were critically related to the amount of silver deposited.52,53 After signal amplification by silver deposition, the slides were detected with ArrayIt SpotWare Colorimetric Microarray Scanner (TeleChem International Inc., Sunnyvale, CA). According to the manufacturer’s preset parameters, all images were collected with a broad-spectrum white-light source. The background originating from the slide was recorded and subtracted from each image prior to evaluation. The mean values and standard deviations of the signals were determined for the 32 spot replicates per sample from a single binding assay. For generation of each microbe’s fingerprint, corresponding mean values and standard deviations of lectin-microbe data were normalized by the relative mean value for the GS II-microbe sample.

spotted and immobilized onto epoxide-modified glass slide surfaces by a standard robotic procedure.55 The primary amines on the lectin surfaces act as nucleophiles, attacking epoxy groups and coupling the lectins covalently to the surface. Then, the lectin microarrays are incubated with the microbe of interest, washed, labeled by gold nanoparticles conjugated with a second lectin. This labeling strategy might have a few of drawbacks; for example, it requires expression of the appropriate glycan ligands on the cell surface, and it could also be affected by the distribution of the glycans. However, these limitations could be overcome by lectin selection and/or use of multilectin-conjugated GNPs. Here, GS II@GNP was employed as a label because GS II enables the binding of most microbes with high affinity by N-acetylglucosamine (GlcNAc) residues on cellular surfaces.56,57 It is wellknown that N-acetylglucosamine (GlcNAc) residues are a widespread expression by bacteria and distributed over the entire cellular wall.56,57 We also found that GS II@GNPs enable binding with microbes with high affinity in solution (see Figure S1 in the Supporting Information). Subsequently, a silver enhancement step was applied to the microarray for signal amplification because the light scattering properties of gold nanoparticles by themselves are relatively poor, if the particles are smaller than ca. 40 nm.42,43,45 After treatment with the silver enhancement solution, the microarrays were readily detected by the RLS microarray scanner. To evaluate the performance of the assay, 128 identical lectin- (i.e., GS II-) microbe reactions were analyzed, and the signal-to-noise ratios (S/N) and quality Z′ factors were calculated (as shown in Figure 2 and in Figure S2 of the Supporting Information). Reasonable assay performance (S/N > 3 and Z′ > 0.5 (except for the E. cloacae assay) was obtained at optimized experimental conditions (i.e., for a spotting lectin concentration of 1 mg/mL, a microbe concentration of 108 cells/mL, and a GS II@GNP concentration of 2 nM in the labeling solution). Therefore, the optimized experimental conditions were fixed for subsequent assays except for the quantitative assay.

RESULTS AND DISCUSSION Binding Patterns of Microbes to Lectins. The methodology of our assay is schematically shown in Figure 1. Lectins are first

(56) Slifkin, M.; Doyle, R. J. Clin. Microbiol. Rev. 1990, 3, 197–218. (57) Ingraham, J. L.; Ingraham, C. A. Introduction to Microbiology, 2nd ed.; Brooks/Cole Publishing Co.: Pacific Grove, CA, 2003.


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Figure 2. Evaluation of the assay performance. The observed RLS intensities (black squares) are shown in comparison with control measurements (red dots). The mean values and standard deviations extracted from the data were used to calculate the S/N ratio and Z′ factor. The spots without immobilized lectins were used as control measurements. The data were derived from four slides that were run on 2 days. The concentration of spotting GS II was 1 mg/mL, the concentration of E. coli was 1 × 108 cells/mL, and the concentration of GS II@GNPs was 2 nM in the labeling solution.

