Automated Gene Sequencer

1
Photonic Technologies for Early Detection of
Human Disease Mark G. Allen, Physical Sciences
Inc.
Biomolecular Systems Research Program
Description
The proposed photonic sensor platform addresses
the following 1. Novel molecular recognition
devices suitable for in-vivo use The photonic
sensor platform directly measures expressed
biomolecular processes as revealed through trace
gases in exhaled human breath. Through this
linkage, it can be at once remote (outside the
body) and in-vivo (within the body) in its
concept. 2. Novel strategies for in-vivo signal
generation and amplification The use of
resonant optical cavities to enhance the IR
absorption signature and recently available,
room-temperature quantum cascade lasers in a
modular configuration is a novel extension of
state-of-the-art photonic technologies. 3.
Non-invasive dynamic signal acquisition systems
suitable for non-invasive, dynamic signal
acquisition from deep tissues and systems of
reduced scale suitable for manned space
missions The photonic sensor platform is
ideally suited for long duration space missions,
capable of continuous monitoring with near- zero
cost per test and no consumption of consumable or
hazardous materials. It is completely
non-invasive. 4. New tools for feature
definition and extraction, including
computational and mathematical approaches Optima
l wavelength selection of the multi-wavelength
sensor will allow selected target gas features to
be extracted from the high-sensitivity, coarse
wavelength spectra.
Innovative Claims/NASA Significance
Plans
Year One 1. The proposed list of target gases and
relevant concentrations will be refined and
ranked according to biophysiological
significance. 2. High sensitivity detection of
broadband absorbing trace breath gas species will
be demonstrated using a conventional multi-pass
optical cell configuration. 3. Various
cavity-enhanced detection methods will be
evaluated according to their suitability for the
proposed application based on detection of small
molecular weight trace breath gas species. 4.
Compact mirror designs will be developed and
components purchased for use in the tasks of
years two and three. Year Two 1. High
sensitivity detection of broadband absorbing
trace breath gas species will be demonstrated
using a dual-wavelength optically resonant
cavity. 2. Time-resolved measurements of small
molecular weight trace breath gas species will
be demonstrated using a compact resonant cavity
design. 1.1.2.3 Compact, dual-wavelength optical
cavities and associated control electronics will
be fabricated and tested. Year Three 1.
Time-resolved measurements of broadband absorbing
trace breath gas species will be demonstrated
using a compact, dual-wavelength resonant cavity
design. 2. Time-resolved, multi-species
measurements of trace breath gases will be
demonstrated using multiple, dual-wavelength
resonant cavity modules. 3. An engineering design
of a complete prototype breath analyzer for
subsequent commercial development will be
completed and documented.
The proposed photonic platform technology
represents an innovative solution to the
technical challenges associated with
multi-signature analysis and dynamic monitoring
of human physiologies for early disease
detection. It also uniquely and specifically
addresses the limitations on potential
technologies that are imposed by the requirements
for long-duration manned space missions.
2
Development of an in-vivo Sensing Technology
for Cancer Signature Sanford A. Asher, U. of
Pittsburgh
Biomolecular Systems Research Program
Description
This research and development program is highly
interdisciplinary and utilizes the most recent
advances in physics, chemistry, biology and
nanoscience. The research utilizes 1. Novel
molecular recognition chemistries, materials,
chemical composites, nanoparticles,
nanostructures, agents and devices suitable for
in vivo use. 2. Novel strategies for in vivo
signal generation. 3. New approaches and
multifunctional technology platforms to create an
interface between in vivo detection and targeted
intervention, including nanostructures/devices
and novel materials and composites.
Innovative Claims/NASA Significance
Plans
Milestones Each of the entries in the Statement
of Work above constitute milestones in the
proposed research program. These milestones
are Year 1 1 Attach PSA to IPCCA 2 Attach
BLCA-4 to PCCA 3 Demonstrate IPCCA response to
PSA and BLCA-4 in saline 4 Optimize sensors Year
2 5a Test IPCCA PSA sensor in fetal calf serum 5b
Test IPCCA PSA sensor with plasma from prostate
cancer patients 6a Test PCCA BLCA-4 sensor in
fetal calf serum 6b Test PCCA BLCA-4 sensor with
urine from bladder cancer patients 7 Refine
sensors for implantation 8 Implant PCCA beneath
skin of mice and rats to determine
biocompatibility 9 Determine visibility of
diffraction fi-om implanted IPCCA Year 3 10
Construct reflectometer 11 Fabricate near IR PCCA
sensor for implantation 12 Demonstrate in vivo
sensing by implanted PCCA to protein signatures
of cancer
Our research and development program will create
a biomolecular sensor technology platform to
enable non-invasive or minimally invasive early
detection of signatures of disease, especially
cancer. We will utilize our recently invented
intelligent polymerized crystalline colloidal
array (IPCCA) material" for in vivo sensing.
