Two-Photon Photoexcited Photodynamic Therapy ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Two-Photon Photoexcited Photodynamic Therapy and Contrast Agent with Antimicrobial Graphene Quantum Dot Wen-Shuo Kuo, Chia-Yuan Chang, Hua-Han Chen, Chih-Li Lilian Hsu, JiuYao Wang, Hui-Fang Kao, Lawrence Chao-Shan Chou, Yi-Chun Chen, SheanJen Chen, Wen-Tsan Chang, Shih-Wen Tseng, Ping-Ching Wu, and Ying-Chih Pu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12014 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 25, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

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

ACS Applied Materials & Interfaces

Two-Photon Photoexcited Photodynamic Therapy and Contrast Agent with Antimicrobial Graphene Quantum Dot Wen-Shuo Kuo,*,†,‡,§,⊥,∆ Chia-Yuan Chang,§,⊥,∆ Hua-Han Chen,¥,∆ Chih-Li Lilian Hsu,& Jiu-Yao Wang,‡,&,○ Hui-Fang Kao,∥ Lawrence Chao-Shan Chou,§ Yi-Chun Chen,¶ Shean-Jen Chen,†, ⊥ Wen-Tsan Chang,○ Shih-Wen Tseng,§ Ping-Ching Wu*,#,$ and Ying-Chih Pu*,◆ †

Advanced Optoelectronic Technology, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan (R.O.C). ‡ Allergy & Clinical Immunology Research Center, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan (R.O.C). §

Center for Micro/Nano Science and Technology, National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan (R.O.C)..

⊥

College of Photonics, National Chiao Tung University, Tainan 711, Taiwan (R.O.C).

¥

Department of Food Science, National Penghu University of Science and Technology, Penghu 880, Taiwan (R.O.C). &

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

∥

Department of Nursing, National Tainan Junior College of Nursing, Tainan 700, Taiwan (R.O.C).

¶

Department of Physics, National Cheng Kung University, Tainan 701, Taiwan (R.O.C).

○

Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan 701, Taiwan (R.O.C). # Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan (R.O.C). $

Medical Device Innovation Center, Taiwan Innovation Center of Medical Devices and Technology, National Cheng Kung University, Tainan 701, Taiwan (R.O.C).

ACS Paragon Plus Environment

1


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

Page 2 of 26

◆

Department of Materials Science, National University of Tainan, Tainan 700, Taiwan (R.O.C).

∆

These authors contributed equally to this work.

KEYWORDS

graphene quantum dot, two-photon photodynamic therapy, reactive oxygen

species, three dimensional two-photon bioimaging, multidrug resistant

ABSTRACT A graphene quantum dot (GQD) used as the photosensitizer with high two-photon absorption in the near-infrared region, a large absolute cross section of two-photon excitation (TPE), strong two-photon luminescence and impressive two-photon stability could be used for dual modality two-photon photodynamic therapy (PDT) and two-photon bioimaging with an ultrashot pulse laser (or defined as TPE). In this study, a GQD efficiently generated reactive oxygen species coupled with TPE, which highly increased the effective PDT ability of both gram-positive and negative bacteria, with ultra-low energy and an extremely short photoexcitation time generated by TPE. Because of its two-photon properties, a GQD could serve as a promising two-photon contrast agent for observing specimens in-depth in three-dimensional biological environments, while simultaneously proceeding with PDT action to eliminate bacteria, particularly in multidrug-resistant (MDR) strains. This procedure would provide an efficient alternative approach to easily cope with MDR bacteria.


2

Page 3 of 26

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. INTRODUCTION Multiphoton microscopy is a form of laser-scanning microscopy that uses localized nonlinear excitation to induce fluorescence, which has been applied to various imaging studies.1 This type of microscopy is normally coupled with near-infrared (NIR) laser excitation to take advantage of maximum tissue transmission for bioimaging; the slight scattering, low energy absorption, optimal irradiation penetration, and reduced photobleaching of NIR have made it possible to observe or investigate the thick tissue and deeper biological specimens,2 as well as in other therapies involving photoexcitation.3,4 However, multiphoton microscopy (also called TPE laser microscopy) also includes ultra-low energy requirements and a short photoexcitation period, and is an alternative approach in PDT. PDT is a process in which a photoexcited photosensitizer (PS) reacts with molecular oxygen and forms reactive oxygen species (ROS) upon exposure to an appropriate wavelength and energy level of light. The ROS generated by PDT are singlet oxygen (1O2) and superoxide radical anion (O2.−), which can cause irreversible damage to cells or bacteria through oxidative reactions with surrounding biological substrates.5 Creating a larger TPE cross section of PS would be the first priority for efficiently achieving PDT action, albeit certain toxic PSs should be avoided.6,7 When two-photon techniques are exploited to evaluate molecular activity in photoproperties, a large cross section is particularly attractive. Because the ratio of the energy absorbed to the input energy flux in a specimen is high, reducing probable photodamage, high values of TPE cross section of PSs enable the PSs to be used efficiently to conduct nonlinear microscopic investigation.8 However, much recent attention has been paid to the use of materials possessing two-photon properties for their many promising application, not only in the field of bioimaging but also in PDT.1,2,4 Some novel materials with two-photon properties are graphene-based.9 Graphene is a


