Nanosensor dosimetry of mouse blood
proteins after exposure to ionizing
Dokyoon Kim1, Francesco Marchetti8,9, Zuxiong Chen1, Sasa Zaric1, Robert J. Wilson1, Drew A. Hall2,
Richard S. Gaster3, Jung-Rok Lee4, Junyi Wang2, Sebastian J. Osterfeld5, Heng Yu5, Robert M. White1,
William F. Blakely6, Leif E. Peterson7, Sandhya Bhatnagar8, Brandon Mannion8, Serena Tseng8,
Kristen Roth8, Matthew Coleman8, Antoine M. Snijders8, Andrew J. Wyrobek8& Shan X. Wang1,2
1Department of Materials Science and Engineering,2Department of Electrical Engineering,3Department of Bioengineering,
4Department of Mechanical Engineering, Stanford University, Stanford, California 94305,5MagArrayInc.,1230Bordeaux Drive,
Sunnyvale, CA 94089,6Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences,
Bethesda, Maryland 20889,7Center for Biostatistics, The Methodist Hospital Research Institute, Houston, Texas 77030,8Life
Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA,9Environmental Health Science
Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada.
in blood for medical diagnosis. Using an in vivo mouse radiation model, we developed protocols for
measuringFlt3 ligand(Flt3lg) andserum amyloidA1(Saa1)insmallamountsofbloodcollectedduring the
first week after X-ray exposures of sham, 0.1, 1, 2, 3, or 6 Gy. Flt3lg concentrations showed excellent dose
discrimination at $ 1 Gy in the time window of 1 to 7 days after exposure except 1 Gy at day 7. Saa1 dose
response was limited to the first two days after exposure. A multiplex assay with both proteins showed
improved dose classification accuracy. Our magneto-nanosensor assay demonstrates the dose and time
responses, low-dose sensitivity, small volume requirements, and rapid speed that have important
advantages in radiation triage biodosimetry.
management as well as effective medical triage3. Although personal dosimeters such as film badges or thermolu-
minescent dosimeters provide accurate radiation dosimetry in the facilities using a regulated radiation source,
alternative methods are required to assess exposure dose in large-scale nuclear incidents and radiation accidents.
Several biological methods have been proposed to assess an individual’s absorbed dose retrospectively4–6.
are not well correlated with the absorbed dose4. The dicentric chromosome assay remains the gold standard for
assessing radiation dose. It measures chromosome aberrations in peripheral blood lymphocytes that are highly
correlated with the absorbed dose5. However, cytogenetic methods are time-consuming, and thus are not well
suited for incidents involving mass casualty where immediate triage decisions are required6. Alternative
molecular approaches, such as the analyses of transcriptomic and proteomic changes (so-called molecular
biomarkers) of radiation exposure, have been studied extensively due to their potential for high-throughput
and rapid assays5,6.
A biomarker is a biomolecule whose concentration varies in a predictable manner with changes in biological
date are generally limited in their performance due to insufficient sensitivity, poor scalability, lack of multiplex
capability, and/or physiologically incompatible working conditions12,16,20. For example, ELISA, the most widely
used immunoassay, makes use of colorimetric or fluorescent read-out that suffers from inherent optical absorp-
tion or autofluorescence of many biological species coexisting in samples20. Therefore, there remains a need for
xposure to ionizing radiation can, depending on dose and dose rate, induce immediate and persistent
damage to internal organs including the lymphatic, haematopoietic, gastrointestinal, and central nervous
systems1,2. An accurate and rapid measurement of dose is crucial for informed medical response and
18 February 2013
1 July 2013
19 July 2013
requests for materials
should be addressed to
lbl.gov) or S.X.W.
SCIENTIFIC REPORTS | 3 : 2234 | DOI: 10.1038/srep02234
innovative ways to overcome the disadvantages of the current detec-
to ionizing radiation.
