- Email: [email protected]
A low-cost, monometallic, surface-enhanced Raman scattering-functionalized paper platform for spot-on bioassays
Accepted Manuscript Title: A low-cost, monometallic, surface-enhanced Raman scattering-functionalized paper platform for spot-on bioassays Author: Wan-Sun Kim Jae-Ho Shin Hun-Kuk Park Samjin Choi PII: DOI: Reference:
S0925-4005(15)30208-2 http://dx.doi.org/doi:10.1016/j.snb.2015.08.030 SNB 18885
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-6-2015 20-7-2015 8-8-2015
Please cite this article as: W.-S. Kim, J.-H. Shin, H.-K. Park, S. Choi, A low-cost, monometallic, surface-enhanced Raman scattering-functionalized paper platform for spot-on bioassays, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.08.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A low-cost, monometallic, surface-enhanced Raman scattering-functionalized paper platform for spot-on bioassays
Wan-Sun Kima, Jae-Ho Shinb, Hun-Kuk Parka,c, Samjin Choia,c*
ip t
a. Department of Medical Engineering, Kyung Hee University, Seoul 130-701, Korea b. Department of Ophthalmology, College of Medicine, Kyung Hee University, Seoul 130-701, Korea
cr
c. Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul 130-701, Korea
us
*Address for correspondence: Samjin Choi, Ph.D.,
an
Department of Biomedical Engineering, College of Medicine,
M
Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu,
Tel: +82 2 961 0290
Ac ce pt e
Fax: +82 2 6008 5535
d
Seoul 130-701, Republic of Korea
E-mail: [email protected]
English Language Editing
This manuscript was checked by the native English speakers of the E-World Editing service (USA) of Kyung Hee University Medical Center (Code No. HEW1505-03).
-1Page 1 of 17
Highlights
A simple and facile SERS-functionalized paper platform was designed for POC diagnostics of biofluids. The optical property of surface plasmon resonance was implemented using monometallic gold NPs.
ip t
The viscous property of a sodium CMC solution was used to improve the uniformity and reproducibility of SERS. The low-cost SERS paper sensor showed an effective signature for classifying adenoviral and herpes simplex
M
an
us
cr
conjunctivitis.
Abstract
d
We demonstrate the fabrication and spot-on bioassay application of a low-cost, monometallic, surface-enhanced Raman
Ac ce pt e
scattering (SERS) paper platform using gold nanoparticle (AuNP)-loaded screen printing inks. The AuNPs were used to enhance the Raman intensity through a surface plasmon resonance (SPR)-driven optical property and sodium carboxymethylcellulose (CMC) was used as a viscous ink to create uniform distribution of the AuNPs on the paper substrate. To minimize the coffee ring effect and uniformly disperse the nanoparticles, the size of citrate-capped AuNPs, the CMC concentration, the volume ratio of CMC solution and AuNPs, and the printing cycles were optimized. Two printing cycles of optimized ink with a 7:1 mixture of 2-wt% CMC and AuNPs produced the strongest SERS effect and an enhancement factor of 1.8×104 with a SERS paper platform based on a Rhodamine B probe molecule. The platform exhibited high reproducibility, with less than 5% spot-to-spot variation in Raman intensity. Furthermore, the SERS spectra of normal and two virus-infected tear biofluids were comparable to the SERS spectral findings of golddeposited SERS substrates. The enhanced SERS activity and high reproducibility of a low-cost, flexible, portable, and lightweight paper platform suggest the potential of point-of-care applications of biofluids in fields ranging from clinical analysis to industry.
Keywords: SERS; paper substrate; AuNPs; CMC solution; screen printing; tear biofluids -2Page 2 of 17
1. Introduction Raman spectroscopy is a technique to provide molecule-specific information on biological and chemical samples [1–3]. Since a Raman signal is inherently very weak, various studies have been performed with the aim of signal enhancement. Surface-enhanced Raman scattering (SERS) activity can dramatically improve the intensity of the Raman spectrum due to the absorption energy on the surface [4,5]. The enhancement factor (EF), which is used as a measure of the
ip t
magnitude of SERS, is commonly in the range of 104–108 and can be as high as 1014, allowing for detection at the single-molecule level [6]. Most studies aiming to increase the SERS EF have concentrated on substrate-related issues by
cr
modifying the materials and nanostructure patterns of the surface [7–11]. Most SERS-active areas are fabricated using complex and sophisticated methods including lithography or a high-temperature process. These SERS fabrications
us
undergo expensive, time-consuming, and complicated steps, while the use of metal nanoparticles as a SERS substrate provides easy synthesis at low-cost, with controllable size and shape via the reaction conditions, and very strong
an
enhancement of aggregated nanoparticles with up to single-molecule sensitivity [12]. These nanoparticles absorb the wavelength used in a Raman laser source, known as surface plasmon resonances (SPRs) [1,13]. In particular, Ag, Au,
M
and Cu nanoparticles have been shown to demonstrate a 103-fold higher SERS enhancement compared to other metal substrates [14]. Silver nanoparticles (AgNPs) exhibit superior SERS enhancement compared to gold nanoparticles
d
(AuNPs). However, AgNPs show rapid degradation of SERS activity due to oxidation in air, while AuNPs show stable
Ac ce pt e
SERS activity due to formation of an oxide layer [15].
Paper substrate, which is low-cost, portable, flexible, easy to handle, and harmless, has been highlighted as a novel platform for analytical detection in biomedical and environmental applications [16–18]. These user-friendly advantages of paper offer various applications including colorimetric, electrochemical, and biochemical analyses [19– 21]. These advantages of a paper substrate are well-suited for point-of-care (POC) applications, however it still suffers from limited detection of analytical materials such as enzymes and redox dyes as well as involvement of complex soluble compounds. Since the Raman spectrum of the sample clearly shows the chemical nature and changes of the internal materials, the nanoparticle-driven SERS paper platform is likely to overcome this limitation [22–24]. Some studies have reported the use of colloidal AuNP-deposited SERS paper platforms for environmental and biochemical monitoring [25–27]. The high surface roughness of paper composed of hydrophilic cellulose fibers helps to maintain uniform distribution of AuNPs compared to smooth surface materials such as glass and silicon wafers. Additionally, paper can be easily modified with coating materials to demonstrate various degrees of hydrophobicity. The AuNPs in the low-viscosity colloid aggregate at the edge of a droplet form a coffee ring on the paper substrate that prevents the SERS-active area from maintaining a homogeneous SERS effect. A screen printing technique using nanoparticles within -3Page 3 of 17
a viscous ink has shown improved reproducibility of the SERS analysis compared to other printing techniques [15,28]. Since there is a limited amount of tear biofluid that can be reasonably collected from one patient, few analytical studies have been performed for human tear biofluids, with most analytical methods intended for human urinal and bloody biofluids. Clinical situations often involve long waiting periods to receive analytical results through conventional analytical techniques such as enzyme-linked immunosorbent assay (ELISA), direct immunofluorescence assay (IFA), and polymerase chain reaction (PCR). Although rapid diagnosis is imperative for prescription of
ip t
appropriate treatments to prevent further infections of viral or bacterial infection-driven conjunctivitis and corneal damage, conventional time-consuming procedures are still used in ophthalmological clinics [29].
cr
Therefore, we introduce the fabrication of a monometallic SERS-functionalized paper platform using a mixture of AuNPs and viscous ink based on a screen-printing technique. The SERS effect was optimized by adjusting
us
the amount of synthesized AuNPs, and its reproducibility was verified by adjusting the viscosity of CMC-AuNP screen printing inks. In particular, to expand the biomedical application of POC diagnosis, the performance of the fabricated
an
SERS paper platform was evaluated using in vivo human tear biofluids collected from patients with adenoviral
M
conjunctivitis and herpes simplex conjunctivitis.
2. Material and methods
d
2.1. Reagents and materials
Ac ce pt e
Hydrogen tetrachloroaurate (HAuCl4, >99%), trisodium citrate dehydrate (99%), sodium carboxymethylcellulose (CMC), and Rhodamine B (RhB, >95%) were purchased from Sigma Aldrich (St. Louis, MO, USA). All reagents were of analytical grade, and all solutions were prepared using 18.3 MΩ·cm-1 distilled water. Whatman® cellulose chromatography paper (Grade 1) with a 0.18 mm thickness and a linear flow rate of 72.22 µm/sec was purchased from Sigma Aldrich.
2.2. Instrumentation
The size and shape of the AuNPs were characterized by a JEM-2100F field emission transmission electron microscope (FE-TEM; JEOL, Tokyo, Japan) operated at an accelerating voltage of 200 kV. The UV-Vis absorption spectra of the colloidal AuNPs were produced by a CARY 300 UV-Vis spectrophotometer (Agilent, Santa Clara, CA, USA) using a 1cm quartz cell. The viscosity of the CMC solution was measured using a DV-II+Pro viscometer (Brookfield, Middleboro, MA, USA) at room temperature (RT). The morphologies of the paper substrates according to the presence of CMC-AuNPs were characterized using a S-4700 field emission scanning electron microscope (FE-SEM; Hitachi, Tokyo, Japan) at an accelerating voltage of 5 -4Page 4 of 17
kV. A 7200-H energy-dispersive X-ray spectroscope (EDX; HORIBA, Northampton, England) was used to examine the elemental compositions of the synthesized products. The Raman spectrum was obtained using a SENTERRA confocal Raman system (Bruker Optics, Billerica, MA, USA). A 785-nm diode laser source with a 10-mW power was focused to a spot size of ~2.4 µm with a 20× objective lens (N.A.=0.4). The spectrum of each point was recorded in the range of 417–1782 cm-1 with a spectral resolution of 5 cm-1 and twice the acquisition time of 30 sec at RT. The Raman spectrum of each sample was measured
ip t
as 10, randomly-printed spots on the SERS-active area to ensure reliability. A 2-µL analytic droplet of the sample was
cr
used to evaluate the Raman spectrum.
