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Gold Nanoprobe-Enabled Three-Dimensional Ozone Imaging by Optical Coherence Tomography Xueqin Jiang, Peijun Tang, Panpan Gao, Yu Shrike Zhang, Changqing Yi, and Jianhua Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04785 • Publication Date (Web): 19 Jan 2017 Downloaded from http://pubs.acs.org on January 22, 2017

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Analytical Chemistry

Gold Nanoprobe-Enabled Three-Dimensional Ozone

1

Imaging by Optical Coherence Tomography

2 3 4 5

Xueqin Jiangaǂ, Peijun Tangaǂ, Panpan Gaoa, Yu Shrike Zhangb, Changqing Yia*, and

6

Jianhua Zhoua*

7 8 9

a

X. Q. Jiang, P. J. Tang, P. P. Gao, Prof. C. Q. Yi, and Prof. J. H. Zhou

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Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong

11

Province, School of Engineering, Sun Yat-sen University, Guangzhou 510275, China

12 13 14

b

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Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139, USA

Dr. Y. S. Zhang

16 17 18 19

*Corresponding author:

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Tel.: +86 20 39387890; Fax: +86 20 39387890.

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E-mail: [email protected] (C. Q. Yi); [email protected] (J. H. Zhou)

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ǂThese authors contribute equally to the work.

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1

ACS Paragon Plus Environment


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Abstract:

2

Ozone (O3) would be harmful to human skin for its strong oxidizing property,

3

especially when stratum corneum or corneal epithelium is wounded. Imaging the

4

penetration and distribution of ozone at depth is beneficial for studying the influence of

5

ozone on skin or eyes. Here, we introduced a facile method for three-dimensional (3D)

6

imaging of the penetration of O3 into the anterior chamber of an isolated crucian carp

7

eye by using optical coherence tomography (OCT) combined with gold triangular

8

nanoprisms (GTNPs) as the contrast agent and molecular probe. We illustrated the

9

specific response of GTNPs to ozone and demonstrated that GTNPs can function as an

10

efficient nanoprobe for sensing O3. The stabilities of GTNPs in different biologic

11

solutions, as well as the signal intensity of GTNPs on OCT imaging system, were

12

investigated. Visualization of 3D penetration and distribution of O3 in the biologic tissue

13

was proved for the first time. The quantitative analysis of O3 diffusion in the anterior

14

chamber of the fish eye revealed a penetration depth of 311 µm within 172 min. Due to

15

the strong scattering, near-infrared extinction band, and easy functionalization of

16

GTNPs, they could further serve as nanoprobes for 3D OCT or multi-modal imaging of

17

other molecules or ions in the future.

18 19 20

Keywords: 3D molecular imaging; gold nanoparticles; ozone; optical coherence

21

tomography.

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Introduction

2

Ozone (O3) has been recognized as a gas with strong oxidizing capability and has

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attracted much attention in applications of sterilization and disinfection. However, the

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growing ground-level O3 is recently becoming a potential threat to public health.

5

Long-exposure to O3 is toxic and harmful to human skin1-6, respiratory tract7 and

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exposed ocular tissue resulting from excessive oxidation. For example, it has been

7

reported that O3 mainly reacts within the stratum corneum which acts as the outer

8

barrier of the skin8-10. Once the skin is wounded and the stratum corneum pierced, O3

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may penetrate into deeper layers of the skin, react with the biomolecules and cells, and

10

induct the onset of inflammatory genes which may contribute to disorders of skin such

11

as skin cancer8. Moreover, it has been reported that high concentrations of atmospheric

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O3 can cause the ocular surface damage and inflammation in vivo11. These reactions are

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mainly occurred in the cornea. If the cornea was impaled or damaged in some

14

conditions12 such as the corneal abrasion caused by the contact lenses, accident injury,

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or the corneal incision during photorefractive keratectomy, O3 may penetrate through

16

the cornea and react with the inner structure such as irises, crystalline lenses and so on.

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Hence, monitoring the process of O3 penetration at depths would be highly beneficial to

18

understand and monitor the influence of O3 on human skin wounds or ocular tissues.

19

Although various techniques for the detection of O3 have been developed13-20, the

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reports on direct visualization of the penetration and distribution of O3 are rare.

