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DOI: 10.1002/chem.201104069
Saccharide-Modified Nanodiamond Conjugates for the Efficient Detectionand Removal of Pathogenic Bacteria
Mirja Hartmann,[a] Patrick Betz,[b] Yuchen Sun,[b] Stanislav N. Gorb,[c]
Thisbe K. Lindhorst,*[a] and Anke Krueger*[b, d]
Introduction
Bacterial contamination of water for human consumption orirrigation of crops is one of the major global problems.[1–3]
Numerous bacterial species endanger the health and well-being of millions of people all over the world in developingcountries, involving bacteraemia, meningitis and respiratorytract infections. The problem also occurs in Europe, such asin the case of recent EHEC infections.[4] Therefore, the effi-cient detection and removal of such contaminations is an
urgent necessity. Most of the available techniques for thesensing of bacterial contaminations require advanced labo-ratory equipment or skills, and often several days for theproduction of reliable results, which is critical in cases ofacute infections. Standard techniques for detection of bacte-rial contamination include cultivation on agar plates,[5] meth-ods based on DNA amplification,[6,7] such as PCR and LCR,mass spectrometric detection[8] or immunological meth-ods.[9,10]
Recently, nanoparticles (NP) have been reported to behighly efficient in applications for sensing of microbes andto require less costly equipment and/or time. Rotello andcollaborators developed a protocol based on functionalizedgold NPs (Au-NPs) carrying quaternary ammonium saltstriggering an enzyme-enhanced pathogen detection.[11]
Other reports on Au-NPs include the detection of viruses,[12]
bacteria[13] and proteins.[14] But not only Au-NPs can be ap-plied for sensing, also macromolecular NPs[15] or NPs fromother materials, such as silver, gadolinium sulfide and ironoxides or carbon nanomaterials have been applied for thedetection and capture of pathogenic bacteria.[16–24]
However, many of the existing methods rely on purelyelectrostatic interactions of the NPs with the respective vi-ruses, microbes or proteins. This interaction is usually strongbut unselective. In other words, selectivity for certain typesof bacteria cannot be achieved. To solve the selectivity prob-lem in sensing and removing bacteria an NP-based platformis required that is conjugated to molecules that are species
Abstract: The detection and removalof bacteria, such as E. coli in aqueousenvironments by using safe and readilyavailable means is of high importance.Here we report on the synthesis ofnanodiamonds (ND) covalently modi-fied with specific carbohydrates (glyco–ND) for the precipitation of type 1 fim-briated uropathogenic E. coli in solu-tion by mechanically stable agglutina-tion. The surface of the diamond nano-particles was modified by usinga Diels–Alder reaction followed by thecovalent grafting of the respective gly-cosides. The resulting glyco–ND sam-ples are fully dispersible in aqueous
media and show a surface loading oftypically 0.1 mmol g�1. To probe the ad-hesive properties of various ND sam-ples we have developed a new sand-wich assay employing layers of twobacterial strains in an array format.Agglutination experiments in solutionwere used to distinguish unspecific in-teractions of glyco–ND with bacteriafrom specific ones. Two types of precip-itates in solution were observed and
characterized in detail by light andelectron microscopy. Only by specificinteractions mechanically stable agglu-tinates were formed. Bacteria could beremoved from water by filtration ofthese stable agglutinates through 10 mmpore-size filters and the ND conjugatecould eventually be recovered by addi-tion of the appropriate carbohydrate.The application of glycosylated NDallows versatile and facile detection ofbacteria and their efficient removal byusing an environmentally and biomedi-cally benign material.
Keywords: bacterial recognition ·carbohydrates · diamond · nanopar-ticles · surface chemistry
[a] Dr. M. Hartmann, Prof. Dr. T. K. LindhorstOtto Diels Institute for Organic ChemistryChristiana Albertina University KielOtto-Hahn-Platz 3-4, 24098 Kiel (Germany)E-mail : [email protected]
[b] P. Betz, Y. Sun, Prof. Dr. A. KruegerInstitute for Organic Chemistry, W�rzburg UniversityAm Hubland, 97074 W�rzburg (Germany)E-mail : [email protected]
[c] Prof. Dr. S. N. GorbDepartment of Functional Morphology and BiomechanicsZoological Institute, Christiana Albertina University Kiel24098 Kiel (Germany)
[d] Prof. Dr. A. KruegerWilhelm Conrad Roentgen Research Center for Complex MaterialsSystemsW�rzburg University, 97074 W�rzburg (Germany)
Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/chem.201104069.
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specific. Carbohydrates are especially useful for this pur-pose, as a majority of microbes show a pronounced specifici-ty of certain saccharides. Thus, we set out to design suitablenanoparticles that are terminated with specific saccharidemotifs (see below) for selective detection and/or removal ofdifferent bacterial strains.
