Published on: Mar 3, 2016
Transcripts - Nanoparticle assay
Nanoparticle-based detection ofactivity of enzymes (and more) James Ghadiali, Phil Howes, Molly Stevens Department of Materials, Imperial College London
Detecting Enzyme Activity – Why?• Enzymes are biological molecules catalysing a wide range of chemical reactions in the body• Abnormal activity level of enzymes are a symptom/cause of a range of diseases• Simple detection could be helpful in clinical diagnosis or screening of drug targets• But more generally, this could be a platform for detection of chemical changes through simple colour-shifts of a solution!
How we started: Kinase & Acetyl Transferase EnzymeActivity Assays•Kinases transfer phosphate group from ATP onto specific amino acids;Acetyl transferases add an acetyl group to amino acids. It’s thischemical change we want to detect.• Important role in cell signalling, cancer and diseases includingAlzheimer’s & Diabetes. Acetyltranferase Acetyl enzyme coA COCH3
Quantum Dots for Enzyme BiosensingBioconjugates of quantum dot nanocrystals (QDs)possess unique optical properties that allow them toserve as exceptional biological sensing reagents.• Fluorescence: absorb at a certain (high) frequency and emit at a lower frequency related to their size• Ability to selectively tune QD emission to red- shifted wavelengths provides a convenient means to minimize signal interference due to sample autofluorescence.• Simple and rapid assay does not require separation or washing steps and can be carried out without specialized instrumentation (just a standard fluorescence reader)• Multiplexing capabilities (i.e. can run multiple assays in the same solution when using different- sized QDs for each)
Enzyme activity causes a shift in the emissionspectrum of QDs Fluorescently- tagged antibody Without enzyme Peptide and antibody QD absorbs high-frequency light and emits remain separate at a lower frequency, X (fluorescence). Dye Add test does not absorb high-frequency light. solution Add quantum Enzyme Bound dots present antibody Peptide substrate Modified peptide Emission is now transferred to dye-molecules bound to the antibody which in turn emit at a different frequency Y < X (a process called Förster Resonance Energy Transfer, FRET)
Analysing the emission shift to quantify enzymeactivity 1.0 No enzyme Emission only at frequency X PL (a. u.) 0.5 λ (nm) 0.0 550 600 650 700 750 Ratiometric quantification 1.0 Enzyme present to limit well-to-well variation PL (a. u.) •Calculate ratio of dye Decrease in QD emission emission at Y to QD 0.5 emission at X Increase in dye emission •Use this to quantify amount of active enzyme λ (nm) 0.0 550 600 650 700 750 Ghadiali, Cohen, Stevens, ACS Nano 2010
The assay is sensitive to subnanomolar enzymeconcentrations (comparable to current state-of-the-art) Increasing Enzyme Plot of photoluminescence Activity intensity (a.u.) vs. ratio of dye to quantum dot emission Demonstrating detection of sub- nanomolar concentration of enzymes (kinase) Ghadiali, Cohen, Stevens, ACS Nano 2010
Example application: enzyme inhibitor screening • Create a system as explained in the previous slides where enzyme activity causes an emission shift • Adding an inhibitor causes a dose- dependent reduction in the dye- emission • Can quantify activity level of inhibitor targets over a wide concentration range • Example on the right: Staurosporine as a kinase inhibitor – results in agreement with literature Ghadiali, Cohen, Stevens, ACS Nano 2010 Lowe, Dick, Cohen, Stevens, ACS Nano 2012
What can we do with this system? • Simple fluorescence-based detection of chemical changes cause by enzymes. • What biomarkers could we use for disease detection in the clinic? • Can use the system to screen drug candidates for enzyme inhibitors. • What clinically important diseases could be treated with such enzyme inhibitors? What should we be looking at? • We could adapt the assay to detect chemical changes of things other than peptides bound to the QDs (as long as we can get antibodies or something else to bind to only the modified version). • What might this be useful for? We’d love your help in finding some ideas for what we can do with our technique and thoughts on how to make it possible – either based on the above or any other crazy ideas of yours
That’s our group
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