Carbon Dot-functionalized Interferometric Optical Fiber Sensor for

Jul 16, 2019 - An optical microfiber is a genre of interferometric optical fiber sensor and can ... For a uniform beating region, the phase difference...
1 downloads 0 Views 1MB Size
Subscriber access provided by BUFFALO STATE

Surfaces, Interfaces, and Applications

Carbon dots-functionalized interferometric-based optical fiber sensor for detection of ferric ions in biological samples Stephanie Hui Kit Yap, Kok Ken Chan, Gong Zhang, Swee Chuan Tjin, and Ken-Tye Yong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b08934 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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 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 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.

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 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

ACS Applied Materials & Interfaces

Carbon dots-functionalized Interferometric-based Optical Fiber Sensor for Detection of Ferric Ions in Biological Samples Stephanie Hui Kit Yap ‡a; Kok Ken Chan ‡a; Gong Zhang a; Swee Chuan Tjin a; Ken-Tye Yong *a a

School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore. ‡ S. H. K. Yap and K.K Chan contributed equally to this work. * Corresponding author E-mail: [email protected] Keywords: carbon dots, heavy metal ion, optical microfiber sensor, chelator, nanomaterial Abstract This work reports an interferometric-based optical microfiber sensor functionalized with nitrogen and sulfur co-doped carbon dots (CDs) for the detection of ferric ions (Fe3+). As compared to other carbon dots-based ferric ion sensor, the sensing mechanism of this presented sensor is dependent on the refractive index modulations due to selective Fe3+ adsorption onto the CDs binding sites at the tapered region. This is the first study at which CDs-based sensing was performed at solid phase as a chelator which does not rely on its fluorescent properties. The detection performance of the proposed sensor is not only comparable to conventional fluorescence-based carbon dots nanoprobe sensor but also capable of delivering quantitative analysis results and ease of translation to a sensor device for on-site detection. The presented sensor exhibits Fe3+ detection sensitivity of 0.0061 nm / (g/L) in the linear detection range between 0 to 300 g/L and a detection limit of 0.77 g/L based on Langmuir isotherm model. Lastly, the potential use of the CDs-functionalized optical microfiber sensor in real environmental and biological Fe3+ monitoring applications has also been validated in this work. 1. INTRODUCTION Iron is geologically abundant and biologically essential to every living entity. 1 It appears as a vital micronutrient in human diets and is commonly exist in its most stable form of ferrous (+2) and ferric (+3) oxidation states. Aside from its primary role in the formation of heme, iron plays an important part in the formation of iron-sulfur clusters which serve as electron transfer groups that mediate redox reactions in the biological system. 2 Both deficiency and overaccumulation of iron in human body have been reported to elevate the risk for serious disease conditions such as anemia 3, ischemia and neurodegenerative diseases. 4 For these reasons, iron detection in water, food and biological fluids has been attracting great interest from researchers to seek novel functional materials and systematic iron sensing mechanisms. Carbon dots, also known as carbon nanoparticles, is the latest addition to the carbonbased fluorescent nanomaterial family and have been increasingly popular due to their attractive properties such as facile synthesis, biocompatible, good aqueous stability, and unique optical characteristics. 5 Carbon dots favor a broad range of applications such as sensing,

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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 19

cellular imaging, cancer therapeutic, optoelectronics and others. 6 The potential use of carbon dots for iron detection has been demonstrated in many recent works. These include using citric acid and tris as the precursor to synthesized carbon dots for Fe3+ detection with a limit of detection (LOD) of 1.3 M 7, a facile preparation of carbon dots-based Fe3+ nanoprobe sensor using DL-malic acid as carbon source with a linear detection range of 6 - 200 M and LOD of 0.8 M 8, and a nitrogen-doped carbon dots which exhibit fluorescence quenching response toward Fe3+ over a concentration range of 0 to 1.6 M with a LOD of 0.05 M 9. Despite the attractive sensing performance, these nano-sensors are generally carried in liquid phase. Moreover, these sensors require a bulky and expensive spectrometer to read the changes in fluorescence emission intensity which further hinder carbon dots widespread use for on-site detection. In regard to these, carbon dots-based colorimetric test strip was proposed to overcome these challenges, however, this solution appears to be more pronounced for qualitative measurement. 10 To circumvent this, optical fiber rises as an alternative by offering a wide variety of sensing system designs which includes variants of fiber structures, optical interrogation methods as well as the versatility to incorporate nanomaterial as the transducer layer to achieve selective and sensitive detection of specific target molecules. For instance, optical tapered fiber, or simply microfiber, is known for its superior sensitivity for trace analysis of chemical analyte via the interaction between the evanescent wave and the analyte binding scheme at the receptor-solute interface. 11 On top of that, optical microfiber exhibits great surface chemistry versatility that allows various kinds of biological or chemical receptors to be coated at the outer surface of the tapered region. For example, a tapered fiber platform demonstrated its high degree of flexibility to functionalize different chelating agents on the tapered region to target Pb2+, Cu2+, Zn2+, and Cd2+ individually in aqueous samples. 12 Another study implemented a cascade fiber taper design which incorporated multi-layer film comprising chitosan/carbon nanotubes/polyacrylic acid for the detection of Ni2+ was able to achieve a LOD of 0.3 mM. 13 Apart from the widespread use of optical microfiber for metal ion sensing applications, its viability to be translated into a practical handheld device has also been reported recently, further increasing the popularity of optical microfiber-based sensor. 14 Unlike the conventional use of carbon dots-based nanoprobes which primarily focus on the changes in fluorescence intensity, this work employed optical microfiber as a platform to functionalized nitrogen and sulfur co-doped carbon dots (CDs) onto the sensing region for selective detection of Fe3+ in both aqueous and biological environment. The CDs serve as a chelator to induce optical spectrum shifts upon detection of Fe3+ as a result of the minute refractive index change at the sensing region. This approach offers a simpler and more economical experimental setup where a high-resolution CCD spectrometer is not required. Moreover, CDs-functionalized optical microfiber-based sensor allows facile detection protocol and portability of the sensing system for on-site measurement. The proposed sensor was utilized to sensitively and selectively detect Fe3+ in aqueous and biological samples. 2.