3


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

Page 4 of 26

two-dimensional monolayer of graphite and carbon atom bonds forming a hexagonal lattice. Graphene-based materials are being studied in almost every field; however, some studies have reported that when they are used in combination with PSs, they can induce PDT mechanisms10 and have directly used GQDs as PSs to generate ROS.11,12 A major shortcoming of these studies is that they have paid no attention to exploiting PDT through two-photon processes. Additionally, surface groups illuminate GQDs, which might contribute to phenomena associated with intrinsic and defect state emissions and involve a photoluminescence (PL) mechanism.13,14 In this study, GQDs were used and designed as a two-photon PS coupled with TPE to achieve high efficacy with ultra-low-energy (2.64 mW, 264 nJ pixel-1, the calculation of power please see supporting information; for the laser system, the x-y axis focal spot is around 375.38 nm, and the z axis resolution is around 0.84078 µm, Figure S1) irradiation from a femtosecond laser and a photoexcitation of only 15 s (Ex: 800 nm), leading to the almost 100% elimination of both Escherichia coli (E. coli) and methicillin-resistant Staphyococcus aureus (MRSA). Furthermore, strong two-photon luminescence (TPL) and high photostability mediated by laser irradiation enables GQDs to serve as a promising contrast probe with which to track and localize GQDtreated bacteria at a deep depth in a three-dimensional (3D) environment, and provide additional information on the status of bacteria irradiated through TPE, as well as simultaneously operate in PDT to completely eliminate bacteria at this depth. Integrating the advantages of GQDs with dual-modality two-photon PDT and two-photon contrast agents has great potential to eliminate bacteria and track targeted bacteria to manifest the effects of therapeutic approaches.

2. MATERIALS AND EXPERIMENTS GQD preparation


4

Page 5 of 26

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


A modified Hummers method was used to prepare graphene oxide from graphite (Bay carbon, SP-1, USA).15 Graphite (8.5 M) and NaNO3 (0.6 M) (Merck, Germany) were mixed with H2SO4 (Wako, Japan). KMnO4 (2.0 M) (J. T. Baker, USA) was slowly added with continual stirring at 35 °C overnight. Then, the ddH2O was gradually added and continued to be stirred. Adding H2O2 (Shimakyu, Japan) was the method used to terminate the reaction. Washing and centrifugation with ddH2O several times were carried out and the graphene oxide was collected. The asprepared graphene oxide was placed in a tube furnace and heated to 400-500 °C in the presence of argon for 3 h; it was then introduced to concentrated HNO3 (Wako, Japan) and stirred for 18h. The mixture was put into a sonicator for at least 1 d and then put it in an oven at 160 °C to vaporize all the liquid. Washing and centrifugation (83000 rpm) (Optima TLX Ultracentrifuge, BECKMAN, USA) with ddH2O several times were carried out. The resulting black suspension had the pH tuned to 7.4 with NaOH (Merck, Germany). The solution remained in a dialysis bag (retained molecular weight: 100 kDa) over 12 h; the GQD was obtained, and then it was placed into an oven at 160 °C to vaporize all the liquid. After that, its weight was measured and it was dissolved in solvent (e.g. ddH2O, PBS or serum) to prepare the GQD solution with working concentration as the experiment required. Characterization The samples were then subject to transmission electron microscopy (TEM, JEOL 1400 and JEOL 2100F, Japan) observation. The height profile diagram, thickness and size of samples were determined by Atomic force microscopy (AFM, multimode 8, Bruker, Germany). The crystalline structures of the samples were identified using X-ray diffraction (XRD, Bruker AXS Gmbh, Germany/ D2 Phaser) with Cuκα radiation at 30 kv and 30 mA. Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible (UV-vis), zeta potential spectra and dynamic light


5


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

Page 6 of 26

scattering (DLS) of samples were recorded by the spectrometers：PerkinElmer RX1 USA, U4100 Hitachi Japan and Manern Nano-ZS90 UK, respectively. Raman spectroscopy (DXR, Thermo Scientific, USA) was used to examine the crystallinity of samples with 532 nm laser. Xray photoelectron spectroscopy (XPS, PHI 5000, VersaProbe, USA) was employed to examine the surface chemistry of the materials. The PL signal was recorded by the spectrophotometer (F7000, Hitachi, Japan). Bacterial cultures E. coli, obtained from our own laboratory were grown in nutrient agar of LB (per liter: tryptone 10g, yeast extract 5g, sodium chloride 8g, agar 15g, and pH tuned to 7.5) (Sigma Aldrich Co., USA) and incubated at 37 °C. MRSA (ATCC 27659) were grown in pH 7.2 of brain heart infusion (DIFCO 0418, BD, USA) and incubated at 37 °C. Coating antibody The absorbance of a quantity of antibody (anti-lipopolysaccharide (LPS) antibody (AbLPS) or anti-protein A (Abprotein A) (Antagene, USA)) was recorded via UV-vis spectroscopy (Abs: approximately 276 nm). By the electrostatic interaction, the nanomaterials were mixed with the same quantity antibody for 30 min of incubation at 4 ℃ in the dark and centrifuged (83000 rpm) to remove excess antibody; the nanomaterial-AbLPS or -Abprotein A was then prepared. On the other hand, the supernatant was retained and its absorbance measured. The difference in absorbance between the collected supernatant and the original antibody was estimated. Consequentially, the quantity of the antibody absorbed on the nanomaterials was calculated by Lambert-Beer's law. In the working solution of 1×PBS buffer, there was approximately 0.09 µg of AbLPS absorbed on 1 µg of nanomaterial, which meant the efficiency of absorption was