21

Transverse two-dimensional (2D) imaging of O3 has been achieved by confocal laser

22

scanning microscope (CLSM) and live-cell microscopy using fluorescent molecules as

23

probe of O321,22. These methods were capable of measuring O3 in living cells but failed

24

to offer real-time three-dimensional (3D) information on the penetration process of O3,

25

due to the limited imaging depth and narrow imaging region. Therefore, more effective

26

methods are needed for in-depth studies of this harmful gas.

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Optical coherence tomography (OCT) is an established medical imaging technique 3



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that uses light to capture 3D images in optically scattering media (e.g., biological tissues)

2

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it offers capability to obtain real-time cross-section images of samples at depths up to

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2-3 mm with a resolution of 5-15 µm24. However, the OCT imaging technique is

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typically incapable of imaging the distribution of specific molecules in tissues due to the

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inherent drawback of its imaging mechanism. For OCT to recognize the distribution of

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specific molecules in a tissue, utilization of a contrast agent that selectively responds to

8

the molecules of interest is essential. The contrast agents used for intensity enhancement

9

and molecular recognition for OCT imaging can be divided into two types (Table S1).

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One is to offer the absorption signal that cannot be directly detected by OCT but

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depends on the signal extraction and algorithm analysis25-31. This method is based on

12

dual light sources with two wavelengths to excite the contrast agent, and obtain the

13

difference value of absorption coefficient by calculating the intensity signals under

14

different excitation wavelengths. The contrast agents for this type of functional OCT

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include conductive polymer (e.g. polypyrrole nanoparticles25), fluorescent agent (e.g.

16

indocyanine green26 , methylene blue27) and photosensitive proteins (e.g. phytochrome

17

and bacteriorhodopsin28). However, this type of contrast agents suffers from complex

18

post-imaging algorithms and relatively low contrast efficacy. The other type is to offer

19

scattering signals that can be directly detected by OCT32-36. Metal nanoparticles

20

constitute a typical example of this type of contrast agents, such as gold

21

nanoparticles37-40. Compared with the first type of OCT contrast agents, gold

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nanoparticles are more photostable and offer a direct and effective contrast enhancement

23

for OCT imaging37-40.

. It is a promising technique for monitoring of dynamic process inside tissues because

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Among gold nanoparticles with different morphologies41-45, gold triangular

25

nanoprisms (GTNPs) stand out because of their excellent structure-dependent optical

26

features. The high refractive index sensitivity and strong scattering locating at the NIR

27

window in biological tissue, make GTNPs as new kind of promising material for wide

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biomedical applications. Moreover, we have previously found that GTNPs have a 4


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specific response to O3, which allows GTNPs to be a potential sensing element for

2

detecting O346. All these factors make GTNPs an excellent molecular nanoprobe for 3D

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imaging the distribution of O3 in biological tissues by OCT.

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In this study, we demonstrated the feasibility of 3D mapping of O3 by OCT using

5

GTNPs. In our demonstration, GTNPs were employed as the OCT contrast agent and O3

6

nanoprobe. When the GTNPs were exposed to O3, their plasmon peaks experienced a

7

significant blue-shift up to ~200 nm, resulting from the morphological change of

8

GNTPs from triangular nanoprisms to circular nanodiscs by O3 oxidation. Compared

9

with the GTNPs, the gold circular nanodiscs could provide a stronger scattering signal

10

and offer an increased contrast enhancement for an 830-nm OCT imaging system.

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Therefore, the enhanced OCT signal of the GTNPs directly correlated with the presence

12

of O3. Based on this principle, we developed a method for 3D imaging of the

13

penetration of O3 in an isolated crucian carp eye by using GTNPs as gold nanoprobe,

14

and demonstrated the capacity to quantify its diffusion process in real-time.

