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Biosensors for Real-Time Monitoring 
of Radiation-Induced Biologic Effects 

in Space 




August 2002 Report 



U.S. National Aeronautics and Space Administration 

NAS2-02069 



Principal Investigator: 
James R. Baker Jr., M.D. 

Center For Biologic Nanotechnology 

Division of Allergy 

Department of Internal Medicine 

School of Medicine 

University of Michigan 

Phone: (734) 647 2777 

Fax: (734) 936 2990 

Email: jbakerjr@umich.edu 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



I. Executive Summary 

This work seeks to develop cellular biosensors based on dendritic polymers. Nanoscale 
polymer structures less than 20 nm in diameter will be used as the basis of the biosensors. 
The structures will be designed to target into specific cells of an astronaut and be able to 
monitor health issues such as exposure to radiation. Multiple components can be assembled 
on the polymers including target directors, analytical devices (such as molecular probes), and 
reporting agents. The reporting will be accomplished through fluorescence signal 
monitoring, with the use of multispectral analysis for signal interpretation. These 
nanosensors could facilitate the success and increase the safety of extended space flight. The 
design and assembly of these devices has been pioneered at the Center for Biologic 
Nanotechnology in the University of Michigan. 

This period, synthesis of the test-bed biosensors continued. Studies were performed on the 
candidate fluorescent dyes to determine which might be suitable for the biosensor under 
development. Development continued on producing an artificial capillary bed as a tool for 
the use in the production of the fluorescence signal monitor. Work was also done on the in 
vitro multispectral analysis system, which uses the robotic microscope. 



August 2002 Report 



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NAS2-02069 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



II. Overall Progress: Organized by technical objective and task group. 

Technical Objective I: Development of dendritic polymer based biosensors capable of 
monitoring radiation-induced changes in lymphocyte populations. 

Synthesis of Biosensor 

Balazs Keszler, Ph.D., Istvan Majoros, Ph.D. 

We have designed the chemical structure of a fluorescence resonance energy transfer (FRET) 
reagent, which will detect apoptosis via the cleavage of the peptide linker by Caspase 3. 

Design of a FRET: 

• Donor and acceptor dye molecules must be in close proximity (10-1 00 A). 

• The absorption spectrum of the acceptor must overlap the fluorescence emission 
spectrum of the donor. 

• Donor and acceptor transition dipole orientations must be approximately parallel. 

The distance at which energy transfer is 50% efficient (i.e., 50% of exited donors are 
deactivated by FRET) is defined by Forster radius (Ro). The magnitude of Ro is dependent 
on the spectral properties of the donor and acceptor dyes: 

Ro = [8.8x10"* K^*n'**QYD*J(>.)]'"' A 

Where k^ = dipole orientation factor (range to 4; k^ = 2/3 for randomly oriented donors 
and acceptors) 
QYd = fluorescence quantum yield of the donor in the absence of the acceptor 

n = refractive index 
J{X) = I(eA(^)*FD(A,)*A.'*)dA. ,cm^M"' — spectral overlap integral of the absorption 
spectrum of the acceptor and fluorescence emission spectrum of the donor 
where Sa = extinction coefficient of acceptor 

Fd = fluorescence emission intensity of donor as a fraction of the 
total integrated intensity 

The structure of the proposed apoptosis FRET reagent is: 



H 

DONOR — N — N= 



o o 

H II II 

C — C— GDEVDGVKC — ACCEPTOR 



Where our donor dye is 6-carboxy-2',7'-dichlorofluorescein hydrazide (Ex: 505 nm and 
Em:525 nm) and our acceptor dye is 5-carboxytetramethylrhodamine (Ex: 550 nm and 
Em: 5 70 nm). They have a 55 A Forster radius. 



August 2002 Rqjort 



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NAS2-02069 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



/NH2 

HN 



COOH 




^O' "^ "O (H3C)2N 

DONOR 




^_ ACCEPTOR 

Figure"l. The structure of the donor and acceptor molecules. 

