Item 1.

Identification and Significance of the Opportunity

Background
The need for improved cancer treatments has driven a tremendous amount of effort aimed at expanding
the knowledge base concerning the underlying mechanisms of the disease. With the sequence of the
human genome nearly complete, there has been a significant shift to the post-genome science of
proteomics. Verification of a gene product by proteomic methods is an important first step in
annotating the genome.1 Modifications that are not apparent from the DNA sequence, such as
isoforms and post-translational modifications, can be determined only by proteomic methodologies. 2
Moreover, because mRNA levels do not necessarily correlate with protein levels, it is crucial to assess
protein expression directly. In addition, mechanisms such as the regulation of protein function by
proteolysis, recycling, and isolation in cell compartments, affect gene products, not genes.3
As a result, the study of proteomics holds tremendous promise for cancer research. For example, Page
et al.4 presented the most extensive study to date of the protein expression map (PEM) of the normal
human breast, which can be compared to the PEMs of breast cancer cells in further studies.
Additionally, a strategy for proteomic analysis of human tumors was described by Emmert-Buck et al. 5
Comparison of the microdissection protein profiles of normal squamous epithelium and corresponding
tumor cells from two patients with esophageal cancer pinpointed seventeen proteins with tumorspecific alterations, including ten that were uniquely present in the tumors and seven that were
observed only in normal epithelium. In the future, comparison of protein profiles will be used to
analyze suspected malignant tumors, diagnose patients with cancer, and design patient-specific
therapeutics.
The Problem
Despite this tremendous potential, the lack of a tool for high-throughput, high-resolution peptide
separation prior to direct mass analysis is clearly limiting advancements in this field. 6 The difficulty of
analyzing the proteome of a cell or serum is that a human cell type, for example, may express up to
20,000 proteins at any time7 with a predicted dynamic range of up to five orders of magnitude 8.
Furthermore, the proteome is dynamic, changing in response to the genome, the environment, and even
its history.9 No existing methodology is adequate to resolve and detect proteins present at such different
levels of concentration. Consequently, a great deal of focus by analytical chemists in recent years has
centered on developing multidimensional fractionation and separation techniques in an attempt to
effectively resolve complex protein mixtures.10
If anything has promised to completely transform the sample preparation portion of protein
analysis, it is the development and use of high performance liquid chromatography (HPLC). In
HPLC multidimensional approaches, the separated proteins are, in most cases, digested into
peptides prior to further separation. The advantage of this is that peptides are initially more
soluble in a wider variety of solvents, and hence easier to separate than the parent proteins; this is
especially the case for hydrophobic and membrane proteins. 11 The disadvantage in working with
peptides is the increase in the number of species that have to be resolved. HPLC
multidimensional methods are necessary to allow for the greatest number of species to be
successfully identified by subsequent MS analysis. While MS technology has and will continue to
rapidly improve, its dynamic-range measurement capability is still 2-3 orders of magnitude less
than the dynamic range of proteins found within a mammalian cell. The ability to lower the
density of peptides introduced to the spectrometer at any one time is directly proportional to the
successful comprehensive characterization of a proteome sample. 12

3*

One of the main advantages of HPLC techniques is that they allow the analyst the freedom to
resolve a complex mixture by employing different separation mechanisms based on the physical
and chemical properties of the solute mixture. For example, two or more different HPLC
separation mechanisms can be combined, potentially in conjunction with gel electrophoresis and
other separation techniques. The advantages of HPLC multidimensional procedures are that they
are automatable, sensitive, reproducible, fast, quantitative, and allow for the use of multiple
separation mechanisms. HPLC separations can also be coupled with a large number of different
detection systems, including ultraviolet (UV), MS or laser-induced fluorescence (LIF).

