Bioscience Innovation for Health and Security Measurement and Analysis of Nano Scale Materials by Flow Cytometry Steven W. Graves National Flow Cytometry Resource Bioscience Division Los Alamos National Laboratory Bioscience Innovation for Health and Security
27
Embed
Measurement and Analysis of Nano Scale Materials by ... - UNMchtm.unm.edu/incbnigert/Presentations/Graves.pdfMeasurement and Analysis of Nano Scale Materials by Flow Cytometry Steven
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
BioscienceInnovation for Health and Security
Measurement and Analysis of Nano Scale Materials by
Flow CytometrySteven W. Graves
National Flow Cytometry ResourceBioscience Division
Los Alamos National Laboratory
BioscienceInnovation for Health and Security
A traditional flow cytometerSample Inlet
SheathInlet
Laser Beam
SampleStream
SheathStream
CollectionOptics
Photomultipliertubes
Filters andMirrors
Scale
of particles analyzed by flow cytometry
Particles in the flow stream
Background depends onInterrogation volumeConcentration of free dye
Signal is determined by the number of bound fluorophores on the particleReduce background
Stream diameterBeam height
Diameter of the laser
Diameter of the sample stream ( ) 0NCVV
NBS
freepi
b
−=
Nb = # of fluorophores on the particleVi = interrogation volume (~ π(DS /2)2DL )
Vp = volume of the particleCfree = concentration of free fluorophore
N0 = Avogadro’s number
Applications of flow cytometryClinical Biochemical
CD4+ cell counting for AIDS assessment
Single Nucleotide Polymorphism analysis
Reticulocyte
counting for anemia patients
Cellular Apotposis
Differential leukocyte counting Kinetic analysis of cellular adhesion
CD34+
cell counting for transplantation Multivalent receptor affinity studies
Cancer diagnosis Molecular assembly analysis
Minimal Residual Disease analysis in Leukemia Treatment
High-throughput screening of peptide-
protein interactions
Diagnosis of infectious diseases Mechanistic study of DNA cleavage by endonucleases
Cytokine expression analysis Evolution of single chain antibodies
Pathogenic bacteria detection E. coli protease optimization
Advantages of flow cytometry
•
High sensitivity•
Homogenous assays (ability to resolve all assemblies and their products without separation steps)
•
A range of detection modalities•
High temporal resolution (kinetic analysis)
•
Population analysis (particle by particle data)•
High throughput analysis
capability
Flow cytometry and
nanotechnology
•
Nano particles can serve as reporter elements–
Flow cytometry is still mainly optical
•
Scatter is limited from nano-particles•
Fluorescence is mostly utilized•
Raman is of interest
–
Other modes of detection are under development (e.g. magnetic)
•
Characterization of
nano materials is possible by flow cytometry
Simple biological ‘nano-reporters’
•
Advantages–
Can adapt this system for most proteases
–
Attachment of
proteins to microspheres enables multiplexing
for multiple subtrates in a single reaction
•
Disadvantages–
Must purify the portein
–
End product not well controlled
Proteases-critical to human health and biodefense
•
Human Health–
HIV proteases (AIDS)
–
Thrombin/blood coagulation enzymes (Thrombosis)
–
Caspases (Cancer treatment)•
Biothreat–
Lethal Factor
–
Botulinum Toxin–
Other secreted proteases (Burkholderia pseudomallei, Clostridium perfringens uses a protease during secretion of epsilon toxin)
Light chain of Botulinum toxin A •
Works as
the second
part of a bipartite A/B toxin
•
‘A’
part–
the ‘heavy chain’–
delivers toxin to the cell
•
‘B’
part–
the light chain–
a protease–
cleaves a cellular protein (Snap25) to interrupt neurotransmission
Barr JR, et al. Botulinum neurotoxin detection and differentiation by mass spectrometry. Emerg Infect Dis 2005 Oct
Available from http://www.cdc.gov/ncidod/EID/vol11no10/04-1279.htm
ŅpreferredÓŅnon-productiveÓ?
Washbourne et al. FEBS Letters 418 (1997) 1-5.
Multiplex assay for the light chain protease of Botulinum Toxin A
#11
SNAP 25 S1-S3 deleted (D1)Full Length SNAP 25
#5
LF consensus CSNegative control
#9
#7
SNAP 25 S4 deleted (D2)
Observation, deletion of S1-S3 gives faster cleavage