Novel Fluorescent Microemulsion: Probing Properties, Investigating

Subscriber access provided by University of Pennsylvania Libraries

Article

Novel Fluorescent Microemulsion: Probing Properties, Investigating Mechanism and Unveiling Potential Application Mengna Hou, Leping Dang, Tiankuo Liu, Yun Guo, and Zhanzhong Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05819 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 15, 2017

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 32

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

Novel Fluorescent Microemulsion: Probing Properties, Investigating Mechanism and Unveiling Potential Application *

Mengna Hou#, Leping Dang#, Tiankuo Liu, Yun Guo, Zhanzhong Wang

School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China.

ABSTRACT: Nanoscale microemulsions have been utilized as delivery carriers for nutraceuticals and active biological drugs. Herein, we designed and synthesized a novel oil in water (O/W) fluorescent microemulsion based on isoamyl acetate， polyoxyethylene castor oil EL(CrEL), and water. The microemulsion emitted bright blue fluorescence, thus exhibiting its potential for active drug detection with label-free strategy. The microemulsion exhibited excitation-dependent emission and distinct red-shift with longer excitation wavelengths. Lifetime and quantum yield of fluorescent microemulsion were 2.831 ns and 5.0 %, respectively. An excellent fluorescent stability of the microemulsion was confirmed by altering pH, ionic strength, temperature, and time. Moreover, we proposed a probable mechanism of fluorochromic phenomenon, which in connection with aromatic ring structure of polyoxyethylene ether substituent in CrEL. Based on our findings, we concluded that this new fluorescent microemulsion is a promising drug carrier that can facilitate active drug detection with label-free strategy. Although further research is required to understand the exact mechanism behind its fluorescence property, this work provided valuable guidance to develop new biosensors based on fluorescent microemulsion.

1

ACS Paragon Plus Environment


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

KEYWORDS: fluorescent nano-microemulsion, label-free strategy, nutraceutical delivery carrier, fluorescence mechanism 1. INTRODUCTION Microemulsions are isotropic liquid mixtures based on oil, surfactant, and water. Due to their extraordinary physicochemical characteristics, such as nanosize, transparency, thermodynamic stability, easy preparation, and low viscosity,1,2 they have drawn intensive attention.3,4 Microemulsions with double layered structure can function as membrane-mimetic system because of their compartmentalized construction. Researches based on this membrane-mimetic system, such as the interaction of jatrorrhizine with human gamma globulin, and the interaction of esculin with human serum albumin as well, have been widely conducted.5-9 While the inner nanopool inside a microemulsion droplet possesses high solubilization capacity to contain hydrophilic or hydrophobic bioactive materials,3,10 the outer layer plays significant role in protecting the system from environmental changes. This feature enhances its applicability in diverse areas from advanced materials to concrete application. For example, microemulsions have been identified as delivery carriers for various nutraceuticals and active biological drugs.4,11,12 Microemulsions not only protect active ingredients, such as carotenoids, phytosterols and polyunsaturated fats, from undesired degradation, but also improve bioavailability.1,11 Furthermore, nanoscale microemulsions are regarded as micro-reactors to improve the efficacy of enzymatic and chemical reactions.3,11 Fluorophores based transducers transfer energy from chemical, physical or 2


Page 2 of 32

Page 3 of 32

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


biological events into light that can be detected and analyzed to interpret otherwise inaccessible information.13 Developing fluorescent nanomaterials as sensing probe to peruse bioactive ingredients and to enable optical bioimaging in vitro and in vivo, such as nutrient delivery, drug targeting and diagnostic imaging, has become an important field of research drawing increasing attention.1,11,14 These endeavors are driven forward to clarify and to gain control over the interaction characteristics of active nutraceuticals and drug molecules with biological systems. To date, various fluorescent materials have been exploited including dye-loaded polymers, dye-doped silica, fluorescent proteins, and carbon quantum dots.15-19 Although these fluorophores were reported to have high fluorescence efficiency, they were not much explored because of some disadvantages associated with these materials.15,16 For instance, the fluorescent sensing based on labeling strategy, such as dye-loaded polymers and dye-doped silica, showed some crucial shortcomings including labeling inefficiency and expensiveness.20-23 Inorganic fluorescent nanoparticles, like carbon quantum dots, require a specially adapted organic shell for their stabilization.24,25 These nanoparticles are also not intrinsically biodegradable.16,26 Organic fluorescent materials, such as fluorescent proteins and conjugated polymer nanoparticles, usually lack flexibility in terms of tuning emitter properties as well as surface chemistry. These fluorophores frequently exhibit drastic fluorescence quenching due to aggregation while binding with biomolecules, resulting fluorescence “turn-off” during detection, strong background noise, and poor sensitivity.15,16,26 Furthermore, these fluorophores are usually synthesized using complex method and are partly poisonous,27-31 thus greatly 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

restricting their applications in food and biomedicine industry. Literature survey reveals that microemulsion research is primarily focused on the physical and chemical properties till date.32-34 Rare reports on microemulsion, which emit fluorescence as fluorophor without any additives, have been published. In order to improve the sensitivity and selectivity of fluorescent microemulsion based assays, to understand the interactions between active biomolecules and fluorescent microemulsion, and to enhance applicability, the properties and sensing mechanism of fluorescent microemulsion need to be explored. In the current scenario, highly fluorescent microemulsion, reported in the present work, might be a promising alternative as novel fluorescent probe owing to its non-toxic, biodegradable, and biocompatible nature. To determine the performance of our fluorescent microemulsion probe, we performed a series of investigation. The polyoxyethylene castor oil EL and isoamyl acetate used in this work have been shown to comply with the food and drug administration. To address special requirements and to engineer an analytically versatile system for applications in active biomolecules detection during delivery, we introduced here an original methodology of preparing fluorescent microemulsion. Subsequently, we characterized its properties, such as size, brightness, excitation and emission spectra, Stokes shift, lifetime, quantum yield, and stability. On the basis of the interaction of polyoxyethylene castor oil EL and water, the fluorescence mechanism of this microemulsion was investigated. In addition, α-linolenic acid-loaded microemulsion was synthesized as an application model, and a steadily emissive fluorescence was observed. This work could offer some guidelines 4


