Detection of Extremely Low Concentrations of Biological Substances Using Near-Field Illumination

www.nature.com/scientificreports OPEN received: 26 August 2016 accepted: 21 November 2016 Published: 19 December 2016 Detection of Extremely Low Concentrations of Biological Substances Using Near-Field Illumination Masato Yasuura & Makoto Fujimaki An external force-assisted near-field illumination biosensor (EFA-NI biosensor) detects a target substance that is propelled through an evanescent field by an external force. The target substance is sandwiched between an antibody coupled to a magnetic bead and an antibody coupled to a polystyrene bead. The external force is supplied by a magnetic field. The magnetic bead propels the target substance and the polystyrene bead emits an optical signal. The detection protocol includes only two steps; mixing the sample solution with a detection reagent containing the antibody-coated beads and injecting the sample mixture into a liquid cell. Because the system detects the motion of the beads, the sensor allows detection of trace amounts of target substances without a washing step. The detection capability of the sensor was demonstrated by the detection of norovirus virus-like particles at a concentration of ~40 particles per 100 μl in contaminated water. Immunoassay-based methods for detection of biological substances, such as enzyme-linked immunosorbent assays and immunochromatography, have been widely used in various applications1–3. These methods, referred to as “sandwich assays”, employ the formation of a “sandwich” of the target substance between two different antibodies4,5. One of the antibodies captures the target substance on a sensing substrate, and the other provides a label. Such detection methods simultaneously exhibit high sensitivity and specificity. To achieve high sensitivity, a variety of electrical, mechanical, and optical sensors have been developed, such as the field effect transistor type sensor6, the microelectric-mechanical system type sensor7, and the surface plasmon resonance sensor8. They can detect changes in physical properties at the surface of a sensing substrate with high sensitivity. These sensing methods are highly sensitive because the capturing antibody concentrates target substances on a surface and the second labeled antibody enhances the signal. However, sensors that utilize surfaces have a serious disadvantage: non-specific adsorption of contaminants, which decreases their sensitivity and specificity9. To reduce non-specific adsorption, countless blocking and washing protocols have been developed, and various blocking and washing reagents are currently available. However, complete suppression of non-specific adsorption has not been achieved. To detect biomolecules at extremely low concentrations, a minimum volume of the sample solution must be applied so that the sample solution does contain the target substance. Thus, for sample solutions with lower concentrations, higher volumes must be applied. An ideal immunosensor that can overcome the limitations of the current methods should have a wide effective sensing area and detect trace amounts of target substances in the presence of non-specific adsorption. In this study, we developed an “external force-assisted near-field illumination biosensor” (EFA-NI biosensor). The EFA-NI biosensor detects target substances that are propelled through an evanescent field by an external force. Candidates for the application of external force include magnetic force, electric force, and gravity. In the work described in the present report, we employed magnetic force as the external force. The sensing area of the EFA-NI biosensor is not a surface, but a free space near the surface. Using “moving signals” for detection, signal from the target substance can easily be distinguished from noise. Thus, the EFA-NI biosensor has the advantages, but not the disadvantages, of surface-based sensing methods. Electronics and Photonics Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan. Correspondence and requests for materials should be addressed to M.F. (email: ) Scientific Reports | 6:39241 | DOI: 10.1038/srep39241 1 www.nature.com/scientificreports/ Figure 1. Configuration of the EFA-NI biosensor system. (a) Schematic of the sensor setup. A Xe lamp provides incident light via the optical fiber, collimator lens, and polarizer to the trapezoidal prism parallel to the sensor chip. The optical detection unit is a microscope equipped with an objective lens with a magnification of 5x. (b) Schematic showing the detection mechanism of the sensor. The particles T, M, and O are the target substance, the magnetic bead, and the bead for optical signal, respectively. The region marked with the red gradient is the region where the enhanced electric field is generated. Results EFA-NI biosensor. The EFA-NI biosensor utilizes an enhanced electric field created by using near-field optics. It has been reported that a layered waveguide structure can generate a stronger and thicker enhanced electric field10–12. Therefore, a sensor chip with a 36-nm thick Si layer and 364-nm thick SiO2 layer placed on a 0.725-mm thick SiO2 substrate was employed in the present system. When S-polarized light is transmitted on to the sensor chip through a prism that is optically attached to the sensor chip at an incident angle of 67.6°, an enhanced electric field with a central wavelength of 644 nm is generated on the surface of the sensor chip. Figure S1 shows the calculated intensity of the electric field generated around the surface of the sensor chip under irradiation with S-polarized 644-nm light. The surface was assumed to be immersed in water. The intensity of the electric field at the sensor surface is 124 times stronger than that of the incident light. The intensity decreases with an increase in the distance from the surface. At a distance of 1200 nm from the surface, the intensity of the electric field is almost equal to that of the incident light. Figure 1a shows the setup of the EFA-NI biosensor. The sensor chip is placed on the bottom surface of a trapezoidal SiO2 glass prism with a bottom angle, α, of 32°. The surface of the sensor chip is illuminated with light from a Xe lamp through an optical fiber, a collimator lens, a polarizer, and the prism. The incident light is S-polarized. As shown in Fig. 1a, the light is transmitted on to the prism parallel to the sensor chip, and the incident angle, θ, becomes 67.6° at λ = 644 nm. A microscope equipped with an objective lens with a magnification of 5x and a cooled charge-coupled device (CCD) camera constitute the optical detection unit, as shown in Fig. 1a. The field of view of the system is approximately 2.5 mm × 2.0 mm. A 5-mm thick silicone rubber plate with an 8-mm diameter through-hole is placed on the sensor chip as a liquid cell. Two neodymium magnets (remanent flux density: 1.13 T) are used to apply the external magnetic force. One is placed under (...truncated)