Microsphere-Based Rolling Circle Amplification Microarray for the Detection of DNA and Proteins in a Single Assay

Abstract

We describe a high-density microarray for simultaneous detection of proteins and DNA in a single test. In this system, Rolling Circle Amplification (RCA) was used as a signal amplification method for both protein and nucleic acid detection. The microsphere sensors were tested with synthetic DNA and purified recombinant protein analytes. The target DNA sequence was designed from a highly conserved gene that encodes the outer membrane protein P6 (OMP-P6) of both typeable and nontypeable strains of Haemophilus influenzae. The proinflammatory mediators IL-6 and IL-8 were selected as target proteins. Capture antibodies were first immobilized on fluorescently-encoded microspheres. The microspheres were then loaded into the etched microwells of an imaging optical fiber bundle. A sandwich assay was performed for target proteins IL-6 and IL-8 using biotin-labeled secondary antibodies. Biotinylated capture DNA probes were then attached to the detection antibodies via an avidin bridge. A padlock probe, complementary to the target sequence, was subsequently hybridized to the capture probe. In the presence of the target sequence, the padlock probe was ligated and this circular sequence was used for RCA. Following RCA, multiple fluorescently-labeled signal probes were hybridized to each amplified sequence and the microarray was imaged using an epi-fluorescence microscope. With this assay, detection limits down to 10 fM and 1 pM were achieved for proteins and target DNA, respectively. In addition to this new approach for detecting both protein and DNA in a single test using RCA, the limit of detection for IL-8 and IL-6 was improved by three orders of magnitude compared to similar microsphere-based assays.

Microfluidics for DNA and Protein Analysis with Multiplex Microbead-Based Assays

“…For example, Li et al developed a platform for quantification of single-nucleotide polymorphisms (SNPs) in clinical samples by microbead-based rolling circle amplification assay (RCA) [50]. Similarly, the presence of a protein and DNA could also be simultaneously detected on microbead surfaces [51]. In this approach, a two-layer sandwich assay was performed on the antibody-modified microbead for detection of protein and the presence of DNA in the same sample could be captured and detected by RCA strategy (Fig.…”

“…Walt's group developed an optic fiber platform and demonstrated its feasibility for genomic and proteomic analysis of clinical samples [51,[65][66][67][68]. The core of individual fibers was selectively etched to generate wells of predefined depth, which were used to assemble microbeads in array on the distal end of the fiber.…”

Particles and microfluidics merged: perspectives of highly sensitive diagnostic detection

“…Employing fiber bundles comprised of thousands of individually addressable fibers enables massively parallel detection capabilities in this approach. Both DNA [32, 36, 38, 39] and protein [34, 40] detection protocols were developed for multiplexed analysis of human saliva utilizing this approach. For the DNA detection protocol, a high-density array was fabricated to detect and quantify the hybridization of fluorescently labeled targets.…”

Analytical Chemistry on the Femtoliter Scale

“…[51] Beads were functionalized with antibodies against the proinflammatory target proteins IL-6 and IL-8. Then, a second biotin-labeled antibody was bound to the target proteins.…”