6

Page 7 of 26

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


approximately 9% (zeta potential of nanomaterial-AbLPS: 6.8 mV), whereas it was 8.7% for Abprotein A (zeta potential of nanomaterial-Abprotein A: 7.4 mV). In culture medium of E. coli, NGQD was approximately 8.8% for AbLPS (zeta potential of nanomaterial-AbLPS: 7.1 mV) whereas it was 9.2% for Abprotein A in that of MRSA (zeta potential of nanomaterial-Abprotein A: 7.6 mV). Since there is not much different between the zeta potential of nanomaterial-Ab in 1×PBS buffer and culture mediums, it meant that the biomolecules would be absorbed on neither nanomaterial and Ab nor nanomaterial-Ab. In other words, the interaction among nanomaterial-Ab, Ab and bacteria would not be influenced by biomolecules in cultural mediums, leading to no subsequently effect in the specific binding among them. Additionally, the positively charged nanomaterial-Ab was favorable for absorbance or internalization by the negatively charged bacterial surface. The above results have proven the successful absorption of Ab on the surface of materials. Biocompatibility assay with colony forming unit (CFU) counting method E. coli and MRSA (OD600 ~0.05) were added with GQD-AbLPS and -Abprotein A (0-1 µg mL−1), respectively, and incubated for 3 h at 37 °C. After incubation, the mixture was centrifuged and the pellets of bacteria were diluted (OD600 ~0.05). A dilution factor of 10-5 to 10-8 was then conducted in the incubated bacteria and plated on the agar plates. The plates remain in an incubator (at 37 °C) overnight. The number of surviving bacteria was determined and expressed as a percentage (%) that corresponded to the unit of CFU mL−1 after incubation. Data are means ± SD (n=6). The other materials and methods associated with this article can be found in supporting information.


7


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

Page 8 of 26

3. RESULTS AND DISCUSSION GQDs were synthesized using an ultrasonic shearing reaction method on graphene oxide sheets prepared with a modified version of Hummers’ method.15 High-magnification high-resolution (HR) TEM exhibited the mean lateral size of GQDs to be approximately 7.1 ± 0.6 nm, as determined by DLS, indicative of the GQD interlayer spacing prepared as 0.213 nm (Figure 1a). The surface chemistry of GQDs, which predominately contains carbon atoms, was examined using XPS. The deconvoluted C(1s) spectra of GQDs showed a non-oxygenated ring (C－C/C＝C, 286.1 eV), C－O bonds (286.9 eV), and carbonyl (C＝O, 287.6 eV) and carboxylate groups (O＝C－O, 289.4 eV) (Figure 1b), as well as exhibited the oxygen-containing functional groups and the O(1s)/C(1s) ratio to be approximately 24.5 % (Supporting information, Table S2). The C－O bond might be due to tertiary alcohol, epoxy functional groups of basal plane and phenol in the periphery, as well as to the carboxylic and ketone groups in the graphene periphery indicative of O＝C －O and C＝O bonds. The C bonding composition showed that most oxygen-containing functionalities were situated at the graphene edge sites of the GQDs, confirming that the surface components of the GQDs had been successfully obtained. Because of the existence of carboxylic groups, the surface charge of GQDs was approximately −22.5 mV by zeta potential. The related results of other characterizations are presented in Figure S2 (Supporting information). The aforementioned characterizations confirmed that GQDs had been successfully synthesized.


8

Page 9 of 26

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


a

b

Figure 1. (a) HRTEM image of a single GQD, showing a mean lateral size of approximately 7.1 ± 0.6 nm, interlayer spacing of 0.213 nm, and a histogram of size distribution by DLS. (b) XPS was used to examine the changes in the chemical states of the GQD. The deconvoluted C(1s) spectra and fitted peaks using Gaussian function are C－C/C＝C, C－O, C＝O, and O＝C－O.

The toxicity of GQDs should be examined before conducting antimicrobial experiments to determine whether other factors that contribute to bacterial elimination will confound the interpretation of the results. In addition, to avoid the possibility of confounding PDT action when GQDs are inadvertently exposed to white light,6,7 the following experiments were conducted in the dark. In this study, gram-negative E. coli and gram-positive MRSA bacteria were chosen as the experimental templates. LPS is the major component of the outer membrane of E. coli, and Protein A is a surface protein in the cell wall of MRSA. In this study, to enhance the specificity and efficiency, the AbLPS and Abprotein A were coated with materials to form materials-AbLPS and Abprotein A, respectively. The number of surviving bacteria was determined using CFU counting assaying and is expressed as the percentage (%) corresponding to the unit of CFU mL−1. The


