Surface Plasmon Resonance (SPR) Sensing Based on DNA Elongation by Site-Selective Surface Plasmon (SP) Field Heating toward Ultra-High Sensitive Detection of Single Pathogenic Particles

Abstract

The purpose of this work is to realize a novel enhancement method for surface plasmon resonance (SPR) sensing based on DNA elongation by surface plasmon (SP) field heating which shows promise for the detection of single pathogenic particles such as viruses and bacteria. The biosensor based on SPR is a powerful analytical tools for biological studies because it can monitor molecular interactions with high sensitivity and in a quantitative manner in real-time. The principle of a SPR sensor is based on the detection of the change in the dielectric constant on the surface due to the ligand-analyte binding. However, the change upon the 1:1 binding may be too small for detection of analytes at high dilutions. To address this limitation, some additional procedures have been proposed and applied: for example, the use of secondary antibody with gold colloid and DNA elongation from the analyte by using rolling circle amplification (RCA) induced by homogeneous heating (1). However, most of these methods have their associated limitations with respect to sensitivity enhancement mainly due to the non-specific binding of the additives, thereby increasing the background signal level. In the present study, we propose a novel sensitivity enhancement method for SPR analysis based on the DNA elongation by site-specific SP heating. Our concept is summarized in Fig. 1(a). At the SPR condition, the almost incident light energy is transferred to the SPs and finally converted to heat. Practical SPR biosensor systems employ a dim excitation light because the resulting heat disturbs the detection. In contrast, we intentionally employ a high-power laser as a light source to generate enough heat to drive the DNA polymerase. The SPR angle increases with increasing the amount of the bound analyte. Accordingly, when we illuminate the high power p-polarized laser at an incident angle (q’) greater than the SPR angle for the ligand (q), the SP fields should be effectively generated at the analyte binding site and the resulting heat can induce site-selective DNA elongation from the primers immobilized on/around the analytes. The amplified DNA (amplicons) elongating on/around the analytes could more effectively contribute to the shift of the SPR angle than the analytes themselves initially bound by the ligands. The amplicons can be detected not only by the shift of the SPR angle but also by SP enhanced fluorescence measurement, in which the number of the incorporated fluorophore is several thousands times larger than that of the analytes. We have employed RCA as a method for DNA elongation, in which the replication isothermally occurs from a small primer using a circular template and a polymerase with a strand displacement activity. A 50 nm gold film on a cover slip was immersed in a 1 mM dimethylformamide (DMF) solution of dithiobis-(succinimidyl undecanoate) and the resulting succinimidyl self-assembly monolayer (SI-SAM) was used for immobilization of the molecules. Site-selective SP heating was carried out by the multispot analysis. Anti-bovine serum albumin antibody (anti-BSA) and anti-mouse IgG were used as the ligand and the control, respectively. The four spots, consisting of two sets of two spots (one ligand and one control) with a 1-mm diameter, one ligand and one control, were prepared on the SI-SAM. The spots were covered with a 6-mm silicone rubber well and immersed in the solution of the analyte, which is primer-immobilized BSA. Then, the mixture solution of a circular template (M13mp18), 96-7 polymerase, and the nucleotide mixture was injected into the well. SP field heating was performed by illumination for 60 min with p-polarized laser (808 nm, 300 mW) at the upper-half area of the well at an offset angle of approximately +2° from the SPR angle. After the SP heating, the amplicons were stained with SYBR-Green I and observed by the confocal microscope and the SP enhanced fluorescence imager. The fluorescence from the analyte-bound and SP heated ligand spot was more intense than that of the control spot (Fig. 1(b) and (c)). This result indicates that, upon binding BSA, the ligand spots absorbed the excitation light and got heat from SP field more effectively than the control spot, which rapidly induces enzymatic DNA elongation giving the enhanced SPR and fluorescence signals. We have applied this method to the pathogenic particle detection. The intense fluorescence spots of the amplicons were observed around the 3-micrometer polystyrene latex beads as the dummy pathogen suggesting the site-selective heating around the particles. Further experimental data and detailed discussion will be presented. Reference 1. Y. Huang, H. Hsu, and C. C. Huang, Biosens. Bioelect., 22, 2007, 980-985. Figure 1