EXPERIMENTAL SECTION 2.1. Reagents and materials. Citric acid (> 99.5 %), thiourea (> 99 %), sulfuric acid, H2SO4, (97-99 %), hydrogen peroxide, H2O2, (15 %), ethanol (95 %), 3

ACS Paragon Plus Environment

2

Page 3 of 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

ACS Applied Materials & Interfaces

aminopropyltriethoxysilane, APTES (99 %), N-(3-Dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride, EDC, N-hydroxysuccinimide, NHS (98 %), phosphate buffered saline, Dulbecco’s Modified Eagle Media reagent were bought from Sigma Aldrich. Iron(III) chloride (98 %) was purchased from Acros Organics. Fetal bovine serum was purchased from Thermo Fisher Scientific and horse serum was purchase from ATCC. All chemicals were used as received without further purification. Deionized (DI) water used was purified by Mili-Q water purification system. 2.2. Preparation of CDs. The nitrogen and sulfur co-doped carbon dots were prepared according to an earlier report. 15 Briefly, 0.5 g of citric acid and 0.5 g of thiourea were dissolved in 25 ml of DI water to form a clear homogeneous solution. The solution was then heated up using a conventional domestic microwave oven at 800 W for 6 minutes. The resultant blackish brown precipitates were then let to cool to room temperature before being added with 30 ml water. The mixture was then subjected to ultrasonication in an ultrasonic water bath for 30 minutes to fully disperse the carbonized precursors. The solution was centrifuged at 15000 rpm for 15 minutes, filtered using 0.22 µm membrane filter followed by dialysis against DI water for 24 hours to remove unreacted precursors before use. 2.3. Fabrication of optical microfiber. Single mode optical fiber (SMF-28) was partially declad at the center part and the fabrication of the optical microfiber was assisted by Vytran machine (GPX-3000). The system was preset with normalized power filament of 44 W and the taper profile was fixed at 6 mm of taper waist length, 3 mm of down taper length, 2 mm of up taper length and 7.9m of taper waist diameter. 2.4. Deposition of carbon dots onto tapered region. Prior to functionalizing the assynthesized CDs onto the tapered region, microfbers were dipped into piranha solution (3:1 v/v ratio of concentrated sulfuric acid, H2SO4 and 15% hydrogen peroxide, H2O2) at room temperature for 10 minutes. Following this, the microfibers were rinsed thoroughly with deionized water and dried completely. This prepares a hydroxylated surface for the subsequent interaction with silane coupling agent, APTES which was prepared with ethanol (0.625% v/v). The silanization step was carried out for one hour at room temperature with continuous stirring at 300 rpm. The silanized microfibers were then rinsed with ethanol to remove weakly bonded molecules and dried completely. In the meantime, 0.225 mg/ml of as-synthesized CDs was prepared in phosphate buffered saline and the conjugation reactions between the active amine groups on the tapered region and the surface moieties of the as-synthesized CDs were performed using EDC (147.6 M)-NHS (147.7 M) coupling route. Finally, the CDsfunctionalized microfibers were removed from the solution, rinsed with deionized water and left to air dry before use. 2.5. Characterization of CDs and CDs-functionalized microfibers. Transmission electron microscopy (TEM) image was captured using JEOL 2010UHR and the size of the CDs was analyzed using ImageJ. CDs- functionalized optical microfiber’s image, surface elemental mapping, and analysis were performed using scanning electron microscope, JEOL JSM 6360 and energy-dispersive X-ray spectroscopy, JEOL JED-2300. Surface functional groups on the surface of as-synthesized CDs and CDs- functionalized optical microfiber were determined using Shimadzu IRPrestige-21 Attenuated Total Reflection Fourier Transform Infrared Spectrophotometer.

ACS Paragon Plus Environment

3

ACS Applied Materials & Interfaces 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 19

2.6. Instrument setup and measurement. A broadband super luminescent light emitting diode (1525 – 1570 nm) was used as the incident light source connected to one end of the microfiber which was secured in place using a pair of magnetic fiber clamp. Another end of the microfiber was connected to an optical spectrum analyzer (OSA) for the real-time visualization of the output transmission spectra. Deionized water was introduced twice to the tapered region to ensure the output spectrum is stable. Next, the sample solution was drop cast onto the tapered region and left undisturbed for 4 minutes, followed by rinsing the microfiber and introduced deionized water to the tapered region again to obtain post-metal binding output spectrum of the microfiber. Finally, the changes in the spectrum shift were recorded accordingly. All measurements were performed at ambient temperature ~24 C, neutral pH environment and all CDs- functionalized optical microfibers used in this work were meant for one-time use only. 3.