Page 4 of 32

Page 5 of 32

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


in developing label-free, turn-on fluorescent probe for active biomolecule detection. 2. EXPERIMENTAL SECTION 2.1. Materials. Isoamyl acetate of AR grade was purchased from Chemical Reagent Supply and Marketing Co. Ltd., Tianjin, China. Polyoxyethylene castor oil EL (CrEL) was purchased from Source Leaf Biotechnology Co. Ltd, Shanghai, China. The α-linolenic acid was purchased from Sigma-Aldrich Co. LLC, USA. The mass fraction purity of α-linolenic acid was more than 99%. Ethanol was purchased from Rionlon Bohua Pharmaceutical Chemical Co. Ltd, Tianjin, China. Other analytical reagents were obtained from Mobi Biotechnology Co. Ltd, Shanghai, China. Deionized-water was used for preparing all the microemulsion samples. 2.2. Preparation of Fluorescent Microemulsion. The microemulsion samples were prepared by taking appropriate amounts of isoamyl acetate (oil phase), CrEL(surfactant), ethanol(co-surfactant), and water in vials and mixing them by

mechanical agitation. The phase diagram has already been constructed at room temperature in our work for an overview of the phase behavior of the different systems. The boundaries for the microemulsion region were first determined and then refined by a titration process involving homogeneous mixtures of surfactant, co-surfactant, oil, and water. For the titration process, water was added dropwise to a CrEL/oil/ethanol blend with varying ratios of CrEL to oil ranging from 9:1 to 1:9 (w/w).

Samples were left for equilibration between each addition of water until transparency were examined visually. One of the optical recipes (isoamyl acetate/ethanol/CrEL/water = 2:1:12:60) was chosen to form microemulsion. The 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

following microemulsion systems were constructed based on the same preparation method. 2.3. Characterization of Fluorescent Microemulsion. Particle size analysis was performed using a dynamic light scattering (DLS) apparatus (Zetasizer Nano ZS90, Malvern Instruments Ltd, UK). The size measurements were carried out at a fixed angle of 90̊. To observe microstructures of the microemulsions, transmission electron microscopy (TEM) on a Tecnai G2 20 ST electron microscope (operating at 200 kV) was used; the instrumental magnification ranged from 2×104 to 10×106. The sample was deposited on a copper grid and coated with a holey carbon film. The micrographs (scale bars = 500 nm and 100 nm) were taken, and the representative images were chosen from at least three similar images. 2.4. Morphology of Fluorescent Microemulsion. Microemulsion was taken into a quartz cell. The micrographs were recorded when the cell was put in the bright field and ultraviolet lamp, respectively. Meanwhile, confocal laser scanning microscopy (CLSM FV1000, Olympus Ltd, Japan) was also used to examine the microscopic image of microemulsion. The image was collected at 400 nm of excitation wavelength. 2.5. Spectrometric Determination. The excitation and emission spectra of microemulsion were recorded using a F-2500 fluorescence spectrophotometer equipped with a 150 WXe lamp, which was manufactured by the Japanese Hitachi Corporation (EM=2.5nm, EX=2.5nm, and PMT=700V). The apparatus operates in a broad spectral range of 220–800 nm. As a contrast, spectra of solvent water were 6


Page 6 of 32

Page 7 of 32

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


determined under the same settings. To preliminarily explore the mechanism behind the fluorescence emission borne out of the microemulsion, the following experiments were performed. Firstly, the emission spectrum of each constituent of the microemulsion was recorded. The emission spectrum of the micelles being composed of CrEL and H2O, and the mixture of CrEL and ethanol, with same proportion as microemulsion, were also measured as a contrast. Furthermore, with invariable ratio of isoamyl acetate to ethanol as 2:1, the stabilized microemulsions with different CrEL concentrations were prepared to compare the emission spectra. Table 1 lists the corresponding ratios of CrEL to water in the microemulsion samples. Table 1. Microemulsion with different concentration of CrEL and the corresponding weight ratio of CrEL to H2O. Concentration of

6

7

8

9

12

14

16

18

21

24

27

6:90

7:90

8:90

9:90

12:80

14:80

16:80

18:80

21:70

24:70

27:70

CrEL (wt %)

Weight ratio of

CrEL to H2O

2.6. Lifetime and Quantum Yield Determination. Fluorescence lifetime measurements are generally carried out in either time-domain or frequency domain.35 In this work, fluorescence lifetime measurements were carried out by FLS920 Fluorescence Spectroscopy based on time-correlated single photon counting (TCSPC). With excitation wavelength of 360 nm and emission wavelength of 440 nm, nanosecond lamp (hydrogen) was set as light source to measure the lifetime. The 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 32

experimental data were analyzed by fitting the fluorescence decay model. Quantum yields (QY) was also calculated for microemulsion. An excitation range of 350–370 nm was used, while a luminescence range from 370 to 650 nm was monitored. 2.7. Fluorescence Stability Determination. The influences of temperature, pH, ionic strength and time on the fluorescence stability of the microemulsion were investigated. The effect of pH on fluorescence stability was carried out by replacing the aqueous phase of microemulsion with phosphate buffer solution of different pH (3.0–11.0). The effect of ionic strength on fluorescence stability was investigated by changing ionic strength (0–1.0M) of the aqueous phase of the microemulsion. The effect of temperature on the fluorescence stability of the microemulsion was studied at different temperatures ( refrigerator temperature 4 ℃ , room temperature 25 ℃ , processing temperature 50℃, and Pasteurization temperature 70℃). The effect of time on fluorescence stability was examined by extending storage time (7–28 days); the fluorescence measurement was performed once in 7 days for each interval.