No gel interactions or removal steps

Fewer size limitations
No visualization (staining) problems
Greater sensitivity

Ease of automation
Multiple types of separation possible
Ability to interface directly with MS

Despite these attractive features, even the best HPLC systems currently available are
fundamentally limited in their ability to effectively separate the full complement of peptides
found in any digested protein spot. Since many of the peptides are left unresolved and currently
available MS instruments are limited in dynamic range, a great deal of potentially crucial
information is lost. This limitation of current HPLC technology is a result of two sets of
observations, described below.
a- Peak Capacity
The separation power of HPLC is limited in large part because the peaks of the various
components of a mixture are separated along a single axis, on the basis of their thermodynamic
constant of equilibrium between the mobile and the stationary phases. 13, 14 Unfortunately, there is
room for only a limited number of zones on a straight line. Because of axial dispersion and mass
transfer resistances, chromatographic bands have a finite width. Assuming a Gaussian profile for
these peaks, their baseline width is four times their standard deviation, a function of retention and
column efficiency. It was shown that the number of peaks resolved with a resolution unity, i.e., all
touching each other, that can be eluted between the holdup time (moment when an unretained
compound is eluted) and the peak of a compound with a retention factor k 015 is given by
n=1+(

N
) ln(1+ko)
2

where n is the column efficiency . In practice, analyses are considered long when the retention
factor of the last compound eluted is of the order of 5 to 10. A value of 6.4 gives a peak capacity
equal to 1 + N1/2. An extreme value of 20 would give a peak capacity only 50% larger. Since it is
exceptional to have a column with an efficiency of 25,000 theoretical plates, we are limited to
peak capacities of approximately 160.
b Statistics of Peak Overlap
The statistics of band overlap has been developed by Giddings who showed that the retention
factors of the components of a complex mixture are randomly distributed. Statistics show that if a
50 component mixture is analyzed on a column with a peak capacity of 100, the most probable
result is that 27 peaks will be recorded, 18 of them corresponding to pure components, the other
nine peaks being multiplets (i.e., peaks corresponding to the coelution of several components, the
bands of which interfere). Systematic investigations, the selection of the most appropriate
column, and optimization of the experimental conditions allow experienced analysts to fare better
than random but the odds are poor, which explains why the pharmaceutical industry is forced to

4*

employ so many analysts for method development. Peak capacity theory and the statistics of
peak overlap demonstrate that chromatography requires a far more powerful tool to confront
the difficult analytical problems raised by proteomics.

The Innovation
QGENICS BIOSCIENCES, INC. (QGENICS) proposes a solution that could be developed into
an automated, high-throughput peptide analysis tool with the potential for substantial advances in
separation resolution. The solution would use true two-dimensional chromatography (TTDC)
carried out on a planar chip, capable of separation and subsequent mass analysis through direct
integration with a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer as the
target plate. TTDC offers the potential of improving peptide separation resolution and lowering
the density of peptides introduced to the spectrometer at any one time, which is directly proportional to
the successful comprehensive characterization of a proteome sample. The unique benefits of the
technique would include:

Extremely high resolving power

Full automation
High throughput (speed)
Ease of operation
Two-dimensional
visualization
prior to MS

Seamless MADLI integration

Fewer size/concentration limitations
Separation patterns similar to 2DE
(allowing for use of comprehensive and
powerful databases built on 2DE data.

The Key Innovation associated with this project, should it proceed to both Phase I and Phase II
efforts, is to devise, design and prototype an experimental peptide analysis system based on
TTDC. The principle of the technique is to design and develop a wide, flat column that can be
developed successively in two perpendicular directions and operate it as a column. 16,17,18,19,20 This
concept is shown in Figure 1 below. The sample would be injected into a microfluidic port on the
corner of the 5 cm x 5cm chip and electro-osmotically transported to the corner of a flat column.
The first dimension would separate the peptides by ion exchange and the second by
hydrophobicity. Once the chip was fully developed, the computer-controlled scanner would create
a 2D image of the separated spots, release the hinge on the cover plate and eject the chip from the
scanner. The chip would be placed directly into the MALDI instrument and the entire plate would
be analyzed. Alternatively, the 2D coordinates of the separated spots could be recorded by the
scanner and used by the MALDI instrument to target specific locations on the chip.
The Phase I goal of the project is the design,
development, and demonstration of a suitable
TTDC chip for high separation resolution, a
means of pumping a chip with the suitable
characteristics, and a means of integrating such a
chip with a MALDI source. The solution that is
offered is the development of a planar chip
constructed from glass and a stationary phase
material of modified silica gel. The mobile
phases will be pumped electrically in both
directions. Although outside the scope of this
Phase I project, it is envisioned that twodimensional detection of the separated spots prior
to MS analysis will be accomplished in Phase II