RESULTS AND DISCUSSION

Figure 1. A) Schematic of the sensing system, B) Sensing principle and C) molecular structure of the as-synthesized CDs 3.1. Sensing principle and numerical analysis. The Fe3+ sensor was implemented using a CDs-functionalized optical microfiber as the sensing probe. Optical microfiber is a genre of interferometric-based optical fiber and can be fabricated using common single-mode fiber 16, multimode fiber 17 or even special fibers such as photonic crystal fiber 18 and hollow core fiber 19. In this work, standard SMF-28 fiber was used to fabricate the bare microfiber. The overall sensing system (Figure 1A) comprised of a broadband super luminescent light emitting diode (SLED), the optical microfiber sensor which was secured in place on an x-translational stage, followed by an OSA. The sensing and the reference arms are self-contained within the tapered

ACS Paragon Plus Environment

4

Page 5 of 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

ACS Applied Materials & Interfaces

region. Interference of light occurs by virtue of the optical path length difference due to waveguide dispersion despite the fixed physical tapered length. 20 Figure 1B depicts incident light from the SLED coupled into the microfiber propagates as fundamental mode. At the down-taper region, fundamental mode, HE11, was excited to the first dominant higher order mode, HE12. 21 As the light reaches the up-taper region, higher order modes will interfere or couple back into the fundamental mode, resulting in a modal interference spectrum which can be expressed as, I = I1 + I2 + 2 I1I2 cos  (1) where I is the total intensity, I1and I2 are the intensities of HE11 and HE12 modes, respectively, and  is the phase difference between these two modes. For a uniform beating region, the phase difference is given as,

 =  L

(2)

where L is the length of beating region while  is the difference between the propagation constants of the two modes which can be represented as, 22

∆β =

∞2 λ (U∞2 2 - U1 )

4πncladding ρ

2

( V2)

exp -

(3) ∞ ∞ where U∞ m is the asymptotic values of the modes (U1 corresponds to HE11 = 2.405 and U2 corresponds to HE12 = 5.520) 21,  is the radius of the tapered region, and V is the normalized frequency given as,

V =

2πρ 2 (ncladding - n2medium)1/2 λ

(4)

where  is the light wavelength, ncladding and nmedium are the refractive indexes of the fiber cladding and the surrounding medium of the tapered region respectively. Based on the above equations, it can be predicted that the output spectrum of the optical microfiber is similar to a sinusoidal from as a function of wavelength. From Eq. (2) - (4), spectrum shift due to the change of refractive index at the cladding-medium interface can be derived as, 23

( (

∆λ = λ exp

2 V'

-

) - 1)

2 V

(5)

where V’ is the revised normalized frequency is,

ACS Paragon Plus Environment

5

ACS Applied Materials & Interfaces 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

𝑉' =

Page 6 of 19

2πρ (n2cladding - (nmedium + ∆nmedium)2)1/2 λ + ∆λ (6)

As seen in Eq. (5) and (6), ∆λ is found to be a function of nmedium and  which directly related to the detection sensitivity of the optical microfiber sensor. The sensitivity of an optical microfiber is primarily dependent on the interaction between the evanescent wave and the interface surrounding the tapered region. Hence, evanescent wave penetrating further into the surrounding medium will results in the optical microfiber being more responsive to small changes that occur at the surface interface of the tapered region. The penetration depth, Dp of the evanescent wave is delineated as the distance away from the interface at which the magnitude of the electric field at the surface begins to decay to its 1/e value, and is mathematically described as, Dp =

λ 2π(n2cladding sin2θ - n2medium)

1/2

(7)

where 𝜃 is the angle of incidence at the core-cladding interface. Based on the principle above, we simulated the optical microfiber used in this work using the Finite-Difference Time-Domain (FDTD) method. Figure 2A-2D illustrates the simulated intensity mode profiles for HE11 and HE12 modes at the cross-section of the 7.9 m tapered waist region. Figure 2E shows the enlarged R1 and R2 regions which were marked as dashed boxes in Figure 2C and 2D, respectively. It was found that the evanescent field of HE12 mode has a larger amplitude than HE11 mode, indicating HE12 mode has a larger penetration depth than HE11 mode. The penetration depth of HE12 mode is calculated by determining the 1/e value of its amplitude and was found to be approximately 540 nm which suffice for ferric ion sensing. On the other hand, Figure 2F shows the light power of HE11 and HE12 modes at the tapered region with different down taper length, Z. It can be seen that HE12 mode does not exist for untapered fiber (Z = 0 m), but present when Z > 0, where the fiber tapering was initiated. HE12 mode was excited when the fiber radius decreases abruptly, which justify stronger HE12 mode at shorter Z length. As the Z length increases, it can be understood that the fiber radius decreases gradually, hence HE12 mode is generally weaker owing to lesser energy coupling from HE11 mode. Based on the simulated result, strongest energy coupling was found at Z = 1840 µm, where the power intensity of HE11 mode was at its minimum while the power of HE12 mode was at its maximum. Therefore, the ideal down taper length of the optical microfiber was 1840 µm which will result in the strongest evanescent field for the interaction with the surrounding medium. However, due to the limitation of our tapering machine as well as to ensure the ease of handling, the down taper length was fixed at 3000 m. Further, Figure 2G depicts the effective refractive indices for HE11 and HE12 modes across different refractive index value at the cladding-medium interface of the tapered region and the corresponding phase difference between the two modes. As described in Eq. (1) – (5), the phase difference will

