discoverbright · elisa assay. the positive control signal (cpn-gar +rigg) is clearly distinct from...
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• Exceptionally bright, magnetic and ultra-stable molecular bioimaging probes
• Covering the visible and near infrared spectrum
• Offering a variety of surface chemistries for bespoke conjugation to targeting molecules
Conjugated Polymer Nanoparticles (CPNs™)
DISCOVERBRIGHT™
CONJUGATED POLYMER NANOPARTICLES (CPNs™) FOR MULTIPLE CELLULAR APPLICATIONS
CPNs are exceptionally fluorescent labelling probes that can be utilised in cellular imaging and diagnostics applications. They are highly stable at a wide range of pHs and temperatures, and produce very intense light emissions that are immensely brighter than conventional technologies. CPNs do not photo-bleach, (>12 months stability), allowing significantly more sensitive detection of analytes than other fluorophores. They can be utilised in techniques such as immunocytochemistry, ELISA and flow cytometry. This greater stability ensures results are highly reproducible. CPNs can be taken up by cells through endocytosis or targeted to specific regions via linkage to antibodies, proteins, nucleotides or other targeting moieties.
Fluorescent hydrophobic polymer core
Iron oxide or gadolinium (magnetic manipulation / MRI)
Conjugated targeting moieties bound to surface: e.g. antibody / oligonucleotide / protein / Fab fragments / azide / streptavidin
NIR CPNs™
- 700
- 650
- 600
- 550
- 500
- 450
- 400
CPNs™ 1130
CPNs™ 900
CPNs™ 680
CPNs™ 610
CPNs™ 550
CPNs™ 510
CPNs™ 475
CPNs™ 420
CPNs™ 435
• Increases the sensitivity of assays
• Improves robustness of test performance
• Flexibility in storage and analysis conditions with regards to temperature and lighting
• Wider range of sample acquisition and isolation options using both centrifugation and magnets
Derived from display screen organic LED technology, the polymer core was originally designed to cope with years of electrical excitation, making it extremely stable. Quantum yield is frequently taken as a measure for brightness while the extinction coefficients are often ignored. It is the extremely large extinction coefficients, for fluorophores, that give CPNs their incredible brightness, sensitivity and robust stability.
HIGH PERFORMANCE, STABLE FLUORESCENCE
BRIGHTNESS = QUANTUM YIELD x EXTINCTION COEFFICIENT
• Range of colours matching standard filter sets and laser lines
• Range of colours extending applications into IR and in vivo imaging
• Link to standard antibodies, protein and nucleotides
• Replace existing fluorophores with an improved performance
READILY COMPATIBLE WITH EXISTING ASSAY PLATFORMS
INCREASED BRIGHTNESS, SENSITIVITY AND STABILITY
IMAGING
Flow cytometry
Microscopy
1°/2° antibodies
FISH
FRET
Fluorescent ELISA
Western blotting
Lateral flow assays
DNA Extraction
qPCR
QUANTIFICATION
CD-4 on lymphocytes labelled by CPN475 or CPN900 directly or via biotin/streptavidin showing detection with a 380nm or 808nm laser excitation.
Detection of analyte in fluorescent ELISA assay. The positive control signal (CPN-GAR +RIgG) is clearly distinct from the negative control wells (CPN-GAR –RIgG, CPN +RIgG, CPN –RIgG).
ELISA 50000
40000
30000
20000
10000
0
Fluo
resc
ence
at 4
70/5
50nm
CPN-GAR +RlgG
CPN-GAR -R
lgG
CPN +RlgG
CPN -RlgG
FLOW CYTOMETRY
HEK 293T cells endocytotically loaded with CPN475 analysed by flow cytometry.