2.3. Preparation of CMC-AuNP inks
us
The AuNPs were obtained through citrate capping based on the Turkevich method of chemical reduction [30]. A 1-mL aliquot of 38.8 mM trisodium citrate dihydrate was added to 10 mL of 1 mM HAuCl4 and heated at 90°C for 20 min on
an
a hot plate under magnetic stirring. The color of the solution changed from a pale yellow to a deep violet. The reaction flask was immediately removed from the hot plate, and stirring was maintained until the solution had cooled to RT.
M
To prepare the screen-printing ink solutions, 1 mL of colloidal AuNPs was concentrated by centrifugation at 6000 rpm for 10 min, and 99% of the supernatant was removed. The concentrated AuNPs were dispersed in sodium
Ac ce pt e
optimize the inks.
d
CMC with distilled water. The concentration of the CMC and the ratio of the AuNPs and CMC were adjusted to
2.4. Fabrication of the SERS platform
The SERS-functionalized paper platform consisted of two parts, the SERS-active area and the labeling area. Figure 1A shows that a 2-mm hydrophilic circular reservoir and a labeling area designed by AutoCAD (Autodesk, San Rafael, CA, USA) were printed on an 8×20-mm2 paper using a Xerox ColorQube 8570N printer (Fuji Xerox, Tokyo, Japan). Uniform impregnation of wax on the paper was performed in a drying oven at 130°C for 45 sec, after which the paper was dried at RT.
-5Page 5 of 17
ip t cr
Fig. 1. Scheme of the SERS platform. (A) Design of the SERS paper platform. The gray color (hydrophobic)
us
indicates the decreased dimension due to the wax impregnation. (B) Cross-sectional view of the SERS-active area on the paper. (C) Photo before and after printing CMC-AuNP inks on the paper substrate. Scale bar=5 mm.
an
Since the hydrophilic SERS-active area has a 2-mm diameter, the size of the stencil hole with a 3-mm diameter was designed to be much wider in order to produce uniform SERS signals; the coffee ring effect caused by the
M
surface tension acting on the ink and the side of the mask wall drives ink to the outer hydrophobic wax part (Fig. 1B). A 4-µL aliquot of the CMC-AuNP ink was dropped on the stencil hole, and a blade was moved across a 0.18-mm thick
d
stencil. The substrate was dried on a hot plate at 50°C for 120 sec (one cycle). Figure 1C shows a photograph of the
Ac ce pt e
SERS-functionalized substrate after printing CMC-AuNP ink on the wax-impregnated paper.
2.5. Human tears
Tear biofluids were collected from the nasoinferior conjunctival sac without additional stimulation using a 025 ISO standard size, a 29-mm-long paper point (Henry Schein Inc, Melville, NY, USA). After a maximum of 5 min, the points were withdrawn from the eye and placed into a 1.5-mL Eppendorf tube. Tear fluids were extracted from the saturated points by centrifuging at 8,000 rpm for 10 min. The points were carefully removed, and the tear fluid was aspirated. Tear samples were stored in an Eppendorf tube sealed with Parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA) at −70°C for <24 h before they were analyzed. Informed consent was obtained from each subject at Kyung Hee University Medical Center. All procedures involving humans adhered to the Declaration of Helsinki and were approved by the Ethics Committee of Kyung Hee University College of Medicine (KMCIRB1401-02).
3. Results and discussion 3.1. Characterization of the AuNPs -6Page 6 of 17
ip t cr us an
Fig. 2. Characterization of the AuNPs and the CMC solution. (A) TEM image of the synthesized AuNPs. solutions with four different concentrations.
M
Scale bar=50 nm. (B) Size of the AuNPs. (C) UV-Vis absorption spectrum of colloidal AuNPs. (D) Viscosity of CMC
d
The characteristics of the citrate-capped AuNPs were evaluated using TEM images and UV-Vis spectra (Fig. 2). Based
Ac ce pt e
on the FE-TEM images, the AuNPs exhibited a stable monodispersity with an average diameter of 25±8 nm. The UVVis spectrum of the AuNPs colloids showed maximum absorption at a wavelength of 525 nm (λ0). Generally, a particle size ranging from 25 to 100 nm is determined from the following equation:
λmax − λ0 = L1 exp ( L2 d )
(1)
where λ0 indicates the maximum absorption wavelength, λmax indicates the maximum value of λ0, and d indicates the particle size d>25 nm (L1=6.53 and L2=2.16×10–2 are constants determined from the experiment results) [31]. The calculated average particle size was 31.9 nm when the peak wavelength of the AuNPs was 525 nm (Fig. 2C). The AuNPs might slightly aggregate in a colloid, but the calculated particle size was within the range of the TEM-measured particle sizes. The size of the AuNPs is a critical factor to determine SERS enhancement [27,32]. Some studies have revealed that metallic nanoparticles in a size range of 10–100 nm produce strong Raman intensities compared to other sizes [33,34].
3.2. Viscosity of the CMC solutions Since direct printing of only concentrated AuNPs colloids might not be suitable for uniform dispersion due to the low -7Page 7 of 17
viscosity, the AuNPs were added in a CMC solution, which is a conventional viscous agent with properties of water solubility and concentration-dependent viscosity, in order to optimize the viscosity of the printing inks [28]. Since CMC has a much higher viscosity than water, it is expected to play a critical role in AuNPs uniformity on paper. The viscosity of four different concentrations of the CMC solution was measured using a viscometer. The viscosity of the CMC increased exponentially with increasing concentration (Fig. 2D). A 3-wt% CMC solution produced too high of a viscosity to allow proper handling of the inks, while the low viscosity of the 0.5-wt% CMC solution resulted in
ip t
inhomogeneous dispersion of the AuNPs on the paper (Fig. S1, Supplementary Information). Both 1- and 2-wt% CMC solutions allowed proper adherence and distribution of AuNPs on the paper substrate. In addition, the high surface
cr
roughness of the paper substrate led to a synergistic effect on adhesion of the CMC solution, acting as a glue. The 1-
us
and 2-wt% CMC concentrations were selected as primarily screen printing ink conditions.
3.3. Surface of the CMC-AuNP ink-printed paper
an
Figure 3 shows SEM images of the printed CMC-AuNP on the paper substrate. The presence of disordered cellulose fibers was observed (Fig. 3A), and the porous structure between the cellulose fibers was filled with the sodium CMC
M
solution, demonstrating the viscosity of the bare and wax-impregnated papers (Fig. S2, Supplementary Information). The AuNPs were well distributed on the cellulose fibers (Fig. 3B) and within the range of a 2.4-μm focal spot size for
d
Raman spectrum measurement. The blurred SEM image of the surface of the CMC-AuNP-printed paper was due to its
Ac ce pt e
mixture with sodium CMC. In order to determine whether these particles (Fig. 3B) were AuNPs, EDX-based molecular observation of the particle composition was performed (Fig. S3, Supplementary Information). The EDX mapping suggested that the CMC-AuNP-loaded paper could be used as a SERS substrate.
Fig. 3. Distribution of the AuNPs on the paper substrate. (A) SEM images of the CMC-AuNP ink loaded on the paper substrate. Scale bar=50 μm. (B) Distribution of AuNPs (yellow arrow) on the paper substrate. Scale bar=500 nm.
3.4. Optimization of the CMC-AuNP inks The condition of the CMC-AuNP ink was optimized by controlling the ratio of AuNPs and CMC solution. A 2-μL -8Page 8 of 17
aliquot of 1 mM RhB (probe molecule of Raman spectroscopy) was dropped onto the SERS-active area to determine the optimal condition of the CMC-AuNP ink. The dominant Raman peaks of RhB were measured at 620 cm–1 (aromatic bending), 1201 cm–1 (aromatic C–H bending), and 1356 cm–1 (aromatic C–C stretching) [9]. The prominent Raman peak at 1356 cm–1 was selected as the reference peak of RhB analysis. The optimal CMC concentration with the maximum Raman intensity was estimated using the 1- and 2-wt% CMC concentrations. Ink solutions with a 5:1 volume ratio of CMC solution to AuNPs were prepared at two CMC
ip t
concentrations. Three printing cycles of the CMC-AuNP ink (Fig. S4, Supplementary Information) showed that the combination with a 2-wt% CMC solution exhibited better SERS intensity and less spectral deviation than the 1-wt%
cr
CMC solution as the higher-viscosity CMC solution inhibited movement and better secured the AuNPs (Fig. S1, Supplementary Information). Furthermore, the rapid drying process of the CMC-AuNP inks at 50ºC during the printing
us
cycles prevented the AuNPs from spreading to the edges and allowed them to adequately disperse over SERS-active area. Therefore, 2-wt% CMC-AuNP was selected as the optimal printing ink combination.
an
The effect of AuNP amount on Raman intensity was investigated by adjusting the volume ratio of AuNPs in the screen printing ink solution. For this purpose, 2-wt% CMC was mixed with AuNPs at three volume ratios; 5:1, 7:1,
M
and 10:1. Figure 4A shows the Raman intensity of the three CMC-AuNP ink solutions with different volume ratios according to printing cycle. The 7:1 CMC-AuNP ink solution exhibited a good SERS effect compared to the other
d
volume ratios. The maximum SERS intensity was observed in two printing cycles with a 7:1 ratio of CMC-AuNPs. The
Ac ce pt e
resonance of nanoparticles at the paper surface is caused by the incident energy in the few nanometers between the particles. The reflected energy is amplified by the scattering according to the resonance, and this energy has a significant impact on the SERS effect. The space between the particles is influenced by the particle number. When the size of the nanoparticles is constant and their number increases in a limited area, the distance between particles decreases, and the Raman scattering increases. However, when the number of particles increases above a certain limit, the AuNPs demonstrate film characteristics rather than acting as separate particles. Under such conditions, the resonance of nanoparticles decreases, and the Raman intensity decreases [11]. These changes were observed in the present study; two printing cycles led to two-fold increases in SERS intensity compared to that after one printing cycle. However, additional printing cycles on the paper substrate resulted in a decrease in SERS intensity and a larger deviation of Raman peaks. In order to validate the reliability of the measurement, the SERS spectra were analyzed randomly at 10 different positions utilizing the optimized two cycles of a 7:1 CMC-AuNP ink solution. The mean intensity of the spot-to-spot variations in the SERS peaks at 1356 cm–1 was 5,858±279 (Table S1, Supplementary Information), and the corresponding relative standard deviation (RSD) was 4.7%. The intensity of the SERS signals was affected by the SERS effects, laser source focusing, biosamples, and several other variables. Although the Raman -9Page 9 of 17
intensity varied among the detection sites, the overall less than 5% variance affirms the high reproducibility of the
M
an
us
cr
ip t
SERS method.