The donor dye molecule will be covalently attached to the oligopeptide through *e 
hydrazide group, while the acceptor molecule will be attached to the pnmary amme of lysme. 




H3N 



Figure 2. The structure of the peptide linker. 

Conjugation of MitoTrack er® dves to G5 dendrimer: ^ 

In this period we have conjugated MitoTracker^ Red CMXRos (M-7512) and MitoTracker 
Deep Red (M-22426) mitochondrion-selective dyes to the dendnmer. We have followed the 
procedure we developed for the model reaction described in our previous report. We have 
prepared these biosensors with folic acid assuring specific targeting. We also prepared OS- 
dye conjugates without specific targeting moiety. These materials serve as control matenal. 

Conjugation of MitoTracker® Red CMXRos (M-7512) to G5 dendrimer: We used partially 
acylated G5 dendrimer for the conjugation, with 86 out of 1 10 primary amine groups ^ylated. 
0.00182 g (6.14xl0-« mol) of G5-(NH2)24-(OCH3)86 was dissolved in 1 ^L of PBS buffer 
rt>H=8) First 0.00026 mL (1.84x10-^ mol, lOx molar excess of the dye) of EtsN, then 100 ^g 
1 84x10-^ mol) of dye dissolved in 0.5 mL of DMSO was added to the dendnmer solf on^ 
The mixture was stiired for 48 hours, dialyzed in water-methanol (2:1), then in DI water. After 
dialysis, TLC showed no traces of free dye. The dye-to-dendrimer ratio was determined by 
UV spectroscopy to be 1:1. 

Conjugation of MitoTracker® Deep Red (M-22426) to G5 dendrimer: We used partially 
acylated G5 dendrimer for the conjugation, with 86 out of 1 10 primary amine groups 
acylated. 0.00186 g (6.27xl0-« mol) of G5-(NH2)24-(OCH3)«6 was dissolved m 1 "^L of PBS 
buffer (pH=8). First 0.00026 mL (1.84x10"' mol, lOx molar excess of the dye) of EtjN, then 

August 2002 Report P^g^ ^ 



NAS2-02069 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



0.0001 g (1.84x10"^ mol) of dye dissolved in 0.5 mL of DMSO was added to the dendrimer 
solution. The mixture was stirred for 48 hours, dialyzed in water-methanol (2:1), then in DI 
water. After dialysis, TLC showed no traces of free dye. The dye-to-dendrimer ratio was 
determined by UV spectroscopy to be 1 : 1 . 

Conjugation of MitoTracker® Red CMXRos (M-7512) to G5-(NH2)2i(OCH3)86-FA3 
dendrimer: The G5 dendrimer had 3 folic acid units for specific cell targeting. 0.00192 g 
(6.21x10"^ mol) of G5-(NH2)2i-(OCH3)86-FA3 was dissolved in 1 mL of PBS buffer (pH=8). 
First 0.00026 mL (1.84x10'^ mol, lOx molar excess of the dye) of Et3N, then 0.0001 g 
(1 .84x10"^ mol) of dye dissolved in 0.5 mL of DMSO was added to the dendrimer solution. 
The mixture was stirred for 48 hours, dialyzed in water-methanol (2:1), then in DI water. 
After dialysis, TLC showed no traces of free dye. The dye-to-dendrimer ratio was 
determined by UV spectroscopy to be 1 : 1 . 

Conjugation of MitoTracker® Deep Red (M-22426) to G5-(NH2)2-(OCH3)86-FA3 dendrimer: 
The partially acylated G5 dendrimer had 3 folic acid units as specific targeting moiety. 
0.00195 g (6.22x10-^ mol) of G5-(NH2)2i-(OCH3)86-FA3 was dissolved in 1 mL of PBS 
buffer (pH=8). First 0.00026 mL (1.84x10"^ mol, lOx molar excess of the dye) of Et3N, then 
0.0001 g (1.84x10"^ mol) of dye dissolved in 0.5 mL of DMSO was added to the dendrimer 
solution. The mixture was stirred for 48 hours, dialyzed in water-methanol (2:1), then in DI 
water. After dialysis, TLC showed no traces of free dye. The dye-to-dendrimer ratio was 
determined by UV spectroscopy to be 1 : 1 . 