In this approach, the utilized nanoscale material needs tofulfill several requirements: broad availability, easy andstable surface functionalization, as well as dispersibility inaqueous solution. In addition, it should be environmentallyand medically benign. A material that complies with allthese prerequisites is nanoscale diamond (ND). It is hydro-philic, available in large quantities; it can be functionalizedin many ways and is considered a biocompatible and safematerial.[25–28] Nanodiamond possesses several advantagesover other nanoparticles and objects (like carbon nanotubesor polymer NP), such as being a completely inert particle,not swelling in any solvents, inherent fluorescence from lat-tice defects (applicable in labeling applications), thermal(up to 450 8C) and mechanical long-term stability and verylow toxicity. There are several sources for nanoscale dia-mond particles, such as high temperature high pressure(HTHP) synthesis, chemical vapor deposition (CVD) anddetonation synthesis. The latter method can be used for theproduction of large quantities of very small (5 nm) nanodia-mond particles, which are typically strongly agglomerateddue to the highly functionalized surface. This is caused bythe oxygenation during synthesis (e.g., by reaction with thecoolant) or the purification process by using mineralacids.[26, 27] The surface of the nanodiamond can be function-alized in many ways, and can lead to stable colloids in aque-ous solution and a high flexibility in functional surface ter-mination.
Here we report on the synthesis and application ofa novel, saccharide-modified ND material for the detectionand agglutination of bacteria. The synthetic construct con-sists of a nanoscale diamond core (Figure 1 a) that is modi-
fied by a linker moiety to allow covalent conjugation witha specific saccharide motif (Figure 1 b). The glycosylatedND can then interact with bacterial cells, such as fluorescingE. coli (Figure 1 c, d), through specific protein–carbohydrateinteractions. Binding to carbohydrates is a typical feature ofbacteria, which they utilize to attach to the glycosylated sur-face of their target cells. Bacterial adhesion to cell surfacescan eventually lead to the formation of biofilms and infec-tion of the host organism.[29]
To accomplish adhesion effectively, many bacteria areequipped with hairy protein appendages, called fimbriae orpili. These adhesive organelles expose protein domains,which function as lectins and thus mediate the molecular in-teraction with specific carbohydrates for which they are se-lective.[30] The best characterized pili are type 1 fimbriae,which recognize terminal a-d-mannoside units in high-man-nose type glycoproteins on the host-cell surface with highspecificity.[31] The lectin domain, which is known to be re-sponsible for mannoside binding, is located at the fimbrialtips and called FimH.[32] Type 1 fimbriae are commonthroughout the family of Enterobacteriaceae, such as E. coli,including highly pathogenic strains of UPEC and EHEC.We, therefore, chose mannose specificity of bacterial bindingfor the investigation of glycosylated ND in sensing and re-moval of bacteria.
Results and Discussion
For the synthesis of the ND conjugates 1–3 (Scheme 1)a suitably functionalized ND surface is needed. We appliedan efficient two-step strategy using a Diels–Alder cycloaddi-tion as its central reaction recently reported by some ofus.[33] Thermally annealed detonation ND (8)[34] was treatedwith in situ generated ortho-quinodimethane to yield a cova-lently arylated ND (9) with surface loading of 0.14 mmol g�1.After a classical aromatic sulfonation, one third of the sul-fonic acid groups was reduced by treatment with triphenyl-phosphine and iodine (according to X-ray photoelectronspectroscopy (XPS), Figure S6 in the Supporting Informa-tion).[33] This leaves a sufficient amount of charged surfacegroups for the colloidal stabilization of the NPs in aqueousenvironments. The thiol functions of compound 11 wereused as anchor groups. They allow the conjugation of thesaccharide moieties on the diamond surface by “thiol–enereaction” to the allyl aglycon of glycosides 6 and 7.[35] Theallyl glycosides 6 and 7 and the trivalent cluster mannoside14 were synthesized according to published protocols.[36, 37]
Allyl glycosides were ligated to the thiolated ND 11 to yieldthe conjugates 1 and 2 after removal of the protectinggroups (see the Supporting Information). The trivalent gly-cocluster 14 was attached to an arylated ND carrying car-boxylic acid moieties (15)[38] by peptide coupling employingthe amino group at the focal point of the cluster mannosidewith EEDQ as the coupling agent (see the Supporting Infor-mation). All ND conjugates were characterized by FTIR,TGA, and z potential measurements as well as elemental
Figure 1. Interaction entities: a) TEM image of nanodiamond (ND);b) schematic representation of the glyco–ND conjugate; c) TEM imageof the GFP-tagged type 1 fimbriated E. coli strain PKL1162;[39] d) sche-matic image of the same bacteria.
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analysis (Figures S1–S4, Table S1 in the Supporting Informa-tion). Their full dispersion as isolated nanoparticles wasshown by dynamic light scattering (DLS) and AFM meas-urements (Figure 2, Figures S7–S9 in the Supporting Infor-mation).
The glycosylated NDs can be regarded as mimetics of thehost cells of bacteria, as the carbohydrate decoration of theemployed ND conjugates resembles parts of the eukaryoticglycocalyx. Hence, it could be predicted that the interactionof fimbriated bacteria with glycosylated ND should be com-parable to their interaction with living cells. This should
allow for the application ofthese ND glycoconjugates indetection and agglutination ofsuch pathogens.