Fig. 4. Optimization of the CMC-AuNP inks. (A) The Raman intensity of CMC-AuNP ink solutions with three
d
volume ratios of CMC solution and AuNPs of CMC:AuNPs=5:1, 7:1, and 10:1, according to printing cycle. (B) High
Ac ce pt e
reproducibility of a 2-wt% 7:1 CMC-AuNP ink solution. RSD=relative standard deviation.
In additional, we evaluated the distribution of CMC-AuNP inks within the SERS-active area on the paper substrate. The Raman signals of six points from the center zone to the ring zone of the SERS-active area (a 1-mm diameter with the interval of 200 μm) were measured (Fig. S5, Supplementary Information). The spectral profile of Raman peaks at 1356 cm–1 showed a low RSD regardless of the zones. This indicates that the proposed fabrication method leads to homogeneous dispersion of the AuNPs on the paper substrate, the absence of the coffee ring effect. Therefore, the noise-independence, uniformity, and reproducibility of the SERS spectral signals suggest that the proposed SERS assessment has the potential to be used as a highly sensitive and selective assessment for biological fluids.
3.5. SERS activity of the fabricated paper platform To quantify the SERS activity of the screen printing ink, the Raman spectra of the SERS-functionalized (CMC-AuNP ink-printed) and bare (intact) paper were measured using 1-mM and 1-M RhB, respectively (Fig. S6, Supplementary Information). The Raman intensity of the 1356 cm-1 band (5,858) of a 1-mM RhB on the SERS paper demonstrated an - 10 Page 10 of 17
approximately 18-fold increase compared to that of 1-M RhB on bare paper (328). Therefore, the EF was calculated as the difference in Raman intensities between two different substrates; ⎛I ⎞⎛ N ⎞ EF = ⎜ SERS ⎟ ⎜ bare ⎟ ⎝ I bare ⎠ ⎝ N SERS ⎠
(2)
where ISERS and Ibare indicate the Raman intensity of the molecule on the SERS and bare papers, respectively, and NSERS and Nbare indicate the average number of adsorbed molecules in the scattering volume for the SERS and non-SERS
ip t
areas, respectively [6]. Assuming that the probe molecules are uniformly distributed on the substrates, the number of adsorbed molecules can be estimated as ⎞ ⎟⎟ Alaser ⎠
cr
⎛ Vdroplet N = ⎜ NA ⋅c⋅ ⎜ Aspot ⎝
(3)
us
where NA is Avogadro’s constant, c is the concentration of the probe molecule, V is the volume of the molecule droplet, Aspot is the size of the substrate, and Alaser is the size of the laser spot [8,9]. Since the same methods for assessing the
an
Raman measurement were applied to two substrates, the parameters of NA of RhB, V, Aspot, and Alaser were the same. Hence, Eq. (2) can be written as
M
⎛I ⎞⎛ c ⎞ EF = ⎜ SERS ⎟ ⎜ bare ⎟ ⎝ I bare ⎠ ⎝ cSERS ⎠
(4)
d
where cSERS and cR indicate the concentration of RhB on the SERS and bare papers, respectively. The final EF at the
Ac ce pt e
1356 cm-1 band was 1.8×104. This result indicates the presence of SERS activity on the CMC-AuNP ink-printed paper. Although the EF value was smaller than those of other materials and structures, such as bimetallic nanoparticles [35], noble metal-coated nanowires [7,9], and sophisticated nanoscale pattern arrays [10], the CMC-AuNP ink-printed SERS paper substrate with a simple and low-cost fabrication is likely to be a useful platform with great potential for application in POC diagnostics.
- 11 Page 11 of 17
ip t cr us an
M
Fig. 5. Representative SERS spectra and analytical peak assignments for normal (control), (A) adenoviral conjunctivitis-infected, and (B) herpes simplex conjunctivitis-infected human tear fluids. The intensity of three Raman peaks at 1003 cm–1 (the ring of phenylalanine), 1242 cm–1 (amide III β-sheet), and 1342 cm–1 (C–H deformation in proteins) could be used for classifying the differences among tears of normal, adenoviral
Ac ce pt e
d
conjunctivitis, and herpes simplex conjunctivitis subjects.
3.6. In vivo application
Figure 5 shows the typical SERS spectra of normal and two infected tears acquired using the fabricated SERSfunctionalized paper sensor platform. Each SERS peak shows the distinct vibration characteristics of human tear fluids (Table S2, Supplementary Information). Without an additional smoothing algorithm, all tear-induced SERS spectra were normalized by setting the variance of the Raman spectral signal to a value of 1.0, with the intense peak located at 1003 cm–1 (the ring of phenylalanine). There was no shift in these SERS peaks between normal and infected tears. However, the SERS intensity of the two prominent peaks at 1242 and 1342 cm–1 varied between the normal and infected tears. Hence, the intensity ratio of C–H deformation at 1342 cm–1 to the amide III β-sheet at 1242 cm–1 could be used as a marker to detect the presence of adenovirus virus and herpes simplex virus,
δ VIRUS =
I1342 I1242
(5)
where I1242 and I1342 indicate the SERS spectral intensity of the amide III β-sheet and C–H deformation at the 1242 and 1342 cm–1 peaks, respectively [29]. - 12 Page 12 of 17
For example, the normal tears showed intensities of 0.3830 for the 1242 cm–1 peak and 0.3075 for the 1342 cm–1 peak, while the tears infected with adenovirus showed an intensity of 0.3782 at 1242 cm–1 and 0.7057 at 1342 cm–1, and the tears infected with herpes simplex virus showed an intensity of 0.4579 at 1242 cm–1 and 1.1307 at 1342 cm–1. The I1342/I1242 ratio was 0.80 for normal tears, 1.86 for adenovirus-infected tears, and 3.73 for herpes simplex virusinfected tears (Fig. S7, Supplementary Information). Therefore, the threshold value, δVIRUS>1, can be used as a marker for detecting the presence of virus in tears. Furthermore, the difference between two virus-infected tear fluids can be
ip t
classified according to the following rule: if δVIRUS>2 and I1342>1, then the infection is herpes simplex conjunctivitis. This significant difference indicates that the tear-induced Raman spectral assessment using a fabricated SERS-
cr
functionalized paper sensor platform is an effective signature for detecting and classifying adenoviral conjunctivitis and herpes simplex conjunctivitis. Finally, the supporting vector machine rule-based classification system supported by the
us
principal component analysis (PCA) algorithm is likely to show enhanced, early detection of adenoviral and herpes
an
simplex conjunctivitis.
4. Conclusions
M
A SERS-functionalized paper sensor platform for analyzing biofluids based on a solution of AuNP-loaded screen printing ink was introduced. For the SERS functionalization of a simple paper, the optical property of SPR was
d
implemented using the citrate-capping method based on monometallic gold nanoparticles. The viscous property of
Ac ce pt e
sodium CMC solution was used to improve the uniformity and reproducibility of the SERS spot-on assay of biological fluids. A paper-based SERS sensor was fabricated with simple and facile processes that do not require professional knowledge of chemical engineering or expensive equipment. The EF of the proposed SERS sensor platform using the Raman spectroscopy probe molecule RhB was approximately 2×104 under optimized conditions consisting of two screen printing cycles of a viscous ink with a 7:1 mixture of 2-wt% CMC and AuNPs. The in vivo application of two representative ocular infection diseases (conjunctivitis) showed clear Raman peaks and SERS effects to support rapid POC diagnosis of biofluids in clinics. Based on this promising paper platform, the bimetallic core-shell structure of AgNPs and the AuNPs lead to superior SERS enhancement compared to a substrate with only AuNP.
Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant 2014R1A1A2054452).
Supplementary data - 13 Page 13 of 17
Ac ce pt e
d
M
an
us
cr
ip t
Supplementary data associated with this article can be found in the online version.
- 14 Page 14 of 17
References
S. Zeng, D. Baillargeat, H.-P. Ho, K.-T. Yong, Nanomaterials enhanced surface plasmon resonance for biological and chemical sensing applications., Chem. Soc. Rev. 43 (2014) 3426–52. doi:10.1039/c3cs60479a.
[2]
L. Polavarapu, J. Pérez-Juste, Q.-H. Xu, L.M. Liz-Marzán, Optical sensing of biological, chemical and ionic species through aggregation of plasmonic nanoparticles, J. Mater. Chem. C. (2014) 7460–7476. doi:10.1039/C4TC01142B.
[3]
X. Liu, Y. Shao, Y. Tang, K.-F. Yao, Highly Uniform and Reproducible Surface Enhanced Raman Scattering on Air-stable Metallic Glassy Nanowire Array, Sci. Rep. 4 (2014) 5308. doi:10.1038/srep05835.
[4]
K. Kneipp, Y. Wang, H. Kneipp, L.T. Perelman, I. Itzkan, R.R. Dasari, et al., Single molecule detection using surface-enhanced Raman scattering (SERS), Phys. Rev. Lett. 78 (1997) 1667–1670. doi:10.1103/PhysRevLett.78.1667.
[5]
A. Campion, P. Kambhampati, Surface-enhanced Raman scattering, Chem. Soc. Rev. 27 (1998) 241. doi:10.1039/a827241z.
[6]
E.C. Le Ru, E.J. Blackie, M. Meyer, P.G. Etchegoin, Surface Enhanced Raman Scattering Enhancement Factors: A Comprehensive Study, J. Phys. Chem. C. 111 (2007) 13794–13803. doi:10.1021/jp0687908.
[7]
J. Hu, S. Chen, R.P. Johnson, X. Lin, Z. Yang, A.E. Russell, Surface-enhanced raman scattering on uniform Pd and Pt films: From ill-defined to structured surfaces, J. Phys. Chem. C. 117 (2013) 24843–24850. doi:10.1021/jp4081433.