15 16 17

Experimental section Synthesis of GTNPs

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GTNPs with extinction peak located within the NIR region were prepared

19

following a seed-mediated method in aqueous solutions47. All glassware were washed

20

with aqua regia (3:1 ratio by volume of HCl and HNO3; CAUTION: Aqua Regia is

21

highly toxic and corrosive), and rinsed copiously. Deionized water was employed

22

throughout the experiments. Specifically, the seed suspension was made by adding a

23

freshly prepared, ice-cold NaBH4 solution (0.1 M, 1 mL) into a mixture solution made

24

of HAuCl4 (0.01 M, 1 mL) and trisodium citrate (0.01 M, 1 mL) as well as water (36

25

mL). The resultant solution was mixed by rapid inversion for 2 min and then kept at

26

room temperature for 4 h before use for the hydrolysis of the surplus unreacted NaBH4.

27

Then three growth solutions (A, B, and C) were prepared for the seed-mediated growth.

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Solutions A and B were identical, contained cetyltriethylammonium bromide (CTAB, 5



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0.05 M, 9.0 mL), KI (0.1 M, 4.5 µL), NaOH (0.1 M, 0.05 mL), ascorbic acid (0.1 M,

2

0.05 mL) and HAuCl4 (10 mM, 0.25 mL). Solution C was made by mixing CTAB (0.05

3

M, 45 mL), KI (0.1 M, 0.023 mL), NaOH (0.1 M, 0.25 mL), ascorbic acid (0.1 M, 0.25

4

mL), and HAuCl4 (10 mM, 1.25 mL). Note that all these solutions were prepared by

5

adding agents in the sequence listed above. The formation of GTNPs was initiated by

6

adding 1 mL of as-prepared seed solution into solution A, followed by gently shaking.

7

Then 5 mL of growth solution A was quickly added to solution B and shacked slightly.

8

2.5 mL of solution B was added in to solution C. The reaction mixture was subjected to

9

gentle inversion for 15 s and then left undisturbed for at least 1 h.

10

The resulting 45 mL mixture contained expected GTNPs and byproduct gold

11

spherical nanoparticles. The purification was conducted by following a procedure

12

modified from previously reported method47. NaCl solution (4.0 M, 2 mL) was added

13

into the resulting mixture, and the mixed solution was left undisturbed for overnight.

14

Then, the supernatant suspension was gently discarded, while the GTNPs with a color

15

of green remained sticking at the bottom. 20 mL water was added to disperse the

16

sediments. Finally, the purified GTNPs suspension with an extinction peak located at

17

~1010 nm was obtained.

18 19

Stability of GTNPs in solutions containing different biomolecules and gases

20

Solutions

containing

different

biomolecules

were

prepared,

including

21

phosphate-buffered saline solution (0.01 M, 1 mL, pH 7.4, PBS), medium solution (1

22

mL, Dulbecco’s modified Eagle’s medium/high glucose supplemented with 1%

23

antibiotics and 10% new born bovine serum), bovine serum albumin solution (5% w/w,

24

1 mL, BSA), gelatin solution (1% w/w, 1 mL). 1 mL of GTNPs suspension was gently

25

added into each biological solution and was incubated for 30 min at room temperature.

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Another 1 mL of GTNPs suspension was diluted by 1 mL of water and was exposed to

27

each kind of gas, including O3, N2, O2, and CO2. O3 gas was produced by an ozone

28

generator (M Fresh High-Tech Co. Ltd, YL-12984316) and other gases are purchased 6


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from a gas company. The optical extinction spectra of different solutions were recorded

2

by a UV-Vis spectroscopy.

3 4

Preparation of polydimethylsiloxane (PDMS) chips

5

A PDMS chip with microchannels was generated using standard soft lithography.

6

Briefly, the microchannel layer composed of PDMS was mold from an aluminum

7

master fabricated by laser ablation. A flat PDMS mold (as the upper layer) was sealed to

8

the PDMS layer with microchannels using semi-cured PDMS as adhesive, and the

9

whole chip was formed.

10 11

Imaging an isolated crucian carp eye using spectral domain OCT (SD-OCT)

12

system

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SD-OCT (TEK SQRAY, HSO-2000) was used in this experiment. The light source

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was a low-coherence light with a center wavelength at 830 nm and a full width at half

15

maximum (FWHM) of 45 nm. The power of the light source was 5 mW. The scanning

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rate of the B-scan was 18 kHz. The imaging resolution and depth were normally 10 µm

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and 3 mm, respectively.