2.8. Loading α-Linolenic Acid in Microemulsion: A Typical Application. To study the influence of active biomolecule on fluorescence of microemulsion, α-linolenic acid, as an active ingredient, was loaded into microemulsion. The proportion of α-linolenic acid in isoamyl acetate was increased from 3 to 18%.

2.9 In-vitro Release of α-Linolenic Acid from Microemulsion. In-vitro release experiments

were

performed

according

to

reported

methods

with

slight

modifications.2,12 The release profiles of α-linolenic acid from microemulsion were 8


Page 9 of 32

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


assessed by dialysis at pH 1.9 and 7.4 buffers (PBS) containing 0.1 %(w/v) Tween 80. The α-linolenic acid-loaded microemulsion were placed in the dialysis tubes (MWCO 3.5 kDa). Dialysis tubes were placed in each buffer (200 mL) and incubated in a shaking bath at 37 ℃with an agitation speed of 100 rpm. Aliquots of dissolution media (2.0 mL) were withdrawn at specific times (0.5, 1.5, 3.0, and 8 h), and an equivalent volume of fresh buffer was added. The concentration of α-linolenic acid in release medium was determined by UV-vis spectrophotometer at 320 nm.

3. RESULTS AND DISCUSSION Particle size and turbidity are effective parameters to identify the formation of a microemulsion system.3 Microemulsion with excellent transparency was synthesized in this work. The particle size and polydispersity index (PDI) of microemulsion were analyzed using dynamic light scattering (DLS) (Figure 1). Table 2 summarized the relevant data and sample appearance. The hydrodynamic diameters (Dh) of the particles in microemulsion were in the range of 5-100 nm with a particle diameter of 14.02 nm, and the average PDI was 0.264. Transmission electron microscope (TEM) images in Figure 2a, 2b revealed the size and shape of the microemulsion. Before observation, the sample was well dyed, and the TEM images were taken under different magnifications. Under relatively small magnification (Figure 2a), we found uniformly dispersed small spherical droplets with no aggregation. While under large magnification, we could clearly observed that the droplets were far away from each other due to the strong repulsive force. We found a relatively narrow size distribution ranging from 10–20 nm with an average size about 15 nm, which was in well 9



agreement with that obtained by DLS method (14.02 nm). The particles in TEM image also exhibited uniform dispersion, representing the good polydispersity of microemulsion. Both the results of DLS and TEM confirmed that the formation of stable microemulsion of small size, without droplet aggregation and flocculation.

30

Microemulsion 20

Intensity(Percent)

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 32

10

0 1

10

100

1000

10000

Size(nm)

Figure 1. Size distribution of microemulsion. Table 2. The hydrodynamic diameter (Dh) and appearance of the microemulsion. Sample

Dh(nm)

PDI

Appearance

Microemulsion

13.78 ± 0.77

0.294 ± 0.023

Transparent

(a)

(b) 10


Page 11 of 32

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 2. TEM images of microemulsion: (a) scale bar: 500 nm, (b) scale bar: 100 nm.

Figure 3. Fluorescence images of microemulsion observed under (a) bright-field, (b) CLSM, and (c) UV light at room temperature, respectively. Figure 3 illustrated the emission images of the fluorescent microemulsion under natural light, CLSM and UV light at room temperature, respectively. As evident from Figure 3a, the microemulsion was transparent and nonluminous under natural light, while Figure 3c showed bright blue fluorescence under UV lamp, thus visually confirming emissive property of the microemulsion. Figure 3b presented the microscopic image of microemulsion under 20×magnification detected by confocal laser scanning microscopy (CLSM), and the results were consistent with TEM results. Additionally, blue spherical beads further confirmed the fluorescence property of the microemulsion.

11



10000 EX=280 EX=300 EX=320 EX=340 EX=360 EX=380 EX=400 EX=420 EX=440 EX=460

8000

6000

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

Page 12 of 32

4000

2000

0 350

400

450

500

550

600

Wavelength(nm)

Figure 4. Fluorescence spectra of the microemulsion at different excitation wavelengths ranging from 280 nm to 460 nm. It is generally believed that the shape of a fluorescence emission spectrum is independent of the excitation wavelength.36 Interestingly, we found that the spectral profile and fluorescence intensity of the microemulsion changed with the changing excitation wavelength and observed an excitation-dependent fluorescence behavior in the emission spectra recorded in the excitation wavelength range of 280–460 nm. The emission peak in Figure 4 clearly showed a red-shift with increasing excitation wavelength. Combined with results of solvent water shown in Figure S1 and Table S1, spectral profile and fluorescence intensity of the microemulsion might be affected by solvent scattering peak and the interaction of different molecules in the microemulsion system.37,38 We have further analyzed this in the following discussion. Additionally, we observed the brightest fluorescence emission with a peak at around 440 nm upon excitation at 360 nm. 12