[8]
L. Zhang, X. Lang, A. Hirata, M. Chen, Wrinkled nanoporous gold films with ultrahigh surface-enhanced raman scattering enhancement, ACS Nano. 5 (2011) 4407–4413. doi:10.1021/nn201443p.
[9]
X. Liu, Y. Shao, Y. Tang, K.-F. Yao, Highly Uniform and Reproducible Surface Enhanced Raman Scattering on Air-stable Metallic Glassy Nanowire Array., Sci. Rep. 4 (2014) 5835. doi:10.1038/srep05835.
[10]
F. De Angelis, F. Gentile, F. Mecarini, G. Das, M. Moretti, P. Candeloro, et al., Breaking the diffusion limit with super-hydrophobic delivery of molecules to plasmonic nanofocusing SERS structures, Nat. Photonics. 5 (2011) 682–687. doi:10.1038/nphoton.2011.222.
[11]
E.D. Diebold, N.H. Mack, S.K. Doom, E. Mazur, Femtosecond Laser-Nanostructured substrates for SurfaceEnhanced raman scattering, Langmuir. 25 (2009) 1790–1794. doi:10.1021/la803357q.
[12]
X.M. Lin, Y. Cui, Y.H. Xu, B. Ren, Z.Q. Tian, Surface-enhanced raman spectroscopy: Substrate-related issues, Anal. Bioanal. Chem. 394 (2009) 1729–1745. doi:10.1007/s00216-009-2761-5.
[13]
S. Zeng, X. Yu, W.-C. Law, Y. Zhang, R. Hu, X.-Q. Dinh, et al., Size dependence of Au NP-enhanced surface plasmon resonance based on differential phase measurement, Sensors Actuators B Chem. 176 (2012) 1128– 1133. doi:10.1016/j.snb.2012.09.073.
[14]
B. Ren, G.-K. Liu, X.-B. Lian, Z.-L. Yang, Z.-Q. Tian, Raman spectroscopy on transition metals., Anal. Bioanal. Chem. 388 (2007) 29–45. doi:10.1007/s00216-007-1141-2.
[15]
L.L. Qu, Q.X. Song, Y.T. Li, M.P. Peng, D.W. Li, L.X. Chen, et al., Fabrication of bimetallic microfluidic surface-enhanced Raman scattering sensors on paper by screen printing, Anal. Chim. Acta. 792 (2013) 86–92. doi:10.1016/j.aca.2013.07.017.
[16]
A.W. Martinez, S.T. Phillips, M.J. Butte, G.M. Whitesides, Patterned paper as a platform for inexpensive, lowvolume, portable bioassays, Angew. Chemie - Int. Ed. 46 (2007) 1318–1320. doi:10.1002/anie.200603817.
Ac ce pt e
d
M
an
us
cr
ip t
[1]
- 15 Page 15 of 17
W. Zhang, B. Li, L. Chen, Y. Wang, D. Gao, X. Ma, et al., Brushing, a simple way to fabricate SERS active paper substrates, Anal. Methods. 6 (2014) 2066. doi:10.1039/c4ay00046c.
[18]
L. Polavarapu, A. La Porta, S.M. Novikov, M. Coronado-Puchau, L.M. Liz-Marzán, Pen-on-paper approach toward the design of universal surface enhanced Raman scattering substrates, Small. 10 (2014) 3065–3071. doi:10.1002/smll.201400438.
[19]
R.S.J. Alkasir, M. Ornatska, S. Andreescu, Colorimetric paper bioassay for the detection of phenolic compounds, Anal. Chem. 84 (2012) 9729–9737. doi:10.1021/ac301110d.
[20]
C. Renault, M.J. Anderson, R.M. Crooks, Electrochemistry in hollow-channel paper analytical devices, J. Am. Chem. Soc. 136 (2014) 4616–4623. doi:10.1021/ja4118544.
[21]
Z. Zhang, W. Xu, N.E. Manicke, R.G. Cooks, Z. Ouyang, Silica coated paper substrate for paper-spray analysis of therapeutic drugs in dried blood spots, Anal. Chem. 84 (2012) 931–938. doi:10.1021/ac202058w.
[22]
W.W. Yu, I.M. White, Inkjet printed surface enhanced raman spectroscopy array on cellulose paper, Anal. Chem. 82 (2010) 9626–9630. doi:10.1021/ac102475k.
[23]
B. Li, W. Zhang, L. Chen, B. Lin, A fast and low-cost spray method for prototyping and depositing surfaceenhanced Raman scattering arrays on microfluidic paper based device, Electrophoresis. 34 (2013) 2162–2168. doi:10.1002/elps.201300138.
[24]
A.K. Yetisen, M.S. Akram, C.R. Lowe, Paper-based microfluidic point-of-care diagnostic devices., Lab Chip. 13 (2013) 2210–51. doi:10.1039/c3lc50169h.
[25]
Y.H. Ngo, D. Li, G.P. Simon, G. Garnier, Gold nanoparticle-paper as a three-dimensional surface enhanced Raman scattering substrate., Langmuir. 28 (2012) 8782–90. doi:10.1021/la3012734.
[26]
Y.H. Ngo, W.L. Then, W. Shen, G. Garnier, Gold nanoparticles paper as a SERS bio-diagnostic platform, J. Colloid Interface Sci. 409 (2013) 59–65. doi:10.1016/j.jcis.2013.07.051.
[27]
G. Zheng, L. Polavarapu, L.M. Liz-Marzán, I. Pastoriza-Santos, J. Pérez-Juste, Gold nanoparticle-loaded filter paper: a recyclable dip-catalyst for real-time reaction monitoring by surface enhanced Raman scattering, Chem. Commun. (2015) 4572–4575. doi:10.1039/C4CC09466B.
[28]
L.-L. Qu, D.-W. Li, J.-Q. Xue, W.-L. Zhai, J.S. Fossey, Y.-T. Long, Batch fabrication of disposable screen printed SERS arrays, Lab Chip. 12 (2012) 876. doi:10.1039/c2lc20926h.
[29]
S. Choi, S.W. Moon, J. Shin, H. Park, K. Jin, Label-Free Biochemical Analytic Method for the Early Detection of Adenoviral Conjunctivitis Using Human Tear Biofluids, Anal. Chem. 86 (2014) 11093–11099. doi:10.1021/ac5025478.
[30]
C.-Y. Lin, C.-H. Liu, W.-L. Tseng, Fluorescein isothiocyanate-capped gold nanoparticles for fluorescent detection of reactive oxygen species based on thiol oxidation and their application for sensing glucose in serum, Anal. Methods. 2 (2010) 1810. doi:10.1039/c0ay00428f.
[31]
W. Haiss, N.T.K. Thanh, J. Aveyard, D.G. Fernig, Determination of size and concentration of gold nanoparticles from UV-Vis spectra, Anal. Chem. 79 (2007) 4215–4221. doi:10.1021/ac0702084.
[32]
C. Shen, C. Hui, T. Yang, C. Xiao, J. Tian, L. Bao, et al., Monodisperse noble-metal nanoparticles and their surface enhanced raman scattering properties, Chem. Mater. 20 (2008) 6939–6944. doi:10.1021/cm800882n.
[33]
C. Wang, C. Yu, Analytical characterization using surface-enhanced Raman scattering (SERS) and microfluidic sampling, Nanotechnology. 26 (2015) 092001. doi:10.1088/0957-4484/26/9/092001.
[34]
K. Kim, K.S. Shin, Surface-enhanced Raman scattering: a powerful tool for chemical identification., Anal. Sci. 27 (2011) 775. doi:10.2116/analsci.27.775.
Ac ce pt e
d
M
an
us
cr
ip t
[17]
- 16 Page 16 of 17
[35]
L. Bi, J. Dong, W. Xie, W. Lu, W. Tong, L. Tao, et al., Bimetallic gold-silver nanoplate array as a highly active SERS substrate for detection of streptavidin/biotin assemblies, Anal. Chim. Acta. 805 (2013) 95–100. doi:10.1016/j.aca.2013.10.045.
*Authors CVs Wan-Sun Kim earned a dual M.S. degree from the Department of Information Display at Kyung Hee University,
ip t
South Korea, and the Sciences and Technologies at Ecole Polytechnique, France, in 2007. From 2012, he studied nBioMechatronics and biosensors on a doctorate path in the Department of Medical Engineering, College of Medicine
cr
at Kyung Hee University.
us
Jae-Ho Shin received an M.D., Ph.D. from the School of Medicine at Kyung Hee University, South Korea, in 1999. He received a Ph.D. in Ophthalmology from Kyung Hee University, South Korea, in 2013. Since 2013, he has worked as an Assistant Professor in the Department of Ophthalmology, Kyung Hee University Hospital at Gangdong, South
an
Korea. Dr. Shin’s main research interests include application of biomedical tools and techniques in ophthalmology,
M
especially oculoplastic surgery.
Hun-Kuk Park received an M.D. and B.M. from the School of Medicine at Kyung Hee University, South Korea, in 1982. He received a Ph.D. in Biomedical Engineering from Rutgers University and the University of Medicine and
d
Dentistry of New Jersey, USA, in 1993. Since 2008, he has worked as the Chairman and Professor in the Department of Biomedical Engineering, School of Medicine at Kyung Hee University, South Korea. Dr. Park’s main research interests
Ac ce pt e
include neuroscience and neurosurgery, biomechanics, nano-biosensors, pain mechanisms and analysis, nanoacupuncture techniques, bioinformatics/pharmacogenomics, and AFM applications for medical science.
Samjin Choi received a Ph.D. in Mechanical Engineering (Electrical Systems) from Yamaguchi University, Japan, in 2006. He served as an Assistant Professor (2006–2008) in the Department of Mechanical Engineering at Yamaguchi University, Japan. Since 2013, he has worked as an Assistant Professor in the Department of Medicine, College of Medicine at Kyung Hee University, Korea. Dr. Choi’s main research interests are nBioMechatronics (http://biomed.khu.ac.kr/xe/prof05). He has published more than 70 papers in peer-reviewed SCI-indexed international journals.