Blood is a preferred tissue for radiation biodosimetry because
a well-accepted approach in medicine to guide personalized treat-
in tissues or cell culture after exposure to ionizing radiation, and our
ment of radiation dose21.
Major challenges in the use of protein biomarkers for radiation
biodosimetry are the technical limitations in evaluating radiation
responses of many blood proteins simultaneously with small blood
lab has developed magneto-nanosensors with multiplex capability
for ultra-sensitive and matrix-independent detection of protein bio-
markers using arrays of GMR spin-valves20,22–24. GMR is a quantum
mechanical effect in which a change in the external magnetic field
induces a change in the electrical resistance of a thin-film layered
structure composed of at least two ferromagnetic layers. GMR spin-
valve sensors are promising for handheld diagnostic devices due to
their cost-effective microfabrication technology and existing man-
ufacturing infrastructure in the hard disk drive industry25. Similar to
target analyte between the capture and detection antibodies.
However, measurable signal, the resistance change of the spin-valve
sensors, is generated by changes in the local magnetic fields from
subsequently added superparamagnetic nanoparticles. The signal is
in real-time, electrically processable, and more convenient to integ-
rate with electronic read-out system than the optical signal from
The mouse is a well accepted mammalian model for identifying
and validating biomarkers for radiation dosimetry after whole or
partial body exposures to ionizing radiation26–28. For our current
study, we developed a novel magneto-nanosensor immunoassay
for Flt3lg and Saa1 in peripheral blood and tested it using the in vivo
mouse radiation model. Flt3lg is an early-acting cytokine that
regulates proliferation and differentiation of various haematopoietic
cells, such as myeloid, lymphoid progenitor, and dendritic cells29–32,
and may be very useful in discerning the functional status of bone
marrow31,32. Saa1 is a member of serum amyloid A acute-phase pro-
tein family whose plasma level increases as much as a thousand-
fold with active inflammation33–35and after exposure to ionizing
In this paper, we measured the dose and time dependent changes
in Flt3lg and Saa1 concentrations in serial samplings of small
amounts of blood (,25 ml) collected from live animals during the
first week after exposure. Our magneto-nanosensor assay results are
highly correlated with concurrent ELISA data, and our nanosensor
platform responds linearly over wide concentration ranges. The
nanosensor showed dose separation for each exposure group in the
range of 1 to 6 Gy during the first week after exposure, and detected
of its sensitivity at low doses. Our findings demonstrate the utility of
our novel magneto-nanosensor platform for selecting candidate
plasma proteins for radiation biodosimetry and for performing triage
on large numbers of persons rapidly to identify individuals with
radiation exposures that require immediate medical intervention.
Detection scheme of magneto-nanosensor. The detection strategy
for blood proteins (Fig. 1) relies on a sandwich approach that uses a
well-characterized capture antibody immobilized onto a nanosensor
surface22. Blood proteins in solution that bind to the capture antibody
are tagged with magnetic nanoparticles using biotin-streptavidin
chemistry. When an external oscillating magnetic field is applied,
stray fields induced from the bound magnetic nanoparticles induce
resistance changes in the GMR spin-valve sensor located beneath the
platform surface. The resistance changes are proportional to the local
concentration of nanoparticles on the sensor surface, and are
measured in real-time using a custom designed electric read-out
system37,38. A typical binding curve obtained by spiking Flt3lg into a
dilution buffer at a concentration of 500 pg/ml and measuring signals
from each sensor for about a 10 minute period is shown in Fig. 1e.
Signals from Flt3lg capture antibody-coated sensors (blue) showed a
magneto-resistance change of ,180 ppm, while negative control
sensors coated with BSA (orange) and reference sensors covered by
epoxy (red) showed almost no signals. Additionally, biotin-coated
sensors were used as positive functional control sensors and
showed a signal around ,1,100 ppm (Supplementary Fig. S1).