5*

1st
Development

Sample
2nd
Development

Fig. 1 The sample is injected in a corner of the plate,

it is developed along the short dimension until the
spot of the interesting component that is less retained
gets close to the plate edge. Then, the column is
developed in the perpendicular direction.

using a two-dimensional diode array (that permits on-line spectroscopic detection as is used in
diode-array detectors in conventional HPLC instruments). However, in this Phase I effort,
preliminary verification of spot location will be accomplished with florescence labeling and a
CCD camera. These separation/instrumentation considerations are more fully described in Item 3
below.
It is important to note that this is a unique undertaking with respect to peptide analysis and
reflects a significant shift in how protein expression profiling might be performed, if successful.
There is currently no available sample preparation tool that is completely automated, can be
seamlessly integrated with a MALDI ionization source, promises high throughput and offers
excellent resolution. The path proposed is simple in concept, but highly challenging in execution.
It is important to note that the technical challenge is not necessarily in the development of the
TTDC separation technique or the MALDI integration alone, but rather in the design, fabrication,
and operation of a single device capable of repeatable operation encompassing both. This is the
focus of the work to identify and demonstrate a technique for constructing an inexpensive,
reproducible, and powerful tool for proteomics applications.
TTDC Principle
A column which, for sake of convenience will be assumed to be square but does not need to be, is
packed entirely with a stationary phase of conventional use in HPLC. Here we will use a layer of
modified silica gel. A narrow strip along one side of the column is packed with a different
material of identical size (see Figure 2). A first solvent is pumped in the direction parallel to that
strip; after steady flow is achieved the sample is injected near the far corner of the column and
one column volume is pumped or more if the first compounds of interest are retained on this
first chromatographic system. After the flow of this first solvent is stopped, a second solvent is
pumped in the perpendicular direction and the sample components are separated across the plane.
Subsequently, the separated spots are visualized and their locations are digitally recorded. Finally,
the silica gel is exposed, the chip is ejected from the system, and the open chip is placed
directly into the MALDI source for ionization and ensuing analysis.
Glass
P

Solvent

Adsorbents

Indium Tin
Oxide Glass

Waste

Fig. 2 Principle of operation of true two-dimensional chromatography chip (see text).

TTDC Theory
The separating power of a chromatographic column is best expressed by its peak capacity, that is,
the number of peaks with resolution unity which are eluted between k = 0 and a final value,
usually 6.4 but rarely exceeding 10. Conventional column chromatography has a peak capacity,
which, in practice, can exceed 150 to 200 only in exceptional cases. Peak capacity of 500 or more
would require the use of the best capillary columns, which are very long, difficult to handle,
require very small samples and are hardly compatible with very sensitive detection techniques. In
fact, an excellent HPLC column would have an efficiency of approximately 25,000 theoretical
plates, with a limited peak capacity of approximately 160. It has been shown that such a column
cannot be used to separate many more than approximately 50 peaks, including a number of
multiple peaks where corresponding compounds will interfere. Previous work has shown that