- 17 Page 17 of 17
S0925-4005(15)30208-2 http://dx.doi.org/doi:10.1016/j.snb.2015.08.030 SNB 18885
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-6-2015 20-7-2015 8-8-2015
Please cite this article as: W.-S. Kim, J.-H. Shin, H.-K. Park, S. Choi, A low-cost, monometallic, surface-enhanced Raman scattering-functionalized paper platform for spot-on bioassays, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.08.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A low-cost, monometallic, surface-enhanced Raman scattering-functionalized paper platform for spot-on bioassays
Wan-Sun Kima, Jae-Ho Shinb, Hun-Kuk Parka,c, Samjin Choia,c*
ip t
a. Department of Medical Engineering, Kyung Hee University, Seoul 130-701, Korea b. Department of Ophthalmology, College of Medicine, Kyung Hee University, Seoul 130-701, Korea
cr
c. Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul 130-701, Korea
us
*Address for correspondence: Samjin Choi, Ph.D.,
an
Department of Biomedical Engineering, College of Medicine,
M
Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu,
Tel: +82 2 961 0290
Ac ce pt e
Fax: +82 2 6008 5535
d
Seoul 130-701, Republic of Korea
E-mail: [email protected]
English Language Editing
This manuscript was checked by the native English speakers of the E-World Editing service (USA) of Kyung Hee University Medical Center (Code No. HEW1505-03).
-1Page 1 of 17
Highlights
A simple and facile SERS-functionalized paper platform was designed for POC diagnostics of biofluids. The optical property of surface plasmon resonance was implemented using monometallic gold NPs.
ip t
The viscous property of a sodium CMC solution was used to improve the uniformity and reproducibility of SERS. The low-cost SERS paper sensor showed an effective signature for classifying adenoviral and herpes simplex
M
an
us
cr
conjunctivitis.
Abstract
d
We demonstrate the fabrication and spot-on bioassay application of a low-cost, monometallic, surface-enhanced Raman
Ac ce pt e
scattering (SERS) paper platform using gold nanoparticle (AuNP)-loaded screen printing inks. The AuNPs were used to enhance the Raman intensity through a surface plasmon resonance (SPR)-driven optical property and sodium carboxymethylcellulose (CMC) was used as a viscous ink to create uniform distribution of the AuNPs on the paper substrate. To minimize the coffee ring effect and uniformly disperse the nanoparticles, the size of citrate-capped AuNPs, the CMC concentration, the volume ratio of CMC solution and AuNPs, and the printing cycles were optimized. Two printing cycles of optimized ink with a 7:1 mixture of 2-wt% CMC and AuNPs produced the strongest SERS effect and an enhancement factor of 1.8×104 with a SERS paper platform based on a Rhodamine B probe molecule. The platform exhibited high reproducibility, with less than 5% spot-to-spot variation in Raman intensity. Furthermore, the SERS spectra of normal and two virus-infected tear biofluids were comparable to the SERS spectral findings of golddeposited SERS substrates. The enhanced SERS activity and high reproducibility of a low-cost, flexible, portable, and lightweight paper platform suggest the potential of point-of-care applications of biofluids in fields ranging from clinical analysis to industry.
Keywords: SERS; paper substrate; AuNPs; CMC solution; screen printing; tear biofluids -2Page 2 of 17
1. Introduction Raman spectroscopy is a technique to provide molecule-specific information on biological and chemical samples [1–3]. Since a Raman signal is inherently very weak, various studies have been performed with the aim of signal enhancement. Surface-enhanced Raman scattering (SERS) activity can dramatically improve the intensity of the Raman spectrum due to the absorption energy on the surface [4,5]. The enhancement factor (EF), which is used as a measure of the
ip t
magnitude of SERS, is commonly in the range of 104–108 and can be as high as 1014, allowing for detection at the single-molecule level [6]. Most studies aiming to increase the SERS EF have concentrated on substrate-related issues by
cr
modifying the materials and nanostructure patterns of the surface [7–11]. Most SERS-active areas are fabricated using complex and sophisticated methods including lithography or a high-temperature process. These SERS fabrications
us
undergo expensive, time-consuming, and complicated steps, while the use of metal nanoparticles as a SERS substrate provides easy synthesis at low-cost, with controllable size and shape via the reaction conditions, and very strong
an
enhancement of aggregated nanoparticles with up to single-molecule sensitivity [12]. These nanoparticles absorb the wavelength used in a Raman laser source, known as surface plasmon resonances (SPRs) [1,13]. In particular, Ag, Au,
M
and Cu nanoparticles have been shown to demonstrate a 103-fold higher SERS enhancement compared to other metal substrates [14]. Silver nanoparticles (AgNPs) exhibit superior SERS enhancement compared to gold nanoparticles
d
(AuNPs). However, AgNPs show rapid degradation of SERS activity due to oxidation in air, while AuNPs show stable
Ac ce pt e
SERS activity due to formation of an oxide layer [15].
Paper substrate, which is low-cost, portable, flexible, easy to handle, and harmless, has been highlighted as a novel platform for analytical detection in biomedical and environmental applications [16–18]. These user-friendly advantages of paper offer various applications including colorimetric, electrochemical, and biochemical analyses [19– 21]. These advantages of a paper substrate are well-suited for point-of-care (POC) applications, however it still suffers from limited detection of analytical materials such as enzymes and redox dyes as well as involvement of complex soluble compounds. Since the Raman spectrum of the sample clearly shows the chemical nature and changes of the internal materials, the nanoparticle-driven SERS paper platform is likely to overcome this limitation [22–24]. Some studies have reported the use of colloidal AuNP-deposited SERS paper platforms for environmental and biochemical monitoring [25–27]. The high surface roughness of paper composed of hydrophilic cellulose fibers helps to maintain uniform distribution of AuNPs compared to smooth surface materials such as glass and silicon wafers. Additionally, paper can be easily modified with coating materials to demonstrate various degrees of hydrophobicity. The AuNPs in the low-viscosity colloid aggregate at the edge of a droplet form a coffee ring on the paper substrate that prevents the SERS-active area from maintaining a homogeneous SERS effect. A screen printing technique using nanoparticles within -3Page 3 of 17
a viscous ink has shown improved reproducibility of the SERS analysis compared to other printing techniques [15,28]. Since there is a limited amount of tear biofluid that can be reasonably collected from one patient, few analytical studies have been performed for human tear biofluids, with most analytical methods intended for human urinal and bloody biofluids. Clinical situations often involve long waiting periods to receive analytical results through conventional analytical techniques such as enzyme-linked immunosorbent assay (ELISA), direct immunofluorescence assay (IFA), and polymerase chain reaction (PCR). Although rapid diagnosis is imperative for prescription of
ip t
appropriate treatments to prevent further infections of viral or bacterial infection-driven conjunctivitis and corneal damage, conventional time-consuming procedures are still used in ophthalmological clinics [29].
cr
Therefore, we introduce the fabrication of a monometallic SERS-functionalized paper platform using a mixture of AuNPs and viscous ink based on a screen-printing technique. The SERS effect was optimized by adjusting
us
the amount of synthesized AuNPs, and its reproducibility was verified by adjusting the viscosity of CMC-AuNP screen printing inks. In particular, to expand the biomedical application of POC diagnosis, the performance of the fabricated
an
SERS paper platform was evaluated using in vivo human tear biofluids collected from patients with adenoviral
M
conjunctivitis and herpes simplex conjunctivitis.
2. Material and methods
d
2.1. Reagents and materials
Ac ce pt e
Hydrogen tetrachloroaurate (HAuCl4, >99%), trisodium citrate dehydrate (99%), sodium carboxymethylcellulose (CMC), and Rhodamine B (RhB, >95%) were purchased from Sigma Aldrich (St. Louis, MO, USA). All reagents were of analytical grade, and all solutions were prepared using 18.3 MΩ·cm-1 distilled water. Whatman® cellulose chromatography paper (Grade 1) with a 0.18 mm thickness and a linear flow rate of 72.22 µm/sec was purchased from Sigma Aldrich.
2.2. Instrumentation
The size and shape of the AuNPs were characterized by a JEM-2100F field emission transmission electron microscope (FE-TEM; JEOL, Tokyo, Japan) operated at an accelerating voltage of 200 kV. The UV-Vis absorption spectra of the colloidal AuNPs were produced by a CARY 300 UV-Vis spectrophotometer (Agilent, Santa Clara, CA, USA) using a 1cm quartz cell. The viscosity of the CMC solution was measured using a DV-II+Pro viscometer (Brookfield, Middleboro, MA, USA) at room temperature (RT). The morphologies of the paper substrates according to the presence of CMC-AuNPs were characterized using a S-4700 field emission scanning electron microscope (FE-SEM; Hitachi, Tokyo, Japan) at an accelerating voltage of 5 -4Page 4 of 17
kV. A 7200-H energy-dispersive X-ray spectroscope (EDX; HORIBA, Northampton, England) was used to examine the elemental compositions of the synthesized products. The Raman spectrum was obtained using a SENTERRA confocal Raman system (Bruker Optics, Billerica, MA, USA). A 785-nm diode laser source with a 10-mW power was focused to a spot size of ~2.4 µm with a 20× objective lens (N.A.=0.4). The spectrum of each point was recorded in the range of 417–1782 cm-1 with a spectral resolution of 5 cm-1 and twice the acquisition time of 30 sec at RT. The Raman spectrum of each sample was measured
ip t
as 10, randomly-printed spots on the SERS-active area to ensure reliability. A 2-µL analytic droplet of the sample was
cr
used to evaluate the Raman spectrum.
2.3. Preparation of CMC-AuNP inks
us
The AuNPs were obtained through citrate capping based on the Turkevich method of chemical reduction [30]. A 1-mL aliquot of 38.8 mM trisodium citrate dihydrate was added to 10 mL of 1 mM HAuCl4 and heated at 90°C for 20 min on
an
a hot plate under magnetic stirring. The color of the solution changed from a pale yellow to a deep violet. The reaction flask was immediately removed from the hot plate, and stirring was maintained until the solution had cooled to RT.