Nanosensor calibration of radiosensitive blood proteins. We pre-
pared standard curves to calibrate the nanosensor measurement of
Flt3lg and Saa1 in mouse plasma samples (Fig. 2). The standard
curves were generated from measurements where each biomarker
was spiked in various concentrations into PBS. The standard
dilutions of mouse plasma samples were chosen so that all mouse
plasma data were within the linear range of the standard curve. The
Flt3lg standard curve had a linear dynamic range of more than two
orders of magnitude (20 , 3,000 pg/ml) with an R2value of 0.98
Figure 1 | (a–d) A schematic of magneto-nanosensor biochip immunoassay: (a) Capture antibodies are immobilized covalently on the sensor surface.
sandwich structure. (d) Streptavidin-coated magnetic nanoparticles bound to the biotinylated detection antibody produce stray magnetic field.
(e) An example of real-time binding curve showing the change in magnetoresistance (MR) in parts per million (ppm) over time for 500 pg/ml Flt3lg
(blue) compared with BSA negative control (orange) and epoxy reference (red). Error bars are 61 standard deviation.
SCIENTIFIC REPORTS | 3 : 2234 | DOI: 10.1038/srep02234
(Fig. 2a). Our magneto-nanosensor assay has a detection limit simi-
and detection antibodies (R&D systems, DY427), but maintained a
linear increase in signal even at concentrations higher than 1,000 pg/
ml. On the other hand, the linear dynamic range of the Saa1 standard
curve was from 3 to 50 ng/ml with an R2value of 0.98 (Fig. 2b), and
signals began to saturate at concentrations higher than ,100 ng/ml.
The red boxes in Fig. 2 indicate the ranges of magneto-nanosensor
chip signal we used for the Flt3lg and Saa1 assay.
Verification of magneto-nanosensor measurements using ELISA.
The magneto-nanosensor immunoassay was significantly correlated
with conventional ELISA measurements (p , 0.0001). ELISA experi-
ments were done using the same capture and detection antibodies
antibody for ELISA was incubated overnight on a polystyrene plate
rather than being covalently immobilized as it was on our nanosensor
chip. Fig. 3 shows the scatter plots of each biomarker in spiked
samples for both ELISA and magneto-nanosensor chip measure-
ments. Spearman’s rank correlation coefficient (r) was calculated to
check the correlation between the ELISA and magneto-nanosensor
measurements, and the calculated r values were 0.915 (p , 0.0001)
for Flt3lg and 0.845 (p , 0.0001) for Saa1, respectively.
Dose response in mouse plasma at day 1 after irradiation. Using
the standard curves shown in Fig. 2, we tested the ability of our
magneto-nanosensor platform to detect the effects of 2 and 6 Gy
total body irradiation (TBI) on Flt3lg and Saa1 concentrations in
peripheral blood plasma obtained from our in vivo mouse radia-
tion model. We performed the immunoassay on a single magneto-
nanosensor biochip because we did not observe any cross-reaction
between Flt3lg and Saa1. A total of 31 mouse plasma samples
collected 24 hours after irradiation (sham, n 5 13; 2 Gy, n 5 9;
6 Gy, n 5 9) were analysed. As shown in Fig. 4a, the concen-
tration of Flt3lg was 275 6 79 pg/ml (range, ,160 to ,410) in the
sham group, increased to 687 6 166 pg/ml (range, ,530 to ,1,050)
inthe2 Gygroup,andfurtherincreasedto14426410 pg/ml(range,
,800 to ,2,000) in the 6 Gy group, with significant concentration
increases for each radiation dose group (p , 0.0001). The
concentration of Saa1 (Fig. 4b) was 3.5 6 3.8 mg/ml (range, ,0.3
Figure 2 | Standard curves measured on magneto-nanosensor chips for mouse (a) Flt3lg and (b) Saa1. Red boxes indicate the ranges of magneto-
nanosensor chip signal and biomarker concentration used in the assays performed in this work. Error bars are 61 standard deviation.
Figure 3 | Correlation scatter plots between ELISA and magneto-nanosensor biochip assay for mouse (a) Flt3lg and (b) Saa1.
