Rapid and Selective Discrimination of Gram-Positive and Gram

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Rapid and Selective Discrimination of Gram-Positive and GramNegative Bacteria by Boronic Acid Modified Polyamidoamine Dendrimer Yuji Tsuchido, Ryosuke Horiuchi, Takeshi Hashimoto, Kanako Ishihara, Nobuyuki Kanzawa, and Takashi Hayashita Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04870 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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Analytical Chemistry

Rapid and Selective Discrimination of Gram-Positive and Gram-Negative Bacteria by Boronic Acid Modified Polyamidoamine Dendrimer Yuji Tsuchido,†,§ Ryosuke Horiuchi,† Takeshi Hashimoto,† Kanako Ishihara,‡ Nobuyuki Kanzawa† and Takashi Hayashita*,† †Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyodaku, Tokyo 102-8554, Japan ‡Cooperative Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan §Present address: Bioengineering Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan ABSTRACT: There is an urgent need to develop a rapid and selective method for the detection of bacteria because delayed diagnosis and the overuse of antibiotics have triggered drug resistance in bacteria. To this end, we prepared boronic acid modified polyamidoamine generation 4 (B-PAMAM(G4)) dendrimer as crosslinking molecules that form aggregates with bacteria. Within 5 min of adding B-PAMAM(G4) dendrimer solution to a bacterial suspension, large aggregates were observed. Interestingly, the aggregate formation with various bacteria was pH-dependent. In basic pH, both Gram-positive and Gram-negative bacteria formed aggregates, but in neutral pH, only Gram-positive bacteria formed aggregates. We revealed that this bacteria-selective aggregation involved the bacterial surface recognition of the phenylboronic acid moiety of B-PAMAM(G4) dendrimer. In addition, we demonstrated that the spherical structure of B-PAMAM(G4) was one of the important factors for the formation of large aggregates. The aggregation was also observed in the presence of < 10 mM fructose. B-PAMAM(G4) dendrimer is expected to be a powerful tool for the rapid and selective discrimination between Gram-positive and Gram-negative bacteria. KEYWORDS: dendrimer, boronic acid, bacteria, selective discrimination, molecular recognition

INTRODUCTION It is well recognized that infections caused by bacteria and viruses spread rapidly worldwide. To prevent the rapid spread of infection, antibiotics are widely utilized; however, the overuse of antibiotics has led to the acquisition of drug resistance in bacteria.1-3 The prevention of infection spread and the acquisition of drug resistance have become significant public health problems.4,5 To effectively diagnose and treat infectious diseases, the development of new techniques for the rapid detection and identification of pathogens is desired.6-9 Although conventional culture-based methods have high sensitivity, such methods require a few days to yield results as well as specialized equipment and species-specific culture protocols.10-13 Other sensitive and specific bacterial detection methods have been developed, including polymerase chain reaction (PCR)-based methods or immunoassays, but these methods also require expensive instruments and a long total assay time because of the long sample preparation procedure.14,15 Therefore, none of the current methods is applicable for the rapid screening of bacteria. Nanomaterials have captivated the attention of many researchers because they are efficient tools for bacterial detection.16,17 Most nanoparticles have a multipoint binding recognition function due to the presence of modified ligands, such as antibody,18 dipicolylamine (DPA),19-21 mannose,22,23 or

antimicrobial agents.24-26 This multipoint binding recognition function enables much stronger and more selective binding to the target bacterial surface than 1:1 binding.27,28 Among them, dendrimers are useful platforms for analytical science. Dendrimers are easy to modify functional molecules, because of many reactive moieties on the surface.29 Besides, both particle size and number of branches can be controlled strictly by the generation of dendrimers. Boronic acid is widely used as a recognition site and a number of boronic acid sensors have been developed.30-36 Boronic acid is known to bind reversibly diol-containing molecules, such as saccharides. Previously, we reported that a boronic acid probe that assembled on the polyamidoamine (PAMAM) dendrimer surface could recognize glucose selectively via aggregate formation.37 Tsai et al. studied the cooperative effects of boronic acid-modified PAMAM dendrimers. They showed that dendrimer platform offer the recognition selectivity to glucose.38 These studies indicated that boronic acid probe/PAMAM dendrimer acts as glue for glucose via multipoint binding. Herein we demonstrate a rapid and selective method for the discrimination of Gram-positive and Gram-negative bacteria, which uses a boronic acid modified PAMAM dendrimer probe. Bacteria have saccharide units on the surface of cell envelope and are classified as Gram-positive and Gram-negative. The