M
To prepare the screen-printing ink solutions, 1 mL of colloidal AuNPs was concentrated by centrifugation at 6000 rpm for 10 min, and 99% of the supernatant was removed. The concentrated AuNPs were dispersed in sodium
Ac ce pt e
optimize the inks.
d
CMC with distilled water. The concentration of the CMC and the ratio of the AuNPs and CMC were adjusted to
2.4. Fabrication of the SERS platform
The SERS-functionalized paper platform consisted of two parts, the SERS-active area and the labeling area. Figure 1A shows that a 2-mm hydrophilic circular reservoir and a labeling area designed by AutoCAD (Autodesk, San Rafael, CA, USA) were printed on an 8×20-mm2 paper using a Xerox ColorQube 8570N printer (Fuji Xerox, Tokyo, Japan). Uniform impregnation of wax on the paper was performed in a drying oven at 130°C for 45 sec, after which the paper was dried at RT.
-5Page 5 of 17
ip t cr
Fig. 1. Scheme of the SERS platform. (A) Design of the SERS paper platform. The gray color (hydrophobic)
us
indicates the decreased dimension due to the wax impregnation. (B) Cross-sectional view of the SERS-active area on the paper. (C) Photo before and after printing CMC-AuNP inks on the paper substrate. Scale bar=5 mm.
an
Since the hydrophilic SERS-active area has a 2-mm diameter, the size of the stencil hole with a 3-mm diameter was designed to be much wider in order to produce uniform SERS signals; the coffee ring effect caused by the
M
surface tension acting on the ink and the side of the mask wall drives ink to the outer hydrophobic wax part (Fig. 1B). A 4-µL aliquot of the CMC-AuNP ink was dropped on the stencil hole, and a blade was moved across a 0.18-mm thick
d
stencil. The substrate was dried on a hot plate at 50°C for 120 sec (one cycle). Figure 1C shows a photograph of the
Ac ce pt e
SERS-functionalized substrate after printing CMC-AuNP ink on the wax-impregnated paper.
2.5. Human tears
Tear biofluids were collected from the nasoinferior conjunctival sac without additional stimulation using a 025 ISO standard size, a 29-mm-long paper point (Henry Schein Inc, Melville, NY, USA). After a maximum of 5 min, the points were withdrawn from the eye and placed into a 1.5-mL Eppendorf tube. Tear fluids were extracted from the saturated points by centrifuging at 8,000 rpm for 10 min. The points were carefully removed, and the tear fluid was aspirated. Tear samples were stored in an Eppendorf tube sealed with Parafilm (Pechiney Plastic Packaging Company, Chicago, IL, USA) at −70°C for <24 h before they were analyzed. Informed consent was obtained from each subject at Kyung Hee University Medical Center. All procedures involving humans adhered to the Declaration of Helsinki and were approved by the Ethics Committee of Kyung Hee University College of Medicine (KMCIRB1401-02).
3. Results and discussion 3.1. Characterization of the AuNPs -6Page 6 of 17
ip t cr us an
Fig. 2. Characterization of the AuNPs and the CMC solution. (A) TEM image of the synthesized AuNPs. solutions with four different concentrations.
M
Scale bar=50 nm. (B) Size of the AuNPs. (C) UV-Vis absorption spectrum of colloidal AuNPs. (D) Viscosity of CMC
d
The characteristics of the citrate-capped AuNPs were evaluated using TEM images and UV-Vis spectra (Fig. 2). Based
Ac ce pt e
on the FE-TEM images, the AuNPs exhibited a stable monodispersity with an average diameter of 25±8 nm. The UVVis spectrum of the AuNPs colloids showed maximum absorption at a wavelength of 525 nm (λ0). Generally, a particle size ranging from 25 to 100 nm is determined from the following equation:
λmax − λ0 = L1 exp ( L2 d )
(1)
where λ0 indicates the maximum absorption wavelength, λmax indicates the maximum value of λ0, and d indicates the particle size d>25 nm (L1=6.53 and L2=2.16×10–2 are constants determined from the experiment results) [31]. The calculated average particle size was 31.9 nm when the peak wavelength of the AuNPs was 525 nm (Fig. 2C). The AuNPs might slightly aggregate in a colloid, but the calculated particle size was within the range of the TEM-measured particle sizes. The size of the AuNPs is a critical factor to determine SERS enhancement [27,32]. Some studies have revealed that metallic nanoparticles in a size range of 10–100 nm produce strong Raman intensities compared to other sizes [33,34].
3.2. Viscosity of the CMC solutions Since direct printing of only concentrated AuNPs colloids might not be suitable for uniform dispersion due to the low -7Page 7 of 17
viscosity, the AuNPs were added in a CMC solution, which is a conventional viscous agent with properties of water solubility and concentration-dependent viscosity, in order to optimize the viscosity of the printing inks [28]. Since CMC has a much higher viscosity than water, it is expected to play a critical role in AuNPs uniformity on paper. The viscosity of four different concentrations of the CMC solution was measured using a viscometer. The viscosity of the CMC increased exponentially with increasing concentration (Fig. 2D). A 3-wt% CMC solution produced too high of a viscosity to allow proper handling of the inks, while the low viscosity of the 0.5-wt% CMC solution resulted in
ip t
inhomogeneous dispersion of the AuNPs on the paper (Fig. S1, Supplementary Information). Both 1- and 2-wt% CMC solutions allowed proper adherence and distribution of AuNPs on the paper substrate. In addition, the high surface
cr
roughness of the paper substrate led to a synergistic effect on adhesion of the CMC solution, acting as a glue. The 1-
us
and 2-wt% CMC concentrations were selected as primarily screen printing ink conditions.
3.3. Surface of the CMC-AuNP ink-printed paper
an
Figure 3 shows SEM images of the printed CMC-AuNP on the paper substrate. The presence of disordered cellulose fibers was observed (Fig. 3A), and the porous structure between the cellulose fibers was filled with the sodium CMC
M
solution, demonstrating the viscosity of the bare and wax-impregnated papers (Fig. S2, Supplementary Information). The AuNPs were well distributed on the cellulose fibers (Fig. 3B) and within the range of a 2.4-μm focal spot size for
d
Raman spectrum measurement. The blurred SEM image of the surface of the CMC-AuNP-printed paper was due to its
Ac ce pt e
mixture with sodium CMC. In order to determine whether these particles (Fig. 3B) were AuNPs, EDX-based molecular observation of the particle composition was performed (Fig. S3, Supplementary Information). The EDX mapping suggested that the CMC-AuNP-loaded paper could be used as a SERS substrate.
Fig. 3. Distribution of the AuNPs on the paper substrate. (A) SEM images of the CMC-AuNP ink loaded on the paper substrate. Scale bar=50 μm. (B) Distribution of AuNPs (yellow arrow) on the paper substrate. Scale bar=500 nm.
3.4. Optimization of the CMC-AuNP inks The condition of the CMC-AuNP ink was optimized by controlling the ratio of AuNPs and CMC solution. A 2-μL -8Page 8 of 17
aliquot of 1 mM RhB (probe molecule of Raman spectroscopy) was dropped onto the SERS-active area to determine the optimal condition of the CMC-AuNP ink. The dominant Raman peaks of RhB were measured at 620 cm–1 (aromatic bending), 1201 cm–1 (aromatic C–H bending), and 1356 cm–1 (aromatic C–C stretching) [9]. The prominent Raman peak at 1356 cm–1 was selected as the reference peak of RhB analysis. The optimal CMC concentration with the maximum Raman intensity was estimated using the 1- and 2-wt% CMC concentrations. Ink solutions with a 5:1 volume ratio of CMC solution to AuNPs were prepared at two CMC
ip t
concentrations. Three printing cycles of the CMC-AuNP ink (Fig. S4, Supplementary Information) showed that the combination with a 2-wt% CMC solution exhibited better SERS intensity and less spectral deviation than the 1-wt%
cr
CMC solution as the higher-viscosity CMC solution inhibited movement and better secured the AuNPs (Fig. S1, Supplementary Information). Furthermore, the rapid drying process of the CMC-AuNP inks at 50ºC during the printing
us
cycles prevented the AuNPs from spreading to the edges and allowed them to adequately disperse over SERS-active area. Therefore, 2-wt% CMC-AuNP was selected as the optimal printing ink combination.
an
The effect of AuNP amount on Raman intensity was investigated by adjusting the volume ratio of AuNPs in the screen printing ink solution. For this purpose, 2-wt% CMC was mixed with AuNPs at three volume ratios; 5:1, 7:1,
M
and 10:1. Figure 4A shows the Raman intensity of the three CMC-AuNP ink solutions with different volume ratios according to printing cycle. The 7:1 CMC-AuNP ink solution exhibited a good SERS effect compared to the other
d
volume ratios. The maximum SERS intensity was observed in two printing cycles with a 7:1 ratio of CMC-AuNPs. The
Ac ce pt e
resonance of nanoparticles at the paper surface is caused by the incident energy in the few nanometers between the particles. The reflected energy is amplified by the scattering according to the resonance, and this energy has a significant impact on the SERS effect. The space between the particles is influenced by the particle number. When the size of the nanoparticles is constant and their number increases in a limited area, the distance between particles decreases, and the Raman scattering increases. However, when the number of particles increases above a certain limit, the AuNPs demonstrate film characteristics rather than acting as separate particles. Under such conditions, the resonance of nanoparticles decreases, and the Raman intensity decreases [11]. These changes were observed in the present study; two printing cycles led to two-fold increases in SERS intensity compared to that after one printing cycle. However, additional printing cycles on the paper substrate resulted in a decrease in SERS intensity and a larger deviation of Raman peaks. In order to validate the reliability of the measurement, the SERS spectra were analyzed randomly at 10 different positions utilizing the optimized two cycles of a 7:1 CMC-AuNP ink solution. The mean intensity of the spot-to-spot variations in the SERS peaks at 1356 cm–1 was 5,858±279 (Table S1, Supplementary Information), and the corresponding relative standard deviation (RSD) was 4.7%. The intensity of the SERS signals was affected by the SERS effects, laser source focusing, biosamples, and several other variables. Although the Raman -9Page 9 of 17
intensity varied among the detection sites, the overall less than 5% variance affirms the high reproducibility of the
M
an
us
cr
ip t
SERS method.