To demonstrate the abilities of this assay, the interactions of 16 lectins [see Tables 1 and S1 (Supporting Information) for details] with four bacteria and one fungus were chosen to be tested. These microbial strains can be separated into three categories: (1) gram-negative bacteria (E. coli and E. cloacae), (2) gram-positive bacteria (B. subtilis and S. aureus), and (3) fungus (S. cerevisiae). In this assay, every slide was printed with eight isolated subarrays, each of which contained four replicate spots per lectin. The spotting map of the subarray is shown in Figure S3 (Supporting Information). The representative differences in both binding patterns and levels for the five different microbes are shown in Figure 3 and in Figures S4-S7 and Table S1 of the Supporting Information. As anticipated, all of these microbes have reasonable binding affinities with GS II, which suggests that all of these microbes express terminal GlcNAc residues in their surface glycoconjugates.56,57 In particular, the fungus, S. cerevisiae, binds only with GS II, which shows obvious differences in glycan expression between the bacteria and the fungus. All four bacterial strains herein have strongly binding behaviors with MAA II and SNA, which indicates that these bacteria express high levels of R-2,3- and R-2,6-sialic acid residues on their cellular surfaces.7,58 The experimental results also indicate that E. coli and B. subtilis express high levels of galactose on their surfaces because they have relatively high binding affinities with GS I. Moreover, S. aureus strongly expresses N-acetylglucosamine complexes, as the binding affinity of S. aureus with AAL is higher than that of S. aureus with UEA. In particular, the difference in the binding patterns of two closely related bacterial strains, E. coli and E. cloacae, has also been demonstrated; for example, both strains show strong binding affinities with RCA 120 and SNA, only E. coli gave a moderate binding to NPL and LcH. This result suggests that E. coli and E. cloacae strains express glycosyl complexes similar to those expressed by galactose residues. However, for mannose residues, only the E. coli strain can express. Therefore, (58) Varki, A. FASEB J. 1997, 11, 248–255.

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Figure 3. (a) RLS images and (b) corresponding lectin binding pattern of E. coli. Sixteen lectins were spotted as 2 × 2 replicates in one subarray (as shown in Figure S3, Supporting Information). The error bars are standard deviations derived from 32 spot replicates on a single slide. The concentration of E. coli in solution was 1 × 108 cells/mL.

NPL and LcH could be used as markers to discriminate the E. coli and E. cloacae strains in the further study. In addition, different binding abilities of AAL and UEA with bacteria were obsevered (in principle, AAL and UEA have similar binding specificities with sugar59,60), suggesting differences in their recognition motifs on the cellular surfaces. This finding is consistent with literature reports.7,61 To demonstrate the reproducibility of the array, the binding affinities of E. coli with lectins were chosen as examples. Six trials were run, using arrays from several batches, printed on different days. The binding patterns of the bacteria with lectins are shown in Figure S8 (Supporting Information). As a result, the assay-assay precision coefficient of variation (CV) of the relative signal intensity was below 8.6% in all experimental data sets. Such precision was considered to be within an acceptable range, indicating that the array has reasonable reproducibility. Although these differences in microarray images clearly indicate the representative binding patterns of the microbes with lectins, it would be of great value if a simple lectin fingerprint could be generated for discriminating microbial types by the lectin microarray platform. Because the RLS intensity is strongly dependent on the binding strength of the GS II@GNPs with the microbe and because all five of the microbes have reasonable binding affinities with GS II on the slide surface, the RLS intensity (59) Hindsgaul, O.; Norberg, T.; Pendu, J. L.; Lemieux, R. U. Carbohydr. Res. 1982, 109, 109–142. (60) Wu, A. M.; Wu, J. H.; Singh, T.; Liu, J. H.; Herp, A. Life Sci. 2004, 75, 1085–1103. (61) Baldus, S. E.; Thiele, J.; Park, Y. O.; Hanisch, F. G.; Bara, J.; Fischer, R. Glycoconj. J. 1996, 13, 585–590.

Figure 4. Lectin fingerprints of microbes. The error bars are normalized standard deviations, and the data points for each microbe were generated from 32 spot replicates in a single binding assay and normalized by the relative mean value of GS II microbe.