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Gram-staining differences are derived from the components of the bacterial cell envelope, including saccharide units on the surface.39 We hypothesize that bacteria can be recognized by comparing aggregate formation through multipoint binding. We evaluate the pH dependence of the bacterial recognition function of the boronic acid modified dendrimer. In addition, we compare aggregate formation of boronic acid modified dendrimer with various bacteria.

EXPERIMENTAL SECTION Reagents. 4-Carboxyphenylboronic acid, sodium chloride, sodium hydroxide, hydrochloric acid, fructose, glucose, galactose, Alizarin Red S (ARS), 4‘,6-diamidino-2phenylindole dihydrochloride n-hydrate (DAPI) and agar were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). 4-Hydroxybenzoic acid and 4-(4,6-dimethoxy1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) were purchased from Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan). Methanol and methanol-d4 were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Polyamidoamine (PAMAM) dendrimer, ethylenediamine core, generation 3.0, 4.0, and 5.0 solutions were purchased from Sigma-Aldrich Japan, Co., LLC. (Tokyo, Japan). Bacto yeast extract and bacto tryptone were purchased from Nippon Becton, Dickinson Co., Ltd. (Tokyo, Japan). All other organic solvents and reagents were commercially available with guaranteed grade and used without further purification. Water was doubly distilled and deionized using a Milli-Q water system (WG222, Yamato Scientific Co., Ltd., Tokyo, Japan and Autopure WR600G, Merck Millipore, MA, USA) before use. Bacteria. Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Staphylococcus pseudintermedius 2012-S-27 was isolated from human nasal swab and identified by PCRRFLP.40 Apparatus. 1H NMR spectra were measured using a JNMECA500 spectrometer (JEOL Ltd., Tokyo, Japan) at 300 K. All pH values were recorded using a Horiba F-52 pH meter (HORIBA, Ltd., Kyoto, Japan). Ultraviolet-visible (UV-vis) absorption spectra were measured using a Hitachi U-3900 UVvis spectrophotometer (Hitachi High-Technologies, Co., Tokyo, Japan) equipped with a Peltier thermocontroller and a 10 mm quartz cell. Fluorescence spectra were measured using a HITACHI F-7000 fluorescence spectrophotometer (Hitachi High-Technologies, Co., Tokyo, Japan) equipped with a Peltier thermocontroller and a 10 mm quartz cell. ζ-potential measurements were carried out at 25 °C using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, United Kingdom). Synthesis of B-PAMAM(G4). PAMAM(G4) (2.0 mL, 11.4 mol), 4-carboxyphenylboronic acid (15.3 mg, 92.2 mol), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (120 mg, 434 mol) were dissolved in methanol (10 mL). The reaction mixture was stirred at room temperature for 8 days. Then, it was transferred into a Spectra/Por® 6 dialysis bag (MW cut-off = 1,000, Spectrum, Houston, TX, USA) and dialyzed against 50% methanol and distilled water. The dialyzed solution was lyophilized to give a white powder (152 mg, 89%). The obtained product was subjected to 1H NMR