Upon conjugation to dendrimer, the absorption maximum of the dyes (Amax) shifted to a 
longer wavelength. 

MrtoTracker* Deep Red '•» ] MitoTracker* Red CMXRos 



1.0 

o.» 




^"0 iOO 400 500 600 700 80 

Wavelength, nm 

Figure 3. The Amax of MitoTracker* Deep Red is 
shifted from 640 nm to 649 nm when it is conjugated 
to dendrimer. 



200 300 400 SOO 600 70O 800 

Wavelength, nm 

Figure 4. The Amax of MitoTracker® Red CMXRos 
is shifted from 578 nm to 592 nm when it is conjugated 
to dendrimer. 



The reaction conditions used in the model reaction must be modified to obtain higher 
dye/dendrimer molar ratios. 

Activities planned for the next reporting period 

In the next period we will modify the donor molecule (to form a hydrazide reactive group), 
purchase oligopeptide, and modify for coupling of the donor molecule. 



August 2002 Report 



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Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



In the next period well increase the mitochondrion-selective dye to dendrimer molar ratio. 
We will also conjugate the apoptosis indicator (Rhodamine 110, bis-(L-aspartic acid) amide) 
dye to the dendrimer. 
Technical Objective II: The analysis and verification of the radiation sensing capability in 

lymphocytes in vitro. ^«;^o^., of 

The main goal of this technical objective is to charactenze and measure the efficacy of 
dendrimer based biosensors as signal carriers. This includes a systematic analysis of single 
or multiple conjugates with intracellular spectral probes. 

Testing of the caspase-3 substrate, rhodamine 110, bis-(L-aspartic acid amide), 
trifluoroacetic acid salt (Rl 10), in vitro as a monitor of apoptosis in vivo 

Andrzej Myc, Ph.D., Jolanta Kukowska-Latallo, Ph.D., Alina Kotlyar, M.S., Katarzyna 
Janczak, M.S., Jeffrey Landers, B.S. 

Caspase-3 is one of the cysteine proteases most frequently activated during the Process of 
apoptosis or Programmed Cell Death (PCD), hi response to pro-apoptotic stimuli the 32 
kba pro-Caspase-3 is processed to an active enzyme consisting of two subunits of 17 and 12 
kDa. Activated caspase-3 is essential for the progression of apoptosis, resulting m the 
degradation of cellular proteins, apoptotic chromatin condensation, and DNA fragmentation. 

The bis-L-aspartic acid amide of Rl 10 contains the rhodamine 110 fluorophore flanked by 
aspartic acid residues and in this form does not fluoresce (Figure 5). Activated caspase-3 



ASP-HN 




NH-ASP 0) 



•2CF,C00H 




350 400 



600 650 



450 500 550 
Wavelength (nm) 

Figure 5. Molecular structure and absorption/emission spectra of rhodanune 110, bis-(L- 
aspartic acid amide), trifluroacetic acid salt. C22H26F6N40,3 Mol. Wt. 788.57 

specifically cleaves the aspartic acid moieties from the compoimd and ^-^l^^^;^ ^^^^f J^ 
fluorogenic rhodamine 110 fluorophore. The fluorescence of rhodamine 1 10 can be detected 
Ld quantified in apoptotic cells by flow cytometry. Since the Rl 10 does not require any 



August 2002 Report 



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NAS2-02069 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



invasive techniques, such as osmotic shock, to gain entrance into the cytoplasm, it may be 
used to detect apoptosis in living cells. 

