A low-cost, monometallic, surface-enhanced Raman scattering-functionalized paper platform for spot-on bioassays
Graphical abstract
Introduction
Raman spectroscopy is a technique to provide molecule-specific information on biological and chemical samples [1], [2], [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 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 modifying the materials and nanostructure patterns of the surface [7], [8], [9], [10], [11]. Most SERS-active areas are fabricated using complex and sophisticated methods including lithography or a high-temperature process. These SERS fabrications 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 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, 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 (AuNPs). However, AgNPs show rapid degradation of SERS activity due to oxidation in air, while AuNPs show stable 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], [17], [18]. These user-friendly advantages of paper offer various applications including colorimetric, electrochemical, and biochemical analyses [19], [20], [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], [23], [24]. Some studies have reported the use of colloidal AuNP-deposited SERS paper platforms for environmental and biochemical monitoring [25], [26], [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 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 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].
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 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 SERS paper platform was evaluated using in vivo human tear biofluids collected from patients with adenoviral conjunctivitis and herpes simplex conjunctivitis.
Section snippets
Reagents and materials
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/s was purchased from Sigma Aldrich.
Instrumentation
The size and shape of the AuNPs were characterized
Characterization of the AuNPs
The characteristics of the citrate-capped AuNPs were evaluated using TEM images and UV–vis spectra (Fig. 2). Based on the FE-TEM images, the AuNPs exhibited a stable monodispersity with an average diameter of 25 ± 8 nm. The UV-Vis 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:where λ0 indicates the maximum absorption wavelength, λmax indicates
Conclusions
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 implemented using the citrate-capping method based on monometallic gold nanoparticles. The viscous property of 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
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).
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).
Wan-Sun Kim earned a dual MS degree from the Department of Information Display at Kyung Hee University, 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 at Kyung Hee University.
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Wan-Sun Kim earned a dual MS degree from the Department of Information Display at Kyung Hee University, 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 at Kyung Hee University.
Jae-Ho Shin received an MD, PhD from the School of Medicine at Kyung Hee University, South Korea, in 1999. He received a PhD 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 Korea. Dr. Shin's main research interests include application of biomedical tools and techniques in ophthalmology, especially oculoplastic surgery.
Hun-Kuk Park received an MD and BM from the School of Medicine at Kyung Hee University, South Korea, in 1982. He received a PhD in Biomedical Engineering from Rutgers University and the University of Medicine and 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 include neuroscience and neurosurgery, biomechanics, nano-biosensors, pain mechanisms and analysis, nano-acupuncture techniques, bioinformatics/pharmacogenomics, and AFM applications for medical science.
Samjin Choi received a PhD 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.