SCIENTIFIC REPORTS | 3 : 2234 | DOI: 10.1038/srep02234
to ,13) in the same sham samples, and increased about 30 fold after
2 Gyirradiation(average,122648 mg/ml;range,,60to,210),but
with no further increase at 6 Gy (average, 128 6 50 mg/ml; range,
,70 to ,210). For Saa1, the sham-irradiated and irradiated (2 or
6 Gy) groups could be fully distinguished since their Saa1
concentration ranges did not overlap. However, the Saa1
concentration difference between 2 and 6 Gy groups was not
significant (p 5 0.8).
Dose response change with time after exposure. We then
investigated the changes in dose response of Flt3lg and Saa1
during the first week after TBI exposure using repeated venous
blood draws from living mice. For this experiment, ,25 ul of
blood was obtained from the saphenous vein of each mouse at
various time points after irradiation, and plasma was isolated by
centrifugation. A total of 210 plasma samples were collected from
Figure 4 | Radiation dose response behaviour of mouse (a) Flt3lg and (b) Saa1 measured using magneto-nanosensor biochips for 0, 2, and 6 Gy
irradiated C57BL/6J mice at 24 hours post-irradiation. Red bars indicate average values, and each black dot represents one animal.
Figure 5 | (a) Radiation time response behaviour of mouse Flt3lg measured using magneto-nanosensor biochips for sham, 0.1, 1, 2, 3, and 6 Gy (blue,
green, orange, magenta, red, and black, respectively) irradiated C57BL/6J mice at 8 days before, 1, 2, 3, 5, and 7 days post-irradiation. Error bars indicate
61 standard deviation for each radiation exposure group. Dose response behaviours of mouse Flt3lg at days (b) 1, (c) 2, (d) 3, (e) 5, and (f) 7 after
exposure to radiation. Red bars indicate average values, and each black dot represents one animal. Red lines are dose response fitting curves.
SCIENTIFIC REPORTS | 3 : 2234 | DOI: 10.1038/srep02234
at 1, 2, 3, 5, and 7 days after exposure) at doses from 10 cGy to 6 Gy
(sham, n 5 7; 10 cGy and 1 Gy, n 5 5; 2 Gy, n5 8; 3 and 6 Gy, n 5
5). All measurements were blinded so that the experimenter
measured magnetic signals without knowing the actual radiation
Fig. 5 shows the time response for Flt3lg. As can be seen in Fig. 5a,
the Flt3lg concentration remained at similar values as the basal level
for sham-irradiated samples, but increased to ,750, 1420, 4520, and
7450 pg/ml as maximum average changes for 1, 2, 3, and 6 Gy irra-
responses at days 1, 2, 3, 5, and 7 after exposure, respectively, and P
values associated with two-tailed Student’s t-test are presented in
Supplementary Table S1 and S2. Exposure groups irradiated at
higher doses showed higher Flt3lg concentrations at all time points,
and significant concentration differences against sham at $1 Gy in
the time window of 1 to 7 days after exposure other than 1 Gy at day
7couldbeseen(Supplementary TableS1,p,0.0001,except1 Gyat
Flt3lg concentration change was not significant for 10 cGy exposure
group. (p 5 0.1). Supplementary Table S2 lists the p values for
comparison of immediately lower doses on the same day.
