Avidin/Biotin Bioinspired Platform for Dual in Vivo 18F-PET/NIRF Molecular Imaging

Article abstract of DOI:10.1021/acs.bioconjchem.7b00536

The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of innovative multimodal PET/NIRF probes a very exciting prospect. Herein, the bioinspired design of novel platform exploiting the strength and specificity of interactions between radioactive and fluorescent biotin derivatives and an avidin core is reported. The combination of an original [¹⁸F]fluoropyridinylated-biotin derivative and commercially available fluorescent biotin derivatives (Atto-425 and Atto-680) is investigated. The in vivo distribution of such a customized platform is also reported, for the first time, in healthy rodent using PET and ex vivo fluorescence imaging.

Full text of DOI:10.1021/acs.bioconjchem.7b00536

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Communication
Avidin/biotin bioinspired platform for dual
in vivo 18F-PET/NIRF molecular imaging
Annelaure Damont, Raphael Boisgard, Frédéric Dollé, Morgane Hollocou, and Bertrand Kuhnast
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00536 • Publication Date (Web): 20 Sep 2017
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Avidin/biotin bioinspired platform for dual in vivo 18F-PET/NIRF molecular imaging
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Annelaure Damont, Raphael Boisgard, Frédéric Dollé, Morgane Hollocou and Bertrand Kuhnast*.
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IMIV, Service Hospitalier Frédéric Joliot, CEA, Inserm, Université Paris Sud, CNRS, Université
ParisꢀSaclay, Orsay, France
* Corresponding author: Bertrand Kuhnast, CEA, 4 place du général Leclerc, 91401 Orsay Cedex,
France, bertrand.kuhnast@cea.fr; + 33 1 69 86 77 36
ORCID iD Bertrand Kuhnast : 0000ꢀ0002ꢀ5035ꢀ4072
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Abstract.
The complementary nature of positron emission tomography (PET) and nearꢀinfrared fluorescence
(NIRF) imaging makes the development of innovative multimodal PET/NIRF probes a very exciting
prospect. Herein, the bioinspired design of novel platform exploiting the strength and specificity of
interactions between radioactive and fluorescent biotin derivatives and an avidin core is reported. The
combination of an original [18F]fluoropyridinylatedꢀbiotin derivative and commercially available
fluorescent biotin derivatives (Attoꢀ425® and Attoꢀ680®) is investigated. The in vivo distribution of
such a customized platform is also reported, for the first time, in healthy rodent using PET and ex vivo
fluorescence imaging.
Introduction.
Molecular medical imaging is today able to provide multiple and complementary information
(anatomic, physiologic, metabolic…) for an accurate diagnosis of a disease, treatment response
prediction and patient follow up. In other words, molecular medical imaging is the cornerstone of the
contemporary stratified and personalized medicine. Imaging is indeed a precious component of
medical diagnosis and patient care either in observing tissue anatomy and morphological changes
using for example Magnetic Resonance Imaging (MRI), Ultra Sound (US) or Computerized
Tomography (CT), or by tracking functional and molecular events with Positron Emission
Tomography (PET), Single Photon Emission Computerized Tomography (SPECT) or Optical Imaging
(OI). In spite of their wide use in preclinical research and clinical diagnosis, these imaging modalities
considered individually present weaknesses. However, cleverly combined, these techniques can not
only concomitantly provide complementary information, but also synergistically conjugate their
advantages in terms of spatial and temporal resolution, depth penetration, sensitivity and cost. Beside
the today classical combination of PET and CT, or more recently PET and MRI, dual PET/near
infrared fluorescence (NIRF) imaging holds great promises. PET is today considered as the gold
standard in functional molecular nuclear imaging without depth limitation (thanks to the high 511 keV
energy photons emitted and tomographic reconstruction) and fluorescence contrast imaging nicely
provides complementary information in terms of spatial resolution at the histologic and superficial
levels. Major advantages of this unique combination are i) both radiolabeled and fluorescent probes
can be detected in the same range of concentrations (nanomolar and even below)1, ii) matched
dynamic whole body (PET) and intraoperative or histologic (OI) images can be produced and iii)
quantitative PET imaging allows for the quantification of fluorescent images. Conceivably, a patient
would undergo a nonꢀinvasive whole body nuclear scan (PET) to assess the distribution of the
radioactive imaging agent to the targeted (diseased) tissue and observe its (dys)function, and
subsequently, the NIRF signal would allow for a refined observation with in vivo endomicroscopy
systems which generate optical biopsies or with ex vivo fluorescence microscopy2ꢀ4. These recent
evolutions and even revolutions in medical molecular imaging lead to reinvent part of the imaging
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agents. The challenge of the implementation of such a combined imaging is to design a probe that
combines in a single monomolecular structure, a targeting moiety, a positronꢀemitting radionuclide,
and a NIRF dye. Such a platform avoids the weaknesses of approaches based on a sequential or
simultaneousꢀ“cocktail” injection of the radioactive and fluorescent probes separately, both suffering
from the possible distinct pharmacokinetics and imaging time windows of each agent leading to
discrepancies in final (combined) images.
