measurement and analysis of nano scale materials by ... -...
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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
#1
SNAP 25 S1-S4 deleted (D4)
Figure courtesy of Matthew Saunders
Differential cleavage of reporter constructs
5ul 0.1mg/ml LC (20nM)
-0.4
0.1
0.6
1.1
0 500 1000
Time after addition (seconds)
Nor
mal
ized
FL1
Consensus CS(control)SNAP 25 Deletion1 (S1-S3 deleted)SNAP 25 D4 (S1-S4 deleted)SNAP 25 D2 (D4deleted)SNAP 25 full lengthGFP
Figure courtesy of Matthew
Saunders
FRET-Based Toxin Recognition
[Cholera Toxin] (nM)
0 1 2 3 4 5 6 7 8
Nor
mal
ized
Acc
epto
r/Don
or R
atio
0.0
0.2
0.4
0.6
0.8
1.0
(Song, Nolan, and Swanson, 1998)
Cholera Toxin
Multiplex flu strain Identification
Blank
A/Kiev
/301/9
4
A/Shandong/9/
93
A/Texas
/1/77
A/Panam
a/200
7/99 (
CDC)
0
1500
3000
4500
6000
7500 Flu A Flu B Flu A-H1 Flu A-H3
Influenza A-H3N2 strains
Med
ian
Fluo
resc
ence
Blank
B/Leningrad
/86/93
B/Qingdao
/102/9
1B/Sich
uan/37
9/99 (
CDC)B/Vict
oria/50
4/200
0 (CDC)
0
1500
3000
4500
6000
7500
Flu A Flu B Flu A-H1 Flu A-H3
Influenza B strains
Med
ian
Fluo
resc
ence
Blank
A/Leningrad
/325/8
8
A/Taiwan
/1/86
A/Beij
ing/262/9
5A/N
ew C
aledonia/
20/99
A/New
Cale
donia/20
/99 (C
DC)
0
2000
4000
6000
8000
10000
12000
14000
Flu A Flu B Flu A-H1 Flu A-H3
Influenza A-H1N1 strains
Med
ian
Fluo
resc
ence
Influenza Virus
MAb to Influenza(Capture antibody)
Fluorochrome-ConjugatedPAb to Influenza A+B(Reporter antibody)
Size or ColorCoated Microsphere
Xiaomei Yan, et al 2003
Nano particles as reporters in flow cytometry
•
Quantum dots–
Commercially available
–
Fluorescent–
Longer lifetimes reduces signal in the short transit times of conventional flow
–
Still fairly broad emission spectra•
SERS
nano particles for flow cytometry
–
Spectral density useful for multiplexing–
Raman signal has an essentially
zero lifetime,
effective for flow cytometry–
Not commercially available
Quantum dots -
summary
•
Convenient–
Already adapted to biological assays
–
Stable–
Size
makes them
excellent reporters•
Bulk spectral properties less than optimal–
Lifetime
–
Emission spectra broad
Figures from Invitrogen.com
New Detection Modalities –
Raman in flow
ORFiber Optic array
connected to individualdetectors
(APDs or PMTs)
Array detectors(APD array or Multianode PMT)
PMTs
Filters and mirrors
Laser
Holographicgrating
Holographic notch filter
Imaging optics
Flow cell
FSCDetector
(photodiode)
Sample inlet
ORCAS(Digital data
Acquisiton system)
Polystyrene microsphere Raman spectra
Raman shift, cm-1500 750 1000 1250 1500 1750 2000
Nor
mal
ized
Inte
nsity
0.00
0.25
0.50
0.75
1.00
SERS
nanoparticles
500 1000 1500
SER
S In
tens
ityRaman Shift (cm-1)
Dye-tagged, glass coatedSERS nanoparticles
500 1000 1500
SER
S In
tens
ityRaman Shift (cm-1)
Dye-tagged, glass coatedSERS nanoparticles
Dye-tagged, glass coatedSERS nanoparticles
Figure courtesy of Stephen Doorn and Leif Brown
Many spectra available
Figure courtesy of Stephen Doorn and Leif Brown
Flow Cytometry -
Multiplex analysis
Full spectral resolution by flow cytometry
Figure courtesy of Gregory Goddar and
Jessica Houston
Characterization of single molecules of phycoerythrin by flow
Figure courtesy of Robert Habbersett and James Jett
Habbersett, RC; Jett, JH Cytometry; August 2004; v.60A, no.2, p.125-134
Evaluation of long linear nano particles (DNA fragments)
Figure courtesy of Robert Habbersett
DNA fragments sized by number of photons in pulse
Hind3 Lambda Digest
Hind3 Lambda Digest **
** 17 & 36 kb fragments added
$7KUniphase
DPSS Laser0.5 mW
$170 LasermateLaser Pointer
0.45 mW
103 105
105102
102
103 104
104
Detectedphotons
**
564 bp
564 bp
48.5 kbp
48.5 kbp
Figure courtesy of Robert Habbersett
Summary
•
Nano
particles are likely an important new class of reporter systems for flow cytometry
•
Flow cytometry can help characterize nano particles on a particle by particle basis for
size and intensity–
DNA, PE, Qdots, etc…
•
Biological molecules represent an important class of nano
materials
AcknowledgementsPROTEASE
•
Matthew Saunders•
Bruce Edwards (UNM)
•
Larry Sklar
(UNM)
SERS/Spectral•
Greg Goddard
•
Jessica Houston•
James Freyer
•
John Nolan (LJBI)•
Stephen Doorn
•
Leif Brown
PE/DNA•
Robert Habbersett
•
James Jett
FUNDING•
The National Flow Cytometry Resource (NIH RR001315)
•
LANL LDRD-ER•
Raman Flow Cytometry (NIH R01EB003824)