Page 13 of 32

10000 Excitation Emission 8000

6000

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


4000

2000

0 300

400

500

600

Wavelength(nm)

Figure 5. Fluorescence excitation (dashed line) and emission (solid line) spectra of the microemulsion. The dashed line in Figure 5 depicted the room temperature excitation spectrum of the microemulsion with maximum at 365 nm and recorded between 285 nm and 420 nm. The solid line in Figure 5 showed the room temperature emission spectrum of the microemulsion with the emission maximum at 440 nm. Excitation wavelength was 360 nm, and the spectrum was recorded from 380 nm to 700 nm. Theoretically, the corrected fluorescence excitation spectra is often a mirror image of the emission spectra.36 However, the spectral profile of excitation and emission can mostly different because of the wavelength factors of transmission coefficient, energy distribution of the light source, and sensitivity of detector.39 As evident from Figure 5, the spectral profiles of excitation and emission were similar, suggesting that the apparatus is suited for our measurement. In addition, the Stokes shift, an important

13



parameter in the measurement of synchronous spectrum and 3D fluorescence spectrum, of the microemulsion was calculated as about 80 nm. To investigate the mechanism behind the fluorescence emission borne out of the microemulsion, we recorded emission spectra of each constituent of the microemulsion (Figure 6). As evident from Figure 7, none of single constituent in the microemulsion is fluorescent. Emission spectrum of micelles of CrEL and water (CrEL+H2O) was quite close to the microemulsion. In comparison, fluorescent intensity of the mixture of CrEL and ethanol was extremely low, which may be attributed to large difference of water and ethanol in polarity. Therefore, the interaction of CrEL and water led to the fluorescence of the microemulsion. To further investigate the source of the fluorescence in the microemulsion, following fluorescence experiments were performed.

10000

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

Page 14 of 32

8000

CrEL+H2O Microemulsion Isoamyl acetate Ethanol CrEL H2O

6000

CrEL+Ethanol CrEL+Oil

4000

2000

0 400

450

500

550

600

Wavelength(nm)

Figure 6. Fluorescence spectra of individual constituents in microemulsion and the micelles being composed of CrEL and H2O/ethanol. 14


Page 15 of 32

Figure 7 presented the fluorescent spectra of the microemulsion with different CrEL concentration. We found that the concentrations of CrEL did not influence the shape of the emission spectra, however, it affected the fluorescence intensity. Figure 8 presented the plot constructed from the changes of emission spectra intensity of the microemulsion with different CrEL concentrations. The fluorescence intensity increased with the increase in CrEL content, i.e., the fluorescence intensity was directly proportional to the concentration of CrEL, and ratio of CrEL to water had no contribution towards the fluorescence property of the microemulsion. When CrEL concentrations are up to 24%, fluorescence intensity reached plateau.

10000 6% 7% 8% 9% 12% 14% 16% 18% 21% 24% 27%

8000

6000

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


4000

2000

0

400

500

600

Wavelength(nm)

Figure 7. Fluorescence spectra of microemulsion at different CrEL concentrations.

15



10000

Intensity 8000

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

Page 16 of 32

6000

4000

6

9

12

15

18

21

24

27

Concentration（ %）

Figure 8. Change in the fluorescence intensity of the microemulsion with increasing CrEL concentration. As reported, carbon dots (CDs) are often applied into optical bioimaging research due to its fluorescence. Nonetheless, the fluorescent mechanism of CDs is not clear till now, a large number of explanations have been developed based on experiments.

28,40-42

Among these explanations, the surface energy levels theory is the most popular. It is generally believed that there might not be only one fluorescence center, but a large number of fluorescence centers coming from different function groups or defections.43 In our work, given the success in constructing this fluorescent microemulsion, a label-free biosensing carrier, it still remains a challenge to further elucidate the mechanism behind the fluorescence. Excitation-dependent fluorescence behavior presented in Figure 4 hinted to the presence of more than one kind of fluorophores existed in the microemulsion. Generally, bright fluorescence of materials can be attributed to the existence of following molecular structure: a) conjugated Π bond 16


Page 17 of 32

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


structure like benzene, naphthalene, and anthracene; b) rigid planar structure like fluorescein; c) the case (Π - Π*) with lowest singlet excited state of S1, e.g., organic phosphor without heteroatom (N, O, S); d) preferable electron donating substituent group, e.g., anisole, formed by substituting H atom of benzene by methoxy group, exhibits higher fluorescent intensity.20,36 The schematic view of fluorescence produced by microemulsion was demonstrated in Figure 9. CrEL, used as surfactant in this work, is a mixture of 83% hydrophobic components and 17% hydrophilic components.44 Scheme 1 illustrated the main structure.

Figure 9. Schematic view of fluorescence produced by microemulsion: a) Aromatic ring structure of polyoxyethylene ether substituent in CrEL. b) Formation of intermolecular or intramolecular hydrogen bond. c) Double layered structure of O/W microemulsion. d) Microemulsion with blue fluorescence emission under UV lamp. Scheme 1. (a) castor oil, (b)-(d) polyoxyethylene castor acid glycerol ester with different degrees of substitution, (e)-(h) polyoxyethylene castor oil ester with different

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 32

degrees of substitution. In (b)-(h), the substituent group is polyoxyethylene ether. (i) polyethylene glycol (PEG), (j) polyethylene glycol glycerol ether.