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To study the effect of O3 exposure on the eyes with corneal damage, we used a

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crucian carp eye as the imaging object. The crucian carp were purchased from a fish

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farm (Guangzhou, China), and were acclimated to laboratory conditions after 14 days

21

feeding commercial feed. The mean diameter of this isolated eye was 214.3 mm. The

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anterior chamber in the fish eye, which is full of aqueous humor, was then loaded with

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0.1 ml GTNPs suspension (~0.0228 mg/mL). Then the cornea was impaled by a scalpel

24

to form a wound. And then this eye was used for the experiment of 3D imaging of

25

penetration process of O3.

26 27 28

Characterization of samples Transmission electron microscopy (TEM, FEI Tecnai G2 Spirit) images were taken 7



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at 120 kV. The samples for TEM studies were prepared by drying a drop of the aqueous

2

suspension of the GTNPs on a piece of carbon-coated copper grid (Zhongjing

3

Technology Corporation). The samples were dried and stored in a vacuum until TEM

4

characterization. The UV-vis extinction spectra were obtained on a UV-vis spectroscopy

5

(Inesa L3S).

6 7

Result and Discussion

8

GTNPs display a specific response to O3, as demonstrated in our previous research46.

9

A schematic diagram in Figure 1A shows the mechanism of this response. As indicated

10

in this scheme, O3 reacts with the gold atoms at sharp corners of the nanoprisms and

11

leads to the shape transformation from prisms to discs. Oxidative etching may play a

12

significant role in this process. Gold atoms at the sharp corners of the GTNPs can be

13

oxidized and dissolved in the environment of O3 as these corners are high-energy sites48.

14

To illustrate the characteristics of this response, we exposed GTNPs to the atmosphere

15

of O3, and monitored their morphologies as well as optical properties by TEM and

16

UV-vis spectroscopy, respectively. Figure 1B-E present the TEM images of GTNPs that

17

had been exposed to the atmostphere containing O3 (~0.08 ppm) for 0, 2.5, 4 and 6 h. It

18

is clearly that significant morphological changes occurred to the GTNPs: the sharp

19

corners of GTNPs were gradually rounded under the exposure of O3, and the GTNPs

20

transformed from triangular nanoprisms to circular nanodiscs at the end. These

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morphological transformations of GTNPs led to obvious changes in their extinction

22

peaks, originating from the localized surface plasmon resonance (LSPR) phenomenon

23

of gold nanostructures. The extinction peaks of these gold nanoplates were at 1011 nm,

24

971 nm, 880 nm, and 810 nm, respectively, as shown in Figure 1F, revealing an obvious

25

blue-shift of their extinction peaks when they were exposed in the environment of O3.

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The maximal shift of wavelength was as significant as ~200 nm. In addition, the

27

extinction intensity of the gold nanodiscs at 830 nm increased by 148%, from 0.768 a.u. 8


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to 1.905 a.u. These optical property changes of the GTNPs caused by reacting with O3

2

make GTNPs a promising nanoprobe for OCT imaging of the molecule.

3

4 5

Figure 1. The response of GTNPs to O3 in the atmosphere (atm.). (A) A scheme

6

showing the shape transformation of GTNPs in the atm. (containing O3 of ~0.08 ppm).

7

The GTNPs eventually became circular nanodiscs when they were exposed to the atm.

8

containing O3. (B-D) TEM images of GTNPs whose corners were gradually rounded,

9

with their extinction peaks located at 1011 nm, 971 nm, 880 nm, and 810 nm,

10

respectively. The scale bars are 100 nm. (F) The UV-vis extinction spectra of the

11

GTNPs suspensions corresponding to (B-E).

12 13

The stability and response specificity of GTNPs in different biologic solutions and

14

gases was investigated. Normalized extinction spectra of GTNPs dispersed in water,

15

medium, 5% BSA solution, and 1% gelatin are shown in Figure 2A. When the GTNPs

16

were dispersed in water and other solutions, the overall shape of the spectrum remained

17

almost the same. However, the extinction peak of each spectrum experienced slight

18

red-shifts of approximately 0.5 to 26.5 nm. The slight red-shifts of the extinction peaks

19

were possibly resulted from the biomolecules adsorbed on the surface of GTNPs, which

20

changed the optical properties of GTNPs and thus the locations their extinction peaks.