To test the specific interac-tion of the new ND glycoconju-gates with bacteria, they wereinitially tested as inhibitors oftype 1 fimbriae-mediated bacte-rial adhesion.[39] However, theknown adhesion-inhibitionassay, which is routinely per-formed to test inhibitors of bac-terial adhesion,[40] could not beapplied to the new ND glyco-conjugates, due to their aggluti-nating properties (see below;Figure S10 in the SupportingInformation). Therefore, a newsandwich assay was developed,that is composed of severallayers: 1) the microtiter platewith the polysaccharidemannan immobilized on its sur-face; 2) a “capture layer” oftype 1 fimbriated bacteria,which is the actual analyte;3) the ND conjugates; and 4) a“detection layer” of fluorescenttype 1 fimbriated bacteria. Inthis setup, the ND conjugatesfunction as “glue” betweena layer of non-fluorescentE. coli and a top layer of fluo-rescing bacteria, which can beanalyzed by fluorescence read-out (Figure 3). In this assay ag-glutination is prevented. Hence,it can be applied to screen theNPs� potential to form a stableadhesive layer between bacteri-al cells, both in an a-d-manno-side-specific manner, as well asby unspecific interactions.Moreover, future applicationswill include the detection of
pathogenic bacteria by this sandwich setup.The assay was performed as follows. E. coli bacteria of
the non-fluorescent type 1 fimbriated strain HB101pPKL4[41] (“capture layer”) were allowed to adhere a-d-mannoside-specifically to mannan-coated 96-well plates inan incubation step at physiological temperature (37 8C).After being washed, serial dilutions of the stably dispersedND sample in PBS buffer were applied and incubated at37 8C. Unbound ND was washed away, before the wellswere coated with a suspension of the GFP-tagged E. colistrain PKL1162[40] that in turn adhere to bound ND. Thus,
Scheme 1. Synthesis of ND conjugates. a) Synthesis of nanodiamond 1 and 2 functionalized with monosacchar-ides: i) 1. allyl alcohol, acetyl chloride, 0 8C to 70 8C, 55%; 2. Ac2O, py, room temperature, 98%; ii) [18]crown-6, KI, toluene, 72 h; iii) SO3HCl, 50 8C, 20 h; iv) PPh3, I2, benzene, reflux, 20 h. b) Synthesis of nanodiamond 3functionalized with a trivalent cluster mannoside: i) 1. BF3·Et2O, CH2Cl2, 0 8C to room temperature, 38 h, 43%;2. NaOMe, MeOH, room temperature, 23 h, 94 %; 3. H2, Pd/C, MeOH, room temperature, 26 h, 73 %; ii) iso-ACHTUNGTRENNUNGamylnitrite, H2O, 80 8C, 16 h; iii) EEDQ, pyridine, reflux, 24 h.
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GFP-tagged E. coli serve as the “detection layer” (Figure 3)and the intensity of the fluorescence signal can be correlatedwith the adhesive properties of ND. This novel sandwichassay showed that ND form adhesive films between twobacterial layers in a concentration-dependent manner (Fig-ure S11 in the Supporting Information).
For all ND samples under investigation the measuredfluorescence was plotted against the respective applied NDconcentration on a logarithmic scale. Sigmoidal dose-re-sponse curves were obtained; these indicate that the adhe-sive properties of the GFP-tagged bacteria in the detectionlayer are strictly dependent on the concentration of appliedND (Figure S11 in the Supporting Information). When theapplied ND concentration was raised over a certain limit,fluorescence did not further increase but leveled out at a pla-teau. This observation indicates that the bacterial detectionlayer does not expand beyond a monolayer but reachesa maximum with maximal adhesion of ND. Correspondingly,uncontrolled agglomeration of bacteria was not observed in
this assay. Negative controls, in which the ND adhesivelayer was omitted, only showed negligibly low backgroundfluorescence signal.
Overall it was shown that our sandwich assay serves asa reliable setup to test the interaction of various functional-ized NPs (not only ND!) with living bacteria. It turned out,that both, the saccharide-modified NDs 1–3, as well as non-glycosylated NDs function as robust adhesives between bac-terial analyte and detection layer. However, mannosylatedND 1 and 3 formed adhesive films already after a very shortincubation time, owing to specific interactions between man-nose and the fimbrial lectin, FimH. Unspecific interactionswith non-mannosylated ND, such as with 2, 8, and 10, onthe other hand, were built up more slowly, as could be seenin incubation-duration studies (Figure S12 in the SupportingInformation). Moreover, sandwich assays with mannoside-modified ND 1 and 3 generated fluorescence values, whichwere 30 % higher than the values obtained with 2, 8, and 10.These non-glycosylated NDs can only establish unspecificinteractions with the bacteria, for example, by hydrophilicinteractions of their surface groups with the pathogens.
Thus, specific and nonspecific interactions of E. coli withND material occur concomitantly on surfaces, as it wasshown in the sandwich assay. In order to investigate ND–bacteria interactions in solution, three-dimensional aggluti-nation experiments appeared to be perfectly suited. Asknown from classical hemagglutination[42] and agglutinationstudies[43] cells can be precipitated through lectin–carbohy-drate mediated interactions. To get deeper insight into theinfluence of specific versus nonspecific adhesion, the type 1fimbriated E. coli strain PKL1162 was used to test mannose-specific interaction with the ND conjugates, and the relatednon-fimbriated strain HB101 was employed to probe unspe-cific interactions of the bacteria with different ND samples.