These IPCCA materials and devices utilize novel
molecular recognition chemistries within
materials containing periodic nanostructures of
composite nanoparticles"". The signal generation
utilizes diffraction of incident light by the
nanoparticle arrays'-338. This diffraction of
light serves as the interface between in vivo
detection and targeted intervention. This
technology platform can be multifunctional, in
that different PCCA sensors for different disease
signatures can be fabricated by utilizing
different molecular recognition elements in these
biocompatible PCCA materials.
3
Fundamental Technologies for the Development
of Biomolecular Sensors James R. Baker, Jr., U.
of Michigan
Biomolecular Systems Research Program
Description
1. Development of Antibody-Dendrimer-Dye
conjugate Biosensors. 2. In-vivo testing of
targeted conjugates using Microscopic
Multispectral Analysis. 3. Development of a
Laser Monitoring Systems.
Multiprobe fluorescence of isolated hepatocytes.
The photograph is an example of the composite
fluorescence of 4 probe with pseudocolor
assigned to the corresponding peak emission
characteristics of each dye. Nuclei are in blue
(Hoechst 33342), lysosomes in green
(lysotracker), mitochondria in orange (TMRM) and
plasma membrane permeability
Innovative Claims/NASA Significance
Plans
We propose to develop a biosensor that would be
loaded into the lymphocytes of astronauts. This
biosensor would measure the viability of
lymphocytes and identify early events associated
with radiation exposure, such as alterations of
mitochondrial calcium mobilization. In addition,
the sensor could also identify activated caspase
activity within lymphocytes an event documenting
irreversible apoptosis. This signal would be
monitored non-invasively using laser light
directed at a laminar-flow stream of blood cells
in a capillary. The system would hnction like a
flow cytometer, but withouthe need for large
equipment. It could be used for additional
non-invasive measurement of other blood
parameters, such as neutrophil activation as a
reliable sign of infection.
4
A Nonlinear Optical Coherence Tomography System
for Biomolecular Detection and
Intervention Stephen A. Boppart, Beckman Inst.
For Advanced Science Technology
Biomolecular Systems Research Program
Description
Objective
We propose the development of a new instrument
that we call the Biomolecular Laser Imaging and
Therapeutic System (BLITS). BLITS combines and
extends several imaging technologies,
including Optical Coherence Tomography (OCT), OCT
spectroscopic, imaging, and Coherent Anti-Stokes
Raman Scattering (CARS) spectroscopy, to Produce
a versatile system capable of noninvasively
measuring Micron-scale structure and molecular
composition of tissues. BLITS is based on OCT,
an imaging sensor that measures micron- Scale
tissue structure in vivo.
Innovative Claims/NASA Significance
Plans
The Biomolecular Laser Imaging and Therapeutic
System (BLITS) will provide an innovative,
integrated platform offering diagnostic
flexibility, on-line treatment capability, and
real-time monitoring. Unlike other noninvasive
imaging modalities, such as ultrasound or
magnetic resonance imaging, OCT provides
micron-scale resolution of cellular and
sub-cellular structural features at real-time
acquisition rates. Unlike the ionizing radiation
used in x-ray computed tomography and PETBPECT,
tissues are imaged with low-power near-infrared
radiation which poses little to no health risk.
Unlike laser-scanning confocal and multi-photon
microscopy, no exogenous agents are required to
provide image contrast. Using semiconductor and
solid-state laser oscillators, the apparatus can
be extremely compact, reliable, and
turn-key. The combination of CARS and OCT in
BLITS adds molecular and spatial discrimination
capabilities not found elsewhere.
5
Translation of Disease Markers into
Bioluminescent Signals Donald Langdry, Columbia
University in the City of NY
Biomolecular Systems Research Program
Description
Specific Aims AIM 1. Selcction of
oligonuclcotides that self-cleave upon of
complexation with thrombin in vitro. AIM 2
Construction of oligonucleotides that release
luciferin upon complexation with thrombin in
vitro and in vivo. AIM 3 Neutralization-of-inhib
ition assays with light-emitting enzymes. AIM 4
Identification of cell-selective
oligonucleotides.
Innovative Claims/NASA Significance
Plans
We propose to detect in yivo the earliest
molecular signatures of disease by translating
individual molecular markers into reporter
molecules that are read-out in urine. Thc
"translation" occurs through an autocatalytic
complex formed between the disease marker and the
recognition elcment of an oligonucleotide-reporter
molecule conjugate. Upon complex formation,
sex-cleavage releases the reporter molecule for
renal excretion.
