Antimicrobial Amino-Functionalized Nitrogen-Doped Graphene

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Biological and Medical Applications of Materials and Interfaces

Antimicrobial Amino-Functionalized Nitrogen-doped Graphene Quantum Dots for Eliminating Multidrug-Resistant Species in Dual-Modality Photodynamic Therapy and Bioimaging under Two-Photon Excitation Wen-Shuo Kuo, Yu-Ting Shao, Chih-Hui Yang, Keng-Shiang Huang, Miao-Hsi Hsieh, Pei-Chi Chen, ChiaYuan Chang, Chia-Hung Huang, Chih-Li Lilian Hsu, Ting-Mao Chou, Jiu-Yao Wang, and Ping-Ching Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01429 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 6, 2018

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ACS Applied Materials & Interfaces

Antimicrobial

Amino-Functionalized

Nitrogen-Doped Graphene Quantum Dots for Eliminating Multidrug-Resistant Species in Dual-Modality Photodynamic Therapy and Bioimaging under Two-Photon Excitation Wen-Shuo Kuo,†,‡ Yu-Ting Shao,§ Chih-Hui Yang,₤ Keng-Shiang Huang,∥ Miao-Hsi Hsieh,& Pei-Chi Chen,& Chia-Yuan Chang,† Chia-Hung Huang,○,$ Chih-Li Lilian Hsu,§, & Ting-Mao Chou,*,⊥ Jiu-Yao Wang,*, §,&,‡ and Ping-Ching Wu*,# †

Advanced Optoelectronic Technology, National Cheng Kung University, Tainan 701, Taiwan (R.O.C). ‡

Department of Pediatrics, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (R.O.C.). §

Department of Microbiology & Immunology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (R.O.C.). ₤

Department of Biological Science and Technology, I-Shou University, Kaohsiung 840, Taiwan (R.O.C). ∥ The School of Chinese Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung 840, Taiwan (R.O.C). &

Institute of Basic Medical Sciences, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (R.O.C.). ○

Metal Industries Research & Development Centre, Kaohsiung 811, Taiwan (R.O.C.).

$

Department of Materials Science Engineering, National Cheng Kung University, Tainan 701, Taiwan (R.O.C.). ⊥ Division of Plastic Surgery, Department of Surgery, E-Da Hospital, Kaohsiung 824, Taiwan (R.O.C.). #

Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan (R.O.C). *To whom correspondence should be addressed. E-mail: [email protected] (T. M. Chou); [email protected] (J. Y. Wang); [email protected] (P. C. Wu).

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KEYWORDS amino-functionalized nitrogen-doped grapheme quantum dots; two-photon photodynamic therapy; multidrug-resistant species; two-photon contrast agent probe; three-dimensional biological environment

ABSTRACT Developing a nanomaterial for use in highly efficient dual-modality two-photon photodynamic therapy (PDT) involving reactive oxygen species (ROS) generation and for use as a two-photon imaging contrast probe is currently desirable. Here, graphene quantum dots (GQDs) doped with nitrogen and functionalized with an amino group (amino-N-GQDs) serving as a photosensitizer in PDT had superior ability to generate ROS than did unmodified GQDs. Multidrug-resistant (MDR) species were completely eliminated at an ultralow energy (239.36 nJ pixel−1) through only 12 s two-photon excitation (TPE) in the near-infrared region (800 nm). Furthermore, the amino-N-GQDs had an absorption wavelength of approximately 800 nm, quantum yield of 0.33, strong luminescence, an absolute cross section of approximately 54356 Göeppert–Mayer units, a lifetime of 1.09 ns, a ratio of the radiative to nonradiative decay rates of approximately 0.49, and high two-photon stability under TPE. These favorable properties enabled the amino-N-GQDs to act as a two-photon contrast probe for tracking and localizing analytes through in-depth 2 ACS Paragon Plus Environment

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two-photon imaging in a three-dimensional biological environment and concurrently easily eliminate MDR species through PDT.

INTRODUCTION In addition to conventional photosensitizers (PSs)1 and several newly synthesized PSs,2 using nanomaterials for PDT has proven to be a desirable alternative approach for achieving improved therapeutic efficacy.3 Graphene-based materials are studied in nearly each area because of their wonderful conductivity and mechanical, optical, and thermal properties. However, some works have demonstrated that when they are used in combination with PSs, they can induce PDT mechanisms4; research on the direct use of GQDs as PSs to produce ROS for PDT has been limited.5 GQDs are a new type of zero-dimensional (0D) quantum dot (QD) converted from 2D graphene sheets and have emerged as a promising optical nanomaterial; GQDs could be applied in biomedical research, including PDT.6 A major drawback of previous relevant studies, however, is that they have not considered the possibility of exploiting PDT through two-photon processes. Using nitrogen dopants to modify the intrinsic properties of GQDs is also considered helpful in performing phototherapy because the carrier density can be noticeably changed, resulting in optical and electrical properties completely distinct from their intrinsic counterparts. Thus, for GQDs exhibiting extraordinary quantum 3 ACS Paragon Plus Environment


confinement and edge effects, N-doped GQDs (N-GQDs) can exhibit enhanced, electrocatalytic, photochemical, and electrochemical activities, providing advantages in optoelectronic and biomedical applications. Furthermore, chemical modifications with primary amine molecules (also called amino-group functionalization) lead to strong electron donation, contributing considerably to the apparent effect on the electronic properties of N-GQDs.7 However, considering the singlet–triplet splitting of amino-N-GQDs, intersystem crossing is so efficacious that it competes with internal conversion among the states with the same multiplicity, leading to the concurrent emission of photoluminescence (PL) and the generation of ROS with PDT action.8 We observed not only that a nitrogen dopant exhibited impressive bactericidal capability in two-photon PDT but also that amino-group functionalization played a critical role in its antimicrobial effects. Moreover, the effect of a large π-conjugated system and strong electron donation in amino-N-GQDs has been proposed to increase the charge transfer efficiency,9 resulting in enhancement of two-photon absorption (TPA), two-photon luminescence (TPL), the TPE cross section, post-TPE stability, and the ratio of radiative to nonradiative decay rates but a shortened lifetime. In this study, amino-N-GQDs served as the two-photon PS coupled with TPE (239.36 nJpixel−1, 2.3936 mW: for calculation of the power after the objective on the sample please see Supporting information; Figure S1, the x–y axis focal spot and 4 ACS Paragon Plus Environment