A suspension of fimbriated E. coli PKL1162 and six dif-ferent ND samples (Figure 4; Table S2 and Figures S13–S17in the Supporting Information) were serially diluted inbidest. water and mixed in test vials at ambient temperature.Equally, a suspension of non-fimbriated E. coli HB101 wasmixed with serial dilutions of mannosylated ND 1. Alreadyafter a short incubation time of 5 min, precipitation was ob-served in all cases, correlating with the ND concentrationsapplied. However, the formed precipitates differed much intheir visual characteristics as well as in their mechanical sta-bility depending on the applied ND. Whereas mannosylatedND 1 and 3 formed well-structured crystal-like agglutinateswith type 1 fimbriated E. coli (Figure 4 b, i), less compactflocculates were formed as a consequence of unspecific in-teractions of non-mannosylated ND (Figure 4 b, ii). More-over, mannosylated ND samples 1 and 3 led to mechanicallystable agglutinates in clear supernatant solution, which re-mained unaffected even after heavy manual shaking of thetest vials. Apparently, strong cross-links were formed thatwithstood shear stress. In contrast, flocculates resulting fromnonspecific interaction of the non-glycosylated ND samples8, 10, or pristine hydrophilic ND (16, see the Supporting In-formation) with the bacteria were mechanically less stable
Figure 2. AFM image of mannosylated nanodiamond 3 dropcasted onhighly ordered pyrolytic graphite (HOPG). The overview (left) shows aneven distribution of the particles. The magnification proves the absenceof agglomerates (right).
Figure 3. Setup of the sandwich assay: type 1 fimbriated E. coli (HB101pPKL4) are immobilized onto polysaccharide (mannan)-coated microtit-er plates. Then, serial dilutions of ND conjugates are applied; this leadsto an adhesive layer, to which fluorescent type 1 fimbriated E. coli(PKL1162) can adhere as the detection layer. Thus, fluorescence intensityis correlated with the adhesive properties of the applied ND conjugates.
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and could be easily disintegrated by manually shaking thevial to form a milky dispersion. The same unstable floccula-tion was observed with non-fimbriated HB101 mixed withmannosylated ND 1 (Figure S17 in the Supporting Informa-tion). These observations again indicate that for the forma-tion of stable agglutinates specific interactions are required.
The glucosylated ND 2 generated precipitates with an in-termediate stability: lower than for the stable agglutinatesof mannosylated samples 1 and 3 and higher than for theflocculates of non-glycosylated ND. Accordingly, agitationof the agglutinate formed with 2 and fimbriated E. coli ledto a cloudy dispersion with some smaller agglutinates re-maining. This can be explained by weak interactions be-tween glucosides and the bacterial lectin FimH, as it hasbeen recently reported by some of us.[44]
As expected, mannose specific agglutination of ND couldbe prevented by addition of a standard inhibitor of type 1fimbriae-mediated bacterial adhesion, such as methyl a-d-mannopyranoside (MeMan; 100 mm). Likewise, agglutina-tion could be reversed by the addition of MeMan (Fig-ure 4 c). Hence, the interaction of the mannosylated ND
with bacteria was shown to parallel with known standardmodels of lectin–saccharide interactions. Glyco–ND canthus be considered a suitable model for the investigation ofcarbohydrate–protein interactions.
In order to test the efficiency of glyco–ND to remove bac-teria from polluted solutions, samples of supernatants afterflocculation/agglutination were grown on agar. For all NDsamples (1, 2, 3, 8, and 16) a reduction of the amount ofE. coli in the remaining supernatant was observed (see theSupporting Information). By far the most potent water de-contaminating agents were shown to be the mannosylatedNDs 1 and 3. In accordance with the multivalency effect,which is often observed in carbohydrate recognition,[45] ND3, functionalized with the trivalent cluster mannoside per-forms better than ND 1, which is conjugated to monovalentmannosides.
After the basic applicability of ND conjugates for the ag-glutination of bacteria had been shown, we set out to gobeyond the proof-of-principle. We aimed at the use of theseagglutinants in a real-world setup for the removal of patho-gens from water. In fact, agglutination-filtration experimentsemploying commercial filters (pore size 10 mm; too big toretain bacteria) and ND 1 or pristine hydrophilic ND 16, al-lowed the removal of different types of E. coli efficientlyfrom various water samples. Retention of bacteria clearlycorrelated with ND concentration and ND type (Table S3,Figures S19–S20 in the Supporting Information). The mostsuitable concentration of ND 1 for the efficient retention ofbacteria was found to lie between 0.08–0.8 mgmg�1 E. coliwith removal rates of 93–97 % (Figure 5). This was shownby cultivation and CFU counting of the generated filtratesin comparison to the original untreated water sample. To ex-
Figure 4. Detection and removal of bacterial cells. a) Agglutination ofbacteria in solution by using surface-modified ND. Depending on the sur-face functionality a reversible flocculation caused by nonspecific interac-tions or an irreversible agglutination due to stable mannose–lectin inter-actions is observed. The latter can be used not only for the specific detec-tion of bacteria, but also for their removal by filtration. b) Mechanicallystable agglutination with mannosylated ND 3 (i) and unstable floccula-tion with glucosylated ND 2 (ii). c) The carbohydrate-specific agglutina-tion of E. coli by mannosylated ND 1 is reversible by addition of a com-peting carbohydrate in solution, such as MeMan. Only in samples withMeMan (+MeMan) the agglutinates can be redispersed by manual shak-ing (control: �MeMan). Pictures in b) and c) were taken 5 min aftershaking the vials. Samples in c) were shaken simultaneously with thesame strength.