In this communication, we describe the preparation of the first fully modular avidin/biotinꢀbased
bioinspired platform, for dual 18FꢀPET/NIRF molecular imaging. To that purpose, we have synthesized
and radiolabeled at high molar activity (MA) with the shortꢀlived positronꢀemitter fluorineꢀ18 (T1/2:
109.8 min), an original fluorinated biotinꢀderivative (3, Scheme 1) of which we have confirmed the
binding potency (affinity, selectivity) to avidin. Then, using two commercially available fluorescent
dyeꢀcontaining biotinꢀderivatives (Attoꢀ425®, a blueꢀfluorescent dye, and Attoꢀ680®, a nearꢀIR
fluorescent dye) and our newly developed radioactive derivative, we have demonstrated the possibility
to decorate in a fully modular fashion avidin, leading to customized 18FꢀPET/NIRF platforms. Finally,
for the first time, we have evaluated the biodistribution in vivo of one of these “naïve” dual probes
with PET imaging in healthy rats, including a set of ex vivo fluorescence images recorded after a
single injection of the 18FꢀPET/NIRF platform.
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Results and discussion.
Chemistry/radiochemistry. Starting from natural biotin (1), the carboxylic acid function was first
esterified with ethanol in acidic conditions (81%) and the resulting ethylester reduced with lithium
aluminum hydride to the corresponding alcohol (93%) (Scheme 1). The latter was then converted to
the tosyloxy keyꢀintermediate 2 (78%) from which both the nonꢀradioactive fluorinated biotin
derivative 3 and its nitro analogue 4 as precursor for labeling with fluorineꢀ18, could be analogously
obtained, from 2ꢀfluoroꢀ and 2ꢀnitroꢀ3ꢀhydroxypyridine, respectively. Using this straightforward
pathway, intermediate 2 was obtained in an overall 59% yield (3 steps), and compounds 3 and 4 in
70% and 54% yields (from compound 2), respectively (see details in ESI §1 and §2). Radiolabeling
with fluorineꢀ18 was performed in standard reaction conditions, involving a nitroꢀforꢀfluorine aromatic
nucleophilic substitution. Precursor 4 (4 to 5 mg) was reacted for 5 min at 160 °C in DMSO with
K[18F]FꢀK222 complex, as source of noꢀcarrierꢀadded and activated [18F]fluoride. The crude was preꢀ
purified using an Alumina NTM cartridge before semiꢀpreparative RPꢀHPLC and final formulation in
an in vivo compatible excipient (0.9% aq. NaCl/EtOH : 80/20 (v/v)). The whole process
(radiosynthesis, purification and formulation) has been fully automated on a TRACERLab Fx N Pro
synthesizer (GE Medical Systems). Average production yields were 44% ± 9% (n>10, decayꢀ
corrected) in a 50 to 55 min synthesis time. Chemical and radiochemical purity of [18F]3 exceeded
95%, with an average decayꢀcorrected MA of 153 ± 45 GBq/µmol (see details in ESI §3).
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Scheme 1: Synthesis/radiosynthesis of the fluorinated biotin derivatives (3 / [18F]3).
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Other groups have described the synthesis of fluorineꢀ18ꢀlabeled biotin derivatives, where the
radionuclide was borne either by a sp3ꢀcarbon or a boron atom. In the first case, Shoup et al.
synthesized two [18F]fluoroalkylꢀbiotin derivatives ꢀ using a mesyloxy precursor for labeling ꢀ but the
yields were lower (12 to 20% nonꢀdecayꢀcorrected) than those we obtained for longer synthesis times
(90 min)5. Blom et al. proposed three [18F]fluoroalkylꢀ and [18F]fluoroalkoxyꢀbiotin derivatives
prepared in moderate to good yields (13 to 35% decayꢀcorrected) and with high MA (320 GBq/µmol)6.
