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July 2004 - Biotin EviFluor Specifications Sheet v1.0
2004 Evident Technologies, Inc. All Specifications subject to change without notice.
216 River Street
Suite 200
Troy, New York 12180
Phone: 518-273-6266
Fax: 518-273-6267
http://www.evidenttech.com
Page 1 of 7
Biotin Activated Quantum Dots
Biotin EviFluors

are activated quantum dot nanoparticles engineered to avoid


many of the shortcomings of organic dye-based fluorophores. They are approxi-
mately 10 to 35 nanometers in size and combine the advantages of quantum dot
fluorophores and nano-sized beads with between 2 to 20 biotin molecules on the
surface. Use of this Biotin EviFluor is similar to traditional biotinylated organic
dye, but offers advantages of long photostability, unique optical and electronic
properties, and multiple biotin moieties.
Quantum Dot Fluor Built Using Three Semiconductor Material Systems
The Biotin EviFluor is the first quantum dot label to be produced using three
semiconductor materials systems to achieve the widest range of wavelengths.
The technology used to create the Biotin EviFluor is extensible and can be mod-
ified to accept other active terminal molecules. Product developers now can
access multiple wavelengths stretching into the near infrared (NIR) using these
EviFluors to enable next-generation life science applications.
EviFluors Built on Evidents EviTag Technologies
EviFluors have all of the same advantages of our water-stabilized quantum dots,
EviTags

, since their underlying technologies are very similar. They are available
in a wide range of colors from blue/green (490nm) to the near-infrared (900nm).
Biotin EviFluors are modified with polyethylene glycol (PEG) to control the sur-
face charge and reduce nonspecific binding. Further, PEG does not illicit an
immune response and is not filtered by the liver providing a platform for in-vivo
applications.
Biotin is a natural co-factor of cellular enzymes involved in fatty acid metabolism
and biosynthesis, as well as gluconeogenic pathways. The ability to detect bioti-
nylated probes in vitro has led to many assays which allow for signal amplifica-
tion through the high affinity binding of labeled streptavidin or avidin. These
assays can be enzyme-linked or fluorescence based.
Quantum Dots as an Advanced Biotin Probe
Since the 1940's the affinity of avidin for biotin has been recognized. In the
1970s, extensive work was accomplished to create affinity cytochemical probes.
Biotin is a flexible platform for numerous forms of assays. Biotin has been used in
Quantum Dot Activated with
Biotin
Biotin EviFluor from 490nm to NIR
900nm
Biotin EviFluors
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July 2004 - Biotin EviFluor Specifications Sheet v1.0
2004 Evident Technologies, Inc. All Specifications subject to change without notice.
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probes for proteins, in nucleic acid probes, in ELISA, blotting and in Immunohistochemistry. The avidin-biotin
interaction is the strongest known noncovalent, biological interaction between protein and ligand. The bond
formation between biotin and avidin once formed, is unaffected by wide extremes of pH, temperature, organic
solvents and other denaturing agents.
Quantum Dot Biotin EviFluor - Benefits & Advantages:
Biotin EviFluors offers distinct advantages over traditional biotin fluors:
1. They do not agglomerate in live cells - By engineering the surface of the nanoparticle with PEG, the sur-
face charge on the quantum dot nanoparticle is reduced which is the major contributor to agglomeration.
2. Fluorescence Resonance Energy Transfer (FRET) - Due to the small size and unique optical and elec-
tronic properties of the underlying quantum dots, the Biotin EviFluors could possibly be used as a FRET
donor.
3. Wide range of fluorescence wavelengths - By utlizing multiple quantum dot materials systems, Biotin
EviFluor spans from 490nm to 900nm, enabling the use of single fluor family in both the visible and Near
