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Design of Surface-coatable NIR-responsive Fluorescent Nanoparticles with PEI Passivation for Bacterial Detection and Killing Zihnil Adha Islamy Mazrad, Cheong A Choi, Yong Min Kwon, Insik In, Kang Dae Lee, and Sung Young Park ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10688 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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ACS Applied Materials & Interfaces

Design of Surface-coatable NIR-responsive Fluorescent Nanoparticles with PEI Passivation for Bacterial Detection and Killing Zihnil Adha Islamy Mazrad1†, Cheong A Choi2†, Yong Min Kwon2, Insik In1,3, Kang Dae Lee4*, Sung Young Park1,2* 1

Department of IT Convergence, Korea National University of Transportation, Chungju 380-

702, Republic of Korea 2

Department of Chemical and Biological Engineering, Korea National University of

Transportation, Chungju 380-702, Republic of Korea 3

Department of Polymer Science and Engineering, Korea National University of

Transportation, Chungju 380-702, Republic of Korea 4

Department of Otolaryngology–Head and Neck Surgery, Kosin University College of

Medicine, Busan 49267, South Korea

*Corresponding authors: E-mail: [email protected] (Kang-Dae Lee) E-mail: [email protected] (Sung Young Park)

†

These authors equally contributed to this work

KEYWORDS: Antibacterial; ionic complex; bacteria detection; polydopamine; photothermal fluorescent carbon nanoparticle

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ABSTRACT The ability to quickly detect and kill bacteria is crucial in the realm of antibiotic resistance. In this study, we synthesized a detection probe consisting of polyethyleneimine (PEI)-passivated polydopamine-based fluorescent carbon (FDA:PEI) nanoparticles, generating a cationic adhesive material for bacterial detection that is surface-coatable, photothermal, and antibacterial. The cationic FDA:PEI nanoparticles effectively bound to the anionic bacterial cell wall, resulting in a dramatic quenching effect visible in fluorescence spectra and confocal images. In this fluorescence on/off system, FDA:PEI nanoparticles showed similar bacterial detection abilities between aqueous- and solid-phase assays. Scanning electron microscopy clearly showed the attachment of FDA:PEI nanoparticles to the surface of bacteria, both in solution and as a coating on the surface of a polypropylene film. In addition to detection, this versatile material was found to have an antibacterial potential, via near-infrared irradiation to induce a heat release, killing bacteria by thermolysis. Thus, by exploiting the cationic and catechol moieties on the surface of polydopamine carbon dots, we developed a novel bacterial-detection platform that can be used in a broad range of conditions.


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1. INTRODUCTION Pathogens, mostly existing in water and including aquatic bacteria, enteric viruses, protozoa, and other microorganisms are a major problem in aqueous environments because of their antibiotic resistance, and several techniques have been developed to address this issue.1 Recently, biosensors and nanotechnology have provided novel methods for detection of bacteria, using simple and rapid technologies.2 Several reports have examined fluorescent, electrochemical, light-scattering, colorimetric, and surface-enhanced Raman scattering analyses as possible bacterial-detection methods.3 The use of a fluorescent on/off system has attracted much interest, but a few problems persist, such as complicated probe preparation, low sensitivity of biomolecular recognition, and unsuitability to solid-phase assays for pathogen detection.4 Fluorescent nanoparticles (FNPs), a new class of fluorescent materials, are useful in life science applications such as in vitro and in vivo imaging, chemical and biological sensing, drug delivery, photodynamic therapy, and nanomedicine, owing to their tiny size, excellent photoluminescent properties, and high biocompatibility.5-7 One of the techniques for obtaining FNPs is a condensation reaction, as well as a carbonization and dehydration system.8 Polydopamine (pDA), a mussel-inspired biomaterial, contains catechol and amine functional groups, which have been extensively employed for surface chemistry owing to their strong adhesive properties.9 Several studies have shown that pDA can easily functionalize various materials through reactions with amino- or mercapto-nucleophiles, providing a potential carbon source with several distinctive, material-independent coating features, including biocompatibility, antibacterial surface properties, a biomineralization potential, and corrosion resistance.10 Previously, we have successfully carbonized pDA (FDA) to obtain an imaging agent with high chemical stability, biocompatibility, optical absorption efficiency, and tunable emission. FNP-pDA has a graphitelike nanostructure, which holds promise in the field of 3 ACS Paragon Plus Environment