21

Figure 2B shows the shifts of the extinction peaks in the biologic solutions compared 9



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with those after exposure to O3. It can be seen that the shifts caused by different

2

biomolecules in those solutions were much less than the shift caused by O3. The reason

3

is that, the change of the surrounding refractive indexes of GTNPs casued by the

4

adsorption of biomolecules has an inferior impact on the plasmonic peak of the GTNPs

5

than the change of the morphology of GTNPs. These results suggest that the GTNPs’

6

spectra are relatively stable when they were dispersed in different biologic solutions.

7

The response of GTNPs to several typical gases in the atmosphere (N2, O2, and CO2)

8

was also investigated in Figure 2B. Only O3 could cause the significant shift of the

9

extinction peak. The shape and the extinction peaks of GTNPs’ spectra had no obvious

10

changes under the influence of other gases in atmosphere, as shown in Figure S1 in

11

more detail.

12

13 14

Figure 2. The stability and response specificity of GTNPs in different biologic solutions

15

and gases. (A) Normalized extinction spectra of GTNPs dispersed in water, medium,

16

5% BSA solution, and 1% gelatin. The extinction peak of each spectrum was relatively

17

stable with a slight red-shift, showing the stability of GTNPs when they were dispersed

18

in different biologic solutions. (B) The shifts of the peak wavelength of the GTNPs in

19

different biologic solutions, O3, and some principal constituents of the atmosphere

20

including N2, O2, and CO2.

21 22

The stability of the scattering signal from GTNPs on an OCT system without the 10


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exposure of O3 was examined. GTNPs suspension with an extinction peak at 830 nm

2

was loaded in a microchannel and was constantly monitored for 90 min under OCT.

3

Figure 3 exhibits the OCT images of the cross-section of the microchannel loaded with

4

GTNPs at different durations. It could be seen that the intensity of scattering signal from

5

the GTNPs remained stable during the entire imaging process. The average intensity of

6

a certain area in each image was measured and shown in Figure S2. The time-depended

7

intensity of the area remains basically steady. This experiment indicates that the

8

scattering property of GTNPs was stable over continuously OCT imaging. The

9

photostability is benefit from the light emission mechanisms of the GTNPs which are

10

based on the LSPR. Compared with most organic fluorescent complexes that suffer

11

from the quenching, GTNPs possess the advantage of maintaining stable scattering

12

signal and may function as a potential contrast agent for optical imaging.

13

14 15

Figure 3. Scattering signal stability of GTNPs under OCT imaging. OCT images of

16

GTNPs in a capillary at the different imaging durations: (A) 0 min, (B) 30 min, (C) 60

17

min, and (D) 90 min. No obvious change of the intensity of GTNPs’ scattering signal 11



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was found over the entire imaging process. The scale bars represent 300 µm.

2 3

We monitored the shift of extinction peaks of GTNPs with different side-legths

4

(106 nm, 93 nm, and 76 nm) in the course of ozone exposure (Figure S3). The GTNPs

5

with the side-legths around 93-106 nm show their terminal extinction peak in the range

6

of 810 nm to 840 nm, which is the central range of the OCT source light (Figure S3D).

7

Therefore, GTNPs with these sizes could have a better enhancing effect for OCT

8

imaging. As a result, we chose the GTNPs with their sizes of 93-106 nm (with their

9

extinction peak around 1000-1050 nm) in the following demonstrations of OCT

10

imaging.

11

The efficiency of contrast enhancement by using GTNPs as contrast agents for

12

OCT imaging was studied. We applied GTNPs with their extinction peak located at

13

1050 nm and their corresponding nanodiscs (which were from the same batch of the

14

GTNPs and were prepared by exposing them to O3) in this experiment. A PDMS chip

15

composed of microchannels was also employed. Water, GTNPs (~0.0342 mg/mL), as

16

well as gold nanodiscs (~0.0335 mg/mL) were successively loaded into the

17

microchannels. Their OCT images were recorded and presented in Figure 4A-C. Due to

18

the lack of the scattering particle in the channel, the microchannel loaded with water

19

presented a low contrast in the OCT image, as shown in Figure 4A. When the

20

microchannel was loaded with GTNPs, the OCT signal slightly increased (Figure 4B).