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the z-axis resolution of the laser system was approximately 0.38 µm, and 0.95 µm). Thus, MDR analytes can be eliminated completely through photoexcitation for only 12 s (Ex: 800 nm), representing highly effective PDT efficiency compared with that of GQDs. Mediated by TPE, the amino-N-GQDs exhibited favorable two-photon properties of luminescence and stability compared with GQDs, enabling them to serve as

an

effective

two-photon

contrast

agent

for

tracking

or

localizing

amino-N-GQD-treated analytes in a 3D environment. Coupled with two-photon PDT actions, they entirely and efficiently killed the MDR species during observation.

MATERIALS AND EXPERIMENTS Coating antibodies. A quantity of antibody (anti-protein A antibody (Abprotein A), anti-lipopolysaccharide antibody (AbLPS) or anti-nucleus and mitotic cell antigen antibody (Abnucleus) (abcam, USA)) was measured and an absorbance wavelength of approximately 204 nm was recorded using ultraviolet-visible (UV-vis) spectroscopy (Supporting information, Figure S2a). The same quantity of Ab was then coated on materials in the dark for 30 min of incubation at 4 ℃ by the electrostatic interaction. The mixture was centrifuged at 83000 rpm (Optima TLX Ultracentrifuge, BECKMAN, USA) to remove excess Ab, after which the supernatant was collected. The absorbance difference between a quantity of pure Ab and the collected supernatant was calculated (Figure S2a), and then the molar concentration of Ab 5 ACS Paragon Plus Environment


coated on materials was estimated using Lambert-Beer's law (A= εbC, where A= absorbance, ε= molar extinction coefficient, b= path length (1 cm), and C= concentration). On the other hand, the characteristic peak of material-Ab was appeared approximately from 200 nm to 240 nm, which was arisen from the surface plasmon resonance of the peak of Ab (approximately 204 nm) and the peak of materials (approximately 224 nm-226 nm; π-π* transition of aromatic C＝C bonds). Besides, the zeta potential and DLS spectrometers were also used to characterize the coating of material-Ab and the results were as follows. Eventually, the material-Abprotein

A,

-AbLPS or -Abnucleus was then well prepared, respectively

(Supporting information, Figure S2b). In the working solution of 1×PBS buffer, there was approximately 8.8 µg of Abprotein A coated on 100.0 µg of amino-N-GQDs, which meant the coating/loading efficiency was approximately 8.8% (zeta potential of amino-N-GQD-Abprotein A ：15.30 eV), whereas it was 13.10% (zeta potential of GQD-Abprotein A：16.30 eV) for GQD. For AbLPS, amino-N-GQD and GQD were 9.30% (zeta potential of amino-N-GQD-AbLPS：15.40 eV) and 13.40% (zeta potential of GQD-AbLPS：17.0 eV), respectively. For Abnucleus, amino-N-GQD and GQD were 8.10% (zeta potential of amino-N-GQD-Abnucleus：15.20 eV) and 12.0% (zeta potential of GQD-Abnucleus：16.40 eV), respectively. In bacterial culture medium, for Abprotein A, amino-N-GQD and GQD were 8.50% (zeta potential of amino-N-GQD-Abprotein A： 6 ACS Paragon Plus Environment

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15.40 eV) and 12.60% (zeta potential of GQD-Abprotein A：16.50 eV), respectively; for AbLPS,

amino-N-GQD

and

GQD

were

9.00%

(zeta

potential

of

amino-N-GQD-AbLPS：15.60 eV) and 13.20% (zeta potential of GQD-AbLPS：17.10 eV), respectively. In the culture medium of human paclitaxel-resistant KB paclitaxel-50 cervical carcinoma cells (KB-50), for Abnucleus, amino-N-GQD and GQD were 8.20% (zeta potential of amino-N-GQD-Abnucleus：15.60 eV) and 12.30% (zeta potential of GQD-Abnucleus：16.70 eV), respectively. These results had proven the successful coating of Ab on the surface of materials. Besides, the relevant data of DLS was shown in Table S1 and Figure S3 (Supporting information). Molecular weight of GQD-based nanomaterials. The theoretical diameter of a benzene is 0.243 nm with the molecular weight of 72 (ignore the H atoms). According to Figure 1a and Figure S4a (Supporting information), the mean lateral size of GQDs and amino-N-GQDs was approximately 7.31 ± 0.50 nm and 7.24 ± 0.70 nm, respectively. For GQDs, assuming there is no leakage from a layer of material and ignoring the exposed functional groups, and then the benzene number and molecular weight are expected to be approximately 631 and 16200 g mol-1 (Supporting information, Table S2), respectively. According to the assumption mentioned above and the atomic ratios and bonding compositions in Figure 2, the benzene number and molecular weight of amino-N-GQDs are expected to be approximately 631 and 16313 7 ACS Paragon Plus Environment


g mol-1 (Supporting information, Table S3), respectively. The following measurement for the cross section of TPE was available using the estimated molecular weights. The whole materials and methods of this article can be found in supporting information.