Figure 5. The agglutination-filtration experiments with E. coli show thatthe addition of just 80 mgmL�1 of the mannosylated ND 1 is sufficient toefficiently remove 1000 mgmL�1 of fimbriated bacteria PKL1162 fromthe solution by filtration through a conventional filter with 10 mm poresize. Further results including findings with non-fimbriated bacteria andunmodified hydrophilic ND 16 are shown in Figure S19 (in the Support-ing Information).
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clude any effect of the filter, the control was equally fil-tered.
In multiply contaminated samples, specific interactionsbetween carbohydrates attached to ND 1 and the bacterialtype 1 fimbriae of E. coli enabled the preferential removalof the fimbriated strain PKL1162 (Figure S19 in the Sup-porting Information). This ND conjugate is hence applicablefor the selective removal of specific pathogens in a verysimple setup. After filtration from the water samples, it waspossible to recycle the glyco–ND from the agglutinates bywashing with MeMan solution. The recovered ND 1 wasreused for another efficient removal of bacteria (Figure S21in the Supporting Information). This showed the practicalapplicability of our material, for example, in filter cartridges.Recovery of the ND material after agglutination of bacteriais of high importance if the material is used in a continuous-flow setup. Thus, in future, for continuous removal of patho-gens, for example, from drinking water, sanitation cartridgescould be reused after regeneration. This method wouldreduce costs and enable the long-term application of glyco–ND in difficult environments.
To get deeper insight into the nature of the observed floc-culates and agglutinates, they were investigated by opticaland electron microscopy. In phase contrast microscopicimages of the different types of flocculates it could be clear-ly seen that the non-fimbriated strain HB101 was not at allagglutinated by the mannosylated ND 1. As expected, bac-terial cells and glyco–ND particles were found mobile andseparated from each other. The fimbriated strain PKL1162,on the other hand, was aggregated in extended lattices. Athird type of interaction with PKL1162 was seen with pris-tine hydrophilic ND 16. Unspecific flocculates were formed,presumably due to electrostatic interactions with the bacte-ria.
Two types of the observed precipitates were also investi-gated by using scanning electron microscopy (SEM, Figure 6and Figures S22–S25 in the Supporting Information): the ag-glutinate that was formed through specific interaction ofglyco–ND 1 and PKL1162, and the flocculates that weregenerated when the non-mannosylated ND 16 interactedwith PKL1162. From the SEM images it could be seen thatnon-mannosylated, surface oxidized (and hence hydrophilic)ND 16 accumulates on the bacterial surface in large aggre-gates (Figure 6 a), presumably through nonspecific hydro-philic interactions. On the contrary, mannosylated ND 1 wasevenly spread around the bacteria and their fimbriae (Fig-ure 6 b). As expected, glyco–ND was seen to specifically in-teract with the fimbrial tip (Figure 6 c), where the mannose-specific lectin domain FimH is located. Individual ND parti-cles were also seen to adhere to the fimbrial shaft (Fig-ure 6 b). Thus, SEM clearly revealed the structural differen-ces between unspecific formation of less stable flocculatesand carbohydrate-specific generation of stable agglutinateswhen bacteria are treated with different NDs.
Conclusion
In conclusion, we have synthesized a series of novel nano-diamond conjugates that are covalently modified with sac-charide moieties using a stable ligation by an aromatic ringintroduced by a Diels–Alder reaction. As the actual surfacedecoration can be flexibly chosen according to the targetedinteraction, the synthesis strategy is suited for a wide rangeof applications. Depending on the respective surface termi-nation the ND conjugates can either interact specifically ornonspecifically with the surface of different pathogenic bac-terial strains. The agglutination experiments with E. coli cor-roborate ND particles as a useful means to flocculate bacte-ria and thereby allow their removal by filtration througha low-cost, high-throughput filter material with large poresize. Decorated with specific carbohydrates, it is possible touse them for the detection and removal of the respectivebacteria in a contaminated sample by the formation of me-chanically stable agglutinates. Once separated from the mix-ture by filtration, glyco–ND can easily be recycled by addi-tion of a low-cost, commercially available inhibitor of bacte-rial adhesion such as MeMan. This makes the procedure ofbacterial decontamination in solution an economic and envi-ronmentally friendly process. Furthermore, our methodologyallows the detection of bacterial contamination by simplevisual inspection of the test solution. A novel sandwichassay applying functionalized ND can be used for the cap-ture and detection of bacteria; this overcomes agglomera-tion issues observed in conventional inhibition assays. Bothsetups enable a multitude of applications, such as detectionof pathogens and/or decontamination of water. Other bio-logical applications, such as the investigation of carbohy-
Figure 6. SEM images of flocculates and agglutinates formed with:a) E. coli and pristine hydrophilic nanodiamond 16. The ND particlesform large agglomerates and settle unspecifically on the cell surface.b) Mannosylated ND 1. The particles are well dispersed and are locatedon the fimbriae. c) Mannosylated ND 1. The image shows a magnificationof ND 1 specifically bound to the bacterial lectin located on the tip ofthe type 1 fimbriae (arrow).
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drate-specific interactions, in vivo and in vitro, can be envis-aged taking advantage, for example, of the non-bleaching lu-minescence of ND carrying N-V centers.[46,47] In the futurewe will study the use of the ND conjugates employing a vari-ety of specific glycosides both in continuous filtering car-tridges as well as in the context of glycobiology.