Fig. 4. Optimization of the CMC-AuNP inks. (A) The Raman intensity of CMC-AuNP ink solutions with three
d
volume ratios of CMC solution and AuNPs of CMC:AuNPs=5:1, 7:1, and 10:1, according to printing cycle. (B) High
Ac ce pt e
reproducibility of a 2-wt% 7:1 CMC-AuNP ink solution. RSD=relative standard deviation.
In additional, we evaluated the distribution of CMC-AuNP inks within the SERS-active area on the paper substrate. The Raman signals of six points from the center zone to the ring zone of the SERS-active area (a 1-mm diameter with the interval of 200 μm) were measured (Fig. S5, Supplementary Information). The spectral profile of Raman peaks at 1356 cm–1 showed a low RSD regardless of the zones. This indicates that the proposed fabrication method leads to homogeneous dispersion of the AuNPs on the paper substrate, the absence of the coffee ring effect. Therefore, the noise-independence, uniformity, and reproducibility of the SERS spectral signals suggest that the proposed SERS assessment has the potential to be used as a highly sensitive and selective assessment for biological fluids.
3.5. SERS activity of the fabricated paper platform To quantify the SERS activity of the screen printing ink, the Raman spectra of the SERS-functionalized (CMC-AuNP ink-printed) and bare (intact) paper were measured using 1-mM and 1-M RhB, respectively (Fig. S6, Supplementary Information). The Raman intensity of the 1356 cm-1 band (5,858) of a 1-mM RhB on the SERS paper demonstrated an - 10 Page 10 of 17
approximately 18-fold increase compared to that of 1-M RhB on bare paper (328). Therefore, the EF was calculated as the difference in Raman intensities between two different substrates; ⎛I ⎞⎛ N ⎞ EF = ⎜ SERS ⎟ ⎜ bare ⎟ ⎝ I bare ⎠ ⎝ N SERS ⎠
(2)
where ISERS and Ibare indicate the Raman intensity of the molecule on the SERS and bare papers, respectively, and NSERS and Nbare indicate the average number of adsorbed molecules in the scattering volume for the SERS and non-SERS
ip t
areas, respectively [6]. Assuming that the probe molecules are uniformly distributed on the substrates, the number of adsorbed molecules can be estimated as ⎞ ⎟⎟ Alaser ⎠
cr
⎛ Vdroplet N = ⎜ NA ⋅c⋅ ⎜ Aspot ⎝
(3)
us
where NA is Avogadro’s constant, c is the concentration of the probe molecule, V is the volume of the molecule droplet, Aspot is the size of the substrate, and Alaser is the size of the laser spot [8,9]. Since the same methods for assessing the
an
Raman measurement were applied to two substrates, the parameters of NA of RhB, V, Aspot, and Alaser were the same. Hence, Eq. (2) can be written as
M
⎛I ⎞⎛ c ⎞ EF = ⎜ SERS ⎟ ⎜ bare ⎟ ⎝ I bare ⎠ ⎝ cSERS ⎠
(4)
d
where cSERS and cR indicate the concentration of RhB on the SERS and bare papers, respectively. The final EF at the
Ac ce pt e
1356 cm-1 band was 1.8×104. This result indicates the presence of SERS activity on the CMC-AuNP ink-printed paper. Although the EF value was smaller than those of other materials and structures, such as bimetallic nanoparticles [35], noble metal-coated nanowires [7,9], and sophisticated nanoscale pattern arrays [10], the CMC-AuNP ink-printed SERS paper substrate with a simple and low-cost fabrication is likely to be a useful platform with great potential for application in POC diagnostics.
- 11 Page 11 of 17
ip t cr us an
M
Fig. 5. Representative SERS spectra and analytical peak assignments for normal (control), (A) adenoviral conjunctivitis-infected, and (B) herpes simplex conjunctivitis-infected human tear fluids. The intensity of three Raman peaks at 1003 cm–1 (the ring of phenylalanine), 1242 cm–1 (amide III β-sheet), and 1342 cm–1 (C–H deformation in proteins) could be used for classifying the differences among tears of normal, adenoviral
Ac ce pt e
d
conjunctivitis, and herpes simplex conjunctivitis subjects.
3.6. In vivo application
Figure 5 shows the typical SERS spectra of normal and two infected tears acquired using the fabricated SERSfunctionalized paper sensor platform. Each SERS peak shows the distinct vibration characteristics of human tear fluids (Table S2, Supplementary Information). Without an additional smoothing algorithm, all tear-induced SERS spectra were normalized by setting the variance of the Raman spectral signal to a value of 1.0, with the intense peak located at 1003 cm–1 (the ring of phenylalanine). There was no shift in these SERS peaks between normal and infected tears. However, the SERS intensity of the two prominent peaks at 1242 and 1342 cm–1 varied between the normal and infected tears. Hence, the intensity ratio of C–H deformation at 1342 cm–1 to the amide III β-sheet at 1242 cm–1 could be used as a marker to detect the presence of adenovirus virus and herpes simplex virus,
δ VIRUS =
I1342 I1242
(5)
where I1242 and I1342 indicate the SERS spectral intensity of the amide III β-sheet and C–H deformation at the 1242 and 1342 cm–1 peaks, respectively [29]. - 12 Page 12 of 17
For example, the normal tears showed intensities of 0.3830 for the 1242 cm–1 peak and 0.3075 for the 1342 cm–1 peak, while the tears infected with adenovirus showed an intensity of 0.3782 at 1242 cm–1 and 0.7057 at 1342 cm–1, and the tears infected with herpes simplex virus showed an intensity of 0.4579 at 1242 cm–1 and 1.1307 at 1342 cm–1. The I1342/I1242 ratio was 0.80 for normal tears, 1.86 for adenovirus-infected tears, and 3.73 for herpes simplex virusinfected tears (Fig. S7, Supplementary Information). Therefore, the threshold value, δVIRUS>1, can be used as a marker for detecting the presence of virus in tears. Furthermore, the difference between two virus-infected tear fluids can be
ip t
classified according to the following rule: if δVIRUS>2 and I1342>1, then the infection is herpes simplex conjunctivitis. This significant difference indicates that the tear-induced Raman spectral assessment using a fabricated SERS-
cr
functionalized paper sensor platform is an effective signature for detecting and classifying adenoviral conjunctivitis and herpes simplex conjunctivitis. Finally, the supporting vector machine rule-based classification system supported by the
us
principal component analysis (PCA) algorithm is likely to show enhanced, early detection of adenoviral and herpes
an
simplex conjunctivitis.
4. Conclusions
M
A SERS-functionalized paper sensor platform for analyzing biofluids based on a solution of AuNP-loaded screen printing ink was introduced. For the SERS functionalization of a simple paper, the optical property of SPR was
d
implemented using the citrate-capping method based on monometallic gold nanoparticles. The viscous property of
Ac ce pt e
sodium CMC solution was used to improve the uniformity and reproducibility of the SERS spot-on assay of biological fluids. A paper-based SERS sensor was fabricated with simple and facile processes that do not require professional knowledge of chemical engineering or expensive equipment. The EF of the proposed SERS sensor platform using the Raman spectroscopy probe molecule RhB was approximately 2×104 under optimized conditions consisting of two screen printing cycles of a viscous ink with a 7:1 mixture of 2-wt% CMC and AuNPs. The in vivo application of two representative ocular infection diseases (conjunctivitis) showed clear Raman peaks and SERS effects to support rapid POC diagnosis of biofluids in clinics. Based on this promising paper platform, the bimetallic core-shell structure of AgNPs and the AuNPs lead to superior SERS enhancement compared to a substrate with only AuNP.
Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant 2014R1A1A2054452).
Supplementary data - 13 Page 13 of 17
Ac ce pt e
d
M
an
us
cr
ip t
Supplementary data associated with this article can be found in the online version.
- 14 Page 14 of 17
References
S. Zeng, D. Baillargeat, H.-P. Ho, K.-T. Yong, Nanomaterials enhanced surface plasmon resonance for biological and chemical sensing applications., Chem. Soc. Rev. 43 (2014) 3426–52. doi:10.1039/c3cs60479a.
[2]
L. Polavarapu, J. Pérez-Juste, Q.-H. Xu, L.M. Liz-Marzán, Optical sensing of biological, chemical and ionic species through aggregation of plasmonic nanoparticles, J. Mater. Chem. C. (2014) 7460–7476. doi:10.1039/C4TC01142B.
[3]
X. Liu, Y. Shao, Y. Tang, K.-F. Yao, Highly Uniform and Reproducible Surface Enhanced Raman Scattering on Air-stable Metallic Glassy Nanowire Array, Sci. Rep. 4 (2014) 5308. doi:10.1038/srep05835.
[4]
K. Kneipp, Y. Wang, H. Kneipp, L.T. Perelman, I. Itzkan, R.R. Dasari, et al., Single molecule detection using surface-enhanced Raman scattering (SERS), Phys. Rev. Lett. 78 (1997) 1667–1670. doi:10.1103/PhysRevLett.78.1667.
[5]
A. Campion, P. Kambhampati, Surface-enhanced Raman scattering, Chem. Soc. Rev. 27 (1998) 241. doi:10.1039/a827241z.
[6]
E.C. Le Ru, E.J. Blackie, M. Meyer, P.G. Etchegoin, Surface Enhanced Raman Scattering Enhancement Factors: A Comprehensive Study, J. Phys. Chem. C. 111 (2007) 13794–13803. doi:10.1021/jp0687908.
[7]
J. Hu, S. Chen, R.P. Johnson, X. Lin, Z. Yang, A.E. Russell, Surface-enhanced raman scattering on uniform Pd and Pt films: From ill-defined to structured surfaces, J. Phys. Chem. C. 117 (2013) 24843–24850. doi:10.1021/jp4081433.