Significant concentration differences (p , 0.05) still could be
observed except between 2 and 3 Gy samples at day 1 (p 5 0.4),
and between 3 and 6 Gy samples at day 2 and 3 (p 5 0.2 and 0.1,
Supplementary Table S3 lists the sensitivity, specificity, positive
predictive values (PPV), and negative predictive values (NPV)
obtained by dose discriminant analysis using Flt3lg for sham, 1, 2,
0.1 Gy samples were excluded from the dose discriminant analysis
because their Flt3lg concentration changes from sham samples were
insignificant. Here, the DISCRIM procedure in SAS software was
the probability of measured concentrations at a given time point
being classified into each radiation dose group. Many of the dose
groups could be classified with high sensitivity (0.8 , 1), specificity
(0.8, 1),PPV(0.7 ,1), andNPV(0.7 ,1), showingexcellentdose
discrimination among the dose groups. There were some radiation
1, 6 Gy at day 2, and sham and 1 Gy at day 7), which were caused by
similar Flt3lg concentrations between these radiation dose groups
and other groups at the same day post irradiation, as can be seen in
Figs. 5b, c, and f, respectively.
sharp increase in plasma concentration of ,80 mg/ml on average
was observed for 2, 3, and 6 Gy irradiated samples at day 1 after
irradiation, after which the concentration decreased to near basal
level by 3 days after irradiation. Fig. 6b and c show the Saa1 dose
responses at days 1 and 2 after exposure, respectively. Saa1 concen-
trations at day 2 for doses $1 Gy still remained higher than sham
levels even though they were not as high as those at day 1.
Supplementary Table S4 presents the p values associated with two-
tailed Student’s t-test. Significant concentration differences between
sham and $1 Gy dose groups could be seen for the first two days
after exposure (p , 0.0001), but the concentration change was not
significant for 10 cGy group (p 5 0.4067 at day 1). Supplementary
on the same day.
Supplementary Table S6 lists the sensitivity, specificity, PPV, and
NPV obtained by dose discriminant analysis using Saa1 for sham, 1,
2, 3, and 6 Gy irradiated samples at day 1 and 2 after irradiation.
Although the dose discrimination power of Saa1 concentrations for
distinguishing each radiation dose groups was not as good as that of
Flt3lg concentrations, Saa1 concentrations still could be used to dis-
criminate sham-irradiated samples from irradiated ($ 1 Gy) sam-
ples with high sensitivity (51), specificity (0.87 , 1), PPV (0.7 , 1),
and NPV (51). Supplementary Tables S7 and S8 list the measure-
ment data presented in Figs. 5 and 6.
We developed a magneto-nanosensor platform based on the GMR
effect to measure concentrations of multiple radiation-responsive
proteins in peripheral blood and then demonstrated the robustness
small volume blood samples collected from the in vivo mouse radi-
Our magneto-nanosensor biochip has a size of 10 mm 3 12 mm
and has an array of 64 individually addressable sensors (each sensor
is made of 48 spin-valve strips with a size of 90 mm 3 0.75 mm)
covered by ultrathin and biochemically stable silicon oxide passiva-
epoxy or coated with BSA or biotin-BSA. Epoxy covered sensors
served as electrical reference sensors. Electrical background noise
was removed by subtracting the average signal of the epoxy-covered
sensors from all the other signals. Bovine serum albumin (BSA)-
coatedsensors were usedas negativecontrol sensors, sincethey were
blocked and thus prevented from specifically capturing either target
antigen or streptavidin-tagged magnetic nanoparticles. The very
small signals that appeared on BSA-coated sensors clearly indi-
cated that the observed signals from the capture antibody-coated
sensors were not a result of non-specific binding of the analyte or
magnetic nanoparticles. Biotin-coated sensors were used as positive
Figure 6 | (a) Radiation time response behaviour of mouse Saa1 measured using magneto-nanosensor biochips for sham, 0.1, 1, 2, 3, and 6 Gy
(blue, green, orange, magenta, red, and black, respectively) irradiated C57BL/6J mice at 8 days before, 1, 2, 3, 5, and 7 days post-irradiation. Error bars
indicate 61 standard deviation for each radiation exposure group. Dose response behaviours of mouse Saa1 at days (b) 1 and (c) 2 after exposure to
radiation. Red bars indicate average values, and each black dot represents one animal. Red lines are dose response fitting curves.
SCIENTIFIC REPORTS | 3 : 2234 | DOI: 10.1038/srep02234
functional controls because of their ability to specifically capture
streptavidin-tagged magnetic nanoparticles, and they showed very
large signals as expected.