RESULTS AND DISCUSSION Amino-N-GQDs were prepared with an ultrasonic shearing reaction method involving a graphene oxide sheet synthesized with a modified Hummers method.10 The mean lateral size of an amino-N-GQD was determined through highly magnified high-resolution transmission electron microscopy (HR-TEM). Favorable crystallinity with a lattice distance was also determined, and the corresponded to the d-spacing of −

the graphene {1 1 00} lattice fringes (Figure 1a), and also showing the size distribution determined by dynamic light scattering (DLS) (Supporting information, Table S1). Other related characterizations are presented in Figure 2 and Figures S5-S6 (Supporting information). The aforementioned characterizations determined that amino-N-GQDs were successfully prepared. An MDR strain of Gram-positive Methicillin-resistant Staphyococcus aureus (MRSA) was used as the experimental template in this study. Because of the surface protein, protein A, in the cell wall of MRSA, the nanomaterial 8 ACS Paragon Plus Environment

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was coated with Abprotein

A

to form nanomaterial-Abprotein

A

to increase the

specificity, selectivity, and efficiency. The amino-N-GQDs exhibited favorable biocompatibility in a colony count assay, in which values are shown as a percentage corresponding to colony forming unit (CFU) per milliliter (Supporting

information,

Figure

S7a-b), and favorable

stability in

physiological environments (Supporting information, Table S4). Results also indicated the toxicity of nanomaterials contributing to bacterial elimination was excluded from the PDT action. In addition, to prevent confounding in evaluations of PDT action due to the amino-N-GQDs being inadvertently exposed to white light, which could compromise the experiment, two-photon PDT-related experiments were conducted in the dark. According to the results, treated amino-N-GQDs were incubated with MRSA (OD600 approximately 0.05) at a relatively low dose of 0.25 µg mL−1 for 3 h at 37 °C in the dark in all of the following experiments. In addition, to investigate the antimicrobial ability of amino-N-GQDs against the MDR strain of bacteria in PDT through TPE,11,12 a favorable TPA of nanomaterials at 800 nm in the near-infrared region probably due to the interband transitions involved,13 enabled detecting deeper specimen or tissue was initially determined (Figure 1b). In the process of PDT through TPE (239.36 nJ pixel−1), the viability of bacteria treated with 9 ACS Paragon Plus Environment


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amino-N-GQD-Abprotein A was evaluated as a function of photoexcitation time (0−12 s). After 1 s of photoexcitation, the viability of the bacteria began decreasing (Figure 1c). Over time, the viability of the bacteria clearly decreased to a low level, and 100% elimination of the bacteria occurred after 12 s of photoexcitation. By contrast, only a moderate decrease was observed in bacteria treated with amino-N-GQDs that were not coated with the antibody. These results also confirmed the materials were successfully coated with the antibody. Unexpectedly, after the same experiment was conducted, the GQDs exhibited a reduced capability to eliminate bacteria compared with the amino-N-GQDs (Supporting information, Figures S3, S8, S9a–b and Figure 1d). Furthermore, because LPS is a major component in the outer membrane of gram-negative Escherichia coli (E. coli), GQD- and amino-N-GQD-AbLPS also could eliminate E. coli (Supporting information, Figures S7c–d, S9c–d, and S10), exhibiting a trend similar to that in Figure 1c–d. These results are believed to be due to the ROS generated from the two-photon PS (i.e., amino-N-GQDs). The oxidative stress caused by the ROS could have contributed

to

the

surrounding

biological

substrates

of

the

photoexcited-amino-N-GQD-treated bacteria failing to function normally in the redox reactions as well as resulting in DNA damage, eventually leading to bacterial death. 10 ACS Paragon Plus Environment

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Consequently, the ROS singlet oxygen (1O2) and the superoxide radical anion (O2.−) involved in PDT through TPE (239.36 nJ pixel−1; 800 nm excitation) were monitored. 1

O2 and O2.− were effectively generated and exhibited a phenomenon dependent on

photoexcitation time (0–12 s) (Supporting information, Table S5). Because false positive signals of ROS might have resulted from the interactions between the materials and ROS reagents (Singlet Oxygen Sensor Green reagent (SOSG), trans-1-(2´-methoxyvinyl)pyrene, XTT, and GSH),14 potentially compromising the results, another method was employed for detecting the amount of ROS formed by treating MRSA with laser-irradiated amino-N-GQD-Abprotein A (Table 1). The results exhibited a trend similar to that in Table S5 (Supporting information) and that of amino-N-GQD-AbLPS-treated E. coli (Supporting information, Table S6), consistent with the signal of 1O2 phosphorescence at 1270 nm emitted from amino-N-GQDs (Supporting information, Figure S11). To confirm the involvement of ROS in the PDT effect of the nanomaterials, α-tocopherol was used for ROS neutralization.15 The amount of generated ROS was reduced after adding α-tocopherol. However, regardless of which method was employed, amino-N-GQDs showed a superior capability to generate ROS than GQDs did; the singlet oxygen QYs (ψ∆) of the amino-N-GQDs and GQDs were measured to be approximately 0.53 and 0.40 (ψ∆ = 0.64 is the QY of TSPP in deuterium oxide as a reference16). Furthermore, the 11 ACS Paragon Plus Environment


generated ROS apparently decreased when the bacteria were treated with the material without the antibody coating (Supporting information, Tables S7–S8). Again, the results indicated that the material was successfully coated with the antibody and that the antibody effectively enhanced the functions of the material. Because intrinsic and defective state emissions possibly involving a PL mechanism were observed,6 the amino-N-GQDs could also serve as a contrast probe through TPE. TPE could help to retain the laser power in a low average and make the excitation wavelength to be lengthened to near-infrared region, enhancing the visibility of TPL in two-proton imaging (TPI) processes. TPL is a nonlinear phenomenon, and its intensity is proportional to the square of excitation power, the cross section of TPE, and QY.17 For monitoring molecular actions, a large cross section is desirable. With a high QY and large TPE cross section, intrinsic fluorophores and enhanced localized excitation power can increase the TPL signal. The TPL spectrum of the amino-N-GQDs exhibited a peak of approximately 672 nm (Figure 1e), corresponding to the emission spectrum of one-photon excitation (OPE) (Supporting information, Figure S5e). This followed the confirmation of a two-photon process in which the PL intensity exhibited quadratic dependence on the excitation power under TPE (Ex: 800 nm),17 with an exponent of 2.01 (Supporting information, Figure S12a). Moreover, the image of the emitted TPL from the 12 ACS Paragon Plus Environment