9


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

Page 10 of 26

absorbance (at 600 nm) of bacteria(OD600 ~ 0.05) was treated and incubated with GQDs-Ab in doses ranging from 0 to 1 µg mL−1 for 3 h at 37 °C in the dark. The GQDs displayed an almost 0 log10 reduction (Supporting information, Figures S3a and S3c) corresponding to almost 100% viability (Supporting information, Figures S3b and S3d), and showed excellent biocompatibility for both types of bacteria, while exhibiting good stability in physiological environments (Supporting information, Table S3). Consequently, a dose of 0.5 µg mL−1 of GQDs incubated for 3 h at 37 °C in the dark was selected for conducting all of the following experiments. To exploit the potential bactericidal capability of GQDs, the relative maximum two-photon absorption (TPA) ratio of GQDs approximately 800 nm was determined (Figure 2a), and used in subsequent experiments; moreover, antimicrobial experiments were performed on E. coli and MRSA with femtosecond laser irradiation (0–15 s photoexcitation with a power of 2.64 mW, 264 nJ pixel-1). Figures 2b-c show that without photoexcitation treatment, there was no antimicrobial effect on either type of bacteria in the two panels, as well as bacteria alone with photoexcitation. For the panel on GQD-treated-bacteria with photoexcitation treatment, bacterial viability sharply decreased as a function of irradiation time, killing almost all bacteria of both types after a 15-s exposure, whereas the bacterial viability was shown to be relatively high in the panel of nanomaterials without coating antibodies. Because GQD was believed to have acted as a PS in PDT and to have possessed the ability to generate ROS, the presence of ROS was monitored as shown in Figure 3. ROS is believed to play a major role in PDT because it induces processes that cause bacterial injury, such as DNA damage, the oxidization of fatty acid amino acids, and enzyme inactivation. When ROS is involved in PDT, its main products are 1O2 and O2.−, formed by the combination of the excited triplet GQDs, oxygen, and light of an appropriate wavelength and energy. This renders it necessary to detect them directly. GQDs alone were irradiated by 0–


10

Page 11 of 26

a

b Bacterial vaibility (%)

Nornalized TPA

1.2 1 0.8 0.6 0.4 0.2

* *

120 100 80 60 40 20 0

0

0

720 730 740 750 760 770 780 790 800 810 820

5

10

15

Time (sec)

Wavelength (nm)

* *

c 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 80 60 40 20 0 0

5

10

15

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

Time (sec)

Figure 2. (a) Relative TPA spectra of the GQDs. TPE with a function of the wavelength (720820 nm) at 1.056 mW (105.6 nJ pixel-1) was utilized to determine TPA signals. The quantified viability following the determined viable count of (b) GQD-AbLPS-treated-E. coli and (c) GQDAbprotein A-treated-MRSA, respectively, through CFU assaying by 5–15 s photoexcitation with 2.64 mW (264 nJ pixel-1) of TPE (Ex: 800 nm). Delivered dose: OD600 ~ 0.05 of bacteria and 0.5 µg mL−1 GQDs. Data are means ± SD (n = 6). For GQD-Ab-treated-bacteria with laser irradiation and GQD-treated-bacteria with laser irradiation, (b) p< 0.0001 and p= 0.0113; (c) p< 0.0001 and p= 0.085, respectively. *p value obtained by Student's t test.

15 s photoexcitation with a femtosecond laser 2.64 mW (264 nJ pixel-1). 1O2 can be detected with SOSG reagent6 and DPBF,16 and determined according to the fluorescence produced or quenched,


11


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

Page 12 of 26

whereas XTT was used to monitor the generated O2.−, and record the absorbance at 470 nm7 (Figures 3a, 3c and 3e). Although GQDs could generate ROS, false positive signals of ROS may be caused by the interactions among GQDs, SOSG reagents, and XTT, unexpectedly confounding the results of PDT.17 In order to eliminate this possibility, bacteria were introduced and treated with GQDs-Ab. The amount of ROS generated by the photoexcited GQD-Abtreated-bacteria was monitored (Figures 3b, 3d and 3f); it revealed that the quantities of generated ROS depended on the photoexcitation time. By contrast, the generated ROS showed an obvious decrease as bacteria were treated with nanomaterials without coating antibodies (Supporting information, Figure S4). The results also demonstrated that the antibodies were successfully coated with nanomaterials, thus enhancing the specificity, selectivity and efficiency. To determine the degree that photoexcitation disrupted GQD-treated bacteria, the bacteria were imaged with TEM. Bare E. coli and MRSA (Figures 4a–b) were incubated with GQDs for 3 h, leading to a large number of GQDs being adsorbed on both types of bacterial surfaces due to the highly specific and efficient binding ability of antibodies, but showing no unusual morphology in shape, indicating normal live bacterial morphology (Figures 4c–d). Yet the images presented distorted E. coli and a severe change in shape for MRSA over a long-term incubation of 5 d (Figures 4e–f), resulting in 0.174 and 0.480 log10 reductions corresponding to almost 67% and 33% viabilities of E. coli and MRSA, respectively (Supporting information, Figure S5). In addition to the specific bound antibodies, the GQD coating the bacterial surface were concluded to have suppressed nutrients essential to their growth, leading to change to the properties of