Ricinoleic acid H2C

O

CO

(CH2)7

CH

CH

CH2

CH2

(CH2)5

CH3

(CH2)5

CH3

(CH2)5

CH3

OH HC

O

CO

(CH2)7

CH

CH

CH2

H2C

O

CO

(CH2)7

CH

CH

CH2

CH2 OH CH2 OH

Glycerin

(a) OH

O H2C

CO

O

(CH2)7

CH

CH

CH2

CH2

(CH2)5

CH3

(CH2)5

CH3

OH HC

H2C

CO

O

(CH2)7

OCH2 CH2

OH

n

CH

CH

CH2

CH2

OH polyoxyethylene ether

O

OH

(b) H2C

O

CO

(CH2)7

CH

CH

CH2

CH2

(CH2)5

CH3

(CH2)5

CH3

(CH2)5

CH3

OH CH

H2C

OCH2 CH2

O

CO

n

OH

(CH2)7

CH

CH

CH2

CH2 OH

(c) H2C

OCH2

CH2

CH

OCH2

CH2

H2C

O

CO

(CH2)7

n

OH OH

n

CH

CH

CH2

CH2 OH

(d)

18


Page 19 of 32

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


H2C

OCH2

CH2

CH

OCH2

CH2

H2C

OCH2

CH2

OH

n n n

OH

CO

O

(CH2)7

CH

CH

CH2

CH2

(CH2)5

CH3

OH

(e) H2C

OCH2

CH2

HC

OCH2

CH2

H2C

OCH2

n

O

CO

(CH2)7

CH

CH

CH2

O

CO

(CH2)7

CH

CH

CH2

n

(f) H2C

OCH2

CH2

HC

OCH2

CH2

H2C

OCH2

CH2

(CH2)5

CH3

(CH2)5

CH3

OH

n

CH2

CH2

CH2 OH

OH

n

CO

O

(CH2)7

CH

CH

CH2

CH2

(CH2)5

CH3

(CH2)5

CH3

(CH2)5

CH3

(CH2)5

CH3

(CH2)5

CH3

OH OH

n n

CO

O

(CH2)7

CH

CH

CH2

CH2 OH

(g) H2C

OCH2

CH2

n

CO

O

(CH2)7

CH

CH

CH2

CH2 OH

HC

OCH2

CH2

n

CO

O

(CH2)7

CH

CH

CH2

CH2 OH

H2C

OCH2

CH2

n

CO

O

(CH2)7

CH

CH

CH2

OH

(h) HO

CH2CH2

H2C

CH

(i)

(j)

CH2

CH2

O

n

H

OCH2CH2O

n-1

CH2CH2OCH2 CH

O

CH2 O

Analyzing the structure of CrEL, it could be deduced that the strong fluorescence in microemulsion might be attributed to the aromatic ring structure of polyoxyethylene ether substituent in hydrophobic components. CrEL is a mixture with different degrees of substitution of polyoxyethylene castor acid glyceride and polyoxyethylene castor oil ester. This indicates the presence of more than one single 19



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 32

fluorophore, thus explaining the observed excitation-dependent FL behavior of the microemulsion. The increased quantity of aromatic ring with increasing degree of substitution, resulted in the red-shift of fluorescence spectra and the fluorescence peak accordingly moves towards to longer wavelength with increasing excitation wavelength. This was in accordance with the results presented in Figure 4. Meanwhile, we also observed that CrEL was non-fluorescent, probably due to the relatively small conjugated system in CrEL molecule. When water was added, intermolecular and intramolecular hydrogen bonds could be formed (as shown in Scheme 2) between CrEL and water molecules due to strong polarity of water. This might lead to the planarization of the molecular conformation, which could greatly improve fluorescence efficiency (as shown in Figure 6). As shown in Figure 7, the increase in CrEL concentration probably increased the number of fluorophores, resulting in the fluorescence enhancement. Whether such mechanism can be extended to all kind of microemulsion

emitting

fluorescence

is

fundamentally

interesting

and

application-wise, which is a highly relevant issue deserving further investigation. Also deserving further investigation is the development of synthesis methods of microemulsion with special feature for the delivery of active biomolecules as exclusive probe. As reported in this study, the microemulsion fabricated was probably not entirely selective in nature, rather dependent on CrEL molecules and their interaction with water molecules as well. This indicated that more and effective fluorescent probes can be harvested with desired properties. Further investigation

20


Page 21 of 32

needs to be carried out to understand the mechanism behind the formation of fluorescent microemulsion.

R

R

O

O

O

O

OH

OH

Scheme 2. Formation of intermolecular or intramolecular hydrogen bond.

150

100

Counts

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


Decay Fitted Residuals 50

0

0

4

8

12

16

t(ns)

Figure 10. Time-resolved emission-decay curves and fitted lifetime decay of microemulsion. Time-resolved emission-decay behaviors of the microemulsion were studied and the time-resolved fluorescence curve and fitted curve were illustrated in Figure 10. The lifetime data were processed according to the literature method.45-47 Decay in the fluorescence intensity (I) with time (t) was fitted with a monoexponential function: 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

I = A + B 1 exp(

− t / τ 1)

Page 22 of 32

(1)

where τ is the lifetime of the microemulsion, B is amplitudes, and A is a constant. Table 3 summarized the lifetime data, and the data indicated the excited molecules in the microemulsion decayed through only one pathway (B = 106.8) with a single lifetime of 2.831 ns. The quantum yield of microemulsion was 5.0%. Different fluorescent materials possess different quantum yield. Compared with quantum yield of other fluorescent materials reported in literatures, such as carbon dots(CDs),27-29,39 their quantum yield are usually between 2.0 and 10 %, while dye-loaded materials,24-26 their quantum yield can reach 50 %. Table 3. Fitted fluorescence decay parameters of the microemulsion obtained from FLS920 based on TCSPC.