There were some discrepancies between the concentrations mea-
sured on magneto-nanosensor chips and ELISA, which might result
fromdifferences in antibody surface conjugation or from differences
in the signal generation mechanism. It has been proposed that dif-
ferentsurfacemodification methodsorassaytechniques couldresult
in a different quantification of analyte39–42. However, the calculated
values of Spearman’s rank correlation coefficient (r 5 0.915 for
Flt3lg and r 5 0.845 for Saa1) clearly show that the concentrations
determined using magneto-nanosensor biochip and ELISA have sig-
ELISA measurements in parallel with the magneto-nanosensor bio-
chip measurements for the in vivo irradiation time response mouse
model samples due to limited blood volume permitted by IACUC
(Institutional Animal Care and Use Committee), the significant cor-
ensures the validity of the magneto-nanosensor for measuring
plasma samples collected from radiation exposure studies.
of plasma Flt3lg and Saa1 in C57BL/6J strain mice during the first
week after radiation exposure. Flt3lg showed elevated levels at all
time points in the time windows of 1 to 7 days after exposure to
radiation doses $ 1 Gy, and their maximum changes increased with
increasing radiation doses. Previous studies also reported the
increase of Flt3lg level after exposure to different radiation doses,
although the maximum and temporal concentration changes varied
among different mice strains31,43. Saa1 showed elevated levels only
during the first two days after exposure exhibiting maximum
changes at day 1. Dose dependent increase of serum amyloid A at
day 1 was reported in a study using BALB/c strain mice, although
temporal changes were not measured44.
In the dose response study, although the general trend of Flt3lg
concentration change was elevation of Flt3lg concentration with
increasing irradiation dose, there was some overlap in the concen-
tration ranges of 2 and 6 Gy dose groups. Saa1 concentration also
increased after irradiation, but Saa1 concentration ranges of 2 and
6 Gy dose groups were very similar. These different levels of Flt3lg
and Saa1 upregulation after irradiation suggest inter-individual var-
iations in radiation response45–47. It has been known that genetically
diverse individuals respond differently to radiation exposure, reveal-
ing less than ,10% of individuals are hyper-radiosensitive, ,70%
are normally sensitive, and the remaining 20% are considered radio-
resistant26,47. However, because the mice used in this study were
genetically identical, the observed variations seem to be caused by
In the time response study, it is worth noting the relatively large
samples, implying relatively much higher damage is expected when
irradiated by doses $ 3 Gy. Since Flt3lg and Saa1 showed different
level changes over time after irradiation in our study (Flt3lg level
increases up to 3 , 5 days before starting to decrease whereas ele-
vated Saa1 level persists for only two days), it is possible to use both
biomarkers together for determining the radiation exposure time
point retrospectively. For example, 2 Gy irradiated samples at day
because of their similar Flt3lg concentrations at day 1 and 7.
However, they would be distinguishable if we measure Saa1 along
with Flt3lg since day 1 samples would have higher Saa1 levels than
enables higher accuracy for diagnostic tests than do single biomar-
and Saa1 can improve the classification, especially on day 1 and 2,
tioned above (Supplementary Table S9). Discrimination error rates
from day 5 were higher than earlier days due to similar Saa1 con-
centrations and decreasing Flt3lg concentration differences among
samples of different doses. For samples exposed to $ 3 Gy, on the
other hand, adding Saa1 as a secondary biomarker did not improve
the classification as Flt3lg had a discrimination power much higher
than that of Saa1 (Supplementary Table S10).