6*

there is little hope of increasing the peak capacity available much beyond several hundreds, even
by accepting very long analysis times and resorting to capillary columns of very small diameter
or coupling multiple columns together (often termed as two-dimensional).
Analysts realized the limitations of one-dimensional chromatography long ago and attempted to
develop truly bidimensional methods. Martin et al. 21 invented and developed in the 1940s a
method for the separation of the proteinic amino acids, using two-dimensional paper
chromatography. Two successive developments of a square sheet of filter paper (20 x 20 inch)
were carried out, at right angles, with an acidic and a basic mobile phase, respectively. In spite of
the crude nature of the separation device, the device separated 15 of the 22 proteinic amino acids
tested yielding a separation power that exceeded all LC columns available at that time. Many
other impressive separations were carried out using the method. 22 Zone capacities in the order of
300 to 400 were achieved rather easily in 1944 while the peak capacities achieved in LC exceed
rarely 150 (see earlier). However, because of the problems associated with the quadratic law of
solvent front migration, the use of capillary actuation limited the technique.
When the two-dimensional chromatographic bed is operated as described in this proposal, the
peak capacity is markedly increased over any other chromatographic technique. The peak
capacity for a two-dimensional column developed in two dimensions is given by:
n=

L
( 1 a a )2
4H

(1)

where a = 2/h and h = f(). 23 Assuming column characteristics of = 0.70, A = 1.0, C = 0.03,
and Dm = 5 x 10-6 cm/sec, one can achieve a peak capacity of 1015 with a 5 x 5 cm column
packed with 3 m particles. This peak capacity significantly exceeds the associated peak
capacities of HPLC, thin layer chromatography (TLC), over pressured layer chromatography
(OPLC), and two-dimensional thin layer chromatography (TDTLC) (Table 1).
Table 1 Peak Capacities of Various Chromatography Methods 24
Development Method
TLC
OPLC
HPLC
TDTLC
TTDC

Peak Capacity
30
80
150 to 200
300
1000

Item 2. Phase I Technical Objectives

It is recognized that the proposed Phase I effort is not of sufficient scope to take this project to
fruition, which is the goal of a Phase II effort. This Phase I effort will therefore focus on two key
issues fundamental to the ultimate goal of developing this powerful, fully automated system: (1)
the materials selection and design issues of a two-dimensional chromatography chip capable of
MALDI integration, and (2) the use of this new separation instrument to demonstrate the basic
performance necessary to demonstrate feasibility of the technique. The successful realization of

7*

these two objectives will lay the fundamental groundwork for a Phase II effort to completely
develop a prototype TTDC peptide analysis system.
Phase I Research Objectives
1. Design, development and fabrication of a system for true two-dimensional
chromatography capable of direct MALDI integration.
a. Selection of materials
b. Chip design
i. Optimization of separation parameters
ii. Optimization of MALDI interface parameters
c. Optimization of pumping mechanism
2. Investigation of Separation and Direct Ionization
a. Resolution issues
b. Repeatability issues
c. Ionization issues
Item 3. Phase I Work Plan
The successful realization of the research objectives outlined above will require a cross-functional team
bringing together expertise in chromatography, protein chemistry, mass spectrometry (specifically
MALDI), electronics, microfluidics and micro instrumentation development. QGENICS has assembled
such a team. Dr. Michel Goedert will serve as the companys Principal Investigator (PI) and brings to
the team more than 30 years of micro analytical instrumentation development, with a focus on the
design, development and ultimate support of microfluidics separation instrumentation at Agilent
Technologies (Palo Alto, CA). Further, the company will draw on the experience and skills of several
expert consultants and collaborators to assist Dr. Goedert in this Phase I effort (see Item 8).
Phase I Task Description
(Target Completion Dates Assume a Start Date of May 1, 2003)
Task 1. Materials Selection and Design
Target Completion Date:
Responsible Personnel:
Task 1 Milestone:

June 15, 2003

Dr. Michel Goedert, Dr. Georges Guiochon, Dr.
David Hachey, and Dr. Alfred Yergey.
Test System Design Complete

The first step of the proposed development, which is the focus of this task, is to successfully
design a system for TTDC capable of MALDI integration. There are essentially two types of
problems to be addressed in this task of the project those related to the separation and those
related to the need to develop a suitable instrument.
Focusing on a relatively large separation system initially, with a size easy to handle by a chemist
in a laboratory, and initially using techniques takes from TLC and possibly from OPLC, the
different problems arising from the decision to implement TTDC will be investigated. A critical
challenge of the device is to select two chromatographic systems which fulfill two conditions: (i)
they must give retention patterns widely different for the components of the analyzed mixture,
such as one that separates by molecular size, the other by polarity and/or polarizability; (ii) they