The Rl 10 was evaluated in vitro to assess 
its potential to detect and quantitate apop- 
tosis in Jurkat cells. Jurkat cells were in- 
cubated with 1 |aM staurosporine for 6 h 
and stained afterwards with RllO for 20 
min. The fluorescence was quantified by 
flow cytometry using the 488 nm excita- 
tion laser (Figure 6). Emission spectra 
were detected using four different photo 
multipliers (FLl- 525 nm, FL2- 575 nm, 
FL3- 620 nm, and FL4 - 675 nm). Al- 
though Rl 10 has a very broad emission 
spectrum (Figure 5), the highest fluor- 
escence was recorded at wavelength of 
525 nm (Figure 7). At this wavelength, 
we also observed the greatest resolution 
between non-apoptotic (control cells) and 
apoptotic cells. In our next experiment. 




10" 10' 10^ 10 

Fluorescence Intensity (525 nm) 
Figure 6. Histograms of control (red) and apoptotic 
(blue) Jurkat cells stained with Rhl 10 (10 ^M). Cells 
were treated with either staurosporine ( 1 jiM) or DMSO 
(control) for 6 h, stained with Rh 110 for 20 min and 
washed. Fluorescence of 10,000 cells was measured 
using flow cytometer at the wavelength of 525 nm. 



10^ 



lFL-1 (525nni+/-20) 

I FL-2 (575 nm +/-20) 

FL-3 (620 nm +/-20) 

I FL-4 (675 nm +/-20) 




50 

_ 45 

a> 

§40 

i 35 

S 
I 30 

c 
I 25 

u 

I 20 

s ^^ 

(A 

g 10 



Lkistained Rh110/10uM Rh110/20uM Sta/unst. 

Treatment 
Figure 7. Detection of apoptosis in Jurkat cells using Rhl 10. Jurkat cells were treated for 6 h with luM of 
Staurosponne to mduce apoptosis and subsequently stained with Rhl 10 for 20 min. Cell fluorescence was 
measured with a flow cytometer using four different photo multipliers (FLl- 525 nm, FL2- 575 nm, FL3- 620 
nm, and FL4 - 675 nm). The controls included cells unstained with Rhl 10 (Unstained), stained with 10 uM 
Rhl 10 (Rhl lO/lO^M), cells stained with 20 mM Rhl 10 (Rhl 10/20mM) and Staurosporine treated unstained 
cells (Sta/unst.) The Staurosponne treated apoptotic cells were stained with 10 uM Rhl 10 (Sta/rhl 10/lOuM^ 
andwidl20^MRhllO(Sta/rhllO/20^M). nllu/lu^M; 



Sta/rhl 1 0/1 OuM Sta/rh110/20uM 



August 2002 Report 



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NAS2-02069 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 




we tested if Rl 10 could be used to measure the kinetics of apoptosis. There is a linear cor- 
relation between the progression of apoptosis and the fluorescence intensity of rhodamme 
110 (Figure 8). 

Figures. Kinetics of apoptosis. Jurkat cells were 
treated with Staurosporine for 2, 4, and 6 hours, 
then stained with Rhl 10 (Sta/Rhl 10). The cell 
fluorescence was measured using a flow cyto- 
meter. Controls included Staurosporine treated 
unstained cells (Sta/unst.), DMSO treated Rhl 10 
stained (DMSO/Rhl 10) and DMSO treated 
unstained Jurkat cells (DMSO/unst.). 



In conclusion, the Rl 10 substrate gains entrance into the cytoplasm of living cells and 
therefore can detect the apoptosis in situ. The Rl 10 will be used to stain pnmary mouse 
splenocytes ex vivo and to examine the detection of apoptosis in vivo using two-photon 
fluorescence measurement through optical fibers. 

Technical Objective III: The development of a noninvasive laser analysis system to 
monitor biosensor signals from lymphocytes with radiation-induced damage. 

The primary goal of this component of the research program is to develop ultrasensitive in 
vivo fluorescence detection technologies and methodologies for real-time mon.tonng of 
radiation-induced biologic effects in space. 

Testing Using Microscopic Multispectral Analysis 

Felix de la Iglesia, MD, Timothy Sassanella, M.S. 

The development of the image quantitation procedure has progressed significantly, and 
should be completed during the next month. Several useful macros have been obtained for 
the hnage Pro software, and progress has been made in the areas of background elimination, 
average pixel intensity determination, and the transfer of data to appropnate analytical 
software for manipulation and graphing. 