[8]
L. Zhang, X. Lang, A. Hirata, M. Chen, Wrinkled nanoporous gold films with ultrahigh surface-enhanced raman scattering enhancement, ACS Nano. 5 (2011) 4407–4413. doi:10.1021/nn201443p.
[9]
X. Liu, Y. Shao, Y. Tang, K.-F. Yao, Highly Uniform and Reproducible Surface Enhanced Raman Scattering on Air-stable Metallic Glassy Nanowire Array., Sci. Rep. 4 (2014) 5835. doi:10.1038/srep05835.
[10]
F. De Angelis, F. Gentile, F. Mecarini, G. Das, M. Moretti, P. Candeloro, et al., Breaking the diffusion limit with super-hydrophobic delivery of molecules to plasmonic nanofocusing SERS structures, Nat. Photonics. 5 (2011) 682–687. doi:10.1038/nphoton.2011.222.
[11]
E.D. Diebold, N.H. Mack, S.K. Doom, E. Mazur, Femtosecond Laser-Nanostructured substrates for SurfaceEnhanced raman scattering, Langmuir. 25 (2009) 1790–1794. doi:10.1021/la803357q.
[12]
X.M. Lin, Y. Cui, Y.H. Xu, B. Ren, Z.Q. Tian, Surface-enhanced raman spectroscopy: Substrate-related issues, Anal. Bioanal. Chem. 394 (2009) 1729–1745. doi:10.1007/s00216-009-2761-5.
[13]
S. Zeng, X. Yu, W.-C. Law, Y. Zhang, R. Hu, X.-Q. Dinh, et al., Size dependence of Au NP-enhanced surface plasmon resonance based on differential phase measurement, Sensors Actuators B Chem. 176 (2012) 1128– 1133. doi:10.1016/j.snb.2012.09.073.
[14]
B. Ren, G.-K. Liu, X.-B. Lian, Z.-L. Yang, Z.-Q. Tian, Raman spectroscopy on transition metals., Anal. Bioanal. Chem. 388 (2007) 29–45. doi:10.1007/s00216-007-1141-2.
[15]
L.L. Qu, Q.X. Song, Y.T. Li, M.P. Peng, D.W. Li, L.X. Chen, et al., Fabrication of bimetallic microfluidic surface-enhanced Raman scattering sensors on paper by screen printing, Anal. Chim. Acta. 792 (2013) 86–92. doi:10.1016/j.aca.2013.07.017.
[16]
A.W. Martinez, S.T. Phillips, M.J. Butte, G.M. Whitesides, Patterned paper as a platform for inexpensive, lowvolume, portable bioassays, Angew. Chemie - Int. Ed. 46 (2007) 1318–1320. doi:10.1002/anie.200603817.
Ac ce pt e
d
M
an
us
cr
ip t
[1]
- 15 Page 15 of 17
W. Zhang, B. Li, L. Chen, Y. Wang, D. Gao, X. Ma, et al., Brushing, a simple way to fabricate SERS active paper substrates, Anal. Methods. 6 (2014) 2066. doi:10.1039/c4ay00046c.
[18]
L. Polavarapu, A. La Porta, S.M. Novikov, M. Coronado-Puchau, L.M. Liz-Marzán, Pen-on-paper approach toward the design of universal surface enhanced Raman scattering substrates, Small. 10 (2014) 3065–3071. doi:10.1002/smll.201400438.
[19]
R.S.J. Alkasir, M. Ornatska, S. Andreescu, Colorimetric paper bioassay for the detection of phenolic compounds, Anal. Chem. 84 (2012) 9729–9737. doi:10.1021/ac301110d.
[20]
C. Renault, M.J. Anderson, R.M. Crooks, Electrochemistry in hollow-channel paper analytical devices, J. Am. Chem. Soc. 136 (2014) 4616–4623. doi:10.1021/ja4118544.
[21]
Z. Zhang, W. Xu, N.E. Manicke, R.G. Cooks, Z. Ouyang, Silica coated paper substrate for paper-spray analysis of therapeutic drugs in dried blood spots, Anal. Chem. 84 (2012) 931–938. doi:10.1021/ac202058w.
[22]
W.W. Yu, I.M. White, Inkjet printed surface enhanced raman spectroscopy array on cellulose paper, Anal. Chem. 82 (2010) 9626–9630. doi:10.1021/ac102475k.
[23]
B. Li, W. Zhang, L. Chen, B. Lin, A fast and low-cost spray method for prototyping and depositing surfaceenhanced Raman scattering arrays on microfluidic paper based device, Electrophoresis. 34 (2013) 2162–2168. doi:10.1002/elps.201300138.
[24]
A.K. Yetisen, M.S. Akram, C.R. Lowe, Paper-based microfluidic point-of-care diagnostic devices., Lab Chip. 13 (2013) 2210–51. doi:10.1039/c3lc50169h.
[25]
Y.H. Ngo, D. Li, G.P. Simon, G. Garnier, Gold nanoparticle-paper as a three-dimensional surface enhanced Raman scattering substrate., Langmuir. 28 (2012) 8782–90. doi:10.1021/la3012734.
[26]
Y.H. Ngo, W.L. Then, W. Shen, G. Garnier, Gold nanoparticles paper as a SERS bio-diagnostic platform, J. Colloid Interface Sci. 409 (2013) 59–65. doi:10.1016/j.jcis.2013.07.051.
[27]
G. Zheng, L. Polavarapu, L.M. Liz-Marzán, I. Pastoriza-Santos, J. Pérez-Juste, Gold nanoparticle-loaded filter paper: a recyclable dip-catalyst for real-time reaction monitoring by surface enhanced Raman scattering, Chem. Commun. (2015) 4572–4575. doi:10.1039/C4CC09466B.
[28]
L.-L. Qu, D.-W. Li, J.-Q. Xue, W.-L. Zhai, J.S. Fossey, Y.-T. Long, Batch fabrication of disposable screen printed SERS arrays, Lab Chip. 12 (2012) 876. doi:10.1039/c2lc20926h.
[29]
S. Choi, S.W. Moon, J. Shin, H. Park, K. Jin, Label-Free Biochemical Analytic Method for the Early Detection of Adenoviral Conjunctivitis Using Human Tear Biofluids, Anal. Chem. 86 (2014) 11093–11099. doi:10.1021/ac5025478.
[30]
C.-Y. Lin, C.-H. Liu, W.-L. Tseng, Fluorescein isothiocyanate-capped gold nanoparticles for fluorescent detection of reactive oxygen species based on thiol oxidation and their application for sensing glucose in serum, Anal. Methods. 2 (2010) 1810. doi:10.1039/c0ay00428f.
[31]
W. Haiss, N.T.K. Thanh, J. Aveyard, D.G. Fernig, Determination of size and concentration of gold nanoparticles from UV-Vis spectra, Anal. Chem. 79 (2007) 4215–4221. doi:10.1021/ac0702084.
[32]
C. Shen, C. Hui, T. Yang, C. Xiao, J. Tian, L. Bao, et al., Monodisperse noble-metal nanoparticles and their surface enhanced raman scattering properties, Chem. Mater. 20 (2008) 6939–6944. doi:10.1021/cm800882n.
[33]
C. Wang, C. Yu, Analytical characterization using surface-enhanced Raman scattering (SERS) and microfluidic sampling, Nanotechnology. 26 (2015) 092001. doi:10.1088/0957-4484/26/9/092001.
[34]
K. Kim, K.S. Shin, Surface-enhanced Raman scattering: a powerful tool for chemical identification., Anal. Sci. 27 (2011) 775. doi:10.2116/analsci.27.775.
Ac ce pt e
d
M
an
us
cr
ip t
[17]
- 16 Page 16 of 17
[35]
L. Bi, J. Dong, W. Xie, W. Lu, W. Tong, L. Tao, et al., Bimetallic gold-silver nanoplate array as a highly active SERS substrate for detection of streptavidin/biotin assemblies, Anal. Chim. Acta. 805 (2013) 95–100. doi:10.1016/j.aca.2013.10.045.
*Authors CVs Wan-Sun Kim earned a dual M.S. degree from the Department of Information Display at Kyung Hee University,
ip t
South Korea, and the Sciences and Technologies at Ecole Polytechnique, France, in 2007. From 2012, he studied nBioMechatronics and biosensors on a doctorate path in the Department of Medical Engineering, College of Medicine
cr
at Kyung Hee University.
us
Jae-Ho Shin received an M.D., Ph.D. from the School of Medicine at Kyung Hee University, South Korea, in 1999. He received a Ph.D. in Ophthalmology from Kyung Hee University, South Korea, in 2013. Since 2013, he has worked as an Assistant Professor in the Department of Ophthalmology, Kyung Hee University Hospital at Gangdong, South
an
Korea. Dr. Shin’s main research interests include application of biomedical tools and techniques in ophthalmology,
M
especially oculoplastic surgery.
Hun-Kuk Park received an M.D. and B.M. from the School of Medicine at Kyung Hee University, South Korea, in 1982. He received a Ph.D. in Biomedical Engineering from Rutgers University and the University of Medicine and
d
Dentistry of New Jersey, USA, in 1993. Since 2008, he has worked as the Chairman and Professor in the Department of Biomedical Engineering, School of Medicine at Kyung Hee University, South Korea. Dr. Park’s main research interests
Ac ce pt e
include neuroscience and neurosurgery, biomechanics, nano-biosensors, pain mechanisms and analysis, nanoacupuncture techniques, bioinformatics/pharmacogenomics, and AFM applications for medical science.
Samjin Choi received a Ph.D. in Mechanical Engineering (Electrical Systems) from Yamaguchi University, Japan, in 2006. He served as an Assistant Professor (2006–2008) in the Department of Mechanical Engineering at Yamaguchi University, Japan. Since 2013, he has worked as an Assistant Professor in the Department of Medicine, College of Medicine at Kyung Hee University, Korea. Dr. Choi’s main research interests are nBioMechatronics (http://biomed.khu.ac.kr/xe/prof05). He has published more than 70 papers in peer-reviewed SCI-indexed international journals.
- 17 Page 17 of 17