Creating Advanced Multifunctional Biosensors with Surface Enzymatic Transformations

Abstract

This article summarizes our recent work on the coupling of surface enzyme chemistry and bioaffinity interactions on biopolymer microarrays for the creation of multiplexed biosensors with enhanced selectivity and sensitivity. The surface sensitive techniques of surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS) are used to detect the surface enzymatic transformations in real time. Three specific examples of novel coupled surface bioaffinity/ surface enzymatic processes are demonstrated: (i) a surface enzymatic amplification method utilizing the enzyme ribonuclease H (RNase H) in conjunction with RNA microarrays that permits the ultrasensitive direct detection of genomic DNA at a concentration of 1 fM without labeling or PCR amplification, (ii) the use of RNA-DNA ligation chemistry to create renewable RNA microarrays from single stranded DNA microarrays, and (iii) the application of T7 RNA polymerase for the onchip replication of RNA from double stranded DNA microarray elements. In addition, a simple yet powerful theoretical framework that includes the contributions of both enzyme adsorption and surface enzyme kinetics is used to quantitate surface enzyme reactivity. This model is successfully applied to SPRI and SPFS measurements of surface hydrolysis reactions of RNase H and Exonuclease III (Exo III) on oligonucleotide microarrays.

Porous SiO2Interferometric Biosensor for Quantitative Determination of Protein Interactions: Binding of Protein A to Immunoglobulins Derived from Different Species

“…The reaction of surface adsorbed protein A with IgG was modeled by the expression (2) where A ads represents the available adsorbed protein A binding sites, IgG represents free IgG in solution, AG represents the surface-bound protein A/IgG complex, and k a and k d are the kinetic rate constants for protein A/IgG binding and dissociation, respectively. The equilibrium dissociation constant (K d ) is defined as (3) and can be represented by the following equation (See Supporting Information): (4) Where ΔOT max represents the signal obtained when all protein A binding sites are occupied and ΔOT f is the steady-state value of ΔOT for a given [IgG].…”

Ultrasensitive Detection of DNA by PNA and Nanoparticle‐Enhanced Surface Plasmon Resonance Imaging

“…[6][7][8] It uses optical detectors for spatial monitoring of localized differences in the reflectivity of the incident light (D%R)-which can be seen as brighter or darker regions in the SPR image-from an array of biomolecules linked to chemically modified gold surfaces. Label-free and real-time analyses can be carried out with high throughput and low sample consumption by coupling microfluidic devices with the SPRI apparatus.…”

Resonant optical transmission through hole‐arrays in metal films: physics and applications

“…A typical sensitivity of prism coupling SPR systems is in the range between 3100 to 8000 nm/RIU (RIU = refractive index unit) [158]. Imaging SPR has also been implemented using the reflection configuration, allowing the multiplexing detection of several biological markers simultaneously [159,160]. However, the reflection geometry introduces distortions to the imaging and this arrangement is not easily miniaturized or integrated in lab-on-chip devices.…”