Microsphere-based rolling circle amplification microarray for the detection of DNA and proteins in a single assay

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biosensing

Background:

• Simultaneous detection of multiple analytes is crucial for comprehensive biological analysis.
• Existing microsphere-based assays have limitations in sensitivity and multiplexing capabilities.
• Haemophilus influenzae infections require sensitive detection of both bacterial DNA and host inflammatory responses.

Purpose of the Study:

• To develop a high-density microarray for simultaneous detection of proteins and DNA.
• To utilize Rolling Circle Amplification (RCA) for signal amplification in a multiplexed assay.
• To achieve enhanced sensitivity for detecting specific biomarkers.

Main Methods:

• Immobilization of capture antibodies on fluorescently encoded microspheres.
• Sandwich assay format for protein detection (IL-6, IL-8) and padlock probes for DNA detection.
• Rolling Circle Amplification (RCA) for signal amplification followed by fluorescent probe hybridization and imaging.

Main Results:

• Achieved detection limits of 10 fM for proteins and 1 pM for DNA.
• Demonstrated simultaneous detection of synthetic DNA (Haemophilus influenzae OMP-P6 gene) and proteins (IL-6, IL-8).
• Improved limit of detection for IL-6 and IL-8 by three orders of magnitude compared to previous microsphere assays.

Conclusions:

• The developed microarray system enables sensitive, simultaneous detection of proteins and DNA.
• Rolling Circle Amplification (RCA) is an effective signal amplification strategy for this multiplexed biosensor.
• This technology holds promise for improved diagnostics of infectious diseases and inflammatory conditions.