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analysis. The degree of substitution of boronic acid was also calculated from the 1H NMR peak area (See Supporting Information, Figure S1). Synthesis of Ph-PAMAM(G4). PAMAM(G4) (2.0 mL, 11.4 mol), 4-hydroxybenzoic acid (12.7 mg, 91.9 mol), and 4(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (120 mg, 434 mol) were dissolved in methanol (10 mL). The reaction mixture was stirred at room temperature for 8 days. Then, it was transferred into a Spectra/Por® 6 dialysis bag (MW cut-off = 1,000, Spectrum, Houston, TX, USA) and dialyzed against 50% methanol and distilled water. The dialyzed solution was lyophilized to give a white powder (153 mg, 91%). The obtained product was subjected to 1H NMR analysis. The degree of substitution of boronic acid was also calculated from the 1H NMR peak area (See Supporting Information, Figure S2). Boronic acid function of B-PAMAM(G4) and displacement assay. B-PAMAM(G4) at concentrations ranging from 0 to 0.2 mM was added to ARS solution and the whole was stirred for 1 min. The solution was subjected to UVVis and fluorescence measurements. Then, saccharide (fructose, glucose, galactose) was added to the ARS-B-PAMAM(G4) solution and the solution was subjected to UV-Vis and fluorescence measurements. Bacterial recognition function of B-PAMAM(G4) at basic pH. S. aureus (IAM1011) and E. coli (K12W3110) were cultured in Luria-Bertani (LB) medium at 37 °C overnight and stained with DAPI. The bacterial suspensions were centrifuged three times to remove the LB medium and DAPI. The concentrations of the bacterial suspensions were adjusted to give the optical density at 600 nm (OD600) = 0.6 by water. The bacterial suspension and 66 M B-PAMAM(G4) or PAMAM(G4) in 20 mM CHES buffer (pH 9.0) were mixed in the ratio of 1:1 and vortexed. Time-dependent changes of OD600 were measured. Evaluation of pH dependence and bacterial recognition function of B-PAMAM(G4). Bacteria (S. aureus (IAM1011) and E. coli (K12W3110)) were cultured in LB medium overnight. Bacterial suspensions (S. aureus and E. coli) were centrifuged three times to remove the LB medium. The concentrations of the bacterial suspensions were diluted with water to obtain OD600 = 0.6. The bacterial suspension and 66 M B-PAMAM(G4) in 4 mM phosphate buffer adjusted at (a) pH 7.4 or 9.0, (b) various pH were mixed in the volume ratio of 1:1 and vortexed for 5 min. pH was adjusted by the addition of sodium hydroxide into phosphate buffer and then the OD600 was measured. Evaluation of bacterial recognition function of PhPAMAM(G4). S. aureus (IAM1011) bacterial suspension was centrifuged three times to remove the LB medium. The concentration of the bacterial suspension was adjusted to give OD600 = 0.6. The bacterial suspension and 66 M PhPAMAM(G4) or B-PAMAM(G4) in PBS (pH 7.4) were mixed in the ratio of 1:1 and vortexed for 5 min. OD600 was measured before and after vortex mixing. Selectivity of B-PAMAM(G4) for various bacterial species. Various bacterial species were cultured in MuellerHinton Broth (Oxoid Ltd., Hampshire, UK) or LB medium at 35-36 °C overnight and centrifuged three times to remove the medium. The concentrations of the bacterial suspensions were adjusted to give OD600 = 0.6 with PBS. The bacterial suspension


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Analytical Chemistry and 66 M B-PAMAM(G4) were mixed in the ratio of 1:1 and vortexed for 5 min. OD600 was measured before and after vortex mixing. Evaluation of bacterial detection limit of B-PAMAM(G4). S. aureus (IAM1011) bacterial suspension was centrifuged three times to remove the LB medium. We prepared various concentrations of the bacterial suspension. The bacterial suspension and 66 M B-PAMAM(G4) in PBS (pH 7.4) were mixed in the ratio of 1:1 and vortexed for 5 min. OD600 was measured before and after vortex mixing. In addition, we prepared various concentrations of B-PAMAM(G4). The 108 CFU/mL of bacterial suspension in PBS (pH 7.4) and BPAMAM(G4) were mixed in the ratio of 1:1 and measured OD600. Competitive experiment of bacterial recognition of BPAMAM(G4) in monosaccharide solution. S. aureus (IAM1011) bacterial suspension was centrifuged three times to remove the LB medium. The concentration of the bacterial suspension was adjusted to give OD600 = 0.6. The bacterial suspension and 66 M Ph-PAMAM(G4) and B-PAMAM(G4) in PBS (pH 7.4) were mixed in the ratio of 1:1 and vortexed for 5 min. OD600 was measured before and after vortex mixing.