The IRB protocol for blood use for the project has been submitted. 

KB cells were tested with six mitochondnal monitoring dyes (including MitoTracker® Red 
CMXRos and MitoTracker® Deep Red) to verify cellular uptake and localization of the free 
dye in culture. The six dyes employed were those previously selected as potential candidates 
for conjugation. All dyes fluoresced in live cells under the conditions tested. The optimal 
concentration range was determined. Qualitative analysis indicates mitochondnal 
localization, but the intensity of fluorescence varied significantly, though the dyes were tested 



August 2002 Report 



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NAS2-02069 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



at the same concentrations and with the same methods. MitoTracker® CMXRos appeared to 
be the most intensely fluorescent (Figure 9). The absorbance spectra in water for the six dyes 
was also determined (Figure 10, next page). 



rv3 



OnM 



iOO nVI 



( V 5 



OnM 



IOO n.M 

Mitochondrial 

Dye 
Absorbance 
Kxtinction CoeiT. 
Kmission 




FIK 



( y 3 



MF Ml MF Ml MTRed MF 

Far Red Deep Red Red 594 Red 58<t <MXRos Cnen 

682 641 604 588 578 489 

95K I73.7K 97K 76.1K 124.9K II2K 

702 662 637 643 599 517 



Figure 9. Summary of mitochondria-specific dye testing in KB cells. Six dyes were tested: five 
were in the red range, and the Cy3 and Cy5 filter sets were used; the sixth dye was in the green 
range, and the FITC and Cy3 filer sets were used. The concentration of dye was 100 nM. 

Two dyes, MitoTracker® Red CMXRos and MitoTracker® Deep Red, have been conjugated 
to dendrimers, both with and without a targeting agent, and have been received recently from 
the chemistry group. Experimental preparations are underway to test these conjugates. 

Preparation, function testing, and calibration testing of the robotic microscope and individual 
components continued in August. 



August 2002 Report 



Pages 



NAS2-02069 




0.35 
0.30 
0.25 



80.20 

c 
n 

€0.15 
o 

<0.10 



0.05 



0.00 



Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



Mitochondrial Dye Absorbance^ 



"MF Green 
•MT Red CMXRos 
"MT Red 580 
-MF Red 594 
MT Deep Red 
■MF Far Red 




400 



450 



500 



650 



550 600 
Wavelength (nm) 

Figure 10. Absorbance spectra of the six selected mitochondria-specific dyes. The spectra of fr^^ and 
dendrimer conjugated dyes will be used to select or acquire appropriate excitation, dichroic, and emission 
filters for photomicrography. 



When 



Activities planned for the next reporting period: 

1) The quantitative protocol for micrographs will continue to be developed, 
completed, microscope calibrations will be conducted. 

2) Quantitative analysis on these micrographs will be done in the commg month as the 
technique is developed. ^ . r^ , ®t^ 

3) A photobleaching study using MitoTracker® Red CMXRos and MitoTracker Deep 

Red will be completed. 

4) Cell toxicity under single and dual dye conditions will be tested. 

5 Dendrimer conjugates with MitoTracker® Red CMXRos and MitoTracker Deep Red 
will be tested as soon as the appropriate controls and parameters are determined. 



August 2002 Report 



Page 9 



NAS2-02069 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



Flow Cytometer-Laser System Development 

Theodore Norris, Ph.D., Jingyong Ye, Ph.D., Cheng Frank Zhong, B.S. 

Integrated Microfluidics System 

We are designing a specific microfluidics chip to simulate blood flow through a capillary 
bed. We will use small samples of human blood. The dimension of the channels on the chip 
should be over 20 microns. We have contact with the MEMS-Exchange, an on-campus 
microfrabication facility. They suggest that we use a chip with a single-level depth. Making 
separated wafers and each having channels of a uniform depth, is a straightforward task. The 
process sequence is shown below: 

1 . Contact photolithography 

2. DeepRIE 

3. Si02CVD or thermally grow a thin layer of Si O2 

Both methods of producing a Si02 layer will cause the mouth of the trench to pinch closed 
slightly. The CVD method will also cause the layer to thin towards the bottom of the trench. 
But since the thickness is not so important for us, this should not present a problem. 