Microemulsion

τ1 (ns)

A

B1

χ2

2.831

1.550

106.8

1.135

Good fluorescence stability is a prerequisite for a label-free probe. We further investigated the impacts of temperature, ionic strength, pH and time on the stability of fluorescence signal emitted by microemulsion. Figure S2 showed insignificant effect of temperature on the fluorescence intensity. To analyze the impact of ionic strength on fluorescence intensity, we introduced sodium chloride into the aqueous phase of the microemulsion, and found that the fluorescence intensity coherently increases with increasing ionic strength (Figure S3). Figure S4 showed that the fluorescence intensity of the microemulsion remains almost unaffected by pH of the aqueous phase. We also examined the influence of storage time on fluorescence intensity (Figure S5) 22


Page 23 of 32

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


and found a slightly downtrend in the fluorescence intensity of the microemulsion during the first three weeks but later tend to stabilize. Similar trend was observed under both 4℃ and 25 ℃. The above findings suggested that this new microemulsion with remarkable fluorescence stability has potential for optical bioimaging (in vitro and in vivo) application. To check the effect of active biomolecule on the fluorescent microemulsion as delivery carrier, we investigated the change in fluorescence intensity of the microemulsion with increasing α-linolenic acid-loaded amounts. Figure S6 revealed that no obvious change in the fluorescence intensity of the microemulsion with different loading amounts of α-linolenic acid, suggesting the potential of the microemulsion as a stable fluorescent drug carrier. In order to investigate whether α-linolenic acid can be released from microemulsion, an in-vitro experiment was performed. Figure S7 was the release curves of α-linolenic acid at pH 1.9 and pH 7.4 from microemulsion. From Figure S7, it could be seen that release amounts of α-linolenic acid rose with increasing time. 4. Conclusions In summary, we designed and synthesized a novel fluorescent microemulsion as delivery carrier of active biomolecules. This microemulsion can function as a label-free sensing probe based on harboring active biomolecules into nanopool of the microemulsion. This nanoscale fluorescent microemulsion exhibited uniform particle distribution and bright blue fluorescence. We found that the emission was excitation-dependent, and the emission peak exhibited a clear red-shift with longer excitation wavelength. The concentration of CrEL brought a great impact on the 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

fluorescence intensity of the microemulsion, and the fluorescence intensity increased with increasing CrEL concentration. We attribute the fluorescence of the microemulsion to the aromatic ring structure of polyoxyethylene ether substituent in CrEL. However, further research is required to understand the exact mechanism behind the fluorescence of the microemulsion. In addition, the microemulsion exhibited remarkable stability of 4 weeks under both 4 ℃ and 25 ℃ environments. Therefore, we concluded that this highly fluorescent and robust microemulsion can be a promising delivery carrier for active biomolecules. ASSOCIATED CONTENT Supporting Information Figures, which reveal the impacts of temperature, ionic strength, pH and time on the stability of fluorescence signal emitted by microemulsion, were supplied in Supporting Information. The effect of α-linolenic acid on fluorescence intensity of microemulsion and results of in vitro release of α-linolenic acid were provided in Supporting Information. AUTHOR INFORMATION Corresponding Author *Tel.: +86-022-27400291. Fax: +86-022-27400287. E-mail: [email protected] Author Contributions #

M.H. and L.D. contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS 24


Page 24 of 32

Page 25 of 32

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


This work was funded by the National Natural Science Foundation of China (No.21406158). REFERENCES (1) Amiri-Rigi, A.; Abbasi, S. Microemulsion-Based Lycopene Extraction: Effect of Surfactants, Co-surfactants and Pretreatments. Food Chem. 2016, 197, 1002–1007. (2) Lee, J. J.; Park, J. H.; Lee, J. Y.; Jeong, J. Y.; Lee, S. Y.; Yoon, I. S.; Kang, W. S.; Kim, D. D.; Cho, H. J. Omega-3 Fatty Acids Incorporated Colloidal Systems for The Delivery of Angelica Gigas Nakai Extract. Colloid Surf. B-Biointerfaces. 2016, 140, 239–245. (3) Xu, Z. B.; Jin, J.; Zheng, M. Y.; Zheng, Y.; Xu, X. B.; Liu, Y. F.; Wang, X. G. Co-surfactant Free Microemulsions: Preparation, Characterization and Stability Evaluation for Food Application. Food Chem. 2016, 204, 194–200. (4) Chen, M. W.; Wang, S. P.; Tan, M.; Wang, Y.T. Applications of Nanoparticles in Herbal Medicine: Zedoary Turmeric Oil and Its Active Compound β-Elemene. Am. J. Chin. Med. 2011, 39, 1093–1102. (5) Li, Y.; Wang, C.; Lu, G.H. Interaction of Jatrorrhizine with Human Gamma Globulin in Membrane Mimetic Environments: Probing of The Binding Mechanism and Binding Site by Spectroscopic and Molecular Modeling Methods. J. Mol. Struct. 2010, 980, 108-113. (6) Zhang, Y.H.; Li, J.Z.; Dong, L.J.; Li, Y.; Chen, X.G. Characterization of Interaction between Esculin and Human Serum Albumin in Membrane Mimetic Environments. J. Mol. Struct. 2008, 889, 119-128. 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 32