8*

must be compatible in the sense that the first solvent should not destroy the second adsorbent and
should be either miscible with the second solvent or easy to get rid of. This can be accomplished
by driving a third solvent, miscible with both the first and second and of small elution strength, in
the second direction. The chromatographic systems that will be investigated will be based on
hydrophobic interaction and cation- and anion-exchange. Additionally, the statistics of band
overlap must be considered and investigated in a two-dimensional separation space. Finally, the
solvents will be moved and forced to percolate through the bed using electro-osmotic flow, with
special attention given to the reproducibility and stability of the flow rate.
The chip-based planar chromatography column will be integrated with a MALDI time of flight
(MALDI-TOF) mass spectrometer utilizing a chemically modified silica gel as the stationary
phase. Silica gel is the most widely used stationary phase for TLC and is available as a
conventional or as a high-performance layer. Furthermore, silica gel can function as both the
stationary phase of the chromatographic separation as well as the matrix used in the MALDI-TOF
analysis. In contrast to conventional MALDI-TOF MS, the use of silica gel virtually eliminates
the signal interference of low-molecular analytes contained in the matrix.
The particle size and pore structure of the inorganic silica gel matrix must be optimized for
penetration of the analytes into the pores and the associated effective desorption and ionization of
the analytes. Zhang et al25 found that 5-m silica gel particles could be homogeneously
distributed to produce better mass spectrometer signals. In general, signal intensity increases with
an increase in pore size. Zhang et al 26 demonstrated that silica gels with pores sizes of 100 and
300 both gave quite strong signals for a number of protein and peptide molecules tested. Based
on these results, QGENICS will initially attempt to test the separation system using 5-m silica
gel particles with pores sizes of 300 .
In spite of the success of MALDI using silica gel as a matrix, the signal intensity is not as strong
as that obtained by using conventional matrices such as DHB (2,5-dihydroxybenzoic acid) or
HCCA (a-cyano-4-hydroxycinnamic acid). To alleviate this problem, DHB will be covalently
bound to the surface of the silica gel to improve spectrum intensity (Figure 3).

Fig. 3 Modification of silica gel with (a) DHB or HCCA

Finally, the actual instrument must be designed. It is envisioned that the chip will be substantially
similar in design to that shown in Figure 4 below. The chip is constituted of two main parts the
microfluidics and the separation area.

a Microfluidics

9*

First, the chip must be optically transparent to allow for 2D detection prior to MS in Phase II. As
such, the chip will be constructed of glass using the microfluidics facilities available at the Oak
Ridge National Laboratory (see Item 9). The high voltage supplied to the chip will generate the
osmotic flow necessary to move the solvent and the sample through the various parts of the
system. Channels of approximately 50-100 microns will be ground on the bottom part of the chip.
The wells will be part of the left side part of the cover.
The solvent is supplied and mixed with a small amount of sample using wells A, B1, and B2.
Wells C and D are used for solvent supply and wells E and F are used for waste. The number of
wells could easily be increased should we decide to increase the number of samples. Electrodes
are connected into the wells and used to generate an osmotic flow through the chip. When the
flow of solvent is within expected values, the injection is done (1) by turning off the main flow
(C-E), (2) switching the high voltages to load the sample (B1 and B2), (3) switching the voltage
to establish a flow from C to E and developing in the first dimension. Once the first dimension is
developed properly, the voltage is switched (D-F) and the sample is developed in the second
dimension.
b Separation
The 2 D separation area will be constructed by grinding an area of 5 cm x 5 cm with a depth of 0.5 mm.
These preliminary geometrical dimensions will be carefully reinvestigated to ensure that the total size
of the chip is compatible with the MALDI source. The chip cover will be normally in contact with the
gel inside the chip. This will be achieved by a floating mechanism, which will have to be designed and
tested. The purpose of this mechanism is to eliminate any void between the gel and the cover.