In summary, we have developed a novel protein immunoassay
platform using magneto-nanosensor biochips, and have demon-
strated its utility by measuring Flt3lg and Saa1 biomarkers in an in
vivo mouse model. For the same capture and detection antibodies,
the ELISA and magneto-nanosensor results were highly correlated,
and the magneto-nanosensor showed a large dynamic range20,22. We
also monitored dose dependent and temporal upregulation of Flt3lg
and Saa1 in radiation dose and time response studies. Flt3lg concen-
tration change showed excellent dose discrimination over a week
after radiation exposure, and Saa1 dose response was apparent for
the first two days after exposure. A multiplex assay with both pro-
teins could improve classification accuracy of radiation exposure.
ical diagnosis and clinical research, including but not limited to
radiation biomarker validation and early radiation triage.
Materials. Anti-mouse Flt3lg antibody, biotinylated anti-mouse Flt3lg antibody,
mouse Flt3lg standard (all from R&D systems, DY427), anti-mouse Saa1 antibody
(R&D systems, AF2948), biotinylated anti-mouse Saa1 antibody (R&D systems,
BAF2948), mouse Saa1 standard (ALPCO diagnostics, 41-SAAMS-E01),
poly(allylamine hydrochloride) (Polyscience), poly(ethylene-alt-maleic anhydride)
(Aldrich), 13 phosphate buffered saline (PBS) (Invitrogen), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Thermo scientific), N-
hydroxysuccinimide (NHS) (Aldrich), 1% bovine serum albumin (BSA) (Aldrich),
biotinylated bovine serum albumin (biotin-BSA) (Pierce), Tween 20 (Aldrich), and
streptavidin-coated MicroBeads (Miltenyi, 130-048-101) were used as received, and
without further purification. Mouse (C57BL/6J males and females) plasma samples
were prepared by depositing blood, with heparin added as an anticoagulant, into
Microvette tubes (Sarstedt) and centrifuging.
Mouse radiation model for collection of blood plasma. The use of animals in this
study was approved by the Institutional Animal Care and Use Committee at both
Lawrence Berkeley National Laboratory and Stanford University.
were irradiated and blood sampled similar to that previously described26,27. Briefly
mice were exposed to sham, 2, or 6 Gy irradiation using 320-kVp X-ray (Pantak) at
dose rates of 0.75 Gy/min for 2 Gy and 1.9 Gy/min for 6 Gy. The consistency of the
delivered dose was monitored using a dosimeter in every 2 , 3 radiations. Blood was
collected from euthanized mice by cardiac puncture 24 hr after irradiation using
heparin-wetted syringes and 25G needles and transferred into Eppendorf tubes
containing 10 ml of heparin. Blood was then transferred to another centrifuge tube
and layered on top of 1 ml HISTOPAQUE 1083 (Sigma). Plasma was isolated by
centrifugation and stored at 280uC prior to analysis.
with 0, 0.1, 1, 2, 3, or 6 Gy X-ray of 320-kVp at a dose rate of 0.75 Gy/min except the
6 Gy exposure, which was performed at a dose rate of 1.9 Gy/min. About 25 ml
1, 2, 3, 5, and 7 days after irradiation using an heparinised eppendorf pipette tip and
centrifugation and stored at 280uC prior to analysis. Pre-irradiation samples were
collected from all mice 8 days before irradiation or sham-irradiation to establish
baseline levels of Flt3lg and Saa1 for each mouse.
Magneto-nanosensor chip surface preparation. The magneto-nanosensor chip was
fabricatedbypreviouslyreported method22.Thesensorchip surfacewaswashedwith
exposing to oxygen plasma (Harrick Plasma, PDC-32G) for 3 minutes. Then, the
5 minutes, followed by rinsing with deionised water. The sensor chip was baked at
120uC for 1 hour. After incubation in a 2% aqueous solution of poly(ethylene-alt-
maleic anhydride), the surface was washed again with deionised water and activated
by adding a mixture of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride and N-hydroxysuccinimide in deionised water. A robotic spotter
(Scienion, sciFlexarrayer) was then used to deposit capture antibody solution on the
sensor chip surface. A PBS solution of anti-mouse Flt3lg (0.5 mg/ml) and a PBS
solution of anti-mouse Saa1 (1 mg/ml) were deposited on at least 8 sensors. Also,
SCIENTIFIC REPORTS | 3 : 2234 | DOI: 10.1038/srep02234
electrical background signals. Finally, the sensor chip was stored in a humidity
chamber at 4uC before use.