ACS Paragon Plus Environment

6

Page 7 of 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

ACS Applied Materials & Interfaces

contribute to an output spectrum shift, as shown in Figure 2H. The simulated result (red line) clearly shows an increase in refractive index from 1.33 to 1.37 at the cladding-medium interface will lead to a spectrum red shift. More importantly, it was found that a refractive index difference of 0.002 resulted in a spectrum shift of 5 nm, indicating the theoretical capability of our optical microfiber to detect minute change of refractive index at the fiber cladding-medium interface. We further validated these simulation results with the fabricated microfiber sensor by using glycerin solutions of different refractive indices. The experimental results (blue dot in Figure 2H) are found to be in good agreement with the simulation results. It has been reported that binding of heavy metal ions onto the chelator-modified optical fiber sensor will cause refractive index change at the cladding (metal-bonded on the chelatormodified surface) - medium interface, which can be detected by the resonance wavelength shift in the output signal. 24-25 Likewise, we foresee that the binding of Fe3+ onto the surface of the CDs-functionalized optical microfiber (Figure 1C) will induce a change in the refractive index at the cladding-medium interface, which in turn modulates the phase difference between the modes propagating along the tapered length and eventually translates to wavelength shifts of the interference spectrum. Therefore, the amount of detectable Fe3+ can be quantified by correlating the output spectrum shift.

ACS Paragon Plus Environment

7

ACS Applied Materials & Interfaces 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 19

Figure 2. A-D) Simulated intensity mode profiles for HE11 and HE12 modes at the cross-section of the tapered waist region, E) Enlarged area of the mode intensity profile at cladding-air interface, F) Simulated light power of HE11 and HE12 modes at the tapered region with different down taper length, Z, G) Simulated effective refractive index for HE11 (red: primary x & yaxes) and HE12 (blue: primary x & y-axes) modes at different refractive index of claddingmedium interface and the phase difference between the two modes (green: primary x-axis & secondary y-axis), and H) Spectrum shift as a function of the refractive index of claddingmedium interface; simulated and experimental results.

ACS Paragon Plus Environment

8

Page 9 of 19

B

A

C

Intensity (a.u.)

CDs CDs-immobilized tapered fiber

(S-H)

4000

D 30000

3500

3000 2500 2000 Wavenumber (cm-1)

Si

Element

20000 15000

5000

O K

C K

Fe K

(C=O)

25000

1500

1000

Weight (%)

Atomic (%)

C

K series

Line Type

31.94

44.85

O

K series

34.59

36.47

Si

K series

28.75

17.27

Fe

K series

4.71

1.42

100.00

100.00

Total

10000

Si K (C=C)

(O-H), (N-H)

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

ACS Applied Materials & Interfaces

O C

Fe

Fe

Fe

Si

0 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

keV

Figure 3. A) TEM image of the as-synthesized CDs, B) FT-IR spectrum, C) element mapping, and D) EDS analysis of the CDs-functionalized optical microfiber. Inset shows the SEM image of the microfiber and the area highlighted with red dash box is the measured area. 3.2. Characterization of CDs and CDs-functionalized optical microfiber. The CDs were prepared using a one-step microwave assisted pyrolysis process and the resultant CDs were found to have an average size of 3.27 ± 0.58 nm, as observed Figure 3A. The surface properties of as-synthesized CDs were analyzed using Fourier Transform Infrared Spectroscopy (FT-IR) and is shown in Figure 3B. A strong and broad large peak centered at 3290 cm-1 is associated to the band of O–H and N–H stretching, while the peaks found around 2360 cm-1 are contributed by the S-H moiety. 15 These peaks correspond to the surface functional groups of the as-synthesized CDs. On the lower wavenumber, absorption peaks associated to C=O and C=C moieties were seen at 1637 cm-1 and 1514 cm-1. The presence of CDs on the surface of the optical microfiber was validated using FT-IR and similar peaks were found on the CDs-functionalized microfiber fiber, confirming successful functionalization of CDs on the surface of the tapered region. In addition to this, Figure 3C also shows the scanning electron microscopy (SEM) image of the surface of a CDs-functionalized microfiber which has been used for Fe3+ detection. Elemental analysis using energy dispersive X-ray spectroscopy (EDS) (Figure 3D) validated the formation of Fe3+ chelates on the tapered region upon binding to the as-synthesized CDs.

ACS Paragon Plus Environment

9

ACS Applied Materials & Interfaces 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 19

Figure 4. A) Time vs. sensor’s response at different concentration of Fe3+ solutions, B) calibration curve for the CDs-functionalized optical microfiber sensor and (inset) linear sensing response to changes in Fe3+ concentration (n=5), C) sensor calibration curve fitted to Langmuir adsorption isotherm model (n=5) and d) CDs-functionalized optical microfiber sensor response to various kinds of heavy metal ions (n=5) 3.3. Quantification of Fe3+ in aqueous medium and the detection limit. Figure 4A presents the responses of the sensor to three different concentrations of Fe3+ solutions. Negligible spectrum shift was observed when deionized water was introduced to the sensor, indicating no reaction has occurred. Meanwhile, when 50 and 200 g/L of Fe3+ solutions were introduced to the sensor, incremental spectrum shifts were observed as a result of the interaction between CDs and Fe3+ ions which changed the refractive index at the surface interface. In both cases, the measured output spectrum shift reached a plateau after 4 minutes. Hence, for the subsequent measurements, the reaction time between the sensor and the sample solution was fixed at 4 minutes. The detection capability of the CDs-functionalized optical microfiber sensor was examined with Fe3+ solutions of concentration ranging from 0 – 3000 g/L prepared using deionized water. As shown in Figure 4B, the measured spectrum shift was found to linearly increase with increasing Fe3+ concentration. The functional groups of the as-synthesized CDs serve as the electron donors and form coordinate bonds with Fe3+ ions, which lead to the formation of Fe3+ chelates on the surface of the tapered region. 26 For this reason, the refractive index at the tapered region-ambient interface was altered, resulting in a shift in the output spectrum. The sensor exhibits a linear detection range from 0 – 300 g/L and sensitivity of 0.0061 nm / (g/L).