RESULTS AND DISCUSSION Boronic acid function of B-PAMAM(G4). We synthesized boronic acid modified PAMAM(G4) dendrimer (BPAMAM(G4)) by the condensation reaction of the amino groups of PAMAM(G4) and 4-carboxylphenylboronic acid. First, the diol binding ability of B-PAMAM(G4) was analyzed by using ARS. At pH 10, the UV-Vis spectrum of ARS was blue-shifted and fluorescence maximum at 564 nm was increased as the amount of B-PAMAM(G4) titrant added to ARS solution was increased (Figure 1). The blue shift and the fluorescence emission indicated the formation of the ARSphenylboronic acid (PBA) complex.41-43 Assuming that each PBA binding site independently bound ARS, the apparent binding constant of ARS and the PBA moiety of BPAMAM(G4) was calculated to be (1.45 ± 0.03) × 103 M-1 from the titration curve of B-PAMAM(G4). This binding constant was almost consistent with that of monomeric phenylboronic acid and ARS.41-43 In addition, the reversible binding of BPAMAM(G4) was evaluated by the displacement assay,44,45 which involves the titration of the B-PAMAM(G4)- ARS complex with monosaccharides (fructose, glucose, and galactose). In the titration of the B-PAMAM(G4)-ARS

Figure 1. Titration of B-PAMAM(G4) with ARS solution monitored by measuring UV-Vis spectral and fluorescence emission changes at 564 nm. pH of the measurement solution was adjusted to 10.0. [ARS] = 0.01 mM, [Phenylboronic acid in B-PAMAM(G4)] = 0–0.2 mM, [CAPS buffer] = 10 mM, λex = 492 nm.

complex with saccharide (particularly fructose), the UV-Vis absorption maximum was red-shifted from 460 to 518 nm. Fluorescence intensity decreased with saccharide concentration and saturated at concentrations exceeding 20 mM (See Supporting Information, Figure S3). The fluorescence intensity changes followed the order of fructose > glucose > galactose and the calculated binding constants of B-PAMAM(G4) and the saccharides were (1.43 ± 0.01) × 103 M-1, (7.60 ± 0.10) × 102 M-1, and (1.30 ± 0.03) × 102 M-1. The results demonstrated that as the B-PAMAM(G4)-ARS complex dissociated, BPAMAM(G4) competitively bound saccharide and consequently, free ARS was displaced to the solution. Thus, BPAMAM(G4) exhibited reversible binding of diol compounds. Bacterial recognition function of B-PAMAM(G4) under basic conditions. Synthesized B-PAMAM(G4) has five phenylboronic acid moieties in one molecule and bacteria have saccharide units on the surface of cell envelope. Thus, we hypothesized that the phenylboronic acid moieties of BPAMAM(G4) would bind the saccharide units on the surface of bacterial cell envelope to induce multipoint crosslinking. In this study, S. aureus as Gram-positive bacteria and E. coli as Gramnegative bacteria were used as model bacteria. First, we evaluated the S. aureus binding ability of B-PAMAM(G4) at pH 9.0. To determine the mixing time for the detection of S. aureus, time-dependent changes of the optical density at 600 nm (OD600) were measured after the addition of water (control), PAMAM(G4), or B-PAMAM(G4) to S. aureus suspension (Figure 2). It is known that at basic or neutral pH, the bacterial cell surface is negatively charged as a result of the dissociation of carboxylic acid, phosphoric acid, and saccharide chain.46 Thus, S. aureus was stably dispersed by electrostatic interaction and OD600 as an indication of solution turbidity showed almost no change (Figure 2(a)). When PAMAM(G4) was added to S. aureus suspension and the suspension was mixed for 5 min, OD600 was slightly increased (Figure 2(b)). Niu et. al reported that pKa of the terminal

Figure 2. Time dependence of aggregation of S. aureus stained with DAPI treated with (a) water (control), (b) PAMAM(G4), and (c) B-PAMAM(G4). The solution was adjusted to pH 9.0 with CHES buffer. [S. aureus] = 108 CFU/mL, [B-PAMAM(G4) or PAMAM(G4)] = 33 M, [CHES buffer] = 10 mM.