F
C

B1

B2
Fig. 4 Top view schematic view of TTDC chip showing the various microfluidics connections.

Task 2. Construct Test System

Target Completion Date:
Responsible Personnel:
Task 2 Milestone:

August 1, 2003
Dr. Michel Goedert and Dr. George Guiochon.
Test System Construction Complete

Once the system design is complete, the test system will be constructed. The silica gel will be prepared
using standard techniques. Careful attention will be given to ensure that no bubbles are trapped inside
the chip. The silica gel with the appropriate chemical modifications (enabling ion exchange in direction
1 and hydrophobic interaction in direction 2) will be purchased from a suitable commercial vendor.

10*

Then, the silica gel (0.5 g), DHB (0.1090 g) and 4,4-diphenylmethane diisocyanate (0.16 g) will be
mixed, and dry toluene (15 mL) and pyridine (2 mL) will be added. The mixture will be refluxed at
90C for 24h. The cooled solution will then be filtered and the solid substance will be washed and then
dried under vacuum. The cavity will be filled with the DHB-bound silica gel in the appropriate solvent
(methanol). A slight excess of the gel will be deposited. Once it has settled sufficiently, but before the
solvent has evaporated, this excess will be scraped off by passing a microtome blade over the glass
plate. The bed will then be dried in an oven placed in a hood.27
Task 3. Test TTDC System
Target Completion Date:
Responsible Personnel:
Task 3 Milestone:

September 15, 2003

Dr. Michel Goedert and Dr. Georges Guiochon.
Separation Demonstrated

Once the system construction is complete, various tests will be performed to characterize
performance. The flow profile of the percolating mobile phase must be examined and optimized.
Once the flow profile of the system has been characterized as stabile and repeatable, the systems
separation performance will be examined. Two standard and well-characterized proteins
encompassing a range in molecular weight, myoglobin and bovine serum albumin (BSA), will be
digested separately with trypsin using standard techniques. Then, each of the prepared samples
will be fluorescently labeled using standard techniques and separated using the TTDC test
system. Upon completion of the separation, a CCD camera will be used to visualize and record
the results of the separation. The results from the separation of the myoglobin and BSA will be
compared with existing HPLC methods to characterize system performance and repeatability.
Task 4. Analyze Results and Prepare Optimized Design for Phase II Prototype
Target Completion Date:
Responsible Personnel:
Task 5 Milestone:

November 1, 2003
Dr. Michel Goedert, Dr. George Guiochon, Dr.
David Hachey, and Dr. Alfred Yergey.
Final Report Complete. Phase II Prototype
Design Complete.

At the completion of Task 4, the complete results of the project will be thoroughly analyzed and
compared to theory. These results will demonstrate the feasibility or, alternatively, the lack of
feasibility of the proposed system. The final report will be prepared and submitted.

Item 4. Related Research and Development

Attempts at implementing TTDC were first made in the early 1980s but failed due to
technological and engineering limitations and of the general lack of interest at the time (an
application which required such high resolving power did not exist at the time). Yet it was shown
that the principle was sound and promising initial results were obtained. 28,29,30,31 A separation cell
constructed of steel and Plexiglas was necessary since the mobile phases were pumped
conventionally and high pressures were created. As a result, it was necessary to construct valves
along each edge of the square cell. The development of valves with these dimensions
(approximately 10cm long and 1mm tall) that did not leak proved extremely challenging.