Compared to these two examples, the strategy that we have developed allows the production of an
original [18F]pyridinylꢀbiotin derivative in 30 to 80% higher radiochemical yields. Claesener et al. also
synthesized another [18F]fluoroalkylꢀbiotin derivative, using a tosyloxy precursor too. Whereas
comparable yields (45%) and reaction times (45 min) were reported, the MA they observed was
drastically lower (16 GBq/µmol) than the one we obtained7. None of these studies proposed a whole
body PET image of the [18F]biotin derivative/avidin complex, in spite of the fact that the affinity of all
these radioactive biotinꢀderivatives for the avidin was assessed. The biodistribution of only two
[18F]biotin detivatives was evaluated5,7.
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When a boron center was considered, two examples were reported in the literature8,9. In both cases, the
radiolabeling was performed by a 19F/18F isotopic exchange leading to the radiolabeled biotin
derivatives with expectedly low MA, even if these values are not precisely reported. As a
consequence, the low MA may limit the binding efficiency to avidin. Moreover, it has been recently
observed that defluorination of 18Fꢀboronꢀbased radiotracers may occur in vivo10, restraining therefore
the interest for such labeling processes. Strategically, we have chosen to introduce fluorineꢀ18 on a
sp2ꢀcarbon center and at the alphaꢀposition of a pyridine ring to benefit from i) the high carbonꢀ
fluorine bound stability observed for this motive11 and ii) the high yielding radiofluorination of the
pyridine moiety at the ortho position12,13
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Binding potency to avidin. We have then evaluated in vitro i) the binding kinetics of [18F]3 to avidin,
ii) the binding specificity of [18F]3 regarding other proteins, iii) the stability of the [18F]3/avidin
complex, iv) the affinity of [18F]3 for avidin in competition binding assays and v) the multiplexing of
avidin with [18F]3 and two (commercially available) fluorescent biotinylated dyes. For all these in
vitro experiments, the formation of the biotin/avidin complexes was systematically assessed in a
doubleꢀanalysis approach, using radioꢀiTLC and size exclusion chromatography (SEC) (see details in
ESI: §1.2.2; §1.2.3; §4.1).
Binding kinetics was established by analyses of a mixture of [18F]3 and avidin (in excess), every
minute over a total incubation period of 5 minutes at room temperature. Both iTLCs and SEC analyses
showed that more than 98% of the radioactivity was bound to avidin as early as 1 min of incubation
(iTLC : > 98% of radioactivity detected at Rf = 0.0, corresponding to [18F]3/avidin complex; SEC : >
98% of the radioactivity eluted in the highꢀMW fraction, corresponding to [18F]3/avidin complex; ESI:
§ 4.2). These results not only confirmed that the introduction of a fluoropyridine moiety at the alkylꢀ
terminal position of biotin does not alter the potency of this derivative to bind to avidin, but also
demonstrated that the formation of such avidin complexes is compatible with the short halfꢀlife of
fluorineꢀ18.
The stability of the [18F]3/avidin complex has been evaluated at different time points after complex
formation and purification. More than 97% of the radioactivity was associated with the highꢀMWꢀ
fraction over a period of time of 90 min in a SEC analysis (see details in ESI: §4.3, Table S1). This
results showed that there are neither release of [18F]3 (as expected) nor generation of radioactive lowꢀ
MW byꢀproducts that would be retained in the gel column.
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The nonꢀspecific binding of [18F]3 – e.g. binding of 3 to a protein lacking the binding site of the
characteristic bicyclic motive (oxoꢀtetrahydroꢀ1Hꢀthieno[3,4ꢀd]imidazo) borne by all biotin derivatives
– was evaluated by incubating [18F]3 with a Bovin Serum Albumin (BSA) buffered solution for 5 min
at room temperature. iTLCs and SEC showed that no radioactivity was associated with the protein
(iTLC : radioactivity was only detected at Rf = 0.7, corresponding to free [18F]3; SEC : no
radioactivity was eluted with the highꢀMW fraction and more than 98% of the radioactivity was
retained on the column, which is compatible with free [18F]3; ESI : § 4.4). As natural biotin, the
fluoropyridinilated analogue 3 is devoided of nonꢀspecific binding properties to proteins.