21

After the microchannel was further loaded with gold nanodiscs, the signal was

22

significantly enhanced (Figure 4C), compared to the signal from the microchannel

23

loaded with GTNPs. The intensity of channel loaded with gold nanodiscs is 175%

24

higher than that of water, and is 93% higher than that of GTNPs. To explain such

25

phenomenon, we compared the OCT’s source spectrum and the UV-vis extinction

26

spectra of GTNPs and gold nanodiscs in Figure 4A. Before the exposure to O3, the

27

extinction peak of GTNPs fell at 1050 nm, which is far away from the center of the

28

OCT’s source spectrum; while after the exposure to O3, the extinction peak of the gold 12


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nanodiscs shifted to 829 nm, which well matched the central wavelength of OCT source.

2

The extinction coefficient of the gold nanodiscs at 830 nm (the OCT’s source central

3

wavelength) rose to 1.63 a.u., about four times higher than that of GTNPs. Therefore

4

both GTNPs and gold nanodiscs provided contrast enhancement in OCT imaging, but

5

gold nanodiscs presented a stronger OCT signal. As the extinction intensity of GTNPs at

6

830 nm gradually increased with the presence of O3 (Figure S4), the OCT signal of

7

GTNPs was accordingly enhanced during the O3-exposing process. As a result, GTNPs

8

could be used as nanoprobes for imaging the penetration of O3 by monitoring the signal

9

intensity of GTNPs on an OCT system.

10

11 12

Figure 4. Transverse OCT images of the microchannels loaded with GTNPs, gold

13

nanodiscs, and water. The “Y”-shaped microchannels were loaded with (A) water, (B)

14

GTNPs suspension with the extinction peak locating at 1050 nm, and (C) gold

15

nanodiscs suspension with the extinction peak at 829 nm. (D) UV-vis extinction spectra

16

of GTNPs, gold nanodiscs, and the spectrum of the 830 nm excitation laser of OCT

17

system. The scattering signals were significantly enhanced when the gold nanodiscs

18

were used as contrast agents. Scale bars represent 100 µm. 13



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1 2

To demonstrate the feasibility of using GTNPs as nanoprobes for molecular 3D

3

imaging by OCT, we firstly applied this technique to image the penetration and

4

distribution of O3 into an artificial human skin phantom. The phantom was the

5

homogeneous mixture of the GTNPs and gelatin, and the phantom was covered by a

6

thin polythene film to mimic the human skin. To simulate the wound on the skin, a hole

7

with a radius of 500 µm was produced in the film, as illustrated in Figure S7A. The

8

phantom was placed in atmosphere of ~75 ppm ozone for up to 1 h, and the OCT was

9

used to visualize the penetration of O3 into the phantom. B-scan OCT images of this

10

phantom during O3 exposure were obtained (as shown in Figure S5). It can be seen that

11

the shape transformation of the GTNPs in gelatin started immediately, and achieved an

12

increasement of 1.95% of the OCT signal within 24 s (as shown in Figure S6). Figure

13

S7B-D shows the reconstructed 3D OCT images of the phantom, which were taken at

14

13 min, 20 min, and 46 min in the atmosphere of O3. It can be seen that the region

15

around the hole gradually brighten, and the lightened region expanded spherically as the

16

exposure time increased. Further data analysis is presented in Figure S8. By employing

17

the GTNPs as O3 nanoprobe, we were able to image the penetration and distribution of

18

O3 in the tissue phantom at depths using OCT.

19

We also used an isolated eye of crucian carp (a real biologic tissue) as the imaging

20

object to show the capability of GTNPs for the 3D imaging of the penetration and

21

distribution of O3 at depths into the tissue. The cross-sectional OCT images of the

22

crucian carp eye are shown in Figure 5. The cornea, anterior chamber and the crystalline

23

lens of the eye can be seen clearly in these OCT images. We can see that there are low

24

backscattering signals in the anterior chamber, as shown in Figure 5B. Then a certain

25

amount of GTNPs (~0.0228 mg/mL) was injected into the anterior chamber of the eye;

26

as shown in Figure 5C, the GTNPs were distributed homogeneously in the anterior

27

chamber, and enhanced the OCT scattering signals.

