Applied Science and Convergence Technology 2016; 25(3): 61-65
Published online May 30, 2016
Copyright © The Korean Vacuum Society.
Hoang Hiep Nguyena,c,†, So Yeon Yib,†, Abdela Woubitd, and Moonil Kima,c,d,*
aBioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahangno, Yuseong-Gu, Daejeon 305-806, Korea, bBioNano H-Guard Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahangno, Yuseong-Gu, Daejeon 305-806, Korea, cDepartment of Nanobiotechnology, Korea University of Science and Technology (UST), 217, Gajeongno, Yuseong-Gu, Daejeon 305-350, Korea, dDepartment of Pathobiology, College of Veterinary Medicine Nursing & Allied Health (CVMNAH), Tuskegee University, Tuskegee, AL 36088, USA
Correspondence to:E-mail: firstname.lastname@example.org
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Here, the rapid detection of
Keywords: Biosensor, surface plasmon resonance, portable SPR,
As a label-free technique, the surface plasmon resonance (SPR) system does not require any tag or dye to recognize biomolecules such as nucleic acids, proteins, peptides, small molecules, and cells. SPR angle changes result from alterations in the refractive index upon binding of analytes to the gold surface. To date, a wide variety of SPR biosensors have been developed for various applications including foodborne pathogen detection, environmental monitoring, and medical diagnosis [8?10]. From a practical point of view, miniaturization is important in the development of a portable SPR-type biosensor. Traditional SPR devices are of large size and heavy weight. Thus, they are not portable or cost-effective for use in point-of-care (POC) applications. In this regard, it is noteworthy that current need for the rapid detection of biohazardous agents such as bacterial or viral pathogens that endanger public safety is driving increased demand for a portable SPR system.
A variety of immunoassays based on SPR-type sensors have been developed for detection of
Phosphate buffered saline (PBS, P4417), Bovine serum albumin (BSA, A7906), and thimerosal (T2299) were purchased from Sigma Aldrich Inc. (St. Louis, MO). Anti-Salmonella antibody directed against lipopolysaccharides (LPS) (ab8274) was purchased from Abcam Inc. (Cambridge, MA). The
The growth curve of
The Cys-tagged protein G construct was prepared as described previously . Briefly, the plasmid, which contains the gene encoding the IgG-binding protein G and a Cys-tag, was transferred into
The SPR sensor system using a rotating mirror uses laser diode rather than LED as a light source for emitting the incident light to the gold thin film. The rotating mirror having cylindrical shapes reflects the polarized incident light for radiating light with disk-shape. A cylinder lens help to focus the light rays which pass through a light shielding film, and the gold thin film absorbs the focused light to create surface plasmon resonance. A dielectric medium was provided under the gold thin film, and the light reflected from the metal thin film was captured by a CMOS image sensor.
A pre-cleaned gold chip was rinsed with PBS (pH 7.4), which was used as a running buffer. The cysteine-tagged protein G at a concentration of 0.2 μg/mL was attached to the gold surface at room temperature by 100 μL injection of the solution through the SPR channel at a flow rate of 5 μL/min. Subsequently, anti-
Figure 1 shows the
Surface plasmons (SPs) are composed of oscillating waves of free electrons that travel along the interface between a metal thin film and a dielectric. When incident light enters a thin layer region via a dielectric medium (i.e., a prism), the SPs are excited and cause a plasmon resonance wave if the incident angle exceeds the critical angle of the prism. Surface plasmon resonance (SPR) is characterized by the resonant oscillation of free electrons. SPR angle (or resonance angle) is highly sensitive to variations of the refractive index. Thus, the value of the resonance angle depends on changes in the refractive index of the metal surface. SPR detection modules are based on this theoretical background for detection of a wide range of target analytes. The prism-based Kretschmann configuration, in which a laser is used as a light source because it can deliver a fast-pulse and high-intensity beam to a target, is adopted in most SPR applications to measure the SPR angle. In this mode, reflected light may cause flaring or ghosting, resulting in an interference problem, which can deteriorate the image quality [8,15]. In order to establish an SPR detection system for more elaborate measurement of analytes, an electrical measurement unit with extreme stability and precision is required. For this reason, the use of lasers as light sources is not suitable for the development of compact and portable SPR biosensor systems. To solve this issue, a light-emitting diode (LED) could offer a hopeful alternative to laser-based systems, because LEDs produce no interference problems. SPR sensors normally use an angular reflection spectrum based on a monochromatic light source. However, LED light is not monochromatic, while a laser emits nearly monochromatic light, occurring at a single wavelength. The LED spectral widths range from 20 nm to 170 nm; this range induces a broad angular spectrum in an SPR sensor, lowering the SPR sensitivity. In this case, an additional monochromatic filter should be applied to offer a narrow range of wavelengths. Oscillation mirror-based instruments, in which a mirror adjusts the incident angle at which surface plasmons are excited, also allow the detection of analytes of interest. However, for the mirror format, the oscillation mirror should be exactly synchronized with the optical excitation of the photodiode to read the intensity of the reflected light. For this reason, oscillation mirror-mediated SPR systems may not be suitable for portable applications. To solve the abovementioned problems, a rotating mirror instead of a prism as a dielectric medium was integrated into the SPR system to develop a hand-held SPR device (Figure 2).
The rotating mirror adjusts the angle of incidence, optimizing it for the SPR. Using this portable SPR to modulate the laser beam with a rotating mirror of cylindrical shape, changes in resonance angle were determined by measuring the reflected intensity of light upon binding of
For the purpose of orientation-controlled immobilization of antibodies on the SPR sensor surface, the cysteine-mediated immobilization method, which is based on the interaction between a gold surface and the thiol group (?SH) of cysteine, was adopted. Particularly, protein G fused with Cys-tag was used as an antibody-capturing agent because protein G has the ability to bind to fragment crystallizable regions (Fc regions) of heavy chains of antibodies. The protein G-mediated antibody immobilization strategy is a single step method with no need for antibody pre-modifications such as pre-activation or coupling. Therefore, immobilized antibodies mediated by protein G do not lose their binding strength or specificity. Studies have demonstrated that an SPR immunosensor based on the orientation-controlled immobilization of antibodies mediated by Cys-tagged protein G offered considerably enhanced sensitivity compared with conventional covalent immobilization methods, which are prone to random orientation of probe antibodies. Lee et al
The rotating mirror-based portable SPR device was developed for rapid and specific detection of
The PBS buffer solution (pH 7.4) was introduced to the flow cell of the SPR device; then, 10^7, 10^8, and 10^9 CFU/mL of
Upon return of PBS flow, the SPR signal remained constant. To evaluate the sensing performance of the portable SPR sensor in response to
Here, a portable SPR-based immunosensor for detection of one important food pathogen, in which a rotating mirror modulates a laser beam as an incident light source, was described. Especially, an SPR instrument having a small and lightweight module as a portable monitoring and diagnostic device for point-of-care (POC) application was used for rapid monitoring of
This work was supported by a Creative Allied Project (CAP) grant of the National Research Council of Science & Technology (NST, Korea), and the KRIBB Initiative Research Program (KRIBB, Korea).
Summary of SPR measurement.
|?Cys-Protein G (0.2 mg/mL)?||?Antibody (0.1 mg/mL)?|