11*

Moreover, difficulties were encountered with controlling the flow rate in the two dimensions of
development and with the detection (the implementation investigated then involved development
in the first two dimensions and elution in the second using mechanical pumps). The development
of electrical fluid actuation, micro- and nano-fabrication, and alternative detection technologies
offers great potential to alleviate the problems previously encountered.
Further, the combination of TLC with MS was a very active area in the past two years. Important
publications on TLC/MS include the following: a review of methods for structure elucidation of
saponins32; direct microextraction and analysis of rough-type lipopolysaccharides by TLC/
MALDI-MS33; analysis of cationic pesticides by TLC/MALDI-MS34; a critical, general review of
TLC/MS, including MALDI, surface-assisted laser desorption (SALDI), and the TLCelectrospray interface, with some speculations as to future prospects 35; analysis of the
pharmaceutical compound UK-224,671 and related substances by TLC/MALDI-TOF-MS 36;
direct MALDI-TOF-MS of gly-colipids on thin-layer plates and polymer transfer membranes 37;
separation and detection of carcinogen-adducted oligo-nucleotides by TLC/MALDI-MS 38;
application of TLC/MALDI-TOF-MS to identification of unknown mixtures produced in an
organic synthetic process39; quantification of caffeine by off-line TLC/MS 40; and 2-D TLC
separation and MS identification of anthraquinones isolated from the fungus
Dermocybesanguinea41. A new TLC/MALDI-MS direct coupling method that recovers 100% of
the analyte was described. The method makes use of a hybrid plate in which a silica gel layer and
a MALDI layer are configured adjacently on a common backing. After TLC separation, the plate
is rotated 90, and the separated analyte zones are eluted from the silica gel layer to the MALDI
layer via the capillary action of the latter. Low-femtomole detection limits were demonstrated for
small cyclic peptides42.

Item 5. Relationship with Future Research and Development

The achievement of the Technical Objectives of this Phase I proposal will demonstrate that the
proposed TDCC system is capable of (1) repeatably separating a sample peptide mixture on a
chip-based platform, and (2) integrating the chip-based platform with a MALDI source for
subsequent mass analysis. The Technical Objectives of this Phase I proposal are designed to
develop a complete understanding of the factors controlling the resolution of the chromatographic
separations and the sensitivity of the mass spectrometer quantification. In addition, Phase I will
create the framework for the Phase II work plan by yielding the information necessary to select
the optimum format and design of the separation platform to be prototyped in Phase II.
Moreover, it is anticipated that the Phase II work will commence with the development of a
complete computer model to simulate the operation of the TTDC system and determine the
optimum operating parameters. These parameters will form the basis for the prototype to be
developed. Initially, it is anticipated that the design of the prototype will be developed using a
three-dimensional mechanical design software package, such as SoldWorks, or other similar
design package. Following the design of the prototype system, the platform will be constructed. It
is envisioned that the chip will be microfabricated by a commercial vendor. The material is
envisioned to be glass, although the use of plastics will be thoroughly investigated. Finally, the
performance of the prototype will be extensively tested using a wide range of complex biological
samples.

12*

Item 6. Potential Commercial Applications

Perhaps the most exciting aspect of this innovative technology is its vast commercialization potential.
The TTDC technology has numerous potential applications in several different industries. Applications
for a chip-based chemical analysis system with the characteristics of the device proposed herein
include, but are not limited to, DNA, protein, and peptide screening for drug discovery and academic
research, clinical chemistry and research, medical diagnostics, and environmental monitoring. Based on
an in-depth market analysis, QGENICS will initially target the drug discovery industry and,
specifically, the proteomics sector.
With the human genome nearly sequenced, drug discovery and academic researchers have placed
an increased emphasis on proteomics, which is the study of the diversity and function of proteins.
Proteomics promises to reduce the opportunity costs of drug discovery and improve efficiency by
delivering an increased number of valid drug targets and providing a means of evaluating those
targets prior to costly clinical evaluation. Additionally, the field of proteomics has the potential to
reveal new biochemical markers used to diagnose and monitor disease and illness. As a result, the
market for proteomics technologies is expected to grow to approximately $3B over the next four
years (Figure 5). QGENICS will initially target the separations segment of the proteomics
market. The demand for separation technologies was approximately $400M in 2002 and is
expected to grow to approximately $1B in 2006.