Experimental Section
Synthesis of mannosylated ND 1: Under nitrogen atmosphere thioaryl-ACHTUNGTRENNUNGated nanodiamond 11 (59 mg) was suspended in anhydrous benzene in anultrasonic bath for 15 min. Allyl 2,3,4,6-tetra-O-acetyl-a-d-mannopyrano-side (81 mg, 0.209 mmol) and a catalytic amount of AIBN were added tothe suspension. The reaction mixture was heated, overnight, to 78 8C.After the suspension cooled to room temperature, the diamond particleswere isolated by centrifugation. The precipitate was washed repeatedlywith toluene, acetone and dichloromethane in consecutive washing/cen-trifugation cycles. Ultrasonic treatment was used in every cycle in orderto redisperse the diamond and remove adsorbed impurities. After beingwashed, the sample was dried at 70 8C, in vacuo. The mannosylated nano-diamond 1 (170 mg) was suspended in a mixture of methanol (4 mL),water (5 mL) and potassium hydroxide solution (1 m, 2 mL) in an ultra-sonic bath for 15 min. The suspension was stirred, overnight, at roomtemperature and the diamond particles were isolated by centrifugation.The precipitate was washed repeatedly with water (until the supernatantbecame neutral), acetone and dichloromethane in consecutive washing/centrifugation cycles. Ultrasonic treatment was used in every cycle inorder to redisperse the diamond and remove adsorbed impurities. Afterbeing washed, the sample was dried at 70 8C, in vacuo. FT-IR (vacuum-cell): n =3417, 2934, 2881, 1718, 1583, 1373, 1195, 1120, 1040 cm�1; ele-mental analysis calcd (%): C 90.38, H 1.08, N 2.70, S 0.31; surface load-ing: (calcd from TGA): 0.11 mmol g�1 [Dm (137–489 8C) 4.0 %]; frag-ment: C17H24O6S (356 gmol�1); particle size: (H2O): 10%�44, 50%�59,90%�96 nm.
The synthesis of all other ND conjugates and the saccharide precursors isdescribed in the Supporting Information.
Three-layered sandwich assay by using nanodiamond as adhesive layer:To prove the cross-linking feature of the NDs towards E. coli bacteria,the nanoparticles were assayed in a novel sandwich setup. Black 96-wellpolystyrene microtiter plates (Thermo Fisher Scientific, Nunc, Maxisorp)were coated with mannan solution (1.2 mgmL�1 in carbonate bufferpH 9.5, 120 mL per well) and dried, overnight, at 37 8C. The plates werewashed three times with PBST (150 mL per well). Non-fluorescent type 1fimbriated E. coli (strain pPKL4) were suspended in PBS buffer(2.2 mg mL�1) and incubated in the mannan-coated wells (100 mL perwell) for 2 h at 37 8C to form a first bacterial layer—the capture layer.The plates were washed three times with PBS (150 mL per well). Glyco–ND or ND suspensions in PBS were prepared by applying ultrasound for2 h. These suspensions were serially diluted in PBS and transferred to thepretreated microtiter plate (50 mL per well). The plate was incubated at37 8C for 1 h to allow the formation of the adhesive layer and washedthree times with PBS (100 mL per well). GFP-tagged bacteria (strainPKL1162) were suspended in PBS (2 mg mL�1), dispensed to the wells(50 mL per well) and incubated for 1 h at 37 8C to build up the fluorescingdetection layer. The plate was washed three times with PBS (100 mL perwell) and each well was filled with PBS (50 mL) before bacterial adhesionwas detected by fluorescence readout (excitation wavelength 485 nm,emission wavelength 535 nm). The dose-response curves are shown in theSupporting Information.
Agglutination/precipitation experiments : To evaluate the adhesive poten-tial of mannosylated ND, ND suspensions in double distilled water wereprepared by applying ultrasound for 2 h. The NDs were diluted to givefive samples at different concentrations for each functionalized ND. Theconcentrations used were the same for all hydrophilic NDs (1, 2, 3, 10,16): 5 mg mL�1, 500, 50, 5, and 0.5 mgmL�1. Thermally annealed detona-
tion ND 8 was applied in the following concentrations: 10, 1 mg mL�1,100, 10, and 1 mgmL�1. Type 1 fimbriated E. coli (PKL1162) were sus-pended in bidest. water at a concentration of 2 mg mL�1. Then, this bacte-rial suspension (900 mL) was combined with the respective ND sample(100 mL) and the resulting agglomeration was studied by visual inspection(Table S2 in the Supporting Information). Details of filtration and recy-cling experiments are described in the Supporting Information.
Abbreviations : CFU, colony forming units; DLS, dynamic light scatter-ing; EEDQ, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; E. coli, Es-cherichia coli ; EHEC, enterohemorrhagic E. coli ; FTIR, Fourier trans-form infrared spectroscopy; LCR ligase chain reaction; MeMan, methyla-d-mannoside; ND, nanodiamond; NP, nanoparticle, PCR, polymerasechain reaction; PBS, phosphate-buffered saline; PBST, PBS with 0.05 %Tween-20; TGA, thermogravimetric analysis; TLC, thin layer chromatog-raphy; UPEC, uropathogenic E. coli.