12

Page 13 of 26

a

Fluorescence intensity


b

*

1600 1200 800 400 0 0

10

* *

2000

E. coli 1600

MRSA

1200 800 400 0 0

15

d

*

1500



c

1000

500

0

15

*

*

1500

E. coli MRSA 1000

500

0

0

10

15

0

Time (sec) 1.6

f

*

1.2 0.8 0.4 0 0

10

10

15

Time (sec) Absorbance of 470 nm

e

10

Time (sec)

Time (sec)

Absorbacne of 470 nm

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


*

1.6

*

E. coli MRSA

1.2 0.8 0.4 0

15

Time (sec)

0

10

15

Time (sec)

Figure 3. ROS measurements. After 0–15 s photoexcitation with 2.64 mW (264 nJ pixel-1) of TPE (Ex: 800 nm) to GQDs, direct detection of the generated 1O2 by (a) SOSG reagent (p=


13


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

Page 14 of 26

0.0701) and (c) DPBF (p< 0.0001), and O2.− by (e) XTT (p< 0.0001) were conducted; photoexcitation-generated 1O2 and O2.− from GQD-AbLPS- and GQD-Abprotein A-treated-bacteria, were detected by (b) SOSG reagent (for E. coli and MRSA, p= 0.0782 and 0.0856), (d) DPBF, (for both bacteria, p< 0.0001) and (f) XTT (for both bacteria, p< 0.0001), respectively. SOSG reagents produced the fluorescence (Ex/Em: 488/525 nm) that was utilized to determine the generated 1O2,as well as DPBF (Ex/Em: 403/450-520 nm). XTT was used to monitor the generated O2.− and record the absorbance at 470 nm. Delivered dose: OD600 ~ 0.05 of bacteria and 0.5 µg mL−1 GQDs. Data are means ± SD (n = 6). *p value obtained by Student's t test.

a

c

e

g

b

d

f

h

E. coli

MRSA

Figure 4.TEM images showing bare (a) E. coli and (b) MRSA without any treatment. E. coli and MRSA treated with GQD-AbLPS and -Abprotein

A

for (c, d) 3 h and (e, f) 5 d of incubation,

respectively. (g) The photoexcited GQD-AbLPS-treated-E. coli and (h) GQD-Abprotein A-treatedMRSA, respectively, for 3 h of incubation with 2.64 mW (264 nJ pixel-1) TPE for 15 s (Ex: 800 nm). Delivered dose: OD600 ~ 0.05 of bacteria and 0.5 µg mL−1 GQDs.


14

Page 15 of 26

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


membrane wall permeability, thereby arousing internal osmotic imbalances and constraining bacterial growth, as well as forming an external barrier to further suppress the absorption of essential nutrients.18,19 This results demonstrate that the GQDs also possess antibacterial (defined as bacteriostatic or bactericidal) capabilities after a long-term incubation of 5 d. However, the photoexcited GQD-Ab-treated-bacteria incubated for 3 h showed unusual, severely damaged morphology, with MRSA particularly affected (Figures 4g–h). Figures 2–4 show that MRSA was susceptible to photoexcitation, leading to a higher death rate, higher levels of ROS generation, and a more severe collapse in shape than occurred in E. coli. This result might be attributable to the lack of an outer membrane composed of LPS and protein on gram-positive MRSA bacteria. These experimental results, once inspected and clarified, demonstrated that GQDs were able to generate ROS and induce low-energy PDT to rapidly eliminate bacteria after photoexcitation. This capability provides an alternative, easier approach to killing the MDR bacterial strains of MRSA. The illumination of GQDs by surface groups may contribute to a phenomenon associated with intrinsic and defect state emissions,13,14 and possibly involving a PL mechanism. For this reason, the quantum yield (QY) of GQDs was calculated to be approximately 0.185 (QYref = 0.69 is the QY of rhodamine B in methanol, as a reference;8,20 singlet oxygen QY (ψ∆) of GQDs was approximately 0.51, ψ ∆ = 0.64 is the QY of TSPP dissolved in D2O as a reference,21,22 supporting information) and one-photon excitation or two-photon excitation (TPE) yield the same QY.23 Additionally, the high values of the absolute cross section of TPE make fluorophores effective for multi photon microscopic investigation because this corresponds to a high ratio of