Infrared (NIR) regions.
4. Improved Multiplexing & Lower Signal to Noise - By having such a wide range of wavelengths available,
multiplexing is achievable over a wide spectrum, with improved signal to noise ratio because of less cross
talk. All wavelengths are excited using a single excitation source, simplifying the detection platform.
5. Imaging with NIR Biotin EviFluors - NIR wavelengths are ideal for deep tissue imaging since these wave-
lengths can penetrate much deeper than visible light, permitting a whole new range of in-vivo applications.
Biotin EviFluors are smaller and brighter than other quantum dot conjugates with lower non-specific binding.
Additional brightness is achieved by advanced surface engineering of the nanocrystal that forms the core of the
EviFluor.
EviFluors have a number of advantages over traditional fluors:
1. They are more photostable - Building upon our EviTags technology, EviFluors show little degradation over
hours of constant illumination, enabling, for example, long-term studies under the microscope.
2. Quantum dots possess a greater extinction coefficient than traditional fluors - This results in the ability
to absorb more photons, and presents a brighter signal for imaging.
3. EviFluors are easily excited with broad band excitation sources - By having a broad excitation source
(<400nm), emission of multiple colors of EviFluors is accomplished with very little interference between
the excitation and emission sources.
4. EviFluors have a sharp emission peak and no red side tailing - Since EviFluors are based on quantum dots,
they have Gaussian shaped emission spectra with narrow emission line-widths leading to readily differenti-
ated emission signals.
Biotin EviFluor for Bridging Applications
Biotin EviFluors can be used in a manner similar to other Bridging Applications using well understood proto-
cols:
Endogenous biotin is first blocked with excess unlabeled streptavidin and excess unlabeled biotin.
Tissues, cells or other surfaces can then be probed with a biotinylated oligonucleotide, peptide, or antibody.
This preparation can be treated with unlabeled avidin or streptavidin (biotin-binding proteins).
After washing, detection can be performed in a variety of different platforms depending on your specific applica-
tion, including:
Microscopy
Flow Cytometry
Microtiter plate
Membrane based technology
Detection of endogenous biotinylated proteins:
Endogenous biotinylated proteins can be detected with unlabeled streptavidin as mentioned above.
The streptavidin can be detected with Biotin EviFluor
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July 2004 - Biotin EviFluor Specifications Sheet v1.0
2004 Evident Technologies, Inc. All Specifications subject to change without notice.
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Advanced Applications of Near Infrared Biotin EviFluors
Hemoglobin Differentiation
Differences in the absorption spectra of oxygenated and reduced hemoglobins allow quantitative distinction
between these two states. NIR light is inherently sensitive to tissue blood-oxygenation state without exogenous
contrast agents. Low NIR tissue absorption permits photons to travel several centimeters deep into tissue (see
figure 1). Optical methods can be rapid, noninvasive, portable, and inexpensive.
REF: S. Wray, et. al. Biochimica et Biophsica Acta 933, 184-192, 1988.
In Vivo Cell Imaging
There are many uses in cell imaging from extended color multiplexing to in-vivo cell imaging through layers of
tissue. This could help surgeons define cancer volumes in real time during surgery.
Forensic Detection Reporter
Near Infrared dyes possess all the advantages that make an excellent reporter for forensic detection of latent
chemical residues left at crime scenes. Background interference and scatter are minimal in this wavelength
regime, there are excellent light sources, and detectors are available to detect changes in NIR fluorescence as
the dyes interact with the latent residue.
650 750 850 950
Wavelength (nm)
A
b
s
o
r
p
t
i
o
n