Multi-dve detection 

We changed the optical fiber based detec- 
tion system to a free-space detection sys- 
tem. Using this system, we did a set of 
experiments to compare the fluorescence 
signal of five free dyes, each as a solution 
in DSMO, and at the same concentration 
of 30 fiM. The dyes tested were: Mito- 
Tracker®Red CMXRos, MitoTracker® 
Red 580, MitoTracker® Deep Red 633, 6- 
TAMRA, and FITC (Figure 1 1). Results 
show that MitoTracker® Deep Red 633 
gives the strongest two-photon fluores- 
cence excitation signal. We repeated this 
dye comparison experiment using a 76- 
MHz fs pulse laser with a pulse duration 
of 10 fs and wavelength of 790 nm. The 
result is almost the same, with MitoTrack- 
er Deep Red 633 giving the strongest 
two-photon fluorescence excitation signal. 



-FITC 

- 6-TAMRA 
CMXRos 

- Red580 
Deep Red 633 



3ODOO00 



2500000 



$ 2000000 - 



^ 1500000- 



1000000- 



500000 



480 500 520 540 5B0 580 600 820 840 680 680 700 720 740 760 

Wavelength (nm) 

Figure 11. Two-photon fluorescence excitation spectra. 

Cone. nM 

3000000 

2500000- 
2000000 
1500000- 
1000000- 




500000 



We did another set of measurements on 
solutions of MitoTracker® Deep Red 633 
at different concentrations ranging from 




560 580 600 820 840 6«0 680 700 720 740 



Wavelength (nm) 

Figure 12. Two-photon fluorescence excitation spectram 
of MitoTracker® deep red 633. 



August 2002 Report 



Page 10 



NAS2-02069 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



30 |j,M to 0.385 nM, with each consec- 
utive sample being 80% less concentrated 
than the previous one (Figure 12, previous 
page). This gives us a detailed view of 
the signal level of this dye in solution at 
lower concentrations (Figure 13). The 
two-photon fluorescence excitation sig- 
nals are peaked around 670 nm. Choosing 
the maximum fluorescence signal of each 
spectrum (except for 0.385 nM because 
we did not see any signal), we drew a 
linear fit of relative two-photon induced 
fluorescence vs dye concentration for the 
solutions of MitoTracker®Deep Red 633 
in DSMO (Figure 14). The correlation 
coefficient of 0.997 is very close to 1, and 
the Y-axis intercept value of 2.143 is 
small compared with the signal level. 
This result shows that the two-photon 
fluorescence excitation signal is propor- 
tional to the dye concentration within the 
limits of experimental error. 

The shape of the two-photon fluorescence 
excitation spectrum at lower concentra- 
tions (less than 48 nM, Figure 13) is not 
as expected. From the linearity of the 
logrithmic plot of fluorescense vs dye 
concentration (Figure 14), we expected 
the lower concentrations to give us curves 
similar to the curves for higher concen- 
trations. However, they have a peak 
larger than expected around 700 nm. This 
is due to the high background signal of 
DMSO, relative to the low dye signal. 
DMSO has a peak around 700 nm (Figure 
15). 



^ Cone. nM 

30.000. 

1000000 J 6.000. 

1,200. 

240. 

48 




560 580 600 620 ' frio ' 660 680 700 720 740 760 

Wavelength (nm) 
Figure 13. Logarithmic plots of two-photon fluorescence 
excitation spectrum of MitoTracker® Deep Red 633. 



1000000 




Number of Dala Points: 7 

Y Intercept Value: 2.143 

Slope Value: 0.%2 

Correlation coefficient: 0.997 



10000 



100 1000 

Concentration (nM) 

Figure 14. Logarithmic plot of the dependence of 

relative two-photon induced fluorescence on dye 

concentration for solutions of Mito Tracker Deep Red 

633 in DSMO. 