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optoelectronics. The system retains the catechol moieties even after carbonization, allowing for easy coating of some surfaces, and enabling attachment of a fluorescent material to versatile substrates. However, the fluorescence emission of FNPs is rather poor due to the surface ionic state, which requires passivation using a stabilizing agent.6 Gram-positive and gram-negative bacteria possess different components in their outer membranes, which are easily deprotonated to produce negative charges, increasing their hydrophilicity.11 By understanding the chemical structure of bacterial surfaces, we can overcome the complexity of specific antibody conjugation in detection probes by instead using electrostatic interactions with bacterial surface charges.12 Cationic polymers are proven as bacterial detection probes. Their use is based on the quenching of their fluorescent properties, and a few of them involve conjugated fluorescent dyes in the respective detection system.13 Another possible platform is polyethyleneimine (PEI), a synthetic polycationic polymer that contains primary, secondary, and tertiary amino groups. PEI has several uses, for example, as a drug, and in the development of catalyst supports because of effective neutralization of excess anionic colloidal charges.14 Because of PEI’s cationic sites, its interactions with bacteria are mainly of electrostatic nature, suggesting that it may aid in the immobilization and detection of bacteria.15, 16 The aim of this work was to develop a simple method for detecting and killing bacteria through changes in fluorescent behavior, based on carbonized pDA (FDA), which

has

multicolor

photoluminescent

properties.

We

fabricated FDA:PEI

nanoparticles by surface passivation of FDA nanoparticles with PEI, a cationic surface modification that promotes binding to the anionic surface of bacterial cells. The FDA:PEI nanoparticles possess bright, distinguishable fluorescence and interacted with both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria, resulting in fluorescence quenching upon bacterial detection in a solution phase assay. The retained catechol moiety in FDA:PEI nanoparticles resulted in the 4 ACS Paragon Plus Environment

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easy coating of various surfaces, including a propylene (PP) film, with similar bacterial detection ability in a solid-phase assay. This work focuses on fluorescent characteristics of a composite material when it interacts with bacteria as a biosensor for application to various fields such as microbiology, medicine, and environmental engineering. The novel features of this nanomaterial-based composite (relative to existing methods) include suitability for a solid-phase assay that offers fast sensitive bacterial detection not only in a bacterial suspension model but also in a real-world scenario.

2. EXPERIMENTAL SECTION 2.1 Materials and characterization Trizma base, Trizma HCl, [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (MTT), dopamine hydrochloride, NaOH, PEI, and concentrated H2SO4 were purchased from Sigma-Aldrich (Korea). A penicillin-streptomycin solution, fetal bovine serum (FBS), 0.25% (w/v) trypsin, a 0.05% (w/v) EDTA solution, and the RPMI 1640 medium were acquired from Gibco (Carlsbad, CA, USA). Proton NMR spectra were recorded using a Bruker AVANCE III 400 MHz spectrometer with dimethyl sulfoxide (DMSO) as the solvent. Absorption spectra were acquired on an Optizen 2120UV spectrophotometer (Mecasys, Yuseong-gu Daejeon, South Korea). Fluorescent properties were analyzed by means of a L550B luminescence spectrometer from Perkin Elmer. A near-infrared (NIR) laser (PSU-III-LRD, CNI Optoelectronics Tech. Co., Ltd., China) was set to wavelength 808 nm. Photothermal heating curves were examined using an NEC Avio TH9100 Thermo Tracer infrared camera. Transmission electron microscopy (TEM) (JEM-2100F, JEOL) was carried out with an 80–200 kV electron gun. Field-emission scanning electron microscopy (FE-SEM) 5 ACS Paragon Plus Environment


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micrographs were obtained using a JEOL JSM-6700F SEM (Tokyo, Japan). X-ray photoelectron spectra (XPS) were acquired using an ESCALAB apparatus (Omicrometer, Taunustein, Germany) and a PHI Quantera-II system (Ulvac-PHI, Chigasaki, Kanagawa, Japan). Analysis of live and dead bacteria was performed under a Zeiss LSM 510 confocal laser-scanning microscope (CLSM; Zeiss, Germany) using 405-, 488-, and 543-nm emission filters at 20× magnification. Particle size was measured by dynamic light scattering (DLS) on a Zetasizer Nano (Malvern, Germany). Static water contact angles were measured with a DO3210 instrument (KRUSS Ltd., Germany).