ACS Paragon Plus Environment

10

Page 11 of 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

ACS Applied Materials & Interfaces

The relationship between the measured output spectrum shift and the amount of Fe3+ present was further analyzed based on the Langmuir isotherm model to calculate the sensor detection limit. The non-linear expression of the Langmuir isotherm model can be illustrated as follows: 27

(1 +LCLC)

∆ = ∆ max (8)

where  is the output spectrum shift as a result of Fe3+ ions adsorptions onto the CDsfunctionalized optical microfiber, max is the maximum output spectrum shift induced by the formation of a complete monolayer, C is Fe3+ concentration in the aqueous solution, and L is the Langmuir constant. Using the non-linear curve fitting based on Eq. (8), Figure 4C shows a good correlation of the experimental data (R2 =0.9687), and the Langmuir isotherm parameter values are found to be max = 2.4790 nm and K = 0.0052 (g/L)-1. Omitting the effects of inherent thermal and environmental noise originating from the OSA, the LOD of the proposed sensor is primarily restricted by the OSA resolution. 28 To calculate the LOD, the Langmuir isotherm equation can be rewritten as: 29 C LOD =

∆λ

(9)

K (∆λmax - ∆λ)

Using the K and max determined previously and by substituting  with 0.01nm (resolution of the OSA), the calculated CLOD of the sensor was found to be 0.77 g/L. In summary, the developed sensor exhibited a lower LOD than recently reported CDs-based sensors in Table 1 and the World Health Organization requirement (300 g/L) of Fe3+ in drinking water. Table 1. Previous reported CDs-based sensors for Fe3+ sensing Sensing scheme Linear detection range LOD Fluorescence quenching 4.2 nM 0 – 0.7 M

Reference 30

Fluorescence quenching

0 – 100 M

0.96 M

31

Fluorescence quenching

0 – 16 M

242 M

32

Fluorescence quenching

0 – 30 M

21nM

33

Fluorescence quenching

0.3 – 546 M

90 nM

34

Fluorescence quenching

0 – 30 M

15 nM

35

Fluorescence quenching

1 – 78 M

0.54 M

36

Fluorescence quenching

0.05 – 30 M

13.5 nM

37

Refractive index change using CDs-functionalized optical microfiber

0 – 5.37 M (0 – 300 g/L)

13.8 nM (0.77 g/L)

This work

ACS Paragon Plus Environment

11

ACS Applied Materials & Interfaces 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 19

3.4. Effects of pH and temperature. The stability of the CDs-functionalized microfiber sensor was assessed by subjecting it to elevated temperature at 30, 40 and 50 C for 30 minutes. The annealed microfiber sensors were then used to measure aqueous samples containing 100 g/L of Fe3+. As shown in Figure S1A (Supporting Information), negligible temperature influence was observed, indicating the stability of surface coating. In addition, the sensitivity of the CDs-functionalized microfiber sensor under different pH ranging from pH 4 to pH 9 was also investigated. From Figure S1B, effect of pH variation was negligible from pH 4 to pH 7. However, as the pH increases above pH 7, the sensitivity of the sensor reduces significantly. This can be attributed to the weak solubility of Fe3+ due to the formation of ferric hydroxide in alkaline medium38. Therefore, the optimized medium pH for optimal sensing performance of the fabricated CDs-functionalized microfiber sensor is investigated as weakly acidic to neutral, validating its potential applications in biosensing. 3.5. Selectivity test of CDs-functionalized microfiber sensor. In order to evaluate the selectivity of the CDs-functionalized optical microfiber sensor, the sensor was examined with various metal ion solutions including Al3+, Ca2+, Cd2+, Cs2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+, each at a concentration of 100 g/L at room temperature. Figure 4D illustrates the measured concentration for each cation. Negligible interference from other cations and exceptionally high selectivity toward Fe3+ ion was observed. This result is in accordance with literature which described the existence of hydroxyl, amine and carboxylic groups on the surface of the CDs which can specifically coordinate with Fe3+ ions. 15 In addition to this, a mixed-metal solution containing the above mentioned interfering ions were tested, with and without Fe3+ to investigate the anti-jamming capability of the sensor. Notably, the sensor showed high affinity to Fe3+ despite the presence of interfering metal ions. Slight interference was observed with the mixture solution containing no Fe3+, which could be attributed to the non-specific binding of the interfering ions. Table 2. Determination of Fe3+ in environmental samples, biological buffers and biological serums using CDs-functionalized optical microfiber sensor (n=5) Sample Type Tap water Tap water DMEM DMEM PBS PBS Fetal bovine serum Fetal bovine serum

Spiked Fe3+ content (g/L) 100 200 0 100 100 200 0 100

Measured concentration using CDsfunctionalized microfiber sensor (g/L)