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amino-N-GQDs was illuminated through the two-photon process (Figure 1f). However, the calculated relative QY was around 0.33 (QYref = 0.28 is the QY of Cy5.5 in DMSO as a reference18); additionally, the absolute QY19 was similar, at approximately 0.34, and OPE or TPE yielded the same QY.20 By contrast, GQDs had lower relative and absolute QYs of 0.18 and 0.19, as well as lower TPL intensity (emission: 651 nm) (Figure 1e and Supporting information, Figure S12b). Because of the existence of a carboxylic acid group on the surface of the GQDs, nonradiative recombination of electron–hole pairs was induced, leading to inhibition of the intrinsic state emission. However, the NH2 groups at the edge of N-GQDs have been proposed to have a high highest occupied molecular orbital due to the strong orbital interaction with the primary amine.9 The resonance feature between the molecular orbital in the primary amine and the delocalized π orbital thus would lead to a narrowing of the orbital band gap. Figure 3a–b shows the TPL spectra measurement, where the peaks for amino-N-GQDs and GQDs were appeared from approximately 634 nm to 672 nm and 622 nm to 651 nm, respectively, under TPE wavelengths of 720–800 nm, similar to the emission wavelength for OPE in Figures S4f and S5e (Supporting information). The large absolute TPE cross section make chromophores effective for investigation and particularly attractive for monitoring molecular activities in vitro or in vivo with two-photon techniques because they correspond to a 13 ACS Paragon Plus Environment


high ratio of the energy absorbed to the input-energy flux in a specimen, minimizing the probability of photodamage. The calculated absolute TPE cross section for the amino-N-GQDs was approximately 54854 Goeppert–Mayer units (GM, with 1 GM = 10−50 cm4 s photon−1; rhodamine B was selected as a reference21 to determine the cross section; Table 2 and Supporting information, Figure S11 and Table S9), whereas approximately 48212 GM was chosen for the GQDs. Besides, the absolute TPE cross section for the amino-N-GQDs was more than two orders of extent greater than that of traditional fluorophores and larger than that of semiconductor QDs (Table 2).14 After further investigation, lifetime was also measured; sequentially radiative and nonradiative decay rates were calculated upon the QY and lifetime (Figure 3c and Supporting information, Table S10). The average lifetime of the amino-N-GQDs was approximately 1.09 ns, obtained from the observed lifetimes of 0.158, 0.942, and 3.771 ns, whereas the average lifetime of the GQDs was approximately 1.59 ns (Supporting information, Table S10). Therefore, the ratio of the radiative to nonradiative decay rates of the amino-N-GQDs was approximately 0.49, derived from 2.992 × 108 s−1 and 6.158 × 108 s−1, respectively; the ratio of the GQDs was approximately 0.23 (1.162 × 108 s−1 and 5.119 × 108 s−1). The results suggest that the materials primarily pass through the radiative pathway after TPE instead of through the nonradiative pathway when the QY increases and lifetime decreases. The 14 ACS Paragon Plus Environment

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enhanced two-photon properties of amino-N-GQDs have been proposed to be due to the increased quantum confinement with a highly symmetric bandgap, strong electron donating effect, and large π-conjugated systems of the amino-N-GQDs, thus resulting in efficient intramolecular charge transfer.22 Furthermore, the amino-N-GQDs here exhibited two-photon stability according to TPL intensity determination after photoexcitation involving TPE, resulting in a reduced photobleaching effect (Figure 3d). The superior two-photon properties of absorption, QY, luminescence, the absolute cross section, and stability under TPE together indicate that the amino-N-GQDs can act as promising contrast agents for noninvasively detecting deeper and 3D biological specimens by using a TPE wavelength, which can be extended to the near-infrared region. To imitate the 3D biological environment, bacteria embedded in a collagen matrix14 were used. The PL emitted from amino-N-GQD-Abprotein A-treated MRSA under TPE at 63.36 nJ pixel-1 (0.6336 mW) (Ex: 800 nm) was evaluated as a function of depth at every 28 to 84 µm (Figure 4a–c). The results indicated a clear image and clear localization of bacteria, as well as enhancements in specificity, selectivity, and efficiency attained using the Abprotein A coating. At a depth of 84 µm, photoexcitation was increased to 239.36 nJ pixel−1 for 12 s to induce two-photon PDT. With an increased irradiation time, less TPL was 15 ACS Paragon Plus Environment


emitted by the amino-N-GQD-Abprotein A-treated bacteria (Figure 4d). This indicated either that the bacteria underwent severe morphological damage or that the generated ROS would oxide and deteriorate the surrounding biological substrates on the bacterial surface. This led to rapid atrophy of the bacteria and desorption of the amino-N-GQDs from the bacterial surface, thus reducing PL. In addition, the two-photon autofluorescence (TPAF) image emitted from the intrinsic fluorophores of the bacteria without treatment with the material (unlabeled bacteria) was difficult to observe under TPE at 239.36 nJ pixel−1, which would not compromise TPI (Figure 4e). However, neither the one-photon image of the amino-N-GQD-Abprotein A-treated MRSA exposed to a 612 nm laser (0.24 W cm−2, Coherent, USA) at a depth of 84 µm (Figure 4f) nor the image of the unlabeled bacteria under PDT action at the same location could be observed (Figure 4g). The wavelength of OPE in the visible region exhibited greater scattering, energy absorption, and photobleaching and less irradiation penetration than that in the near-infrared region, making observing or investigating the thick tissue and deeper biological specimens difficult. Human paclitaxel-resistant cervical carcinoma cells (KB-50) were also treated with amino-N-GQDs coated with a specific antibody against nucleus and mitotic cell antigens (amino-N-GQD-Abnucleus) for TPI by using the same TPE method (Figure 5). The amino-N-GQDs exhibited favorable biocompatibility to KB-50 and efficiently 16 ACS Paragon Plus Environment