(
m
M
c
m
)
*
*
-
1
OxyHGb
DeoxyHGb
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
Absorption Spectra of Hemoglobin
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July 2004 - Biotin EviFluor Specifications Sheet v1.0
2004 Evident Technologies, Inc. All Specifications subject to change without notice.
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Biotin EviFluor Specifications:
Biotin EviFluor Detailed Specifications:
Description:
Biotin activated quantum dot modified with polyethylene glycol (PEG)
Compositions: 490nm-620nm: CdSe/ZnS Quantum Dot, with approx. 2-20 biotin molecules
640nm-720nm: CdTe/CdS Quantum Dot, with approx. 2-20 biotin molecules
850nm-950nm: PbS Quantum Dot, with approx. 2-20 biotin molecules
Solvent: DI Water
Storage:
Sealed Container, keep in dark, 4
o
C to 25
o
C, do not freeze.
Shelf Life: 6 months unopened
Volume: 250 microliters, 1ml, 5ml
Color
Emission
Peak [nm]
Material
System
Typical
FWHM
[nm]
Suggested
Excitation
Wavelengt
h [nm]
1st
Exciton
Peak
[nm-
nominal ]
Mol ecular
Weight
[mg/
nmol s]
Concentra
ti on
a
[mg/ml ]
a. Concentration of core nanocrystals is the mass of the underlying core EviDots contained in the Evi-
Tags. This weight excludes the shell of the EviDots and the water stabilized coating.
Mol ar
Extinction
Coeffi cient
b
[10
5
]
b. Measured at first exciton peak, units expressed in l
.
(mol
.
cm)
-1
Quantum
Yield
(approx. )
Lake Placid
Blue
490 10 CdSe <40 <400 ~472 0.015 0.25 0.3 >15%
Adirondack
Green
520 10 CdSe <35 <400 ~504 0.021 0.25 0.5 >15%
Catskill
Green
540 10 CdSe <32 <400 ~526 0.029 0.25 0.6 >15%
Hops Yellow 560 10 CdSe <32 <400 ~547 0.042 0.25 0.8 >15%
Birch Yellow 580 10 CdSe <32 <400 ~569 0.070 0.25 1.4 >15%
Fort Orange 600 10 CdSe <32 <400 ~590 0.13 0.25 2.8 >15%
Maple Red-
Orange
620 10 CdSe <32 <400 ~612 0.29 0.25 5.9 >15%
Cortland Red 640 10 CdTe <40 <425 ~630 - 0.25 2 >40%
Empire Red 680 10 CdTe <40 <425 ~670 - 0.25 2.9 >20%
Adams
Apple Red
720 10 CdTe <40 <425 ~695 - 0.25 ~4.0 -
Gallium NIR 850 50 PbS <125 <500 ~725 - 0.25 - -
Snake Eyes
NIR
950 50 PbS <125 <500 ~825 - 0.25 - -
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July 2004 - Biotin EviFluor Specifications Sheet v1.0
2004 Evident Technologies, Inc. All Specifications subject to change without notice.
Page 5 of 7
Absorption and Emission Graphs:
.'. .'.. /'...)'..
1.1
1.'
!.1
!.'
'.1
'.'
.1
-11 '11 11 l11 11 11 !111
1
1.'
1.-
1.
1.
!.1
!.'
-11 '11 11 l11 11 11 !111
.'. .'.. m....
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July 2004 - Biotin EviFluor Specifications Sheet v1.0
2004 Evident Technologies, Inc. All Specifications subject to change without notice.
Page 6 of 7
Biotin EviFluor Environmental Conditions:
Material Systems
CdSe (490-620nm) CdTe
a
(640-720nm)
a. The stabilities of CdTe Biotin EviFluor may vary significantly.
pH 5-11 6.5-11
Temperature
b
b. At higher temperature, the biotin EviFluor may show some
decrease of the quantum yield.
4-25
o
C 4-25
o
C
Photostability
75mins
c
c. Nikon Eclipse TS 100 fluorescence microscope with UV2A
DM400 filter and 100W mercury lamp.
75mins
d
d. Under 360nm UV lamp.
Percent of Fluorescence Intensity
e
After One Hour
e. Percent of fluorescence intensity remaining after 1hour incuba-
tion at room temperature compared to the initial time point.
B
u
f
f
e
r
s
PBS >97 >80
0.1XTE
f
f. TE= 10mM Tris and 1mM EDTA at pH 8.0.
>77 >69
0.1XTAE
g
g. TAE= 40mM Tris acetate and 1mM EDTA at pH 8.3.
>80 >86
0.1XSSC
h
h. 20X SSC= 0.3M Sodium citrate, pH ~7.0, containing 3M NaCl.
>94 >73
0.1XTNT
i
i. TNT=10mM Tris-HCl, 150mM NaCvl and 0.05% Tween 20.
>99 >54
0.1XTNB
j
j. TNB=100mM Tris-HCl, 150mM NaCl and 0.5% Blocking
Reagent.
>98 >56
0.1M Borate pH 8.4 >96 >82
0.3M NaCl >89 >70
Percent of Fluorescence Intensity
k
After One Hour
k. Percent of fluorescence intensity remaining after 1hour incuba-
tion at room temperature compared to the initial time point.
D
e
t
e
r
g
e
n
t
s
0.1%SDS >99 >62
0.1%Triton >99 >84
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July 2004 - Biotin EviFluor Specifications Sheet v1.0
2004 Evident Technologies, Inc. All Specifications subject to change without notice.
216 River Street
Suite 200
Troy, New York 12180
Phone: 518-273-6266
Fax: 518-273-6267
http://www.evidenttech.com
Page 7 of 7
Open Platform for Assay Development
Evident's proprietary EviTags and EviFluors technologies offer an open platform for the production and devel-
opment of fluorescence assays. Evident Technologies has filed numerous patent applications and negotiated
license agreements with various third parties with respect to the manufacture and use of its semiconductor
nanocrystals and coatings. Additionally, Evident Technologies innovative research is continuing to introduce
new materials and biologically compatible coatings that extend the range of colors available for a wide array of
assay.
Research and Development Use Only and Toxic Substances Control Act (TSCA)
Except as provided by written contracts, products ordered from Evident Technologies are sold for research and
development use only. They are not intended for food, drug, household, agricultural or cosmetic use. The buyer
assumes responsibility to ensure that the products purchased are approved for use under TSCA, if applicable.
Use of Evident Technologies' products shall be supervised by a technically qualified individual. A Material
Safety Data Sheet (MSDS) accompanies products defined as hazardous by the US Occupational Safety and
Health Administration (OSHA) Hazard Communication Standard.
Partner to Product with Evident Technologies
We deliver quantum dots in multiple materials systems when you need to reach beyond the natural limits of
existing materials. We manufacture the highest quality nanocrystals ranging from UV through the visible spec-
trum into the infrared. Evident's quantum dots are available to meet your needs in commercial quantities. At
Evident Technologies, we partner to make products by giving you unprecedented freedom to engineer optical,
electronic, conducting, semi-conducting or insulating properties for your application needs.
Call Evident when nature limits your applications performance.

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