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

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Page 1: DISCOVERBRIGHT · 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

• 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™

Page 2: DISCOVERBRIGHT · 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

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

Page 3: DISCOVERBRIGHT · 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

• 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

Page 4: DISCOVERBRIGHT · 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

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

Page 5: DISCOVERBRIGHT · 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

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

Page 6: DISCOVERBRIGHT · 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

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

Page 7: DISCOVERBRIGHT · 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

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)

Page 8: DISCOVERBRIGHT · 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

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: [email protected]

DISCOVERBRIGHT™