ACS Paragon Plus Environment

92.4  6.9 204.3  6.8 96.1  6.4 206.7  13.0 110.6  11.5 207.0  7.9 31.3  2.6 121.8  12.0

12

Page 13 of 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

ACS Applied Materials & Interfaces

Fetal bovine serum Horse serum Horse serum Horse serum

200 0 100 200

221.5  10.1 18.3  2.6 112.5  11.1 209.1  12.5

3.6. Integration of CDs-functionalized optical microfiber with portable interrogation system for the detection of Fe3+ in tap water and biological samples. In our previous work, we have developed an in-house portable interrogation system which aims to substitute the OSA and promote the usability of the optical microfiber-based sensor for on-site use. Figure S2 depicts the actual image of the in-house portable interrogation system and the step-by-step procedures to perform Fe3+ detection using the CDs-functionalized optical microfiber sensor. Inset of Figure S2A portraits a CDs-functionalized optical microfiber sensor packaged in a cartridge terminated with LC-type connectors and mounted on the interrogation system. A round opening below the sensor window was made to introduce liquid samples to the sensor as well as to minimize perturbation to the microfiber during the insertion and removal of liquid samples. The details of the interrogation system has been previously described in details. 14 Briefly, the linear calibration curve equation in Figure 3B was programmed into the interrogation system to correlate the output spectrum shift to the concentration of Fe3+ present. The interrogation system would then show the detected concentration values on the display screen. To perform the analysis of the real samples, the CDs-functionalized optical microfiber sensor was immersed in deionized water twice to ensure the output spectrum was fixed, followed by immersing with the sample and left undisturbed for 4 minutes to allow the reaction to completely take place. Finally, the measured Fe3+ concentration can be read from the display screen. In order to evaluate the practicability of the CDs-functionalized optical microfiber sensor for the environmental and biological applications, tap water, phosphate buffered saline (PBS), Dulbecco’s Modified Eagle Media (DMEM), fetal bovine serum (FBS) and horse serum samples were employed to conduct the test. FBS and horse serum samples were subjected to a 100-fold dilution before analysis. The iron ion content in each control samples were identified using inductive coupled mass spectroscopy (ICPMS) or based on formulation standards, and were listed in Table S1. Further, the samples were spiked with various concentrations of standard Fe3+ solution. As shown in Table 2, the test results obtained using the presented sensor were in accordance with the known spiked Fe3+ content. However, it is worth noting that for DMEM samples, the Fe3+ concentrations detected were two times more than the spiked concentration. This is attributed to the Sigma-Aldrich’s DMEM formulation which contains trace level of Fe3+, ~ 100 g/L. Likewise, 30 and 23 g/L of iron ion content was found in FBS and horse serum samples (100-fold dilution) respectively after tested using inductive coupled mass spectroscopy. This explains the slight increment of measured concentration using the proposed sensor as compare to the spiked Fe3+ concentration. Overall, good analytical agreement was achieved, further confirmed the reliability and practicability of the CDsfunctionalized optical microfiber sensor for Fe3+ detection in environmental and biological samples.

ACS Paragon Plus Environment

13

ACS Applied Materials & Interfaces 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

4.

Page 14 of 19

CONCLUSION Nitrogen and sulfur co-doped CDs were successfully prepared in a one-step

microwave-assisted pyrolysis method and functionalized onto the tapered surface of an optical microfiber as a chelator to enable metal ion detection at solid phase. The end product was used as a label-free Fe3+ sensor probe and it possesses excellent detection sensitivity, achieving a LOD of 0.77 g/L, calculated using Langmuir isotherm model and a linear detection range of 0 – 300 g/L. More significantly, the established sensor exhibited high selectivity to Fe3+ as compared to other types of metal ions. The presented CDs-functionalized optical microfiber sensor and the inhouse portable interrogation system has demonstrated the practicability of the system to be used as a fast screening tool for on-site detection, particularly for environmental monitoring and biological sensing applications. Apart from the fact that this work has introduced a new avenue of CDs-based sensing mechanism, it is envisaged that the presented work can be further extended to adopt other functional CDs or novel nanomaterials as the sensing element owing to the versatility of chemical modification on the surface of the developed optical microfiber. Overall, this work has demonstrated the feasibility of nanomaterial-based sensors to operate at solid phase via the integration with optical microfiber sensor technology. ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website at DOI: XXXX Temperature and pH effects; Procedures to perform Fe3+ detection using in-house portable interrogation system; ICPMS results for control tap water and biological samples. (PDF) AUTHOR INFORMATION Corresponding Author *Email: [email protected] ORCID Ken-Tye Yong: 0000-0001-7936-2941 Stephanie Hui Kit Yap: 0000-0002-2847-9936 Kok Ken Chan: 0000-0002-0592-4427 Author Contributions S.H.K Yap and K.K Chan contributed equally to this work.