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eliminated KB-50 through TPE in PDT (Supporting information, Figures S14–S15); 1O2 and O2.− were effectively generated and exhibited a phenomenon dependent on photoexcitation time (Supporting information, Table S11), and the treatment of bacteria with the material without an antibody coating apparently reduced ROS generation (Supporting information, Table S12). The results indicated that the material was successfully coated with the Ab and that the Ab effectively enhanced the functions of the material. Further, the amino-N-GQD-treated human peripheral blood mononuclear cells (PBMCs) were also exposed to the laser irradiation (Supporting information, Figure S16). After 12-s photoexcitation at a depth of 84 µm, the red fluorescence emitted from the amino-N-GQDs verified that the nanomaterials were successfully uptaken by cancer cells and retained in the nuclei (Figure 5b). Furthermore, the cancer cells were dead, a morphology indicative of severe nuclear damage, and a destroyed cytoskeleton, which changed their properties and functions. Furthermore, the generated ROS would oxide and deteriorate the surrounding biological substrates, leading cells to become distorted, atrophy, and sustain

severe

morphological

damage.

These

changes

indicated

that

the

amino-N-GQDs were desorbed from the nuclei, compared with the cell alone (Figure 5a). Again, the TPAF image of unlabeled cancer cells would not compromise observations under the same treatment (Figure 5c). Under OPE, neither the signal 17 ACS Paragon Plus Environment


from the amino-N-GQD-Abnucleus-treated KB-50 (Figure 5d) nor that from unlabeled cancer cells could be detected (Figure 5e). Different optical systems have different detection depths. Because of the detection efficiency and the objective used here, the maximal z depth that could be observed using the laser optical system was approximately 100 µm. However, 84 µm provides the optimal resolution for observing the ability of GQDs to act as a two-photon contrast probe for 3D observation and to concurrently eliminate the investigated MDR species. Using TEM to determine the effect of amino-N-GQDs in treating MRSA with TPE action was also necessary to identify the extent to which the amino-GQD-treated bacteria were disrupted after photoexcitation. Bare MRSA (Supporting information, Figure S17a) was used after 3h of incubation with nanomaterials, resulting in substantial amino-N-GQD-Abprotein A being adsorbed on the bacterial surface because of the highly efficient and specific binding capability of the Ab (Supporting information, Figure S17b). Results of the uptake assay indicated that the amount of nanomaterials absorbed on the bacterial surface achieved saturation in the first 3 h of incubation (Supporting information, Figure S18). In addition to specifically bound Abs, bacteria must filter external ions and assimilate nutrients to retain and develop the physiological functions by using the cell wall.23 Thus, the nanomaterials were absorbed and accumulated to become the external barrier on the bacterial surface. 18 ACS Paragon Plus Environment

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However, in the case of an unusual, extreme observation and elimination of both MDR species under TPE, the amino-N-GQDs had the same, substantial efficiency. In summary, treatment of bacteria with photoexcited amino-N-GQD-Abprotein A induced marked morphological damage that corresponded to nearly 100% death after 3 h of incubation (Supporting information, Figure S17c). Results display that the amino-N-GQDs can both act as favorable two-photon contrast agents for deeper observation of bacteria and be employed in PDT to completely eliminate bacteria.

CONCLUSIONS In this work, amino-N-GQDs were effectively prepared and employed as a PS. They exhibited a strong PDT effect, easily eliminating MDR analytes with low-energy delivery of an ultrashot pulse laser (239.36 nJ pixel-1, with 12 s TPE). Moreover, they showed desirable two-photon properties that enabled them to act as a favorable contrast probe for TPI in 3D investigation. Furthermore, GQDs doped with nitrogen and functionalized with an amino group exhibited a stronger PDT effect and more favorable two-photon properties than unmodified GQDs did. The capabilities may provide an efficient approach to easily observe and effectively eliminate MDR species and serve as a guide to design novel compounds with valuable photoproperties.



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FIGURES Figure 1. (a) HR-TEM image of single amino-N-GQD, illustrating the graphene{11 00} lattice planes and the mean lateral size of 7.31 ± 0.50 nm with a −

d-spacing of 0.213 nm. (b) Relative TPA spectra of the amino-N-GQDs and GQDs. TPE with a function of the wavelength (720-820 nm) at 105.60 nJ pixel-1 (1.0560 mW) that was used to monitor the signals. Quantified viability followed the viable count of (c) amino-N-GQD-Abprotein A- and (d) GQD-Abprotein A-treated-MRSA, respectively, through CFU assay by 1–12 s photoexcitation with 239.36 nJ pixel-1 (2.3936

mW)

of

TPE

(Ex:

800

nm).

For

material-Ab-treated-

and

material-treated-bacteria with laser irradiation, (b) p< 0.0001 and p = 0.6135 and (c) p = 0.6308 and p = 0.8792, respectively. *p value obtained by Student’s t test. (e) TPL spectra of materials, exposed to a power of 176.00 nJ pixel-1 (1.7600 mW) by TPE. (f) TPI of the aggregated amino-N-GQDs was observed with 63.36 nJ pixel-1 (0.6336 mW) using TPE. Excitation wavelength: 800 nm. Dose: OD600 ~ 0.05 of bacteria and 0.25 µg mL−1 materials. Data are means ± SD (n = 6). Figure 2. (a) The deconvoluted C(1s) XPS spectra and fitted peaks using Gaussian function: non-oxygenated ring (C－C/C＝C, 285.5eV), C－N bonds (286.3eV), hydroxyl (C－O, 287.5 eV) and carbonyl (C＝O, 288.1eV), respectively; (b) N(1s) spectra and fitted peaks of XPS using Gaussian function: pyridinic N (398.3eV), 20 ACS Paragon Plus Environment