Flt3lg/Saa1 duplex-assay protocol. After washing the sensor chip surface with a
washingbuffer(0.1%BSAand 0.05%Tween20in PBS), thesurface wasblockedwith
1% BSA for 1 hour. Then, the surface was washed again and immersed in a 40003
hours. The sample solution was washed away with the washing buffer and a Saa1
detection antibody solution with a concentration of 5 mg/ml was added. Following 1
hour incubation with the detection antibody, the surface was washed again before
measuring Saa1 signal from the chip. After completing the real-time signal collection
for Saa1, similar procedures excluding the blocking step were used with a 53 diluted
sample and an Flt3lg detection antibody solution before collecting Flt3lg signals.
Analyte quantification. The electrical read-out system for the magneto-nanosensor
chip was implemented according to Hall et al.37,38. Streptavidin-coated magnetic
nanoparticles (Miltenyi, streptavidin MicroBeads) were added to sensor chip to
induce an analyte concentration-dependent signal change. The measured signals
were converted to corresponding concentrations using the standard curves for each
ELISA measurements. Plasma protein biomarkers (i.e., Flt3lg and Saa1) were
E01) according to the manufacturer’s instructions. Three replicate measurements
were determined for each sample and standards. The Flt3lg and Saa1 concentrations
in plasma samples were determined via the generated calibration curve for standard
Two-sided Student’s t-test was used to determine significant difference between two
groups. P values of , 0.05 were considered statistically significant. Correlation
analysis between magneto-nanosensor and ELISA was done using non-parametric
Spearman rank correlation, since it did not require normality assumption and the
number of samples was relatively small. Statistical software SAS was used for the
DISCRIM procedure to classify the radiation exposure time points under a given
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This project has been funded in whole or in part by Federal funds from the Biomedical
Advanced Research and Development Authority, Office of the Assistant Secretary for
Preparedness and Response, Office of the Secretary, Department of Health and Human
Services, under Contract No. HHSO100201000006C. This work was performed under the
auspices of the U.S. Department of Energy by the University of California, Lawrence
Berkeley National Laboratory under DOE contract No. DE-AC02-05CH11231 and with
funding support from BAA-BARDA-09-36. The Armed Forces Radiobiology Research
Institute, under work units RAB4AP and RBB4AR, provided the support for one of the
co-authors involved in this research (W.B.). The views expressed here are those of the
authors; no endorsement by the U.S. Department of Defense or the U.S. government has
been given or inferred. The work at Stanford also benefited from the National Cancer
Institute grants Physical Science Oncology Center (U54CA143907), Center for Cancer
NanotechnologyExcellence (U54CA151459),InnovativeMolecular AnalysisTechnologies
D.K., F.M., W.F.B., A.J.W. and S.X.W. designed the research. D.K., F.M., Z.C., S.Z., S.B.,
B.M., S.T., K.R. and M.C. performed the experiments. D.K., F.M., R.J.W., D.A.H., R.S.G.,
J.L., J.W., S.J.O., H.Y. and A.J.W. contributed new reagents/analytic tools. D.K., F.M.,
R.J.W., R.M.W., W.F.B., L.P., A.M.S., A.J.W. and S.X.W. analysed the data. D.K., F.M. and
S.X.W. wrote the initial draft of the paper and all authors commented on the paper.
Supplementary information accompanies this paper at http://www.nature.com/
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Kim, D. et al. Nanosensor dosimetry of mouse blood proteins after
exposure to ionizing radiation. Sci. Rep. 3, 2234; DOI:10.1038/srep02234 (2013).
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SCIENTIFIC REPORTS | 3 : 2234 | DOI: 10.1038/srep02234