Objective DNA amplification (eg. PCR) advances in forensic, clinical applications Comparable...
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Transcript of Objective DNA amplification (eg. PCR) advances in forensic, clinical applications Comparable...
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
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 sequencesA-TC-GT-AG-C
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
PSA detection and bar-code DNA amplification and identification
Scanometric detection of PSA-specific bar-code DNA
Mol. BioSyst., 2006, 2, 470–476
Bio barcode assay vs. ELISA (gold standard)
Summary
Biobarcode assay offers several advantages over current methods
1. Signal amplification due to high ratio of biobarcodes per protein molecule2. 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 biomolecules5. Potential for multiplexing and simultaneous detection of several proteins