ACS Paragon Plus Environment

14

Page 15 of 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

ACS Applied Materials & Interfaces

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the NRF-ANR Joint Call 2017 project (Grant No. M4197007.640) and MOE Tier 2 project (MOE2017-T2-2-002; Grant No. M4020385.110) References 1. Abbaspour, N.; Hurrell, R.; Kelishadi, R., Review on iron and its importance for human health. Journal of research in medical sciences : the official journal of Isfahan University of Medical Sciences 2014, 19 (2), 164-174. 2. Eid, R.; Arab, N. T. T.; Greenwood, M. T., Iron mediated toxicity and programmed cell death: A review and a re-examination of existing paradigms. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2017, 1864 (2), 399-430. 3. Camaschella, C., Iron-Deficiency Anemia. New England Journal of Medicine 2015, 372 (19), 1832-1843. 4. Minhas, G.; Modgil, S.; Anand, A., Role of iron in ischemia-induced neurodegeneration: mechanisms and insights. Metabolic Brain Disease 2014, 29 (3), 583-591. 5. Chien, Y.-H.; Chan, K. K.; Yap, S. H. K.; Yong, K.-T., NIR-responsive nanomaterials and their applications; upconversion nanoparticles and carbon dots: a perspective. Journal of Chemical Technology & Biotechnology 2018, 93 (6), 15191528. 6. Chan, K. K.; Yap, S. H. K.; Yong, K.-T., Biogreen Synthesis of Carbon Dots for Biotechnology and Nanomedicine Applications. Nano-Micro Letters 2018, 10 (4), 72. 7. Zhou, M.; Zhou, Z.; Gong, A.; Zhang, Y.; Li, Q., Synthesis of highly photoluminescent carbon dots via citric acid and Tris for iron(III) ions sensors and bioimaging. Talanta 2015, 143, 107-113. 8. Lu, W.; Gong, X.; Nan, M.; Liu, Y.; Shuang, S.; Dong, C., Comparative study for N and S doped carbon dots: Synthesis, characterization and applications for Fe3+ probe and cellular imaging. Analytica Chimica Acta 2015, 898, 116-127. 9. Wang, R.; Wang, X.; Sun, Y., One-step synthesis of self-doped carbon dots with highly photoluminescence as multifunctional biosensors for detection of iron ions and pH. Sensors and Actuators B: Chemical 2017, 241, 73-79. 10. Choudhary, R.; Patra, S.; Madhuri, R.; Sharma, P. K., Equipment-Free, Single-Step, Rapid, “On-Site” Kit for Visual Detection of Lead Ions in Soil, Water, Bacteria, Live Cells, and Solid Fruits Using Fluorescent Cube-Shaped Nitrogen-Doped Carbon Dots. ACS Sustainable Chemistry & Engineering 2016, 4 (10), 5606-5617. 11. Ahmad, M.; Hench, L. L., Effect of taper geometries and launch angle on evanescent wave penetration depth in optical fibers. Biosensors and Bioelectronics 2005, 20 (7), 1312-1319.

ACS Paragon Plus Environment

15

ACS Applied Materials & Interfaces 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 19

12. Ji, W. B.; Yap, S. H. K.; Panwar, N.; Zhang, L. L.; Lin, B.; Yong, K. T.; Tjin, S. C.; Ng, W. J.; Majid, M. B. A., Detection of low-concentration heavy metal ions using optical microfiber sensor. Sensors and Actuators B: Chemical 2016, 237, 142-149. 13. Lin, Y.; Dong, X.; Yang, J.; Maa, H.; Zu, P.; So, P. L.; Chan, C. C. In Detection of Ni2+with optical fiber Mach-Zehnder interferometer coated with chitosan/MWCNT/PAA, 2017 16th International Conference on Optical Communications and Networks (ICOCN), 7-10 Aug. 2017; 2017; pp 1-3. 14. Yap, S. H. K.; Chien, Y.-H.; Tan, R.; bin Shaik Alauddin, A. R.; Ji, W. B.; Tjin, S. C.; Yong, K.-T., An Advanced Hand-Held Microfiber-Based Sensor for Ultrasensitive Lead Ion Detection. ACS Sensors 2018, 3 (12), 2506-2512. 15. Chan, K. K.; Yang, C.; Chien, Y.-H.; Panwar, N.; Yong, K.-T., A facile synthesis of label-free carbon dots with unique selectivity-tunable characteristics for ferric ion detection and cellular imaging applications. New Journal of Chemistry 2019, 43 (12), 4734-4744. 16. Yadav, T. K.; Narayanaswamy, R.; Abu Bakar, M. H.; Kamil, Y. M.; Mahdi, M. A., Single mode tapered fiber-optic interferometer based refractive index sensor and its application to protein sensing. Optics Express 2014, 22 (19), 22802-22807. 17. Qiu, H.; Gao, S.; Chen, P.; Li, Z.; Liu, X.; Zhang, C.; Xu, Y.; Jiang, S.; Yang, C.; Huo, Y.; Yue, W., Evanescent wave absorption sensor based on tapered multimode fiber coated with monolayer graphene film. Optics Communications 2016, 366, 275-281. 18. Feng, X.; Feng, W.; Tao, C.; Deng, D.; Qin, X.; Chen, R., Hydrogen sulfide gas sensor based on graphene-coated tapered photonic crystal fiber interferometer. Sensors and Actuators B: Chemical 2017, 247, 540-545. 19. Wang, B.; Wang, Q.; Du, C., Sensitivity-enhanced single-mode fiber-tapered hollow core fiber-single-mode fiber Mach–Zehnder interferometer for refractive index measurements. Instrumentation Science & Technology 2018, 46 (1), 28-42. 20. Harun, S. W.; Lim, K. S.; Tio, C. K.; Dimyati, K.; Ahmad, H., Theoretical analysis and fabrication of tapered fiber. Optik 2013, 124 (6), 538-543. 21. Lacroix, S.; Gonthier, F.; Black, R. J.; Bures, J., Tapered-fiber interferometric wavelength response: the achromatic fringe. Optics Letters 1988, 13 (5), 395-397. 22. Cassidy, D. T.; Johnson, D. C.; Hill, K. O., Wavelength-dependent transmission of monomode optical fibertapers. Appl. Opt. 1985, 24 (7), 945-950. 23. Ji, W. B.; Liu, H. H.; Tjin, S. C.; Chow, K. K.; Lim, A., Ultrahigh Sensitivity Refractive Index Sensor Based on Optical Microfiber. IEEE Photonics Technology Letters 2012, 24 (20), 1872-1874. 24. Cennamo, N.; Alberti, G.; Pesavento, M.; #039; Agostino, G.; Quattrini, F.; Biesuz, R.; Zeni, L., A Simple Small Size and Low Cost Sensor Based on Surface Plasmon Resonance for Selective Detection of Fe(III). Sensors 2014, 14 (3), 4657-4671. 25. Raghunandhan, R.; Chen, L. H.; Long, H. Y.; Leam, L. L.; So, P. L.; Ning, X.; Chan, C. C., Chitosan/PAA based fiber-optic interferometric sensor for heavy metal ions detection. Sensors and Actuators B: Chemical 2016, 233, 31-38. 26. Shi, B.; Su, Y.; Zhang, L.; Huang, M.; Liu, R.; Zhao, S., Nitrogen and Phosphorus Co-Doped Carbon Nanodots as a Novel Fluorescent Probe for Highly Sensitive