2.2 Surface passivation of FDA with PEI (FDA:PEI) The method for the synthesis of carbonized polydopamine (FDA) was described previously.6 To obtain passivated FDA in FDA:PEI ratios 1:10 and 1:100, 1 mg of FDA was dissolved in 30 mL of distilled water in a 250-mL flask, then we added 10 mg and 100 mg of PEI solution (δ= 1.27g/cm³). The mixture was stirred vigorously to obtain a homogenous solution, then transferred to a hydrothermal reactor and heated at 120°C for 72 h in an inert (N2) atmosphere. Next, the solution was allowed to cool at room temperature and was centrifuged at 4000 rpm for 10 min to collect PEI-passivated FDA at FDA:PEI ratios of 1:10 and 1:100. The sample was purified by dialysis against water for 24 h (across a membrane with a molecular weight cutoff of 3500 Da), and freeze-dried.

2.3 Liquid- and solid-phase bacterial detection assays using FDA:PEI nanoparticles For the liquid phase detection assay, S. aureus (gram-positive, strain ATCC 25323) and E. coli (gram-negative, strain ATCC 25922) were cultured in 50 mL of LB and MRS media, respectively, at 37°C for 24 h. Each version of FDA:PEI nanoparticles (1 mg/mL) was added to the bacterial suspension (108 colony-forming units [CFU]/mL) and incubated for 1 h, followed by centrifugation and washing with PBS (pH 7.4) to remove unbound FDA:PEI 6 ACS Paragon Plus Environment

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nanoparticles. To verify the capacity of the FDA:PEI nanoparticles for detection of bacteria, the pellet of cells labeled with FDA:PEI nanoparticles was resuspended in PBS and examined on a L550B luminescence spectrometer at an excitation wavelength of 520 nm and under the LSM 510 CLSM.15, 16 For the solid-phase detection assay, an FDA:PEI nanoparticle-coated PP film was added to each bacterial strain at 108 CFU/mL. The FDA:PEI nanoparticle-coated PP film was prepared as previously reported.15 After a 1-h incubation period, the coated PP film was removed from the solution and washed with PBS. The coated PP was dried at room temperature and its fluorescent properties were measured using the LSM 510 CLSM.15

2.4 Bacterial cell imaging by SEM For SEM imaging, bacterial suspensions cultured as above were centrifuged and washed consecutively with 25%, 50%, 75%, and 99% ethanol. A few microliters of bacteria untreated or treated with nanoparticles was placed on a silicon wafer, dried for 3–4 h, and used to record SEM micrographs. After washing with PBS, the nanoparticle-coated PP film was dried at room temperature, and the interaction of S. aureus and E. coli with coated PP was examined by FE-SEM.

2.5 FDA:PEI nanoparticle bacterial photothermolysis assay We measured the bacterial photothermolytic ability of FDA:PEI nanoparticles in a range of concentrations (0.1–2.0 mg/mL) under NIR irradiation by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. S. aureus and E. coli cells (105 CFU/mL) were incubated with FDA:PEI nanoparticles for 1 h, then irradiated by a NIR laser for 5 min at 37°C. After that, the bacteria were centrifuged, washed with PBS, and incubated for additional 4 h with 20 µL of 5 mg/mL MTT in PBS. The MTT-solubilizing agent (180 µL) 7 ACS Paragon Plus Environment