(7) Zhang, Y.H.; Dong, L.J.; Li, J.Z.; Chen, X.G. Studies on The Interaction of Gallic Acid with Human Serum Albumin in Membrane Mimetic Environments. Talanta 2008, 76, 246-253. (8) Zhang, Y.H.; Dong, L.J.; Li, Y.; Li, J.Z.; Chen, X.G. Characterization of Interaction between Bergenin and Human Serum Albumin in Membrane Mimetic Environments. J. Fluoresc. 2008, 18, 661-670. (9) Zhang, Y.H.; Yue, Y.Y.; Li, J.Z.; Chen, X.G. Studies on The Interaction of Caffeic Acid with Human Serum Albumin in Membrane Mimetic Environments. J. Photoch. Photobio. B. 2008, 90, 141-151. (10) Shu, Y.; Cheng, D. H.; Chen, X. W.; Wang, J. H. A Reverse Microemulsion of Water/AOT/1-butyl-3-methylimidazolium

Hexafluorophosphate

for

Selective

Extraction of Hemoglobin. Sep. Purif. Technol. 2008, 64, 154–159. (11) Duan, X.; Li, M.; Ma, H. J.; Xu, X. M.; Jin, Z. Y.; Liu, X. B. Physicochemical Properties and Antioxidant Potential of Phosvitin–Resveratrol Complexes in Emulsion System. Food Chem. 2016, 206, 102–109. (12) Cheng, N. N.; Lu, T. L.; Chen, J.; Xue, J.; Mao, C. Q. Determination of Drugs in Liposomes Containing Zedoary Turmeric Oil and Application of the Method. Anhui Med. Pharm. 2010, 14, 404–406. (13) Hozer, B.; Medintz, I. L.; Hildebrandt, N. Fluorescence in Nanobiotechnology: Sophisticated Fluorophores for Novel Applications. Small 2012, 8, 2297–2326. (14) Sun, L.; Liu, Z. Y.; Cun, D. M.; Tong, H. H.; Zheng, Y. Application of Nanoand Micro-particles on The Topical Therapy of Skin-Related Immune Disorders. Curr. 26


Page 27 of 32

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


Pharm. Design. 2015, 21, 001–025. (15) Wang, Y. F.; Zhang, T. B.; Liang X. J. Aggregation-Induced Emission: Lighting Up Cells, Revealing Life. Small 2016, 12, 6451–6477. (16) Reisch, A.; Klymchenko, A. S. Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging. Small 2016, 12, 1968–1992. (17) Singh, V.; Sivaramaiah, G.; Mohapatra, M.; Rao, J. L.; Singh, N.; Pathak, M.S.; Singh, P. K.; Dhoble, S. J. Probing The Thermodynamic and Magnetic Properties of UV-BEmitting GdAlO3 Phosphors by ESR and Optical Techniques. J. Electron. Mater. 2017, 46, 1137–1144. (18) Genovese, D.; Rampazzo, E.; Bonacchi, S.; Montalti, M.; Zaccheroni, N.; Prodi, L. Energy Transfer Processes in Dye-Doped Nanostructures Yield Cooperative and Versatile Fluorescent Probes. Nanoscale 2014, 6, 3022–3036. (19) Gnach, A.; Bednarkiewicz, A. Lanthanide-Doped Up-Converting Nanoparticles: Merits and Challenges. Nano Today 2012, 7, 532–563. (20) Kerr, C.; DeRosa, C. A.; Daly, M. L.; Zhang, H.; Palmer, G. M.; Fraser, C.L. Luminescent

Difluoroboron

β‑ Diketonate

PLA−PEG

Nanoparticle.

Biomacromolecules 2017, 18, 551-561. (21) Shi, H.; Ma, X.; Zhao, Q.; Liu, B.; Qu, Q.; An, Z.; Zhao, Y.; Huang, W. Ultrasmall Phosphorescent Polymer Dots for Ratiometric Oxygen Sensing and Photodynamic Cancer Therapy. Adv. Funct. Mater. 2014, 24, 4823−4830. (22) Tarpani, L.; Latterini, L. Plasmonic Effects of Gold Colloids on The Fluorescence Behavior of Dye Doped SiO2 Nanoparticles. J. Lumin. 2017, 185, 27



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

192-199. (23) Zhang, X.; Li, Y. B.; Cai, Y. X.; Zhang, Y.; Tian, Y. Q.; Wang, L.; Gu, H. S.; Chen, W. Pluronic(R) F127 Micelles Template Biomimetic Assembly and Optical Properties of Red Fluorescent Silica Nanocapsules. Sci. Adv. Mater. 2017, 9, 608-615. (24) Peng, H. S.; Chiu, D. T. Soft Fluorescent Nanomaterials for Biological and Biomedical Imaging. Chem. Soc. Rev. 2014, 44, 4699–4722. (25) Wu, C.; Bull, B.; Szymanski, C.; Christensen, K.; McNeill, J. Multicolor Conjugated Polymer Dots for Biological Fluorescence Imaging. ACS Nano 2008, 2, 2415–2423. (26) Wang, H.; Ma, K.; Xu, B.; Tian, W.J. Tunable Supramolecular Interactions of Aggregation-Induced Emission Probe and Graphene Oxide with Biomolecules: An Approach toward Ultrasensitive Label-Free and “Turn-On” DNA Sensing. Small 2016, 12, 6613–6622. (27) Liu, C. J.; Zhang, P.; Tian, F.; Li, W. C.; Li, F.; Liu, W. G. One-Step Synthesis of Surface Passivated Carbon Nanodots by Microwave assisted Pyrolysis for Enhanced Multicolor Photoluminescence and Bioimaging. J. Mater. Chem. 2011, 21, 13163–13167. (28) Zhu, H.; Wang, X. L.; Li, Y. L.; Wang, Z. J.; Yang, F.; Yang, X. R. Microwave Synthesis of Fluorescent Carbon Nanoparticles with Electrochemiluminescence Properties. Chem. Commun. 2009, 1, 5118–5120. (29) Ma, Z.; Ming, H.; Huang, H.; Liu, Y.; Kang, Z. H. One-Step Ultrasonic Synthesis of Fluorescent N-Doped Carbon Dots from Glucose and Their Visible-Light 28