15


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

Page 16 of 26

the energy absorbed to the input-energy flux in a specimen, reducing probable photodamage.8 When two-photon techniques are exploited to monitor molecular activities in vitro or in vivo, a large cross section is especially desirable. However, TPL is a nonlinear phenomenon and its intensity is proportional to the TPE cross section, quadratic excitation power, and PL QY. The strong TPL signal has attracted attention and resulted in several techniques that enhance its intensity, namely the increase of localized excitation power and the modification of intrinsic characteristics of GQDs by increasing PL QY and larger TPE cross section.1,7,8,23,24 Therefore, determining the absolute TPE cross section and TPL spectrum of GQDs was necessary. An improved TPA ratio in the NIR window (Figure 2a) enabled the GQDs to achieve a greater TPL imaging depth and to detect deeper tissue with TPE. By measuring the TPL intensity dependence on the excitation power, the dependence was determined to be the square of the excitation power, with exponents of 2.01 measured with a function of excitation power at 800 nm in wavelength (Figure 5a), demonstrating that excitation is a two-photon process.1,8,23 The absolute cross section of TPE for the GQDs was calculated to be approximately 47903 GM (Goeppert–Mayer units, with 1 GM= 10−50 cm4 s photon−1) at 800 nm of excitation wavelength (with rhodamine B selected as the standard reference for the cross section1,8) (Supporting information, Figures S6S7 and Table S4; Table 1), similar to the value obtained in other studies.9,24 GQDs showed not only higher PL QY but also larger absolute cross section of TPE compared to gold-based nanomaterials,3,6,7 demonstrating potential application as a contrast probe in the two-photon process. Figure 5b shows TPL spectra measured for the GQDs with irradiation; the peaks were observed approximately from 618 nm to 647 nm for 740-800 nm TPE wavelengths. This indicates that the same PL spectrum was yielded by TPE. Moreover, the aggregated GQDs that emitted TPL were observed the same optical system (Figure 5c).


16

Page 17 of 26

a

b 3

740 nm

c

770 nm

2.5

Log intensity

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


800 nm

2 1.5 1 0.5 0 0

0.5

1

1.5

Log excitation power (mW)

2

250 300 350 400 450 500 550 600 650

Wavelength (nm)

Figure 5. (a) The TPL intensity dependence on the excitation power (logarithm) of the GQDs with a slope of 2.01, exposed to from 7.04 mW (704 nJ pixel-1) to 28.16 mW (2816 nJ pixel-1) of TPE. R2> 0.99. (b) TPL spectra of GQDs, exposed to 0.704 mW (70.4 nJ pixel-1) TPE with the excitation wavelength from 740 nm to 800 nm(cut off at 690 nm by the cascading filters). (c) TPL image of the aggregated GQDs was observed with 2.64 mW (264 nJ pixel-1) TPE. Excitation wavelength: 800 nm. Delivered dose: 0.5 µg mL−1 GQD.

Table 1. TPE cross section of GQD at 800 nm of excitation wavelength.a,b

a

Rhodamine B was selected as the standard reference for the cross section. bRhodamine B and GQD were with the same concentration in this measurement.


17


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

Page 18 of 26

Deeper TPE images were observed through NIR laser excitation, taking advantage of maximum tissue transmission for bioimaging caused by slight scattering, low energy absorption, optimal irradiation penetration, and reduced photobleaching via GQD-treated bacteria. Bacteria were embedded in a collagen matrix to mimic the 3D biological environment. Figure 6 shows the TPL images of a layer of GQD-Ab-treated bacteria with different depths attained by photoexcitation. As a function of depth at every 25 µm, the TPL emitted from GQD-AbLPStreated E. coli (Figures 6a–c) and GQD-Abprotein A-treated-MRSA (Figures 6f–h) at 0.704 mW (70.4 nJ pixel-1) of TPE with a depth to75 µm revealed the clear images and localization of the bacteria. The power was increased to 2.64 mW (264 nJ pixel-1) for an additional photoexcitation of 10–15 s to induce PDT. The TPL of both types of bacteria started to decrease during a 10-s photoexcitation, showing that the bacterial cell wall had been changed its original property and function, or surrounding biological substrates on bacterial surface were oxidized and deteriorated by the generated ROS, which would lead bacteria to soon atrophy, become distorted, and sustain severe morphological damage, indicating the desorption of the GQDs from the bacterial surface (Figures 6d and 6i). The TPL intensity sharply dropped to the lowest extent for the 15-s photoexcitation (Figures 6e and 6j). The photothermal effect of the GQDs was not observed following identical laser treatments of the nanomaterials (Figure S8). On the other hand, the images of GQD-Ab-treated bacteria were not able to be observed by one-photon excitation with a 594 nm laser (0.15 W cm-2, coherent, USA) at the same depth (Supporting information, Figure S9). The surviving viabilities of bacteria were also indicated by fluorescence and quantification (Figure 7).7 For the photoexcited bacteria alone panel, results showed nearly no damage, as evidenced by the green fluorescence, indicating live bacteria (Figures7a and 7d). However, the dead bacteria from the GQDs–Ab treatments and laser exposure were distinguishable to a


18

Page 19 of 26

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


25 µm

50 µm

75 µm

75 µm

75 µm

a

b

c

d

e

f

g

h

i

j

10-s photoexcitation

15-s photoexcitation

E. coli

MRSA

Figure 6. TPL images at different depths of (a–c) E. coli and (f–h) MRSA were observed from 25 to 75 µm at a power of 0.704 mW (70.4 nJ pixel-1) by TPE. Images of (d) E. coli and (i) MRSA for extra 10-s photoexcitation and (e) E. coli and (j) MRSA for extra 15-s photoexcitation at a depth of 75 µm with a power of 2.64 mW (264 nJ pixel-1). Excitation wavelength: 800 nm. Delivered dose: OD600 ~ 0.05 of bacteria and 0.5 µg mL−1 GQD-AbLPS and 0.5 µg mL−1 GQDAbprotein A, respectively.