Page 21 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


amino N (NH2, 399.1 eV), pyrrolic N (399.7eV), quaternary N (400.6eV) and amide N (O＝C－N, 401.3 eV), respectively. The atomic ratios and bonding compositions for amino-N-GQDs are summarized in the table. O(1s)/C(1s) and N(1s)/C(1s) atomic ratios are 29.5% and 4.8%, respectively. Figure 3. TPL spectra of (a) amino-N-GQDs and (b) GQDs, exposed to (176.00 nJ pixel-1, 1.7600 mW) TPE wavelengths of 720 nm-800 nm (cut off at 690 nm by the cascading filters). (c) Time-resolved PL decay profile of materials detected the emission with TPE (176.00 nJ pixel-1, Ex: 800nm) at room temperature. (d) Two-photon stability. TPL spectra of amino-N-GQDs, which was subjected to the TPE at 176.00 nJ pixel-1 (Ex: 800 nm) and detected for the amino-N-GQDs with irradiation and the peak was monitored at approximately 670 nm under TPE (Ex: 800 nm). As a function of TPE time (0-90 s), the relative intensity of integrated area from 350 nm to 675 nm in wavelength of TPL maintained almost the same intensity, exhibiting the highly photostability of amino-N-GQDs. Delivered dose: 0.25 µg mL−1 amino-N-GQDs. Data are means ± SD (n=6). Figure 4. TPI at different depths of (a–c) bacteria were observed from 28 to 84 µm at a power of 63.36 nJ pixel-1 (0.6336 mW) by TPE. (d) Image of bacteria for extra 12-s photoexcitationand (e) TPAF image of unlabeled bacteria at a depth of 84 µm with the power of 239.36 nJ pixel-1 (2.3936 mW). Excitation wavelength: 800 nm. Images 21 ACS Paragon Plus Environment


of (f) the amino-N-GQD-Abprotein

A-treated

Page 22 of 38

MRSA and (g) unlabeled bacteria

following OPE with 612 nm laser (maximal and fixed output power: 0.24 W cm-2) at the same depth. Dose: OD600 ~ 0.05 of MRSA and 0.25 µg mL−1 amino-N-GQD-Abprotein A. Figure 5. (a) TPI of KB-50 cancer cells alone that was observed at a depth of 84 µm without amino-N-GQD-Abnucleus treatment. At the same depth with a power of 63.36 nJ pixel-1 (0.6336 mW) by TPE, (b) TPI of cancer cells with treatments of materials and extra 12-s photoexcitation; (c) TPAF image of unlabeled cancer cells without the treatments of materials and the counterstaining with a power of 239.36 nJ pixel-1 (2.3936

mW).

Excitation

wavelength:

800

nm.

Images

of

(d)

the

amino-N-GQD-Abnuclues-treated KB-50 and (e) unlabeled cancer cells under OPE with 612 nm laser (0.24 W cm-2) at the same depth. Dose: 0.25 µg mL−1 amino-N-GQD-Abnucleus. Nuclei and cytoskeleton were counterstained with DAPI (blue) and Alexa 488 phalloidin (green), respectively.


Page 23 of 38

b Normalized TPA

a

1.2

amino-N-GQDs

1

GQDs

0.8 0.6 0.4 0.2 0 720

c

740

Bacterial viability (%)

100 80 60 40 20

760

780

800

820

Wavelength (nm)

d 120

* *

120

Bacterial Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


* *

100

Bacteria alone without laser irradiation Bacteria alone with laser irradiation Material-Ab-treated-bacteria without laser irradiation Material-Ab-treated-bacteria with laser irradiation Material-treated-bacteria with laser irradiation

80 60 40 20 0

0 0

1

2

3

6

0

12

1

e

2

3

6

12

Time (sec)

Time (sec)

amino-N-GQDs

f

GQDs

300

400

500

600

Wavelength (nm)

Figure 1.

*



a

Page 24 of 38

b Raw data 398.3 eV(pyridinic N) Raw data 399.1 eV(amino N, 285.5 eV(C-C/C=C)

399.7 eV(pyrrolic N)

286.3 eV(C-N) 400.6 eV(quaternary N)

287.5 eV(C-O)

401.3 eV(amide N, O=C-N)

288.1 eV(C=O)

Atomic ratio

Carbon bonding composition (%)

O(1s)/C(1s)

C-C/C=C

C-N

C-O

C=O

29.5%

20

19

26

35

Nitrogen bonding composition (%) N(1s)/C(1s)

pyridinic

amino

pyrrolic

quaternary

amide

4.8%

13

16

28

25

18

Figure 2


Page 25 of 38

a

b

720 nm

720 nm 760 nm 800 nm

760 nm 800 nm

200

300

400

500

600

200

300

Wavelength (nm)

c

400

500

600

Wavelength (nm)

d Normalized integration area

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.2 1 0.8 0.6 0.4 0.2 0 0

15

30

45 Time (sec)

Figure 3


60

75

90


28 μm

Page 26 of 38

84 μm

56 μm

a

b

c

84 μm, TPAF image

84 μm, 12-s photoexcitation

of unlabeled bacteria

d

e

f

g

Figure 4.


Page 27 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5.



TABLES Table 1. The amount of ROS generated14 from a 6-12s TPE (239.36 nJ pixel-1, 2.3936 mW; Ex: 800 nm) to material-Ab (0.25 µg mL−1)-treated-MRSA (OD600~0.05) was monitored. Data are means ± SD (n=6).

a

Negative control: only treat reagent and laser irradiation without material (0 µg

mL−1). b

ROS neutralization: with the treatments of nanomaterial, the laser irradiation and 30

ppm of antioxidant α-Tocopherol/methyl linoleate.15 c

SOSG reagent (Ex/Em: 488/525 nm) has a specific reactivity to generate

fluorescence recorded by a PL spectrometer. d

Positive control: the treatment of 50 µM tert-butyl hydroperoxide (TBHP) and laser

irradiation. e

t-MVP (Ex/Em: 352/465 nm) can react with 1O2, forming a dioxetane intermediate

that generates fluorescence upon decomposition to 1-pyrenecarboxaldehyde, and monitored by a PL spectrometer. f

XTT would interact with O2.− and produce the XTT-formazan generating strong

absorption (wavelength of 470 nm).