Page 28 of 32

Page 29 of 32

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


Sensitive Photocatalytic Ability. New J. Chem. 2012, 36, 861–864. (30) Hu, Q. S.; Zhu, C. J.; Xia, Y. Y.; Wang, L. L.; Liu, W. H.; Pan, Z. F. Hydrothermal Synthesis and Luminescent Properties of BaSrMg(PO4)2: Eu3+. Spectrosc. Spect. Anal. 2016, 36, 340–344. (31) Dong, G. S.; Liu, H. B.; Luo, L.; Wang, Y. H. Study on Synthesis and Luminescence Mechanism of Novel Green Sr3Y(PO4)3:Ce3+, Tb3+ Phosphors. Spectrosc. Spect. Anal. 2015, 35, 2189–2193. (32) Dima, S.; Dima, C.; Iordachescu, G. Encapsulation of Functional Lipophilic Food and Drug Biocomponents. Food Eng. Rev. 2015, 7, 417–438. (33) Pankaj-Kumar, S. G. Drug Delivery Using Nanocarriers: Indian Perspective. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 2012, 82, 167. (34) Wu, Z.; Alany, R. G.; Tawfeek, N.; Falconer, J.; Zhang, W.; Hassan, I. M.; Rutland, M.; Svirskis, D. A Study of Microemulsions as Prolonged-Release Injectables through In-Situ Phase Transition. J. Control. Release 2014, 174, 188–194. (35) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, Plenum Press: New York, 2006. (36) Chen, G. Z. Fluorescence Analysis, 2nd ed; Science Press: Beijing, 1990. (37) Clarke, R. J.; Oprysa A. Fluorescence and Light Scattering. J. Chem. Educ. 2004, 81, 705-707. (38) Li, H. L.; Zhang, J. L.; Tian, R. T. Raman Scattering of Solvent in Fluorescence Analysis. Chinese Journal of Health LaboratoryTechnology 2009, 19, 478 – 481. (39) Ma, C. Q.; Chen, G. Q.; Wei, B. L.; Shi, Y. P.; Gu, L.; Gao, S. M.; Zhu, T. 29



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

Study on The Mutation of Fluorescence Excitation Spectra of Red Algae. Spectrosc. Spect. Anal. 2011, 31, l065–1068. (40) Han, B.; Wang, W.; Wu,H.; Fang, F.; Wang, N.; Zhang, X.; Xu, S. Polyethyleneimine Modified Fluorescent Carbon Dots and Their Application in Cell Labeling. Colloid. Surface. B. 2012, 100, 209-214. (41) Tian, R.; Hu, S.; Wu, L.; Chang, Q.; Yang, J.; Liu, J. Tailoring Surface Groups of Carbon Quantum Dots to Improve Photoluminescence Behaviors. Appl. Surf. Sci. 2014, 301, 156–160. (42) Zhu, S.; Zhang, J.; Tang, S.; Qiao, C.; Wang, L.; Wang, H.; Liu, X.; Li, B.; Li, Y.; Yu, W. Surface Chemistry Routes to Modulate The Photoluminescence of Graphene Quantum Dots: From Fluorescence Mechanism to Up–Conversion Bioimaging Applications. Adv. Funct. Mater. 2012, 22, 4732–4740. (43) Wang, S.; Zhu, Z.; Chang, Y.; Wang, H.; Yuan, N.; Li, G.; Yu, D.; Jiang, Y. Ammonium Hydroxide Modulated Synthesis of High-Quality Fluorescent Carbon Dots for White LEDs with Excellent Color Rendering Properties. Nanotechnology 2016, 27, 5202-5210. (44) Gao, P.; Tu, J. S. Research Progress of Polyoxyethylene Castor Oil and Its Safety. Pharm. Clin. Res. 2010, 18, 59–63. (45) Qin, A.; Jim, C. K.; Tang, Y.; Lam, J. W. Y.; Liu, J.; Mahtab, F.; Gao, P.; Tang, B. Z. Aggregation-Enhanced Emissions of Intramolecular Excimers in Disubstituted Polyacetylenes. J. Phys. Chem. B 2008, 112, 9281–9288. (46) Xu, B.; Chi, Z.; Zhang, J.; Zhang, X.; Li, H.; Li, X.; Liu, S.; Zhang, Y.; Xu, J. 30


Page 30 of 32

Page 31 of 32

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


Piezofluorochromic and Aggregation-Induced-Emission Compounds Containing Triphenylethylene and Tetraphenylethylene Moieties. Chem. Asian J. 2011, 6, 1470 –1478. (47) Kundu, N.; Banerjee, P.; Kundu, S.; Dutta, R.; Sarkar, N. Sodium Chloride Triggered the Fusion of Vesicle Composed of Fatty Acid Modified Protic Ionic Liquid: A New Insight into the Membrane Fusion Monitored through Fluorescence Lifetime Imaging Microscopy. J. Phys. Chem. B 2017, 121, 24−34.

31



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

TOC

32


Page 32 of 32

Novel Fluorescent Microemulsion: Probing Properties, Investigating

Recommend Documents