The document describes a nanoparticle-based method for detecting enzyme activity using quantum dots. Specifically: - Enzymes catalyze chemical reactions in the body, and abnormal enzyme activity can indicate disease. The method allows simple detection of enzyme activity. - It uses quantum dots that fluoresce at one wavelength. When an enzyme modifies a substrate linked to the dots, a dye molecule emits at a different wavelength via FRET. - By measuring the ratio of dye to dot fluorescence, the assay can quantify enzyme activity levels down to subnanomolar concentrations, comparable to current methods. It has potential applications in disease screening, drug development, and detecting other chemical changes.

1. Nanoparticle-based detection of
activity of enzymes (and more)
James Ghadiali, Phil Howes,
Molly Stevens
Department of Materials, Imperial College
London

2. 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!

3. How we started: Kinase & Acetyl Transferase Enzyme
Activity Assays
•Kinases transfer phosphate group from ATP onto specific amino acids;
Acetyl transferases add an acetyl group to amino acids. It’s this
chemical change we want to detect.
• Important role in cell signalling, cancer and diseases including
Alzheimer’s & Diabetes.
Acetyltranferase
Acetyl
enzyme
coA
COCH3

4. Quantum Dots for Enzyme Biosensing
Bioconjugates of quantum dot nanocrystals (QDs)
possess unique optical properties that allow them to
serve 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)

5. Enzyme activity causes a shift in the emission
spectrum 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)

6. Analysing the emission shift to quantify enzyme
activity
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

7. The assay is sensitive to subnanomolar enzyme
concentrations (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

8. 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

9. 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 

10. That’s our group

11. Thanks to
For sponsoring
the Challenge:
For research
funding:
Remember: Just use your marbles!