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was added, followed by 15 min of shaking. Finally, the absorbance was measured at 570 nm on a Varioskan Flash microplate reader (Thermo Electron Corporation). Relative cell viability was evaluated by comparison with control cells, which were not subjected to NIR irradiation. 3. Results and Discussion We hypothesized that the PEI amine groups on the surface of FDA:PEI nanoparticles would act as a substantial source of cationic charges, which would be attracted to the highly anionic outer membrane permeability barrier of bacteria. As shown in Scheme 1, we designed pDA-based FNPs through controlled carbonization in a strongly acidic environment, then used surface passivation with PEI to generate cationic fluorescent nanoparticles to detect bacteria via a quenching mechanism. Carbonized FDA has been reported to release heat; thus, the newly developed agent also has a potential to kill bacteria photothermally in response to NIR light. The remaining catechol moieties in FDA provide adhesion, allowing for coating of various surfaces. Figure 1(a) shows the 1H-NMR signals of 1:10 and 1:100 FDA:PEI nanoparticles, with PEI peaks at 2.5–2.7 ppm.17 Absorption analysis of the prepared samples in aqueous solutions yielded peaks at 280 and 320 nm for all three samples, indicating π–π* electron transitions in the phenolic compounds of pDA.18, 19 The addition of PEI only increased the peak area at 220 nm, but not the NIR absorbance area. The unchanged intense peak of FDA at 320 nm after PEI passivation suggests that surface passivation had no effect on the optical properties of FDA (Figure 1(b)). PEI did not show NIR absorbance because the increasing PEI amount decreased the photothermal absorption profile of FDA due to the covering of π– π* electron transitions on the core FDA carbon dots. Fourier transform infrared spectroscopy (FT-IR) data in Figure S1(a) show distinctive peaks at ~1654 cm−1, which might indicate aminate N–H/O–H stretching vibrations. At the same time, the appearance of higher intensity peaks for FDA:PEI (1:10) and FDA:PEI (1:100) nanoparticles denotes surface-passivated PEI. 8 ACS Paragon Plus Environment

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To confirm the results of this analysis, the zeta potentials of FDA:PEI (1:10) and FDA:PEI (1:100) nanoparticles were measured and showed a highly negative charge (+mV) irrespective of pH (ranged from 5.0 to 8.5), indicating the positively charged surfaces because of coating with PEI groups (Fig. S1 (b)). The surface morphology and elemental composition were investigated by TEM, DLS, and EDX measurements. The TEM images of 1:10 and 1:100 FDA:PEI nanoparticles at increasing magnification, shown in Figure 1(c), reveal that after surface passivation, the prepared nanoparticles retained their spherical morphology, with an average size of 50 nm. The lattice separations at both PEI ratios (0.290 to 0.31 nm) confirmed graphene carbon structures, indicating internalized carbon particles of FDA.20, 21 Accordingly, when measured by DLS, the average sizes of FDA:PEI nanoparticles at the 1:10 and 1:100 ratios were predicted to be 42.15 and 51.52 nm, respectively. Generally, FDA:PEI (1:100) nanoparticles were bigger than the 1:10 counterparts, indicating higher passivation of the polymeric surface of FDA at the higher FDA:PEI ratio (Figure 1(d)). In addition to structural analysis, the composite was then analyzed by EDX elemental mapping to understand the variations in carbon (C), oxygen (O), and nitrogen (N) composition. The presence of PEI covering the FDA core was confirmed by an increase in the percentage of N in the composite. The higher ratio of PEI resulted in higher percentage content of N (Figure S2). Fluorescence decay time (τ) values (Figure S3(a)) were 4.78 and 6.30 ns for 1:10 and 1:100 FDA:PEI nanoparticles, respectively, suggesting that surface passivation enhances the fluorescence lifetime of FNPs. Moreover, as shown in Figure S3(b), the carbonized nanomaterials possessed electron donor–acceptor properties, making them a candidate for electroluminescence owing to the optical and electrical phenomena.22 To examine the electron-donating ability based on the electronic energy levels of two interacting materials, we examined the ability of FDA:PEI (1:10 and 1:100) emission intensities to be quenched by the known electron acceptor 2,4-dinitrotoluene (-0.9 V vs. NHE) and the electron donor N,N9 ACS Paragon Plus Environment


