Biosensing Amplification by Hybridization Chain Reaction on Phase-Sensitive Surface Plasmon Resonance

Abstract

Surface Plasmon Resonance (SPR) is widely used in biological and chemical sensing with fascinating properties. However, the application of SPR to detect trace targets is hampered by non-specific binding and poor signal. A variety of approaches for amplification have been explored to overcome this deficiency including DNA aptamers as versatile target detection tools. Hybridization chain reaction (HCR) is a high-efficiency enzyme-free DNA amplification method operated at room temperature, in which two stable species of DNA hairpins coexist in solution until the introduction of the initiator strand triggers a cascade of hybridization events. At an optimal salt condition, as the concentrations of H1 and H2 increased, the HCR signals were enhanced, leading to signal amplification reaching up to 6.5-fold of the detection measure at 30 min. This feature enables DNA to act as an amplifying transducer for biosensing applications to provide an enzyme-free alternative that can easily detect complex DNA sequences. Improvement of more diverse recognition events can be achieved by integrating HCR with a phase-sensitive SPR (pSPR)-tested aptamer stimulus. This work seeks to establish pSPR aptamer system for highly informative sensing by means of an amplification HCR. Thus, combining pSPR and HCR technologies provide an expandable platform for sensitive biosensing.

Keywords: aptamer; hybridization chain reaction; phase-sensitive surface plasmon resonance (pSPR) biosensor.

Figure 1

(A) Setup of the phase-sensitive surface plasmon resonance (pSPR) system. (B) Wavefront division of the birefringent crystal. (C) SPR chip. (D) Phase-sensitive SPR scan steps.

Figure 2

The design of three DNA aptamers that exhibit HCR signal amplification capabilities. (A) Schematic illustration of the HCR-pSPR detection aptamers. (B) Simulative schematics of the bare aptamer and the coupling DNA modified aptamer.

Figure 3

Schematic illustration of the HCR-pSPR self-assembly on gold surface detection platform.

Figure 4

(A) Real-time detection of the HCR reaction. In the pSPR result, after 30 min of the initiator and H1 binding reaction and the 30 min washing step marks A, the time point of 30 min after the addition of the H1 and H2 mixture marks B. (A + B)/A = 6.5, indicating a 6.5-fold of the detection measure at 30 min. (B) Combining pSPR and DNA gel electrophoresis results. The length of the HCR complex at the same reaction time, 30 min, can be observed.

Figure 5

Result of HCR reaction under different aptamer concentrations and ionic strength environments. (A) The concentration of H1, H2 and the subsequent HCR amplification are in direct proportion: while the concentration of H1 and H2 increases, HCR amplification increases. Blue: 1 µM H1+ 1 µM H2, Red: 500 nM H1 + 500 nM H2 and Black: 250 nM H1 + 250 nM H2. (B) Validation of salt concentration of HCR on pSPR with pre-immobilized initiator probe; 1.0 M, 1.5 M and 2.0 M NaCl in 1× PBS were added with 1 µM H1 and H2 mixture at 400 s, and injected 1× PBS buffer at 2530 s. Three HCR reactions showed 0.0007, 0.0018 and 0.0036 in Δ RIU indicating different salt concentrations affected HCR amplification results.