Acknowledgements
We gratefully acknowledge the funding of the Deutsche Forschungsge-meinschaft (T.K.L., S.N.G. and A.K.) and the European Commission(A.K., contract DINAMO). We thank D. Lang for the HRTEM imagesof the NDs, S. Uemura for the AFM, and Pavo Vrdoljak for XPS meas-urements (all W�rzburg).
[1] W. A. Suk, K. M. Ruchirawat, K. Balakrishnan, M. Berger, D. Car-penter, T. Damstra, J. Pronczuk de Garbino, D. Koh, P. J. Landrigan,I. Makalinao, P. D. Sly, Y. Xu, B. S. Sheng, Environ. Health Perspect.2003, 111, 1340 –1347.
[2] a) P. Rochelle, J. Clancey, J. Am. Water Works Assoc. 2006, 98, 163 –166; b) P. Rochelle, J. Clancey, J. Am. Water Works Assoc. 2006, 98,169 – 174; c) P. Rochelle, J. Clancey, J. Am. Water Works Assoc.2006, 98, 177 –191.
[3] Pathogenic Mycobacteria in Water (Eds.: S. Pedley, J. Bartram, G.Rees, A. Dufour, J. A. Cotruvo), World Health Organization, IWA,London, 2004.
[4] A. R. C. Wong, J. S. Pearson, M. D. Bright, D. Munera, K. S. Robin-son, S. F. Lee, G. Frankel, E. L. Hartland, Mol. Microbiol. 2011, 80,1420 – 1438.
[5] B. S. Stevenson, S. A. Eichorst, J. T. Wertz, T. M. Schmidt, J. A.Breznak, Appl. Environ. Microbiol. 2004, 70, 4748 – 4755.
[6] S. Bae, S. Wuertz, Appl. Environ. Microbiol. 2009, 75, 2940 –2944.[7] M. R. Pingle, K. Granger, P. Feinberg, R. Shatsky, B. Sterling, M.
Rundell, E. Spitzer, D. Larone, L. Golightly, F. Barany, J. Clin. Mi-crobiol. 2007, 45, 1927 –1935.
[8] N. J. C. Strachan, F. J. Nicholson, I. D. Ogden, Anal. Chim. Acta1995, 313, 63–67.
[9] S. C. Ricke, S. D. Pillai, Crit. Rev. Microbiol. 1999, 25, 19 –38.[10] I. Safarik, M. Safarikova, S. J. Forsythe, J. Appl. Bacteriol. 1995, 78,
575 – 585.[11] O. R. Miranda, X. Li, L. Garcia-Gonzalez, Z.-J. Zhu, B. Yan,
U. H. F. Bunz, V. M. Rotello, J. Am. Chem. Soc. 2011, 133, 9650 –9653.
[12] J. Griffin, A. K. Singh, D. Senapati, E. Lee, K. Gaylor, J. Jones-Boone, P. C. Ray, Small 2009, 5, 839 –845.
[13] H.-W. Cheng, S.-Y. Huan, H.-L. Wu, G.-L. Shen, R.-O. Yu, Anal.Chem. 2009, 81, 9902 –9912.
[14] C. C. You, O. R. Miranda, B. Gider, P. S. Ghosh, I.-B. Kim, B. Erdo-gan, S. A. Krovi, U. H. F. Bunz, V. M. Rotello, Nat. Nanotechnol.2007, 2, 318 –323.
[15] M. U. Larsen, M. Seward, A. Tripathi, N. C. Shapley, Biotechnol.Prog. Biotechnol. Progr. 2009, 25, 1094 –1102.
[16] G. Naja, P. Bouvrette, J. Champagne, R. Brousseau, J. H. T. Luong,Appl. Biochem. Biotechnol. 2010, 162, 460 –475.
[17] V. Parashar, S. K. Pandey, A. C. Pandey, Chem. Commun. 2010, 46,3143 – 3145.
Chem. Eur. J. 2012, 00, 0 – 0 � 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org
These are not the final page numbers! ��&7&
FULL PAPERSaccharide-Modified Nanodiamond Conjugates
[18] A. Fornara, P. Johansson, K. Petersson, S. Gustafsson, J. Qin, E.Olsson, D. Ilver, A. Krozer, M. Muhammed, C. Johansson, NanoLett. 2008, 8, 3423 –3428.
[19] H. Lee, T.-J. Yoon, R. Weissleder, Angew. Chem. 2009, 121, 5767 –5770; Angew. Chem. Int. Ed. 2009, 48, 5657 – 5660.
[20] A. L. Chun, Nat. Nanotechnol. 2009, 4, 698 – 699.[21] S. Y. Hong, G. Tobias, K. T. Al-Jamal, B. Ballesteros, H. Ali-Boucet-
ta, S. Lozano-Perez, P. D. Nellist, R. B. Sim, C. Finucane, S. J.Mather, M. L. H. Green, K. Kostarelos, B. G. Davis, Nat. Mater.2010, 9, 485 –490.
[22] P. Wu, X. Chen, N. Hu, U. C. Tam, O. Blixt, A. Zettl, C. R. Bertozzi,Angew. Chem. 2008, 120, 5100 –5103; Angew. Chem. Int. Ed. 2008,47, 5022 –5025.
[23] E. Perevedentseva, P.-J. Cai, Y.-C. Chiu, C.-L. Cheng, Langmuir2011, 27, 1085 –1091.