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Affinity of [18F]3 for avidin was finally evaluated in a binding competition assay. [18F]3 was incubated
with a constant amount of avidin (7.5 nmol, thus corresponding to 30 nmol in terms of binding sites
for biotin) and increasing amounts of (nonꢀradioactive) 3 or natural biotin (Figure 1) (see in ESI: § 4.5
Table S2 and Table S3). Competition assays were analyzed according to different methods: two of
them were analyzed by iTLC and the third one by SEC. Graphical determination allowed establishing
the IC50 to 30 µM and 45 µM for biotin and 3, respectively. This result definitively confirmed that the
chemical structure of our biotin derivative and thus the modifications introduced when compared to
natural biotin, do not affect its binding affinity for avidin.
Figure 1: Binding competition assay.
Multiplexing and customized constructions. Finally, we have evaluated in vitro the multiplexing
ability of this bioinspired platform by simultaneous complexation of various biotin derivatives with
avidin (Figure 2): [18F]ꢀ3 as well as two commercially available fluorescent biotins (Attoꢀ680® and
Attoꢀ425®).
Figure 2: Schematic representation of the bioinspired platforms.
In a first set of experiments, [18F]3 (4.06 nmol) and Attoꢀ680® (8.9 nmol) or [18F]3 (4.12 nmol) and
Attoꢀ425® (14.06 nmol) were incubated with an excess of avidin (7.57 nmol avidin corresponding to
30 nmol binding sites). In these conditions the binding of all biotinꢀderivatives exceeds 84%,
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indicating an almost quantitative complexation (Table 1, entries 1/2 and ESI: § 4.6). In a second set of
experiments, avidin (7.57 nmol or 30.3 nmol) was incubated with equimolar amounts of the three
biotin derivatives. When an excess of avidin biotin’s binding sites (121.2 nmol, e.g. 30.3 nmol avidin)
was incubated with a total amount of 36 nmol of biotin derivatives ([18F]3 = 13.3 nmol; Attoꢀ680® =
8.9 nmol; Attoꢀ425® = 14.1 nmol), the binding of each biotin derivative exceeds 92%, demonstrating
again a quantitative complexation of the biotin derivatives (Table 1, entry 3 and ESI: § 4.6). In a third
and last set of experiments, a default of avidin biotin’s binding sites (30 nmol e.g. 7.57 nmol avidin)
was incubated with a 20% excess of biotin derivatives (condition 1: total amount = 36 nmol distributed
according to [18F]3 = 13.1 nmol; Attoꢀ680® = 8.9 nmol; Attoꢀ425® = 14.1 nmol; condition 2: total
amount = 38 nmol distributed according to [18F]3 = 13.1 nmol; Attoꢀ680® = 10.7 nmol; Attoꢀ425® =
14.1 nmol), a partial binding was observed but the values were equally distributed between the
different biotin derivatives (61%, 70%, 65% (condition 1) and 53%, 53% and 52% (condition 2) for
[18F]3, Attoꢀ680® and Attoꢀ425®, respectively (Table 1, entries 4/5 and ESI: § 4.6). These results show
that it is possible to perform a fine tuning of the design of the platform with different biotin derivatives
and to reach the desired balance between imaging dyes and targeting entities in the final construction,
in particular for dual 18FꢀPET/NIRF imaging applications.
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Table 1: Evaluation of multiplexed [18F]3/fluorescentꢀbiotin avidin platforms
biotinꢀderivatives
Attoꢀ425®
(nmol)
0
Avidin % of biotinꢀderivatives bound with avidin
Attoꢀ680®
(nmol)
8.9
[18F]3
(nmol)
4.06
Attoꢀ680®ꢀ
Attoꢀ425®ꢀ
(nmol) [18F]3
biotin
biotin
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4
7.57
7.57
30.3
7.57
7.57
93a
94a
92a
53a
61a
0.240b
ꢀ
86c
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0
8.90
14.06
14.06
4.12
13.3
ꢀ
0.1625b 84c
0.292b 100c 0.1906b 99c
10.7
8.90
14.06
14.06
13.2
13.1
0.176b
0.19b
53c
70c
0.1013b 52c
0.1253b 65c
5
a
% calculated as the ratio between the amount of radioactivity associated with the highꢀMW fraction (γ counting) and the
initial amount of [18F]3 introduced in each assay;
b optical measurement using a UV detector at λ = 680 nm for Attoꢀ680®ꢀbiotin and λ = 425 nm for Attoꢀ425®ꢀbiotin;
c
% calculated as the ratio between the optical measurement associated with the highꢀMW fraction and the standard OD
values for each fluorescent biotinꢀderivative.
