1. Objective DNA amplification (eg. PCR)    advances in forensic, clinical applications Comparable protein amplification tools (?)  aid medical diagnostics, etc. Current approach: Immuno-PCR (tag proteins with DNA, then detect with PCR) Drawbacks: 1. Complex conjugation chemistries to link DNA and protein, 2. PCR requirements, 3. Low sensitivity Need for simple, sensitive assay for protein amplification and detection Nanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection of Proteins Jwa-Min Nam, C. Shad Thaxton, Chad A. Mirkin, Science September 2003

2. Some background info: 1. Model protein detection assay using Prostate Specific Antigen (PSA) PSA – widely used tumor marker in prostate and breast cancer detection Disease relapse after surgery marked by very low levels of PSA Need for ultrasensitive assay to detect PSA levels at early stage of relapse 2. Antibodies (Ab) are a class of proteins that recognize and specifically bind to their target protein 3. DNA hybridization: Based on binding of complementary DNA sequences A-T C-G T-A G-C

3. Probe design and preparation Gold nanoparticles (NP) heavily functionalized with hybridized DNA (biobarcodes + capture DNA) and PSA Ab Magnetic microparticles (MMP, 1 µm polyamine beads with magnetic iron oxide cores) functionalized with PSA Ab

4. PSA detection and bar-code DNA amplification and identification

5. Scanometric detection of PSA-specific bar-code DNA

6. Mol. BioSyst., 2006, 2, 470–476 Bio barcode assay vs. ELISA (gold standard)

7. Summary Biobarcode assay offers several advantages over current methods 1. Signal amplification due to high ratio of biobarcodes per protein molecule 2. Also, assay is homogenous (i.e. MMPs are added in solution) – hence the volume of MMPs can be increased to improve binding kinetics  Faster, more sensitive assay 3. No need for complex conjugation chemistries 4. Direct detection of DNA – hence elimination of background signal from protein or other biomolecules 5. Potential for multiplexing and simultaneous detection of several proteins