28 14


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1 2

Figure 5. Cross-sectional OCT images of a crucian carp eye before and after the

3

injection of GTNPs. (A) A schematic diagram showing the structure of a fish eye. (B)

4

Cross-sectional OCT image of a crucian carp eye before the injection of GTNPs. (C)

5

Cross-sectional OCT image of a crucian carp eye after the injection of GTNPs into the

6

anterior chamber. The scale bars represent 300 µm.

7 8

To study the effect of the O3 exposure on the eyes with corneal damage, the cornea

9

of an isolated crucian carp eye was impaled. After the loading of GTNPs (~0.0228

10

mg/mL), the crucian carp eye was placed in the atmospheric condition for 60 min.

11

During this process, there is no trend towards increase or decrease in OCT signal. Hence,

12

we consider that the scattering background of the eye, as well as the distribution of the

13

GTNPs in the anterior chamber, were both stable over time. Then the eye was placed in

14

an atmosphere containing ~75 ppm O3 for 172 min. The penetration process of O3 was

15

visualized by the OCT system. Figure 6A-D show the cross-sectional OCT images of

16

this crucian carp eye at 0 min, 76 min, 110 min, and 172 min in the presence of O3. It

17

can be seen that the region around the wound gradually lightened up, and the lightened

18

region expanded spherically as the exposure time increased. This is caused by the

19

morphology change of the GTNPs in the presence of O3. Before being oxidized by O3,

20

the GTNPs in the anterior chamber were in the shape of nanoprisms and therefore

21

showed low OCT signal under the 830-nm light source. After the O3 exposure, the O3

22

gas penetrated into the anterior chamber through the wound and reacted with the

23

GTNPs in the anterior chamber, leading to the transformation of GTNPs from prisms to 15



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1

discs; and as the result, the extinction coefficient of gold nanoparticles became higher at

2

the wavelength of 830 nm, leading to the obvious enhancement of the imaging contrast.

3

The extinction would increase along with the formation of the nanodiscs, and the

4

scattering signals in the anterior chamber become stronger. Also, the crystalline lens

5

which was beneath the gold nanoparticle suspenstion can be seen clearly in Figure 6D,

6

which is indicated that the nanodiscs exhibiting strong scattering signals did not block

7

the signals from deeper part of the anterior chamber. Therefore, by employing GTNPs

8

as O3 nanoprobe, we were able to image the penetration and distribution of O3 in an eye

9

at depths using OCT.

10 11

Figure 6. OCT images of an isolated crucian carp eye after the injection of GTNPs in

12

the course of O3 (~ 75 ppm) exposure at different durations: (A) 0 min, (B) 76 min, (C)

13

110 min, and (D) 172 min. The cornea of the eye was impaled (shown as the wound

14

marked in A). The scale bars represent 300 µm.

15 16

The penetration rate and depth of O3 into the anterior chamber of the crucian carp

17

eye was further analyzed quantitatively. Firstly, the morphology changes of GTNPs

18

could be obtained by measuring the changes of signal intensity of the region near the 16


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1

wound. When GTNPs start to transform from GTNPs to gold nanodiscs, the blue-shift

2

of GTNPs’ extinction peak occurs, and their extinction coefficient at 830 nm increases,

3

resulting in the lifting in the intensity of OCT signal. Therefore, the penetration process

4

of O3 in the anterior chamber monitored by OCT is a comprehensive process of the

5

diffusion of O3 into the anterior chamber and the reaction of O3 with GTNPs. We

6

measured the average intensity of the same location near the wound (i.e. location a) in

7

the images at 0 min, 76 min, 110 min and 172 min respectively, as presented in Figure

8

7A. In the first 34 min, the intensity raised quickly up to 49.58%. In the last 58 min, no

9

obvious change of the intensity at the location a was shown. The penetration depth of O3

10

into the anterior chamber of the crucian carp eye was also investigated. We defined the

11

location where the scattering intensity was increased by about 2% as the frontier that O3

12

reached, and took the distance between the frontier and the surface of cornea as the

13

penetration depth of O3. We recorded the depth of this region at different time. The

14

curve of the depth as a function of time is shown in Figure 7B. In this plot, we can see

15

that the depth of O3 penetration was 311 µm within 172 min.