amino group of PAMAM(G4) was approximately 9.2.47 In our experimental conditions, PAMAM(G4) had a positive charge and thus, small aggregates were formed through the electrostatic interaction between PAMAM(G4) and S. aureus. When B-PAMAM(G4) was added to S. aureus solution and the suspension was mixed for 5 min, large aggregates were formed and OD600 was dramatically decreased (Figure 2(c)). In general, pKa of phenylboronic acid is reported to be around 8–948 and the phenylboronic acid moieties of B-PAMAM(G4) are composed of anionic tetrahedral structures at pH 9.0. Our results indicated that the large aggregates were formed through multiple interactions and crosslinking between the phenylboronic acid moieties of B-PAMAM(G4) and S. aureus. In the same manner, the E. coli recognition function of BPAMAM(G4) was investigated. When B-PAMAM(G4) was added to E. coli suspension and the whole was mixed at pH 9.0, E. coli was also aggregated (See Supporting Information, Figure S4). From these experiments, we confirmed that the phenylboronic acid moieties of B-PAMAM(G4) crosslinked with both S. aureus and E. coli at pH 9.0 to form large aggregates that were visible to the naked eye. Bacterial recognition function of B-PAMAM(G4) under neutral conditions. To investigate the pH dependence of the aggregate formation of B-PAMAM(G4) with bacteria (S. aureus and E. coli), OD600 was measured after mixing for 5 min (Figure 3). Figure 3(A) shows the results of mixing BPAMAM(G4) and bacterial suspension at pH 7.4 and pH 9.0. At pH 9.0, both S. aureus and E. coli formed aggregates with B-PAMAM(G4). However, at pH 7.4, different aggregation behaviors were observed; S. aureus formed large aggregates with B-PAMAM(G4), whereas E. coli was dispersed stably. To analyze the pH effect on S. aureus selective aggregate formation, OD600 measurements were performed at various pH after the addition of B-PAMAM(G4) (Figure 3(B)). OD600 of S. aureus was nearly zero in the pH range of 7.0 to 9.5, whereas OD600 of E. coli was nearly zero in the pH range of 8.0 to 9.0, which was narrower and had weaker basic conditions than that of S. aureus. We hypothesized that the pH difference for S. aureus and E. coli aggregation was derived from the binding affinity of phenylboronic acid to cell envelope. It is well known that the surface structures of S. aureus (Gram-positive bacteria) and E. coli (Gram-negative bacteria) are extremely different.49,50 The surface of Gram-positive bacteria is mainly composed of N-acetylglucosamine (GlcNAc) and

Figure 3. (A) OD600 changes after addition of BPAMAM(G4) at pH 7.4 and pH 9.0 for (a) S. aureus and (b) E. coli. (B) Dependence of OD600 on pH after mixing BPAMAM(G4) and bacteria. [B-PAMAM(G4)] = 33 M, [bacteria] = 108 CFU/mL, [phosphate buffer] = 2 mM.

N-acetylmuramic acetylmuramic acid (MurNAc),51 and the surface of Gram-negative bacteria has many lipopolysaccharides. To investigate the binding site of BPAMAM(G4) to the Gram-positive bacteria, we studied the competitive experiments (See Supporting Information, Figure S5). The aggregate formation of B-PAMAM(G4) and S. aureus was not inhibited in spite of the addition of B-PAMAM(G4) to S. aureus suspension with excess amount of GlcNAc and MurNAc. The result indicated that the binding site of BPAMAM(G4) to S. aureus was not GlcNAc or MurNAc. Although the binding site is not cleared yet, it is reasonable to consider that multipoint binding of B-PAMAM(G4) with saccharide units of S. aureus surface enhance the binding ability of phenylboronic acid with S. aureus. To clarify whether the bacterial aggregation was caused by the saccharide recognition of phenylboronic acid, phenolmodified PAMAM(G4) dendrimer (Ph-PAMAM(G4)) was prepared (Figure 4). We evaluated the S. aureus recognition function of Ph-PAMAM(G4) at pH 7.4. S. aureus formed large aggregates with B-PAMAM(G4) and OD600 was decreased, whereas S. aureus was dispersed stably in the presence of PhPAMAM(G4) and OD600 was increased. We also measured the zeta potentials of B-PAMAM(G4) and Ph-PAMAM(G4) at pH 7.4 to be + 3.12 ± 7.51 and + 7.42 ± 2.12 mV, respectively. These results indicated that there was no significant difference in surface charge between B-PAMAM(G4) and PhPAMAM(G4). This result demonstrated that electrostatic interaction was little affected and the phenylboronic acid of BPAMAM(G4) was involved in the formation of aggregates.