Page 28 of 38

Page 29 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


g

GSH containing a thiol-tripeptide can prevent damages to cellular or bacterial

components caused by stress of oxidation. Thiol group from GSH can be oxidized to disulfide bond converting GSH to glutathione disulfide. GSH oxidation was used to confirm the generated O2.−. Loss of GSH (%) = (absorbance difference between of sample and negative control / absorbance of negative control) × 100 %.

Table 2. TPE cross section of nanomaterials (Ex: 800 nm).a

a

Rhodamine B was selected as a reference to determine the TPE cross section.

(Information

was

obtained

from

the

free

website

http://www.drbio.cornell.edu/cross_sections.html, kindly provided by Prof. Chris Xu, Cornell University, USA), and the relevant calculations were shown in Materials and Experimental Section. bInformation was available in Thermo Fisher Scientific, USA.



Page 30 of 38

Table 1. 1

O2 (by SOSG)c

Negative controlac

ROS neutralizationabc

Positive controlcd

6-s two photon excitation

223±11

227±12

1882±101

12-s two-photon excitation

226±14

230±10

3479±114

amino-NGQDs 702±43

1526±38

ROS neutralizationbc 255±12

249±11

GQDs

503±18

739±20

ROS neutralizationbc 250±13

252±15

1

O2 (by t-MVP)e

Negative controlac


Positive controlcd

amino-NGQDs

ROS neutralizationbc

GQDs


6-s two photon excitation

330±15335±18

5911±113 3422±76

349±12

2047±59

345±9


334±19

13455±269 7693±117

353±16

3320±95

346±11

339±15

C -

O2˙ (by XTT) Negative controlac 6-s two photon excitation

0


0


0

C

Positive controlcd

amino-NGQDs


GQDs


0.87±0.08

0.25±0.03

0.04±0.01

0.15±0.02

0.03±0.01

0.05±0.01

0.29±0.03

0.05±0.02


GQDs


84.6±4.9% 51.9±2.8%

3.5±0.4%

28.1±2.6%

2.5±0.6%

99.1±0.9%91.3±4.2%

3.7±0.3%

46.3±4.1%

4.2±0.4%

1.67±0.13

0

f

0.64±0.04

O2˙- (by GSH)g Negative controlac 6-s two photon excitation

0


0


0

0

Positive controlcd

amino-NGQDs


Page 31 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Reference

Integrated emission intensity (counts)

Action cross-section (ησ)

Rhodamine B

279.30

158.20

Sample

Integrated emission intensity (counts)

Relative quantum yield (η)

Absolute cross-section (σ)

amino-N-GQDs

31668.57

0.33

54356.32

GQDs

15746.88

0.18

49551.49

66.56

0.72

24

b

42937.30

40381.07

Fluorescein 565 Qdot ITK carboxyl quantum dots (Q21331MP)

56095.87

0.74

525 Qdot ITK Carboxyl quantum dots (Q21341MP)

57746.71

0.81b

52.36



Page 32 of 38

ASSOCIATED CONTENT Supporting Information. Laser resolution; characterizations of amino-N-GQDs and GQDs; bacterial and cell viabilities; stability in physiological environment; the quantified viability and the amount of generated ROS after photoexcitation; cell viability after TPE; logarithmic plat of TPL intensity; TPE action cross section; lifetime data; TEM images; uptake assay; UV-vis spectra for absorbance of antibody; molecular weights of GQD and amino-N-GQD; Materials and Experiments are included as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

Ting-Mao

Chou

(email:

[email protected]);

Jiu-Yao

Wang

[email protected]); Ping-Ching Wu (email: [email protected]).

Notes

The authors declare no competing financial interest.


(email:

Page 33 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGMENT

This study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. (MOST-106-2811-B-006-011; MOST-104-2314-B-006-076-MY3), and the Ministry of

Education,

Taiwan,

R.O.C.

MOST-106-2221-E-006-002-;

(MOST-106-2119-M-038-001-; MOST-105-2812-8-006-002-;

MOST-106-2119-M-006-008-).

ABBREVIATIONS PDT, photodynamic therapy; ROS, reactive oxygen species; GQD, graphene quantum dot; amino-N-GQD, amino-functionalized nitrogen-doped graphene quantum dot; MDR, multidrug-resistant; TPE, two-photon excitation; PS, photosensitizer; 0D, zero-dimensional; QD, quantum dot; N-GQDs, N-doped with GQDs; PL, photoluminescence; TPA, two-photon absorption; TPL, two-photon luminescence; ddH2O, deionized water; TEM, transmission electron microscopy; AFM, atomic force microscopy;

UV-vis,

ultraviolet-visible;

FTIR,

Fourier

transform

infrared

spectroscopy; DLS, dynamic light scattering; XPS, X-ray photoelectron spectroscopy; MRSA, Methicillin-resistant Staphyococcus aureus; E. coli, Escherichia coli; CFU, colony

forming

unit;

Abprotein

A,

anti-protein

A

antibody;

AbLPS,

anti-lipopolysaccharide antibody; 1O2, singlet oxygen; O2.−, superoxide radical anion; 33 ACS Paragon Plus Environment


Page 34 of 38

SOSG, Singlet Oxygen Sensor Green; TBHP, tert-butyl hydroperoxide; t-MVP, trans-1-(2´-methoxyvinyl)pyrene;

XTT,

2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-

carboxanilide;

GSH,

γ-L-glutamyl-L-cysteinyl- glycine; TSPP, meso-tetra(4-sulfonatophenyl)porphine dihydrochloride;D2O,deuterium oxide; TPI, two-photon imaging; OPE, one-photon excitation; GM, Goeppert–Mayer; TPAF, two-photon autofluorescence; KB-50, human paclitaxel-resistant KB paclitaxel-50 cervical carcinoma cells; Abnucleus, anti-nucleus and mitotic cell antigen antibody; THBP, tert-butyl hydroperoxide.

REFERENCES (1) Levy, J. G. Photosensitizers in Photodynamic Therapy. Semin. Oncol.1994, 21, 4-10.