16

17 18

Figure 7. The response kinetics of GTNPs and the penetration depth of O3 in the

19

anterior chamber of an fish eye with corneal damage. (A) The scattering signal of the

20

GTNPs as a function of time at location a. (B) The depths of the O3 penetration as a

21

function of time.

22 23

It is known that, the effects of O3 on the ocular tissue or different layers of the skin 17



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Page 18 of 22

1

are still not clear. Also, O3, like other drugs, poisons and radiation, can display either a

2

damaging effect on the skin from a long exposure or a beneficial effect after a brief

3

exposure to O349. The study on the penetration of O3 into the anterior chamber of eye

4

and mimic skin phantom would be helpful to reveal the interaction mechanism between

5

O3 and cutaneous tissues.

6

Conclusions

7 8

In conclusion, we have developed a convenient method for 3D imaging of ozone at

9

high resolution based on an OCT system, by using GTNPs as contrast agent and

10

O3-responsive sensing element. GTNPs were stable in different biologic solutions, and

11

their OCT intensity signals were steady, which made GNTPs suitable for OCT imaging

12

in biological systems. The penetration process of O3 into the crucian carp eye was

13

visualized in-situ by using this technique. It is the first time that GTNPs have been

14

demonstrated for 3D OCT imaging, and the technique developed here would be useful

15

for investigating the effects of O3 on the eye and skin. So this method may be a potential

16

approach to study the relative eye diseases. But there are still some limitations of this

17

method. This method can not determine the exact concentration of O3 in a real tissue.

18

And the detection process of O3 is one-shot and irreversible. Also, the attenuation of the

19

signal may occur in the deeper part of the tissue, and the penetration depth of O3 may be

20

underestimated. So this method could only apply to indicate and estimate the depth of

21

penetration of O3. Moreover, when the backscattering signals of the bio-tissue are

22

strong and unknown (such as skin), it is very difficult to distinguish the signals of gold

23

nanoparticles from the

24

photothermal OCT technique may be able to separate the spectral profile of the

25

nanoparticles from the background tissue scattering, and extract measures of O3. It is

26

worth to mention that, with the advantages of strong scattering, easy modification, and

27

biocompatibility, GTNPs could be further functionalized with other sensing materials

background noise.

A

multi-wavelength

18


approach

or

a

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1

(which respond to the molecules or temperature and change their refractive indexes) for

2

the imaging of different molecules or ions, as well as the distribution of temperature, in

3

biologic tissues. In addition, because the plasmon peaks of GTNPs are located at the

4

NIR window in biological tissue, the imaging depth into the tissue should be greater and

5

the heating effect of GTNPs would be higher, which make the GTNPs to be potential

6

multi-function agents for NIR imaging and theranostics in future.

7 8

Supporting Information

9

The list of contrast agents for OCT imaging; UV-vis spectra of GTNPs when exposing

10

to O3 and other gases; UV-vis spectra of GTNPs with different original extinction peaks

11

after exposed to O3 for different time; UV-vis spectra of GTNPs suspensions which

12

were exposed to O3 at different durations; The stability of the scattering signals emitted

13

from the GTNPs; B-scan OCT images of a phantom containing GTNPs during O3

14

exposure; B-scan OCT images of a phantom during short O3 exposure durations; 3D

15

OCT images of a phantom containing GTNPs during O3 exposure; The response

16

kinetics of GTNPs and the penetration depth of O3 in the GTNPs-gelatin phantom. This

17

material is available free of charge via the Internet at http://pubs.acs.org.

18 19

Acknowledgments

20

We thank Yanhong Ji (South China Normal University) for the help in experiments.

21

This work was supported in part by the National Natural Science Foundation of China

22

(No. 21405183), the special support plan for training high-level talents in Guangdong

23

Province (No. 2014TQ01R695), and the Fundamental Research Funds for the Central

24

Universities (No. 16lgjc62). The work was also supported in part by the Australia-China

25

Joint Institute for Health Technology and Innovation.

26 27 19



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