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diethylaniline (0.88 V vs. NHE). The quenching constant (Ksv) values from a Stern–Volmer plot gradually increased from the acceptor to donor in each case, suggesting that FDA:PEI nanoparticles can act as a donor and acceptor at the same time.23, 24 Besides, the FDA has a 1.33% blue quantum yield, which increased after surface passivation at the 1:10 ratio to 2.67%, but when we examined it for the ratio 1:100, the blue quantum yield considerably decreased to 0.17% owing to complete covering of the carbon dot system by PEI. These results therefore revealed excitation-dependent emission spectra with identical photophysical properties, indicative of suitability for different fluorescence-based tools.6 As we can see in the PL profile of FDA in Figure S4, the expected excitation dependence showed multicolor emission with maximum emission at excitation 360 nm, whereas its passivated FDA showed changes of properties in solution with different pH. PEI is pH-sensitive because of its abundance of amino groups, which can be protonated and deprotonated depending on pH.25 To examine the effects of pH at both ratios, we measured the fluorescence intensity of FDA:PEI nanoparticles in PBS at various pH values (Figure S4). For FDA:PEI (1:10) nanoparticles, there were no significant changes in fluorescence intensity, while at 1:100, there was a notable decrease in fluorescence intensity as the solutions became more basic. At pH 9, the least fluorescence intensity was recorded, due to deprotonation of the surface amino groups of PEI.25 The result suggests that the larger amount of PEI present at the 1:100 ratio resulted in pH-sensitive FDA:PEI nanoparticles. Various techniques have been used for detection of bacteria in an aqueous suspension, including fluorescence on/off systems that take advantage of interactions between a cationic material and the anionic bacterial cell wall.13 Figure 2 shows the detection of bacteria in a solution phase assay based on the fluorescence intensity of various concentrations of the two FDA:PEI composites. After excitation at 340 nm, FDA:PEI nanoparticle intensity dramatically changed after incubation with either E. coli or S. aureus, yielding a quenching effect as compared to the control polymer. Detection was more sensitive toward E. coli than S. 10 ACS Paragon Plus Environment

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aureus at all concentrations of FDA:PEI nanoparticles, generating greater quenching with E. coli.26 This phenomenon may be due to the ease of formation of electrostatic bonds with phosphate groups and phospholipids in the E. coli cell wall, compared to the long chains of teichoic acid in the predominantly hydrophobic S. aureus cell wall.15, 26, 27 Furthermore, we evaluated the changes in emission of our cationic nanoparticles after interaction with bacteria using confocal images (Figure 3). Figure 3(a) shows emission of FDA:PEI (1:10) and FDA:PEI (1:100) nanoparticles only for the excitation laser wavelength 405 nm (blue) as appropriate with a maximum emission wavelength in the PL spectra in Figure 2. We also examined the FDA:PEI nanoparticle system incubated with a bacterial suspension for 1 h, as fluorescence quenching occurred with both E. coli and S. aureus. After interaction with the bacteria (E. coli or S. aureus) at the concentration of 108 CFU/mL, cationic FDA:PEI nanoparticles bound to anionic phosphate groups on the surface of bacteria, resulting in a quenching effect due to the complex polyionic interaction. The use of confocal measurements distinguished only the interactions of bacteria and FNPs based on color changes, but could not show direct interactions of both components. Therefore, Figure 3(b) shows evidence for the direct interaction of bacteria and FDA:PEI nanoparticles by SEM imaging, which reveals nanoparticles’ adhering to the surface of bacteria. Therefore, we can visibly see differences after FDA:PEI incubated with bacteria with smooth surfaces (both species) versus control images, clearly indicating the complex interactions between the cationic sites of FDA:PEI nanoparticles and negative charges on the bacterial cell walls. The catechol groups in FDA may attach this unique material to various surfaces for use as a solid-phase bacterial sensor. As depicted in Figure S5, the water contact angle on several substrates decreased for PP and PET surfaces, indicating decreased hydrophobicity, while Si wafers displayed increased hydrophobicity, suggesting that FDA:PEI nanoparticles can successfully coat these substrates.6, 28 Therefore, this material could be used to easily detect bacteria using only a single film. 11 ACS Paragon Plus Environment