ACS Paragon Plus Environment

16

Page 17 of 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

ACS Applied Materials & Interfaces

27.

28. 29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

Detection of Fe3+ in Human Serum and Living Cells. ACS Applied Materials & Interfaces 2016, 8 (17), 10717-10725. Maguis, S.; Laffont, G.; Ferdinand, P.; Carbonnier, B.; Kham, K.; Mekhalif, T.; Millot, M.-C., Biofunctionalized tilted Fiber Bragg Gratings for label-free immunosensing. Optics Express 2008, 16 (23), 19049-19062. White, I. M.; Fan, X., On the performance quantification of resonant refractive index sensors. Optics Express 2008, 16 (2), 1020-1028. Ravikumar, R.; Chen, L. H.; Jayaraman, P.; Poh, C. L.; Chan, C. C., Chitosan-nickel film based interferometric optical fiber sensor for label-free detection of histidine tagged proteins. Biosensors and Bioelectronics 2018, 99, 578-585. Li, S.; Li, Y.; Cao, J.; Zhu, J.; Fan, L.; Li, X., Sulfur-Doped Graphene Quantum Dots as a Novel Fluorescent Probe for Highly Selective and Sensitive Detection of Fe3+. Analytical Chemistry 2014, 86 (20), 10201-10207. Edison, T. N. J. I.; Atchudan, R.; Shim, J.-J.; Kalimuthu, S.; Ahn, B.-C.; Lee, Y. R., Turn-off fluorescence sensor for the detection of ferric ion in water using green synthesized N-doped carbon dots and its bio-imaging. Journal of Photochemistry and Photobiology B: Biology 2016, 158, 235-242. Wang, F.; Hao, Q.; Zhang, Y.; Xu, Y.; Lei, W., Fluorescence quenchometric method for determination of ferric ion using boron-doped carbon dots. Microchimica Acta 2016, 183 (1), 273-279. Sun, X.; He, J.; Yang, S.; Zheng, M.; Wang, Y.; Ma, S.; Zheng, H., Green synthesis of carbon dots originated from Lycii Fructus for effective fluorescent sensing of ferric ion and multicolor cell imaging. Journal of Photochemistry and Photobiology B: Biology 2017, 175, 219-225. Liu, Y.; Duan, W.; Song, W.; Liu, J.; Ren, C.; Wu, J.; Liu, D.; Chen, H., Red Emission B, N, S-co-Doped Carbon Dots for Colorimetric and Fluorescent Dual Mode Detection of Fe3+ Ions in Complex Biological Fluids and Living Cells. ACS Applied Materials & Interfaces 2017, 9 (14), 12663-12672. Sun, Y.; Wang, X.; Wang, C.; Tong, D.; Wu, Q.; Jiang, K.; Jiang, Y.; Wang, C.; Yang, M., Red emitting and highly stable carbon dots with dual response to pH values and ferric ions. Microchimica Acta 2018, 185 (1), 83. Zhang, Y.; Gao, Z.; Yang, X.; Chang, J.; Liu, Z.; Jiang, K., Fish-scale-derived carbon dots as efficient fluorescent nanoprobes for detection of ferric ions. RSC Advances 2019, 9 (2), 940-949. Zhou, X.; Zhao, G.; Tan, X.; Qian, X.; Zhang, T.; Gui, J.; Yang, L.; Xie, X., Nitrogendoped carbon dots with high quantum yield for colorimetric and fluorometric detection of ferric ions and in a fluorescent ink. Microchimica Acta 2019, 186 (2), 67. Luo, X.; Zhang, W.; Han, Y.; Chen, X.; Zhu, L.; Tang, W.; Wang, J.; Yue, T.; Li, Z., N,S co-doped carbon dots based fluorescent “on-off-on” sensor for determination of ascorbic acid in common fruits. Food Chemistry 2018, 258, 214-221.

ACS Paragon Plus Environment

17

ACS Applied Materials & Interfaces 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 19

Table of Content

ACS Paragon Plus Environment

18

Page 19 of 19

ACS Applied Materials & Interfaces

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 Paragon Plus Environment

19