particular degree, as indicated by the red fluorescence (Figures7b and 7e). The viability of bacteria was quantified for additional antimicrobial tests, which showed nearly all nanomaterialAb-treated bacteria to be dead after treatment (Figures 7c and 7f). Similar viability was quantified through the CFU counting method (Figures 2b-c). Additionally, the TPL intensity of the GQDs only slightly decreased as the GQDs induced photoexcitation to exhibit photostability involving TPE (Supporting information, Figure S10); moreover, the photoexcited GQDs exhibited better photostability than other conventional PSs (Supporting information, Figure S11),


19


a

c

b

120

*

Live or dead (%)

100

*

Live % Dead %

80 60 40 20 0 Control

d

e

GQD

f

* 120

*

100

Live or dead (%)

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

Page 20 of 26

Live %

80

Dead %

60 40 20 0 Control

GQD

Figure 7. Images and viability of bacteria. After the 15-s photoexcitation laser photoexcitation of (a,b) GQD-AbLPS-treated-E. coli and (d,e) GQD-Abprotein A-treated-MRSA, Live/Dead kit was used to stain bacteria and the images were obtained, and (c,f) quantification of viability was determined. For Live % and Dead %, (c) p= 0.0003 and 0.0002; (f) p= 0.0002 and 0.0002, respectively. Delivered dose: OD600 ~ 0.05 of bacteria and 0.5 µg mL−1 GQD-AbLPS and 0.5 µg mL−1 GQD-Abprotein A, respectively. Negative control: bacteria and laser irradiation alone without nanomaterial treatment. Data are means ± SD (n=6). *p value obtained by Student's t test.

leading to the reduced photobleaching effect. In summary, the GQDs simultaneously serve as promising two-photon contrast agents for observing bacteria at greater depths and employ PDT to completely eliminate bacteria at these depths.

4. CONCLUSION


20

Page 21 of 26

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


GQDs were prepared successfully in this study. They were able to generate ROS and impressive two-photon properties, including TPA in the NIR region, TPE cross section, TPL emission and two-photon stability, which enabled GQD to contribute to PDT action at ultra-low energy levels and a rapid period of photoexcitation. GQDs also served as promising two-photon contrast agents for observing specimens at a greater depth and 3D biological specimens by TPE and proceeding with PDT action to eliminate bacteria. These capabilities provide an efficient alternative approach for observing and eliminating MDR bacteria.

ASSOCIATED CONTENT Supporting information. Experimental section, molecular weight of GQD, z axis resolution of the laser, characterizations, viability, stability in physiological environment, ROS measurements, plat of TPL intensity, TPL spectra, TPE cross section, temperature dependence as a function of irradiation time, one-photon bioimaging, photostability. This information is available free of charge via the internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author. *Wen-Shuo Kuo, [email protected]; Ping-Ching Wu, [email protected]; Ying-Chih Pu, [email protected]

Present Address. National Cheng Kung University, No. 1, University Rd., Tainan 701, Taiwan.


21


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

Page 22 of 26

ACKNOWLEDGMENT This research was supported by the Ministry of Science and Technology, Taiwan (MOST-1032113-M-006- 010- MY2; MOST-104-2321-B-006-008-; MOST-104-2314-B-006-076-MY3; MOST-104-2113-M-024-002-MY2), the Aim for the Top University Project and the National Science Council of Taiwan (NSC-105-2221-E-006 -088), and Spark Taiwan-ANCHOR UNIVERSITY (MOST 105-2321-B-006-02).

ABBREVIATIONS GQD, graphene quantum dot; TPE, two-photon excitation; PDT, photodynamic therapy; MDR, multidrug-resistant; NIR, near-infrared; PS, photosensitizer; ROS, reactive oxygen species; 1O2, singlet oxygen; O2.−, superoxide radical anion; PL, photoluminescence; E. coli, Escherichia coli; MRSA, methicillin-resistant Staphyococcus aureus; TPL, two-photon luminescence; 3D, threedimensional; TEM, transmission electron microscopy; AFM, Atomic force microscopy; XRD, X-ray diffraction; FTIR, Fourier transform infrared spectroscopy; UV-vis, ultraviolet-visible; DLS, dynamic light scattering; XPS, X-ray photoelectron spectroscopy;AbLPS, antilipopolysaccharide antibody; Abprotein A, anti-protein A antibody; CFU, colony forming unit; HRTEM, high-resolution transmission electron microscopy; TPA, two-photon absorption; SOSG, Singlet Oxygen Sensor Green; DPBF, 1,3-diphenylisobenzofuran; XTT, 2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide; QY, quantum yield; TPSS, meso-tetra(4-sulfonatophenyl)porphine dihydrochloride; TPE, two-photon excitation; GM, Goeppert–Mayer.