Figure 4. Aggregation of S. aureus (108 CFU/mL) with BPAMAM(G4) and Ph-PAMAM(G4) at pH 7.4. (a) The changes of optical density at 600 nm before and after addition of BPAMAM(left) and Ph-PAMAM(right). (b) Optical images of S. aureus aggregate formation with B-PAMAM(G4) (left) and PhPAMAM(G4) (right). To prove our hypothesis, the selectivity of B-PAMAM(G4) for various bacteria was evaluated. B-PAMAM(G4) solution was mixed with bacterial suspension for 5 min and OD600 was measured (Figure 5). Left bars are control (OD600 after mixing without B-PAMAM(G4)) and right bars indicate BPAMAM(G4) addition. Examination of three Gram-negative bacteria, E. coli, P. aeruginosa, and Agrobacterium, revealed almost no difference in OD600 between control and BPAMAM(G4) addition. These results corresponded to those of E. coli. On the other hand, examination of Gram-positive bacteria, namely, S. aureus with a different ATCC number, S. pseudintermedius, S. epidermidis, E. faecalis, and B. subtilis


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Analytical Chemistry denoted that OD600 was decreased by adding B-PAMAM(G4). The rate of turbidity change by B-PAMAM(G4) addition is shown by a line graph in Figure 5. For the Gram-positive and Gram-negative bacteria, the differences of the rate of OD600 change indicated the difference in the envelope composition of saccharide unit between Gram-positive and Gram-negative bacteria. It is plausible that the total number of saccharide units (binding sites) on bacterial envelope is different in bacterial species. In addition, the shape and size of bacteria should affect the OD600 changes. These results demonstrated that BPAMAM(G4) induced selective aggregation and worked as a selective glue for Gram-positive bacteria after mixing for only 5 min. These results are consistent with our hypothesis. Details of the binding sites are under investigation.

To discuss the bacterial recognition function of BPAMAM(G4) under a competitive environment, we investigated the S. aureus recognition function of BPAMAM(G4) in the presence of various concentrations of fructose as the competitor compound for bacterial recognition (Figure 7). The vertical axis shows OD600 before and after the addition of B-PAMAM(G4). When fructose concentration exceeded 100 mM, OD600 was the same before and after BPAMAM(G4) addition, indicating that the aggregate formation of B-PAMAM(G4) and S. aureus was suppressed. However, in the presence of < 10 mM fructose, OD600 was almost the same as that in the absence of fructose. We confirmed that BPAMAM(G4) showed S. aureus recognition function in the presence of < 10 mM order of fructose.

Figure 5. Selectivity of B-PAMAM(G4) for bacterial species at pH 7.4 adjusted with PBS. [B-PAMAM(G4)] = 33 M. To discuss the detection limit of this bacterial recognition system, we investigated OD600 changes with B-PAMAM(G4) addition and their dependence on S. aureus concentration (Figure 6(a)). The detection limit was calculated to be 6.4 × 106 CFU/mL. This detection limit seems unsatisfactory, but OD600 at 106 CFU/mL is approximately 0.003. The detection limit was strongly affected by the sensitivity of the instruments. We also evaluated the bacterial detection limit by changing BPAMAM(G4) concentration (Figure 6(b)). The different concentration of B-PAMAM(G4) were added in the presence of 108 CFU/mL of S. aureus. The concentration limit of BPAMAM(G4) for 108 CFU/mL of S. aureus was calculated to be ca. 700 nM (boronic acid unit of B-PAMAM(G4)).

Figure 6. OD600 changes of S. aureus in the presence of BPAMAM(G4) at pH 7.4 by changing the concentration of (a) S. aureus, (b) B-PAMAM(G4). (a) [B-PAMAM(G4)] = 33 M, (b) [S. aureus] = 108 CFU/mL.