Fluorineꢀ18, as radioactive isotope in dual PET/OI platforms, appeared interestingly in the literature
concomitantly with 18F/boron chemistry9,14 and was notably used to radiolabel the dye BODIPY®,
which contains a BF2 moiety15ꢀ17. Other dual imaging dyes containing B[18F]F3 entities were also
reported14,18. But in all cases, the final imaging dyes were obtained with low MA either due to the need
for nonꢀradioactive fluoride carrier to boost the radiolabeling process or simply due to 18F/19F
exchange. The maximum MA reported was 2.7 GBq/µmol16, a very low value compared to the 100ꢀ
500 GBq/µmol usually reported for nonꢀcarrier added 18Fꢀlabeled radiotracers. Such low MAs
drastically limit the potential applications of these probes in receptorꢀbased imaging strategies due to
the often minute amounts of targets to visualize. Moreover, classical BODIPY® dyes are not emitting
fluorescence light in the NIR spectrum, which is strongly recommended for in vivo imaging. It was
also recently reported that the 18Fꢀboron bond could be less stable than expected10. To date, only one
unique study proposes an alternative to the 18F/boronꢀbased dual PET/NIRF imaging dyes19. It is based
on a dual prosthetic reagent bearing fluorineꢀ18 on a carbon center on the one hand and a fluorescent
dye on the other hand. Its conjugation to a vector and applicability in vitro or in vivo has not been
demonstrated yet. Several examples using positronꢀemitting radiometals have also been reported20,21
and very recently, a 64CuꢀPET/NIRF streptavidin/biotinꢀbased platform was published using a dimeric
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RDGꢀcontaining peptide as targeting entity22. Note that in this example, both imaging entities
([64Cu]CuꢀDOTA and AlexaFluorꢀ680®) were covalently bound to streptavidin, and only the dimeric
RGDꢀcontaining peptide was derivatized with biotin. In this approach, like in most of other examples
reported in the literature so far, the dual PET/fluorescence imaging moiety is a covalent construction
limiting the modular fashion of the bioinspired biotinꢀavidin platform. In our case, all imaging entities
as well as the future targeting entities are biotinꢀderivatives, allowing thus to tune onꢀdemand and in a
flexible manner the design and characteristics of the final platform.
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First in vivo experiments. Biodistribution of a “naïve” platform i.e. a platform free of a targeting
entity, was evaluated in healthy rats (Wistar). For this, [18F]3 (407 MBq, 13 nmol) and Attoꢀ680® (18
nmol) were incubated with avidin (1.34 mg, 20 nmol) for 5 min at room temperature, and the crude
reaction mixture subjected to SEC purification. The resulting platform was then formulated as
described above and injected, in part (10% based on radioactivity: 41 ± 3 MBq, 1.8 nmoles Attoꢀ680®,
135 µg avidin ꢀ ESI, §5.1), to animals (n = 3). In vivo distribution of the platform was followed by
PET imaging (INVEON, Siemens) for 60 minutes. Images showed a rapid initial distribution through
the vascular tree (Figure 3, 1 min) and elimination of the radioactivity through the liver and kidneys in
a lesser extent (Figure 3, 2 min). At later time points (Figure 3, 30 min and 60 min), the radioactivity
was mainly accumulated in the liver and biliary tract suggesting a hepatoꢀbiliary elimination of the
probe. Note the cortical distribution of the radioactivity in the kidneys that will majorly persist till 60
min. Time activity curves (TACs) derived from dynamic PET images confirmed the previous
observations (ESI, §5.2, Figure S1). Radioactivity could be mainly detected in elimination organs
(liver and kidneys) from which a slow washout could be observed. Radioactivity did not accumulate in
lungs and muscles (a rapid clearance was shown for these organs). Absence of any radioactivity in
bones also suggests a good metabolic stability of the radiolabeling and absence of defluorination.