[24] a) B. K. Gorityala, J. Ma, X. Wang, P. Chen, X.-W. Liu, Chem. Soc.Rev. 2010, 39, 2925 – 2934; b) K. Kostarelos, A. Bianco, M. Prato,Nat. Nanotechnol. 2009, 4, 627 – 633.
[25] a) A. Krueger, J. Mater. Chem. 2011, 21, 12571 –12578; b) V. N. Mo-chalin, O. Shenderova, D. Ho, Y. Gogotsi, Nat. Nanotechnol. 2012, 7,11– 23; c) A. Krueger, D. Lang, Adv. Funct. Mater. 2012, 22, 890 –906.
[26] A. M. Schrand, S. A. C. Hens, O. A. Shenderova, Crit. Rev. SolidState Mater. Sci. 2009, 34, 18–74.
[27] A. Krueger, Chem. Eur. J. 2008, 14, 1382 – 1390.[28] V. Vaijayanthimala, H.-C. Chang, Nanomedicine 2009, 4, 47–55.[29] N. Sharon, Biochim. Biophys. Acta 2006, 1760, 527 –537.[30] K. Drickamer, Nat. Struct. Biol. 1995, 2, 437 – 439.[31] G. Zhou, W.-J. Mo, P. Sebbel, G. Min, T. A. Neubert, R. Glockshub-
er, X.-R. Wu, T.-T. Sun, X.-P. Kong, J. Cell Sci. 2001, 114, 4095 –4103.
[32] D. Choudhury, Science 1999, 285, 1061 – 1066.[33] G. Jarre, Y. Liang, P. Betz, D. Lang, A. Krueger, Chem. Commun.
2011, 47, 544 –546.[34] Y. Liang, T. Meinhardt, G. Jarre, M. Ozawa, P. Vrdoljak, A. Schçll,
F. Reinert, A. Krueger, J. Colloid Interface Sci. 2011, 354, 23 –30.[35] A. Dondoni, A. Marra, Chem. Soc. Rev. 2012, 41, 573 – 586.[36] F. M. Winnik, J. P. Carver, J. J. Krepinsky, J. Org. Chem. 1982, 47,
2701 – 2707.[37] H. A. Shaikh, F. D. Sçnnichsen, T. K. Lindhorst, Carbohydr. Res.
2008, 343, 1665 –1674.[38] T. Meinhardt, D. Lang, H. Dill, A. Krueger, Adv. Funct. Mater.
2011, 21, 494 –500.[39] M. Hartmann, T. K. Lindhorst, Eur. J. Org. Chem. 2011, 3583 –3609.[40] M. Hartmann, A. K. Horst, P. Klemm, T. K. Lindhorst, Chem.
Commun. 2010, 46, 330 –332.[41] M. A. Schembri, G. Christiansen, P. Klemm, Mol. Microbiol. 2001,
41, 1419 –1430.[42] H. B. Donald, A. Isaacs, J. Gen. Microbiol. 1954, 10, 457 – 464.[43] N. Firon, I. Ofek, N. Sharon, Carbohydr. Res. 1983, 120, 235 –249.[44] C. Grabosch, M. Hartmann, J. Schmidt-Lassen, T. K. Lindhorst,
ChemBioChem 2011, 12, 1066 –1074.[45] J. J. Lundquist, E. J. Toone, Chem. Rev. 2002, 102, 555 –578.[46] O. Faklaris, D. Garrot, V. Joshi, F. Druon, J.-P. Boudou, T. Sauvage,
P. Georges, P. A. Curmi, F. Treussart, Small 2008, 4, 2236 – 2239.[47] Y.-R. Chang, H.-Y. Lee, K. Chen, C.-C. Chang, D.-S. Tsai, D.-S. Fu,
T.-S. Lim, Y.-K. Tzeng, C.-Y. Fang, C.-C. Han, H.-C. Chang, W.Fann, Nat. Nanotechnol. 2008, 3, 284 –288.
Received: December 29, 2011Published online: && &&, 0000
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Carbohydrates
M. Hartmann, P. Betz, Y. Sun,S. N. Gorb, T. K. Lindhorst,*A. Krueger* . . . . . . . . . . . . . . . . . . . &&&&—&&&&
Saccharide-Modified NanodiamondConjugates for the Efficient Detectionand Removal of Pathogenic Bacteria
Sweet diamond : Novel nanodiamond–saccharide conjugates have been syn-thesized and applied for the detectionand removal of pathogenic bacteria. Anew sandwich assay enables this detec-tion by specific and nonspecific inter-
actions by using agglutinating nanopar-ticles. The specific flocculation ofnanodiamond–mannose conjugateswith bacteria was used for the efficientand repeated removal of these germsfrom water samples (see scheme).
A carbohydrate-modified nanodia-mond……is a valuable new material forthe straightforward detection andefficient removal of pathogenicbacteria from polluted watersources. It is nontoxic and canreadily be used in difficult environ-ments. Furthermore, sugar-specif-icity of bacterial surface proteinsallows for identification andtargeted sectioning of particularvirulent strains through tailoredglycosylation of the nanodiamondsurface. For more details see theFull Paper by A. Krueger, T. K.Lindhorst et al. on page && ff.
Chem. Eur. J. 2012, 00, 0 – 0 � 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org
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