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To ensure successful coating of the PP film with FDA:PEI nanoparticles, further surface chemical characterization was performed by XPS. As shown in Figure S6, the spectra were composed of three elements, including C 1s, N 1s, and O 1s for FDA:PEI (1:10) and FDA:PEI (1:100) nanoparticles. The FDA:PEI C 1s spectra at 284.2, 284.7, 285.3, and 288 eV correspond to C=C, C-C, C-N, and C=O bonds, respectively. The abundance of sp2 and sp3 carbon sites indicates that the expected constitution of carbon dots was retained after surface passivation. As for the spectrum of N 1s, there were two specific N+ peaks at 399.5 eV and an No peak at 400.6 eV for FDA:PEI (1:10) nanoparticles, and N+ peaks at 398.9 eV and an No peak at 400.4 eV for 1:100 FDA:PEI nanoparticles. The No peaks showed decreased intensity for FDA:PEI (1:100) nanoparticles, confirming the successful incorporation of N atoms into the carbon within FDA.28 To determine the fluorescence-quenching response of the PP film coated with FDA:PEI nanoparticles, we analyzed confocal images and direct interactions with bacteria on the surfaces of the nanoparticle-coated PP film by SEM imaging after incubation with bacteria for 1 h. Figure 4(a) displayes the changes in emission of FDA:PEI nanoparticles on the surface of the PP film after detection of bacteria. The blue emission of FDA:PEI nanoparticles on the surface of the PP film disappeared noticeably after interaction with both E. coli and S. aureus, at a high concentration but slightly decreased when interacting with lower bacterial concentration. The coated PP has cationic sites (from PEI) on its surface, where there was a strong interaction with negatively charged surface of bacteria resulting in a quenching effect. When we sampled lower bacterial concentrations, the fluorescence was not totally quenched because the interaction of bacteria with polyionic sites is caused not only by the strong ionic complexes but also by hydrophobic interactions with bacterial surfaces. At coating concentrations of 10 mg/mL on the PP surface, not all cationic FDA:PEI sites bound to bacteria, generating a decrease of quenching effect, enhancing the utility for the detection of bacterial in a solid-phase assay. Figure 4(b) depicts SEM analysis of the nanoparticle-coated 12 ACS Paragon Plus Environment

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PP film incubated with a bacterial suspension. The adherence of both bacterial species to the PP surface after incubation for 1 h clearly revealed that polyion complexation and hydrophobic interactions occurred between the bacteria and the cationic groups of these nanoparticles. After coating, the positive charges of PEI as undergo quaternization of the amino groups which enhance the attachment of bacteria via their negatively charged surfaces (Figure 4).15 To test the potential field use, we adapted the bacterial-detection system to very low bacteria concentrations (103 and 102 CFU/mL) for both aqueous- and solid-phase assays (Figure S7). In the presence of a low bacterial concentrations, the fluorescence was not totally quenched because bacterial interactions with the polyionic sites were caused not only by strong ionic bonds but also by hydrophobic interactions with the bacterial surfaces, which reduced the quenching effect.7 In addition, during the solid-state detection, not all cationic FDA:PEI sites on the PP film were bound to bacteria at the low bacterial concentrations because of the high coating concentration of 10 mg/mL on these PP surface. These results suggest that bacterial levels can be detected by the naked eye both in aqueous and solid phase assays. In the XPS analysis of Figure S8, a phosphorous peak (P) was identified between 132 and 134 eV for both samples, evidencing the as existence of bacteria on the surface of the coated PP. We found a lower intensity of peak P with nanoparticles treated with S. aureus compared to E. coli, which agrees with the smaller quantity of phosphate groups in the cell wall of S. aureus compared to E. coli.15 E. coli surfaces contain lipopolysaccharides (LPS), large molecules consisting of a lipid and a polysaccharide and many phosphate groups which can easily be deprotonated to generate negative charges. The cationic site from FDA:PEI nanoparticles will interact with these sites, generating significant fluorescence quenching. In contrast to E. coli, , we observed only slight fluorescence changes for S. aureus. S. aureus possesses no LPS on its surfaces, but the long chains of teichoic acid which did not strongly aggregate with the carbon carbon. The 13 ACS Paragon Plus Environment


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fluorescence-quenching mechanism here follows the aggregation-caused quenching concept reported elsewhere.29 The aggregation of cationic carbon dots with bacteria via ionic interactions leads to strong π-π stacking interactions with ordered or random structures. This aggregation guides fluorescence decay via non-radiative pathways for emission of light in a condensed phase, thus reducing the fluorescent properties.30 After carbonization, such a material will have enhanced photoelectric properties, compositionally assigned to p states of the sp2 fraction, which allow the nanoparticles to act as chromophores.