103 104 105 106
104
105
106
107
7th Feb 475_1ul added... :All Events
103 104 105 106
104
105
106
107
7th Feb 475_10ul added... :All Events
103 104 105 106
104
105
106
107
7th Feb 475_100ul added... :All Events
100 101 102 103 104 105
Sample Name A1 Negative.fcsA4 CPN-510-strep 0.5ug (CD-4).fcs
1.2k
900
600
300
0
102 103 104 105
100
50
0
CD-4 CPN905(16.98%)
100 101 102 103 104 105
Sample Name A1 Negative.fcsA4 CPN-510-strep 0.5ug (CD-4).fcs
1.2k
900
600
300
0
102 103 104 105
100
50
0
CD-4 CPN905(16.98%)
Unconjugated CPN and CPN-GAR 10ug/ml Optimal Gain: 192
700 720 740 760 780 800 820 840 860 880 900
1.00
0.80
0.60
0.40
0.20
0.00
Excitation wavelength (λ)
Nor
mal
ised
em
issi
on in
tens
ity
The immense brightness of CPNs dramatically increases the sensitivity of applications such as flow cytometry, ELISA, and immunocytochemisty, with single nanoparticles being detectable in flow cytometry and immunocytochemistry. This enables the study of individual proteins in samples and cells using CPNs linked to antibodies or other targeting molecules. Linkage to CPNs occurs via their amine groups, using N-ethyl-N’- dimethylaminopropyl-carbodiimide (EDC) chemistry. Other surface chemistries are also available, such as linkage to thiol groups or click chemistry compatible systems.
Standard fluorescent assays can be enhanced using magnets to locally increase the concentration of the CPN-linked antibodies at their target and then to reduce any non-specific binding in subsequent washing steps.
Scan of excitation wavelengths for the four CPNs showing clear 2-photon excitation to eliciting fluorescence at their typical emission wavelengths.
BIOLOGICAL PROPERTIES
MAGNETICALLY ENHANCED ASSAY SIGNAL
CPNs make ideal labels for 2-photon imaging. The conjugated polymer cores offer a large area over which to absorb the energies from coincident photons.
2-PHOTON IMAGING
Excitation 700-900nm Emission CPN475, CPN510, CPN550, CPN680
CPN475 - Emission 450-550nm
CPN510 - Emission 475-650nm
CPN550 - Emission 490-650nm
CPN680 - Emission 590-695nm
Standard Magnet
25
20
15
10
5
0
Perc
enta
ge o
f tot
al s
map
le b
ound
Significant increase in signal and
assay window using magnetic
pull down
CPN™ PRODUCT WAVELENGTHS
400 500 600 700 800 900 1000 1100 1200 1300
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
1.2
1
0.8
0.6
0.4
0.2
0
CPNs™ 1130
CPNs™ 900
CPNs™ 680
CPNs™ 610
CPNs™ 550
CPNs™ 510
CPNs™ 475
CPNs™ 420
CPNs™ 435
NIR CPNs™
- 700
- 650
- 600
- 550
- 500
- 450
- 400
450nm 510nm
300 350 400 450 500 550 600 650 700 750 800
1.2
1
0.8
0.6
0.4
0.2
0
Absorbance Emission
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
390nm 420nm
300 350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
1.2
1
0.8
0.6
0.4
0.2
0
Absorbance Emission
400nm 680nm
300 350 400 450 500 550 600 650 700 750 800
1.2
1
0.8
0.6
0.4
0.2
0
Absorbance Emission
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
CPN™ 420 (Violet)
CPN™ 510 (Green)
CPN™ 680 (Red)
470nm 550nm
300 350 400 450 500 550 600 650 700 750 800
1.2
1
0.8
0.6
0.4
0.2
0
Absorbance Emission
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
395nm 435nm
300 350 400 450 500 550 600 650 700 750 800
1.2
1
0.8
0.6
0.4
0.2
0
Absorbance Emission
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
650nm 900nm
500 550 600 650 700 750 800 850 900 950 1000 1050 1100
1.2
1
0.8
0.6
0.4
0.2
0
Absorbance Emission
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
CPN™ 435 (Indigo)
CPN™ 550 (Yellow)
CPN™ 900 (IR-I)
480nm 610nm
300 350 400 450 500 550 600 650 700 750 800
1.2
1
0.8
0.6
0.4
0.2
0
Absorbance Emission
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
390nm 475nm
300 350 400 450 500 550 600 650 700 750 800
1.2
1
0.8
0.6
0.4
0.2
0
Absorbance Emission
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
750nm 1130nm
500 600 700 800 900 1000 1100 1200 1300 1400 1500
1.2
1
0.8
0.6
0.4
0.2
0
Absorbance Emission
Wavelength (nm)
Nor
mal
ised
Inte
nsit
y (A
U)
CPN™ 475 (Blue)
CPN™ 610 (Orange)
CPN™ 1130 (IR-II)
Stream Bio Ltd, Alderley Park, Nether Alderley, Cheshire, SK10 4TG, UK Follow us @Stream_Bio
For a list of distributors, please visitwww.streambio.co.uk/where-to-buy I or contact: info@streambio.co.uk
DISCOVERBRIGHT™
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