of the GS II-microbe conjugate (as shown in Figure S9, Supporting Information) was used as a reference value to evaluate a simple lectin fingerprint for discriminating microbial types (as shown in Figure 4). When the RLS intensity of the lectin-microbe conjugate was divided by that of the GS II-microbe conjugate, we found that each species of microbes had a unique fingerprint. The fingerprint shows not only the binding affinities of microbes with lectins but also the dynamic relationship among the lectins. Corresponding lectin fingerprints of the microbes from the same culturing medium are shown in Figures S10-S12 (Supporting Information); clear differences can be observed, which also provides further evidence of the feasibility of the approach in terms of microbe discrimination. For instance, an unknown microbe can be identified in two steps: (1) The microbe is first cultured in a randomly selected culture medium, and the obtained lectin fingerprint with is matched to that of a known microbe under the same culturing conditions. (2) After the primary microbe type has been obtained, the microbe is cultured a second time in the specific culture medium, and the lectin fingerprint is profiled to obtain the exact type of the microbe. Therefore, the lectin fingerprint could be used for discriminating microbial types as well as monitoring dynamic changes of glycoconjugates on the microbial surface. To confirm the veracity of this method, the conventional fluorescence technique was also carried out to obtain binding patterns/fingerprints of the microbes with lectins on the same microarrays by the literature-reported labeling procedure.62 The corresponding results are shown in Figures S13 and S14 (Supporting Information), which are consistent with those of the RLS study. Monitoring Growth Environmental Effects. The differences in lectin binding affinity between bacterium growth in a rich culture medium and bacterium growth in an inhibitory medium (e.g., a milk-rich culture medium) were also studied using our assay because these differences can provide crucial information on how bacteria (e.g., E. coli and S. aureus) live in the mammary (62) Kristi, I. S.; Michael, C. F. Biotechnol. Bioeng. 1996, 52, 340–356.

Figure 5. Changes of lectin binding affinities of (a) E. coli and (b) S. aureus after culturing in milk-containing medium for 16 h. The percentages (v/v) of milk in the culturing medium were ( ) 5%, ( ) 20%, ( ) 50%, and ( ) 100% milk. The curve marked by indicates that the bacteria were first cultured in 100% milk for 16 h and then recultured in fresh LB medium for another 12 h. The RLS intensities of lectins with bacteria cultured in LB only were used as control signals. The error bars are the standard deviations of lectin microbe divided by the relative mean values of the control signals, and the data for each curve were generated from 32 spot replicates in a single binding assay.

gland.63 Furthermore, it is likely that altered glycoconjugates of the pathogenic strain can make the pathogen less detectable by the immune system. In the present study, growth of the bacterial strains E. coli and S. aureus in milk-rich culture medium was studied because E. coli is regarded as a type of common clinical pathogen and S. aureus is the major pathogen in bovine intramammary infections.64 As shown in Figures 5 and S15-S17 (Supporting Information), the lectin fingerprints/binding patterns of E. coli and S. aureus were changed when the concentration of milk in the culture medium was increased. This observation is consistent with previous reports on proteomic changes in E. coli grown in fresh milk.65 Most interestingly, we also found that the culturing environmental (i.e., milk) effects on the binding affinity (63) Javier, O.-B.; Juan, J. V.-A.; Marcos, C.-J.; Alejandra, O.-Z.; Joel, E. L.-M.; Alejandro, B.-P.; Vı´ctor, M. B.-A. J. Infection 2007, 54, 399–409. (64) Gunasekera, T. S.; Attfield, P. V.; Veal, D. A. Appl. Environ. Microbiol. 2000, 66, 1228–1232. (65) Lippolis, J. D.; Bayles, D. O.; Reinhardt, T. A. J. Proteome Res. 2009, 8, 149–158.


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Figure 6. RLS images of lectin binding patterns of E. coli. The concentrations of E. coli in solution were (a) 1 × 108, (b) 1 × 107, (c) 1 × 106, (d) 1 × 105, and (e) 1 × 104 cells/mL. Sixteen lectins were spotted as 2 × 2 replicates in one subarray (as shown in Figure S3, Supporting Information).