Figure 7. Competition of S. aureus and fructose for S. aureus recognition by B-PAMAM(G4) at pH 7.4. [B-PAMAM(G4)] = 33 M, [fructose] = 0-1000 mM, [PBS] = 10 mM. Effect of polymer structure on bacterial recognition. We investigated the structure effect on aggregate formation by comparing PAMAM(G4) dendrimer, which has a spherical structure, with polyethyleneimine (PEI), which is a linear polymer. First, we prepared boronic acid modified PEI (B-PEI) by the condensation reaction of 4-carboxyphenylboronic acid and PEI (Mn = ~60,000). The modification number of 4carboxyphenylboronic acid was calculated to be approximately 10 per molecule (B-PEI) from 1H NMR measurement (See Supporting Information, Figure S6). We measured OD600 changes of S. aureus by the addition of B-PEI at pH 9.0. OD600 was slightly increased by mixing with B-PEI, but was almost the same as that of non-modified PEI or control (no polymer addition). This result was explained as follows. BPAMAM(G4) dendrimer was spherical and had a comparatively hard structure, so binding between the boronic acid moiety of B-PAMAM(G4) and S. aureus proceeded via multipoint intermolecular binding. On the other hand, linear BPEI had a soft structure. Once B-PEI bound S. aureus, other binding interactions were promoted by the cluster effect and 1:1 intramolecular binding proceeded. Thus B-PAMAM(G4) acted as glue for bacteria by crosslinking to form aggregates by mixing. This result strongly demonstrated that nanoparticle structure was one of the important factors in the system design.



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Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS

Figure 8. Chemical structure of B-PEI and time-dependent changes of S. aureus OD600 with B-PEI addition. [B-PEI] = 0.5 mg/mL, [CHES buffer] = 10 mM, adjusted to pH 9.0.

CONCLUSIONS We have designed phenylboronic acid modified PAMAM(G4) (B-PAMAM(G4)) dendrimer for the rapid and selective detection of bacterial species. We demonstrated that B-PAMAM(G4) formed large aggregates in less than 5 min by mixing with bacteria. The aggregates were visible to the naked eye. At pH 9.0, B-PAMAM(G4) formed aggregates with both S. aureus and E. coli. At pH 7.4, B-PAMAM(G4) formed large aggregates by working as a selective glue for Gram-positive bacteria, such as S. aureus. We also revealed that this pHdependent, selective aggregate formation involved the phenylboronic acid moiety of B-PAMAM(G4). The spherical structure was just as important for the aggregate formation. In addition, this bacterial recognition function of B-PAMAM(G4) was operative in the presence of fructose. To the best of our knowledge, this is the first example of a simple and rapid screening method that uses the phenylboronic acid probe to target different saccharide units in bacteria. B-PAMAM(G4) dendrimer is expected to be a powerful tool for the rapid and selective discrimination of Gram-positive and Gram-negative bacteria in solution at physiological pH.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXXXXXXX. Experimental details for 1H NMR, indicator displacement assay by fluorescence spectrometry, E. coli recognition at pH 9.0 and competitive experiments for S. aureus detection. Figures S1-S6 are also included.

AUTHOR INFORMATION Corresponding Author *[email protected]

ORCID Yuji Tsuchido: 0000-0001-5505-5293 Takeshi Hashimoto: 0000-0002-7601-5511 Takashi Hayashita: 0000-0003-1264-9694

Author Contributions Y.T. and T. Hayashita conceptualized the work. Y.T., N.K., T. Hashimoto, and T. Hayashita designed the experiments. R.H., Y.T., and K.I. did the experiments. All authors were involved in the data analysis. Y.T., K.I., and T. Hayashita wrote the manuscript. All authors have given approval to the final version of the manuscript.

This work was financially supported by a Grant-in-Aid for EarlyCareer Scientists (Grant No. 18K14255) and a Grant-in-Aid for Scientific Research (C) (Grant No. 18K05180) from Japan Society for the Promotion of Science (JSPS), and a Grant-in Aid for Scientific Research (A) (Grant No. 26248038) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

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Rapid and Selective Discrimination of Gram-Positive and Gram

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