6
Nanoparticle Delivery of Repair Enzymes for
Radiation Protection/DNA Repair James F. Leary,
University of Texas Medical Branch
Biomolecular Systems Research Program
Description
This program will develop a nanotechnology-based
Biosensor diagnostic and therapeutic system to
detect potentially radiation-damaged cells and to
provide radiation protection and/or DNA repair
enzymes to white blood cells in the peripheral
blood of astronauts exposed to radiation in
space. A multi-disciplinary team of scientists
with expertise in targeting and detection of rare
blood cells, DNA repair, nanoparticle technology,
and in-vivo imaging will develop a minimally
invasive, smart-biosensor nanotechnology that can
be potentially read non-invasively by detecting
the nanoparticle fluorescence as cells flow
naturally through blood vessels in the eyes of
astronauts. The focus of this grant is to provide
as much in-vivo protection as possible against
radiation damage to the blood and bone marrow of
astronauts, to provide in-vivo intra-cellular DNA
repair to damaged cells that can be repaired
before thesecells go on to become radiation-
induced leukemias, or if necessary search out
and destroy (by inducing apoptosis)
radiation-caused cancer cells in blood.
Task 1 Construct DNA repair enzymes and test on
human cells in-vitro and ex-vivo Task 2
Development of Biomolecule-Nanoparticle
biosensors for drug delivery to radiation-damaged
cells Lvov and Kotov Task 3 Characterization
of nanoparticle taweting and target cell
responses Task 4 In-vivo Cellular Distributions
Characterization of Smart Nanoparticles for
Recognition and Targeting and Selective
Killing Of Transformed Cells

Innovative Claims/NASA Significance
A novel strategy of in-vivo signal generation in
response to the sensing of particular types of
radiation-induced damage is proposed using a
non-invasive, in-vivo flow cytometric sensing of
fluorescently-labeled radiation-exposed blood
cells as they flow through a blood vessel in the
eye as measured on astronauts using special eye
goggles with wireless transfer of data to a
portable computer. The proposed data acquisition
system would be non-invasive, allowing periodic
or continuous monitoring of radiation damage
levels. A novel method of delivery of repair
molecules allows targeted and specific, rather
than untargeted and non-specific delivery, of
repair molecules to the affected cells while
preventing their exposure to healthy cells.
Lastly, a novel concept forfuture consideration
involves the genetic engineering of hematopoietic
stem/progenitor cells for increased resistance to
radiation damage.
7
Photonic Technologies for Noninvassive Detection,
Diagnosis, and Treatment of Cancer Timothy M.
Swager, MIT
Biomolecular Systems Research Program
Description
The proposed SPM platform requires thin films of
specially designed SA polymers which are only
available through complex organic syntheses. The
development of polymers that behave well in thin
films is not trivial, as interpolymer
interactions generally have a large effect on
electronic and photophysical properties. In
particular, the aggregation of electronic
polymers greatly reduces amplification while also
raising the threshold for laser behavior
(lasing). The latter is extremely sensitive to
any losses, and having an optimal system is
important. New SA sensory polymers developed at
MIT have the highest thin-film fluorescence
quantum yields measured to date for
semiconducting polymers. As the performance of
the SPM technology will be critically and
exponentially dependent on these materials, the
ability of the MIT group to design the optimum SA
polymers cannot be overstated.
Innovative Claims/NASA Significance
Plans
Milestones Year 1 1. Purchase and set up a
confocal microscope with a time-resolved laser
for analysis of microsphere 2. Synthesize
quantities of necessary polymers (this task will
continue when necessary in Years 2 and 3). 3. Use
surface functionalization chemistry to produce
microsphere lasers based on Self-Amplifying
(SA) 4. Produce tailored multilayer films of
electronic polymers in microsphere lasers. 5.
Establish the conditions for optimal lasing and
sensory behavior. 6. Demonstrate of the
amplification of DNA bindinghybridization with a
microsphere. 7. Begin Chemical Vapor Deposition
(CVD) deposition of SA polymers on
microspheres. lasers and develop a testing
station for fiber optic sensors. polymers.
The development of a versatile and sophisticated
photonic platform based on a new semiconducting
polymer and microsphere (SPM) technology that can
be tailored to both detect and treat cancer. This
approach is not simply an incremental advancement
of fiber-optic sensor and photodynamic therapy
technologies. The sensor SPMs will have
sensitivities many orders of magnitude beyond
those provided by conventional methods. The
enhancements will be provided by Integrating
concepts first reported by Swager in 1995,
wherein he demonstrated that semiconducting
polymers are capable of a signal-amplifying (SA)
sensory response.