500000 - 
4SOO0O . 
400000 
350000 

E 300000 

t 
D 

:d50ooo 

200000 - 
1 50000 

100000 -t — r 




480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 

Wavelength (nm) 

Figure 15. Background spectrum of DMSO. 



August 2002 Report 



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Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



III. Problems encountered and their resolution. 

The existing desktop computer that is us^ to process images generated by the robotic 
microscope takes one day to a day and a half to process one batch of images. Being able to 
process such images in hours rather than a few days will have a significant effect on 
productivity and efficiency. It will also enhance our capability to select and screen different 
dyes Mid conjugated materials for acceptability on a quantitative basis, as well as to 
investigate expeditiously the intracellular localization and effects of the new experimental 
materials. 

We submitted a Contrating Officer's Authorization, on July 22, 2002, to request rebudgeting 
of $10,000 for the purchase of a high-end graphics workstation. The selected workstation 
would decrease deconvolution time by at least 50%, significantly enhancing our ability to 
digitally analyze complex, multispectral images with the multimode fluorescent microscope. 



VI. Copies of manuscripts (published or unpublished) derived from the 
research and copies of all abstracts, manuscripts, preprints and 
publications. 

None. 



August 2002 Report 



Page 12 



NAS2-02069 




Biosensors for Real-Time Monitoring of 
Radiation-Induced Biologic Effects in Space 



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1 . AGENCY USE ONLY ( Leave Blank) 



REPORT DATE 

August 2002 



4. TITLE AND SUBTITLE 

Biosensors for Real-time Monitoring of Radiation Induced Biologic Effects in Space 



6. AUTHOR(S) 

James R. Baker, Jr., MD, Lajos Balogh, Ph.D., Istvan Majoros, Ph.D., 
Balazs Keszler, Ph.D., Andrzej Myc, Ph.D., Jolanta Kukowska-Latallo, Ph.D., 
Theodore Norris, Ph.D., Felix de la Iglesia, MD 
Compiler/editor: Nicholas W. Beeson, Ph.D. 

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 
Center for Biologic Nanotechnology 

University of Michigan 

9220 MSRB 111, 1 150 W. Medical Center Drive 

Ann Arbor, MI 48 109-0648 



9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 

NASA-Ames Research Center 

Paul Fung, Ph.D. 

MS 19-20 

Moffett Field, CA 94035-1000 



3. REPORT TYPE AND DATES COVERED 
Monthly: 07/26/02-08/25/02 



5. FUNDING NUMBERS 
C-NAS2-02069 



8. PERFORMING ORGANIZATION 
REPORT NUMBER 
NAS2-02069-4 



10. SPONSORING / MONITORING 
AGENCY REPORT NUMBER 



1 1 . SUPPLEMENTARY NOTES 
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12 a. DISTRIBUTION / AVAILABILITY STATEMENT 
See Handbook NHB 2200.2 



12 b. DISTRIBUTION CODE 



1 3. ABSTRACT (Maximum 200 words) 

This work seeks to develop cellular biosensors based on dendritic polymers. Nanoscale polymer structures less than 20 nm in diameter will be 
used as the basis of the biosensors. The structures will be designed to target into specific cells of an astronaut and be able to monitor health 
issues such as exposure to radiation. Multiple components can be assembled on the polymers including target directors, analytical devices 
(such as molecular probes), and reporting agents. The reporting will be accomplished through fluorescence signal monitoring, with the use of 
multispectral analysis for signal interpretation. These nanosensors could facilitate the success and increase the safety of extended space flight. 
The design and assembly of these devices has been pioneered at the Center for Biologic Nanotechnology in the University of Michigan. 

14. SUBJECT TERMS '5- NUMBER OF 
Nanotechnology/ Optics PAGES 12 

Polymer-based platforms 
Biosensors (implantable) 



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298-102 



August 2002 Report 



Page 13 



NAS2-02069