22

Page 23 of 26

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


REFERENCES (1) Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W. Multiphoton Fluorescence Excitation: New Spectral Windows for Biological Nonlinear Microscopy. Proc. Natl. Acad. Sci. USA 1996, 93, 10763-10768. (2) Helmchen, F.; Denk, W. Deep Tissue Two-Photon Microscopy. Nat. Methods 2005, 2, 932940. (3) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, A. M. Cancer Cell Imaging and Photothermal Therapy in the Near-Infrared Region by Using Gold Nanorods. J. Am. Chem. Soc. 2006, 128, 2115-2120. (4) Zharov, V. P. Ultrasharp Nonlinear Photothermal and Photoacoustic Resonances and Holes Beyond the Spectral Limit. Nat. Photonics 2011, 5, 110-116. (5) Bechet, D.; Couleaud, P.; Frochot, C.; Viriot M.; Guillemin, F.; Barberi-Heyob, M. Nanoparticles as Vehicles for Delivery of Photodynamic Therapy Agents. Trends Biotechnol. 2008, 26, 612-621. (6) Kuo, W. S.; Chang, Y. T.; Cho, K. C.; Chiu, K. C.; Lien, C. H.; Yeh, C. S.; Chen, S. J. Gold Nanomaterials Conjugated with Indocyanine Green for Dual-Modality Photodynamic and Photothermal Therapy. Biomaterials 2012, 33, 3270-3278. (7) Chang, W. T.; Chen, S. J.; Chang, C. Y.; Liu, Y. H.; Chen, C. H.; Yang.; C. H.; Chou, L. C. S.; Chang, J. C.; Cheng, L. C.; Kuo, W. S.; Wang, J. Y. Effect of Size-Dependence Photodestructuve Efficacy by Gold Nanomaterials withMultiphoton Laser. ACS Appl. Mater. Interfaces 2015, 7, 17318-17329.


23


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

Page 24 of 26

(8) Albota, M. A.; Xu, C.; Webb, W. W. Two-Photon Fluorescence Excitation Cross Sections of Biomolecular Probes from 690 to 960 nm. Appl. Optics 1998, 37, 7352-7356. (9) Liu, Q.; Guo, B.; Rao, Z.; Zhang, B.; Gong, J. R. Strong Two-Photon Induced Fluorescence from Photostable, Biocompatible Nitrogen-Doped Graphene Quantum Dots for Cellular and Deep-Tissue Imaging. Nano Lett. 2013, 13, 2436-2441. (10) 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. (11) Ge, J.; Lan, M.; Zhou, B.; Liu, W.; Guo, L.; Wang, H.; Jia, Q.; Niu, G.; Huang, X.; Zhou, H.; Meng, X.; Wang, P.; Lee, C.S.; Zhang, W.; Han, X. A Graphene Quantum Dot Photodynamic Therapy Agent with High Singlet Oxygen Generation. Nat. Commun. 2014, 5, 4596-4603. (12) Ristic, B. Z.; Milenkovic, M. M.; Dakic, I. R.; Todorovic-Markovic, B. M.; Milivijevic, M. S.; Budimir, M D.; Paunovic, V. G.; Dramicanin, M. D.; Markovic, Z. M. Photodynamic Antibacterial Effect of Graphene Quantum Dots. Biomaterials 2014, 35, 4428-4435. (13) 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. (14) Pan, D. Y.; Zhang, J. C.; Li, Z.; Wu, M. H. Hydrothermal Route for Cutting Graphene Sheets into Blue-Luminescent Graphene Quantum Dots. Adv. Mater. 2010, 22, 734–738. (15) Hummers, W. S.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339.


24

Page 25 of 26

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


(16) Mayada, E. A.; Bard, A. J. Production of Singlet Oxygen in Electrogenerated Radical Ion Electron Transfer Reactions. J. Am. Chem. Soc. 1973, 9, 6223-6226. (17) Lyon, D. Y.; Brunet, L.; Hinkal, G. W.; Wiesner, M. R.; Alvarez, P. J. J. Antibacterial Activity of Fullerene Water Suspensions (nC60) is not Due to Ros-Mediated Damage. Nano Lett. 2008, 8, 1539-1543. (18) 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: Polym Rev. 2003, 43, 223-249. (19) Kong, M.; Chen, X. G.; Xing, K.; Park, H. J.Antimicrobial Properties of Chitosan and Mode of Action: AState of the Art Review. Int. J. Food Microbiol. 2010, 144, 51-63. (20) Weber, G.; Teale, F. W. J. Determination of the Absolute Quantum Yield of Fluorescent Solutions.Trans. Faraday Soc. 1957, 53, 646-655. (21) Shi, L; Hermandez, B; Selke, M. Singlet Oxygen Generation from Water-Soluble Quantum Dot-Organic Dye Nanocomposites. J. Am. Chem. Soc. 2006, 128, 6278-6279. (22) Davila, J.; Harriman, A. Photoreactions of Macrocyclic Dyes Bound to Human Serum Albumin. Photochem. Photobiol. 1990, 51, 9-19. (23) 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. (24) Cao, L.; Wang, X.; Meziani, M. J.; Lu, F.; Wang, H.; Luo, P. G.; Lin, Y.; Harruff, B. A.; Veca, L. M.; Murray, D.; Xie, S. Y.; Sun, Y. P. Carbon Dots for Multiphoton Bioimaging. J. Am. Chem. Soc. 2007, 129, 11318-11319.


25


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

Page 26 of 26

Table of Contents Graphic


26

Two-Photon Photoexcited Photodynamic Therapy ... - ACS Publications

Recommend Documents