Taken together, images and TACs both demonstrated the absence of nonꢀspecific signal for the
platform and strongly suggest that once equipped with a relevant targeting entity (using another biotin
link) such a construction will be amenable to detect in vivo, and at the exception of the perimeter of
the elimination organs, specific targets.
Figure 3: In vivo PET biodistribution in rats of a [18F]3/Attoꢀ680® bioinspired platform (n = 3;
injected dose = 41 ± 3 MBq, equivalent to 1.11 nmol (MA = 20 GBq/µmol at injection time)).
Finally, 60 min after injection, animals were sacrificed and organs (liver, kidney, heart, muscle and
lungs) were withdrawn and exposed in darkness to a Fluobeam 700 camera (Fluoptics). In semi
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quantitative fluorescence images (Figure 4A), liver and kidneys could be detected but not lungs,
muscle and heart, confirming the biodistribution of the radioactivity observed on PET images. The fact
that the fluorescence signal appeared stronger in the kidneys than in the liver ꢀ contrarily to the PET
images ꢀ can be explained by the cortical (superficial) distribution of the platform in this organ as it
can be seen on the transversal PET images (Figure 3 30/60 min and Figure 4B). Combined in vivo
PET and ex vivo fluorescence images demonstrate a good coꢀlocalization of the imaging platform in all
examined organs.
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Figure 4: A: semiꢀquantitative fluorescence images of collected organs at 60 min after injection of the
[18F]3/Attoꢀ680® bioinspired platform (Exposure time 100 ms; equivalent Attoꢀ680® injected dose =
1.8 nmol). B: transversal PET images of the kidneys displaying a strong cortical distribution of the
radioactivity.
This set of imaging experiments demonstrates the possibility of dual PET/optical imaging based on a
multiplexed avidinꢀbiotin platform, where biotin is derivatized with both a radionuclide (fluorineꢀ18)
and a fluorescent dye (Attoꢀ680®).
Conclusions.
The design of a fully modular and monomolecular dual PET/OI platform, exclusively based on the
strong and specific avidinꢀbiotin recognition, was herein reported. Moreover, the combination of an
original radiofluorinated biotinꢀderivative and commercially available fluorescent biotinꢀderivatives,
all crossꢀconnected thank’s to the avidinꢀcore, not only demonstrates for the first time the feasibility
and versatility of such an approach, but also opens the route to almost unlimited combinations and
connections of biotinꢀderivatives with avidin, including the addition of a targeting vector.
Supporting information available.
Additional details on syntheses, radiosyntheses, nonꢀradioactive and radiolabeled compound
characterizations and chromatographies.
Additional information on in vitro assays.
Technical and ethical details on animal experiments (PET and optical), timeꢀactivityꢀcurves derived
from PET images.
Notes.
The authors declare no competing financial interest.
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Figures
Figure 1: Binding competition assay
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Figure 2: Schematic representation of the bioinspired platforms
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Figure 3: In vivo PET biodistribution in rats of a selected 18F-PET/NIRF bioinspired
platform (n = 3; injected dose = 41 ± 3 MBq, equivalent to 1.11 nmol (MA = 20 GBq/µmol at
injection time))
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Figure 4: A: semi-quantitative fluorescence images of collected organs at 60 min after
injection of the [18F]3/Atto-680® bioinspired platform (Exposure time 100 ms; equivalent
Atto-680® injected dose = 1.8 nmol). B: transversal PET images of the kidneys displaying a
strong cortical distribution of the radioactivity.
Schemes
Scheme 1: Synthesis/radiosynthesis of the fluorinated biotin derivatives (3 / [18F]3)
Tables
Table 1: Evaluation of multiplexed [18F]3/fluorescent-biotin avidin platforms
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Figure 2
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Scheme 1
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Table 1
Atto-680®
biotin-derivatives
Atto-425®
(nmol)
0
Avidin % of biotin-derivatives bound with avidin
(nmol) [18F]3
[18F]3
(nmol)
4.06
4.12
13.3
Atto-680®-
biotin
Atto-425®-
biotin
(nmol)
8.9
7
8
9
1
2
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7.57
7.57
30.3
7.57
7.57
93a
94a
92a
53a
61a
0.240b
-
86c
-
-
-
0
14.06
14.06
14.06
14.06
0.1625b 84c
0.292b 100c 0.1906b 99c
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8.90
10.7
8.90
13.2
13.1
0.176b
0.19b
53c
70c
0.1013b 52c
0.1253b 65c
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