(2) Allision, R. R.; Downie, G. H.; Cuenca, R.; Hu, X. H.; Childs, C. J. H.; Sibata, C. H. Photosentizers in Clinical PDT. Photodiagnosis Photodyn. Ther.2004, 1, 27-42. (3) Lucky S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015, 115, 1990-2042. (4) Tian, B.; Wang, C.; Zhang, S.; Feng, L.; Liu, Z. Photothermally Enhanced Photodynamic Therapy Delivered by Nano-Graphene Oxide. ACS Nano 2011, 5, 7000-7009. 34 ACS Paragon Plus Environment

Page 35 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(5) Kuo, W. S.; Chen, H. H.; Chen, S. Y.; Chang, C. Y.; Chen, P. C.; Hou, Y. I.; Shao, Y. T.; Kao, H. F.; Hsu, C. L. L.; Chen, Y. C.; S. J. Chen.; S. R. Wu.; J. Y. Wang. Graphene Quantum Dots with Nitrogen-doped Content Dependence for Highly Efficient Dual-Modality Photodynamic Antimicrobial Therapy and Bioimaging. Biomaterials 2017, 120, 185-194. (6) Loh, K. P.; Bao, Q.; Eda, G.; Chhowalla, M. Graphene Oxide as a Chemically Tunable Platform for Optical Applications. Nat. Chem.2010, 2, 1015-1024. (7) Wang, X.; Li, X.; Zhang, L.; Yoon, Y.; Weber, P. K.; Wang, H.; Guo, J.; Dai, H. N-doping of Graphene through Electrothermal Reactions with Ammonia. Science 2009, 324, 768-771. (8) Li, L.-s.; Yan, X. Colloidal Graphene Quantum Dots. J. Phys. Chem. Lett. 2010, 1, 2572-2576. (9) Tetsuka, H.; Asahi, R.; Nagoya, A.; Okamoto, K.; Tajima, I.; Ohta, R.; Okamoto, A. Optically Tunable Amino-Functionalized Graphene Quantum Dots. Adv. Mater. 2012, 24, 5333-5338. (10) Hummers, W. S.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339-1339. (11) Shen, Y.; Shuhendler, A. J.; Ye, D.; Xu, J. J.; Chen, H. Y. Two-Photon Excitation Nanoparticles for Photodynamic Therapy. Chem. Soc. Rev.2016, 45, 35 ACS Paragon Plus Environment


Page 36 of 38

6725-6741. (12) Bhawaker, J. D.; Kumar, N. D.; Zhao, C. F.; Prasad, P. N. Two-Photon Photodynamic Therapy. J. Clin. Laser Med. Surg. 2009, 15, 201-204. (13) Feng, X.; Li, X.; Li, Z.; Liu, Y. Size-Dependent Two-Photon Absorption in Circular Graphene Quantum Dots. Opt. Express 2016, 24, 2877-2884. (14) Wu, P. C.; Wang, J. Y.; Wang, W. L.; Chang, C. Y.; Huang C. H.; Yang, K. L.; Chang, J. C.; Hsu, C. L. L.; Chen, S. Y.; Chou, T. M.; Kuo, W. S. Efficient Two-Photon

Luminescence

for

Cellular

Imaging

Using

Biocompatible

Nitrogen-doped Graphene Quantum Dots Conjugated with Polymers. Nanoscale 2018, 10, 109-117. (15)

Kinen, M. M.; Kamal-Eldin, A.; Lampi, A.-M.; Hopia, A. Effects of α- and γ-

Tocopherols on Formation of Hydroperoxides and Two Decomposition Products from Methyl Linoleate. J. Am. Oil Chem. Soc. 2000, 77, 801-806. (16) Shi, L.; Hernandez, B.; Selke, M. Singlet Oxygen Generation from Water-Soluble Quantum Dot-Organic Dye Nanocomposites. J. Am. Chem. Soc. 2006, 128, 6278-6279. (17) Albota, M. A.; Xu, C.; Webb, W. W. Two-Photon Fluorescence Excitation Cross Sections of Biomolecular Probes from 690 to 960 nm. Appl. Opt. 1998, 37, 7352-7356. 36 ACS Paragon Plus Environment

Page 37 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18) Umezawa, K.; Matsui, A.; Nakamura, Y.; Citterio, D.; Suzuki, K. Bright, Color-Tunable Fluorescnce Dye in the Vis-NIR Region: Establishment of New "Tailor-Made" Multicolor Fluophores Based on Borondipyrromethene.Chem. Eur. J. 2009, 15, 1096-1106. (19) Würth, C.; Grabolle, M.; Pauli, J.; Spieles, M.; Resch-Genger, U. Relative and Absolute Determination of Fluorescence Quantum Yields of Transparent Samples. Nat. Protoc. 2013, 8, 1535-1550. (20) Lin, C. Y.; Lien, C. H.; Cho, K. C.; Chang, C. Y.; Chang, N. S.; Campagnola, P. J.; Dang, C. Y.; Chen, S. J. Investigation of Two-Photon Excited Fluorescence Increment via Crosslinked Bovine Serum Albumin. Opt. Express 2012, 20, 13669-13676. (21) Velapoldi, R. A.; Tønnesen, H. H. Corrected Emission Spectra and Quantum Yields for a Series of Fluorescent Compounds in the Visible Spectral Region. J. Fluoresc. 2004, 14, 465-472. (22) Trauzettel, B.; Bulaev, D. V.; Loss, D.; Burkard, G. Spin Qubits in Graphene Quantum Dots. Nat. Phys.2007, 3, 192-196. (23) Lim, S. H.; Hudson, S. M. Review of Chitosan and its Derivatives as Antimicrobial Agents and Their Uses as Textile Chemicals. J. Macromol. Sci. Part C: Polymer Rev.2003, 43,223-269. 37 ACS Paragon Plus Environment


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Table of Contents Graphic

720 nm 760 nm 800 nm

200

300

400

500

Wavelength (nm)


600

Antimicrobial Amino-Functionalized Nitrogen-Doped Graphene

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