of GS II with E. coli were much stronger than those of GS II with S. aureus. This finding could be used to identify E. coli and S. aureus in the milk. In addition, lectin binding profiles of the E. coli and S. aureus from different culturing media (yeast nutrient medium and nutrient medium) were also examined. Culturingenvironment-dependent differences in the lectin fingerprints/ binding patterns were also observed (as shown in Figures S18-S20, Supporting Information). Furthermore, the binding affinities of lectins with E. coli and S. aureus could be recovered after they were recultured in fresh LB culture medium for a period of time (see Figure 5 and Figures S15-20 of the Supporting Information). The same environment-dependent behaviors of E. cloacae and B. subtilis were also found (as shown in Figures S21 and S22, Supporting Information). This phenomenon suggests that the change of binding affinities of lectins with bacterial strains should come from the interactions of the components of milk/ culturing medium with bacterial glycans, instead of the genotypic variation. Because the detection signal can be changed by changes in the binding affinity of the GS II@GNP with microbe, the interactions of GS II@GNP with E. coli from different culture media in solution were studied as typical cases. As shown in Figure S1 (Supporting Information), although there are significant differences in the binding affinities among the GS II-E. coli samples on the microarray surface, the GS II@GNPs are able to bind E. coli with nearly the same efficiency. The binding difference between spotted GS II and GS II@GNP in solution might be due to the multivalent interaction of GS II@GNP with the microbe. This result suggests that the change of detection signal is mainly caused by the decreasing binding affinity of the spotted lectin with the microbe. In addition, the environment-dependent changes were also examined by the conventional fluorescence-labeling strategy. The corresponding results are shown in Figures S17 and 9246


Table 2. Detection Limits and Dynamic Ranges of the Proposed Assay analyte E. coli E. cloacae B. subtilis S. aureus S. cerevisiae

detection limit (cells/mL) dynamic range (cells/mL) 105 106 105 105 106

105-108 106-108 105-108 105-108 106-108

The detection limit (3 times the standard deviation) and dynamic range of the proposed assay are based on RLS images and corresponding data analysis of lectin binding patterns of microbes. The concentration of the lectins in the probe solution are 1 mg/mL and the concentration of GS II@GNPs is 2 nM in labeling solution.

S23-S27 (Supporting Information), which are consistent with the results of the RLS study. Quantitative Detection of Microbes. Developing a highly sensitive assay would be of great value for microbe monitoring. A set of experiments was designed to determine the detection limits of the method described herein. As a typical example, the interactions of 16 lectins with different concentrations of E. coli are shown in Figure 6. E. coli could be detected at concentrations as low as 105 cells/mL. Given a reaction solution volume of about 50 µL, this means that the actual amount of E. coli per detection is on the order of 103. This RLS signal increased linearly with the logarithm of the E. coli concentration from 105 to 108 cells/mL, indicating a dynamic range of nearly 3 orders of magnitude (see Figure S28 in the Supporting Information). The corresponding experimental results for the interaction of lectins with the other four microbial strains are shown in Figures S29-S32 of the Supporting Information. For comparison, the detection limits and dynamic ranges of the assays are summarized in Table 2. These experimental results suggest that our approach is readily transferable to real analytical problems

such as monitoring microbial types and amounts in environmental or clinical samples. CONCLUSIONS A gold-nanoparticle-labeled lectin-microarray-based assay has been developed for screening glycoconjugates on microbial surfaces. The experimental results indicate that the microarraybased assay has great promise in fingerprinting a series of microbial strains based on their carbohydrate expressions and evaluating dynamic changes in surface glycoconjugates of microbes in response to environmental stimuli. This would open up possibilities for using this technique to study how microbes actively modulate their glycans to establish pathogenic or symbiotic relationships in living systems. However, this assay also has several limitations, for example, (1) only accessible carbohydrate motifs, rather than the entire glycome, and (2) binding affinity of GS II@GNPs with microbes. Because of rapid developments in glycomic studies and bionanotechnologies (e.g., gold nanoparticle functionalization), these disadvantages could be overcome in the near future.

ACKNOWLEDGMENT The authors thank the NSFC (Grants 20675080, 20875087), Chinese Academy of Sciences (Grant KJCX2-YW-H11), and CAS-Bayer Start-up Fund (2008) for financial support. SUPPORTING INFORMATION AVAILABLE UV-visible spectra of GS II@GNPs stained microbes in solution; evaluation of the assay performance on B. subtilis, E. cloacae, S. aureus, and S. cerevisiae; spotting map of the subarray; RLS images and corresponding analysis of lectin binding patterns of B. subtilis, E. cloacae, S. aureus, and S. cerevisiae; fluorescence-labeled lectin microarray and corresponding results; environmental effect on the binding affinities of lectin with microbes; and the interactions of 16 lectins with different concentrations of B. subtilis, E. cloacae, S. aureus, and S. cerevisiae in solution. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 28, 2010. Accepted October 7, 2010. AC1022309


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Screening Lectin-Binding Specificity of Bacterium by Lectin Microarray

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