This

phenomenon

produces

localized

sp2

π-electron-confining

photoluminescence and NIR absorption.6,30 NIR-responsive materials can serve as photothermal agents, killing bacteria through thermolysis at a minimum of ≥60°C, cells are deformed and aggregated. The heat damages bacteria through denaturation of proteins, which results in cell death or inability to proliferate.31,

32

Figure S9 (a and b) illustrates the

photothermal effect of the FDA:PEI biosensor under NIR irradiation, generating temperature changes of up to 12°C. The photothermal conversion efficiency (η) of FDA:PEI (1:10) and FDA:PEI (1:100) nanoparticles (2 mg/mL) was monitored and calculated under continuous irradiation by a 808-nm laser light (2 W/cm2) according to the energy balance of the system.33, 34

The η values for FDA:PEI (1:10) and FDA:PEI (1:100) nanoparticles in water were found

to be 41.28% and 29.48%, respectively (Figure S10). Based on the absorption peaks, the molar extinction coefficients (ε) of FDA:PEI (1:10) and FDA:PEI (1:100) nanoparticles at 808-nm irradiation were found to be 2.74 × 103 and 0.98 × 103 M−1 cm−1, respectively. The heat release by 5 min of NIR irradiation decreased viability of both bacterial strains, compared with non-irradiated bacteria, through inhibition of bacterial proliferation and via inactivation of many essential bacterial functions.15,

31, 35, 36

Even though the FDA:PEI

composites did not reach the desired 60°C, the light energy converted to heat lead to killing of bacteria through thermolysis, as shown in Figure 5. Moreover, as a function of laser irradiance 14 ACS Paragon Plus Environment

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from 0.5 to 2 W/cm2 (Figure S9 (c and d)), the temperature of both FDA:PEI solutions (2 mg/mL) went up dramatically. We confirmed the photothermal efficiency at different laser irradiance powers (0.5, 1, 1.5, and 2 W/cm2) in the MTT assay (Figure S9 (e and f)). In these experiment, the percentage of bacterial viability decreased as increasing laser power. Live and Dead bacteria were examined by confocal microscopy and SEM to provide additional evidences of photothermal therapy (PTT)-mediated killing of bacteria under NIR irradiation. Figure S11 shows dead bacteria, stained in red and heat-damaged bacteria of irregular shape as a consequence of increasing NIR irradiation. To test the application for biosensor under real-life conditions where there are other particles such as dirt, sand, other benign bacteria, and other particles, students’ hands and water from the Dalcheon river located in Chungju, South Korea, tested in both solid and aqueous phase assays. Firstly, we ensured the availability of bacteria on these alternative substrates through colony plating on LB and MRS media. Next, we attempted to detect bacteria using our system with a 24 h incubation, as presented in Figure S12. Remarkably, the results showed the loss of blue emission by the the solid biosensor, indicating the detection of bacteria in the real-life samples. These data show excellent agreement between detection of bacteria in solution and in real-life samples. Therefore, both types of our nanoparticles appear suitable for detecting bacteria at various environments with high accuracy and specificity. FDA:PEI (1:100) nanoparticles gave a slightly better quenching response because the high PEI ratio results in a higher number of cationic amino groups. Therefore, the bacteria can more easily bind to FDA:PEI (1:100) than FDA:PEI (1:10) nanoparticles. However, in the thermolysis process, the FDA:PEI (1:10) nanoparticles proved to be more efficient than FDA:PEI (1:100). We can thus conclude that FDA:PEI nanoparticles can be an excellent biosensor, combining NIR-mediated bacterial killing and detection by tunable absorption properties. 15 ACS Paragon Plus Environment


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4. CONCLUSIONS We successfully designed a simple and economical method for the detection of bacteria in liquid- and solid-phase assays, by functionalizing PEI-passivated FDA (FDA:PEI) nanoparticles with a photothermal antibacterial potential. Proton NMR, FT-IR, UV-Vis, and TEM provided data on the structure and morphology of the FDA:PEI nanoparticles in two ratios, revealing tunable multicolor emission dependent on the excitation wavelength. Addition of a larger amount of PEI into the system generated pH sensitivity. The fluorescent spectra of FDA:PEI nanoparticles after incubation with bacteria were markedly quenched, and this finding was supported by a loss of blue fluorescence in the presence of bacteria in confocal images. We took advantage of the catechol moieties of FDA to apply these nanoparticles as a coating to a PP film for use as a solid-phase bacterial sensor. The blue emission from the coated PP film disappeared after incubation with E. coli and S. aureus, and this result was confirmed by the presence of bacteria on the PP surface in SEM images and was attributed to ionic interactions between cationic FDA:PEI nanoparticles and the anionic bacterial surface. Therefore, surface-coatable FNPs can be developed into a portable device for detection of bacteria when the nanoparticles are attached as a coating to a substrate. Furthermore, without conjugation to a photothermal agent, this carbonized material had antibacterial properties through thermolysis after NIR irradiation. Because of the cationic and catechol groups on the surface of FDA, this system represents a new platform for bacterial detection, with thermolysis effects in a broad range of bacterial conditions.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.


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Chemical and surface characterization using FT-IR spectroscopy and zeta potential; the corresponding EDX analysis for confirming elemental composition and mapping; fluorescence decay time assays and Stern–Volmer plots for quenching analysis; pH responses of fluorescent properties at varying excitation wavelengths; contact angle data for surface film analysis; XPS spectra for elemental binding on the surface of a film as a coating; bacteria detection at a low concentration of bacteria; the chemical analysis on the surface of a film as a coating after evaluation of performance on bacteria detection; photothermal heating profiles and antibacterial activity under NIR laser irradiation; the photothermal conversion efficiency under NIR irradiation at 808 nm; fluorescence microscopy images showing PTT cytotoxicity toward bacteria; a real-world scenario of bacteria detection. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (S.Y.P). * E-mail: [email protected] (KDL). Notes The authors declare that they have no competing financial interests. ACKNOWLEDGEMENTS This research was supported by Grants No. 10062079 and R0005237 from the Ministry of Trade, Industry & Energy (MOTIE) and Korea Institute for Advancement of Technology (KIAT) through the Research and Development for Regional Industry (No R0005303), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1A2B2002365), and Marine Biotechnology Program (20150220) funded by Ministry of Oceans and Fisheries, Korea. 17 ACS Paragon Plus Environment


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Scheme 1. Synthesis of FDA:PEI nanoparticles for detection and killing of bacteria.



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Figure 1. (a) 1H-NMR spectra of FDA:PEI (1:10) and FDA:PEI (1:100) nanoparticles; (b) UV-Vis spectra of FNP-pDA, FDA:PEI (1:10), and FDA:PEI (1:100) nanoparticles; (c) TEM images; and (d) DLS-based particle size distribution of FDA:PEI (1:10) and FDA:PEI (1:100) nanoparticles.


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Figure 2. Detection of E. coli and S. aureus in the liquid phase at varying concentrations of FDA:PEI (1:10 or 1:100) nanoparticles, as measured by fluorescence emission intensity after excitation at 380 nm. The concentration of bacteria was 108 CFU/mL.



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Figure 3. (a) Confocal images and line-scanning profiles of the fluorescence intensities of 1.0 mg/mL FDA:PEI (1:10 or 1:100) nanoparticles after incubation with E. coli or S. aureus (108 CFU/mL) in PBS pH 7.4. (b) FE-SEM images of S. aureus and E. coli detection using 1 mg/mL FDA:PEI (1:10 or 1:100) nanoparticles. Concentration of bacteria: 108 CFU/mL. 26 ACS Paragon Plus Environment

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Figure 4. (a) Confocal images of PP coated with 10 mg/mL FDA:PEI (1:10 or 1:100) nanoparticles after interaction with varying concentrations of S. aureus or E. coli. (b) SEM images of PP coated with 1 mg/mL FDA:PEI (1:10 or 1:100) nanoparticles after interaction with S. aureus or E. coli. Concentration of bacteria: 108 CFU/mL. 27 ACS Paragon Plus Environment


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Figure 5. A MTT assay of (a) S. aureus and (b) E. coli treated with 0.1–2.0 mg/mL FDA:PEI (1:10 or 1:100) nanoparticles and 5-min NIR irradiation. Data are presented as mean ± standard deviation (n = 3). Concentration of bacteria: 105 CFU/mL.


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Graphical Scheme

Design of surface-coatable NIR-responsive fluorescent nanoparticles made of PEIpassivated carbon nanoparticles based on carbonized polydopamine for detection and killing of bacteria


Design of Surface-Coatable NIR-Responsive ... - ACS Publications

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