$U.S. Billions

$3.5
$3.0
$2.5
$2.0
$1.5
$1.0
$0.5
$2001

2002

Separations

2003

2004

2005

2006

Proteomics Instrumentation

Fig. 5 Proteomics Instrumentation Demand

QGENICS initial target customers will be in the pharmaceutical, biotechnology, and academic
industries. More than half the $26.4B spent on pharmaceutical research and development (R&D) in
2000 was wasted investigating drug targets that will never make it to market. 43 Drug treatments average
15 years to develop from idea to product, costing as much as $900M per drug. Thus, drug companies
are especially focused on increased throughput and cost effectiveness and are eager to adopt new
products that impact these objectives, even at increased prices.44 Further, new tools used in pre-clinical
drug discovery do not require Food and Drug Administration (FDA) approval. QGENICS will seek to
penetrate this market by creating brand awareness and product fulfillment through a combination of
strategic alliances and internal marketing and sales efforts.
QGENICS general corporate strategy will be to focus on the design, development, and
commercialization of the high-resolution, high-throughput peptide analysis systems, collaborating
extensively with other companies to sell and use the instruments in their areas of specialization.
Initially, QGENICS will focus on designing and developing a beta prototype system to include the

13*

chip, scanner, and software package. Once the proprietary system is developed, QGENICS will form
strategic alliances with leading pharmaceutical companies and research institutions to perform beta
testing, system optimization, and further application development. Income from these early adoption
arrangements will be used to fund ongoing R&D and to promote diffusion of the technology into the
market. QGENICS will outsource the capital-intensive chip fabrication process to an established
supplier, e.g. Micralyne (Alberta, Canada), and focus on the design, marketing, and sales of the TTDC
systems. QGENICS business model centers not on an initial, one-time equipment sale (the benchtop
computer system) but on repeat sales of the high-margin consumables (the chips).
As shown in Table 2, QGENICS expects revenue to grow to about $250M in 2009, which will
consist of product sales and corporate sponsored research. QGENICS was awarded
approximately $200,000 in technology development grants in 2001 and has recently signed a
term sheet with a venture capital firm, Memphis Biomed Venture Partners (Memphis, TN), which
specializes in early stage, biotechnology companies. The terms of the proposed Series A financing
would provide QGENICS with approximately $1M in technology development funding over the
next year. Supporting documentation is available at QGENICS upon request. Revenues from
early adoption agreements are projected to begin in 2003 and grow to $12M by 2009. Based on
conservative assumptions regarding market penetration and industry growth rates, product sales
are projected to begin in 2004 and grow to $238M by 2009.
Table 2 - Summarized Projected Income Statements

Revenue
COGS
R&D
SG&A
EBITDA

2002
210
0
252
244
(286)

2003
2,060
0
2,301
1,326
(1,567)

2004
6,210
310
4,453
3,786
(2,339)

2005
13,665
2,546
8,034
9,633
(6,548)

2006
35,824
11,307
11,598
14,471
(1,551)

2007
69,123
24,600
14,788
19,906
9,829

2008
139,958
53,207
19,451
31,761
35,539

2009
249,673
96,441
31,720
49,010
72,503

Item 7. Key Personnel

The mission of QGENICS is to become the leading supplier of high performance, lab-on-a-chip
tools for the proteomics industry. Since the companys inception in early 2001, QGENICS has
assembled an inspired management team consisting of proven self-starters and winners.
Currently, the company employs two individuals with M.B.A. degrees, two employees with Ph.D.
degrees, and an experienced technician. In addition, QGENICS has recruited highly qualified
individuals who have taken technology companies from idea to IPO or acquisition for its Board
of Advisors. The core management personnel form a cross-disciplinary team that has experience
in business development, marketing, operations, solid-state physics, molecular biology,
chromatography, and instrumentation development. Below is a short description of each member
of the management team to be involved in this proposed Phase I project.
Michel G. Goedert, Ph.D. is the Principal Investigator for QGENICS. Dr. Goedert earned a
Ph.D. in Physics from the University of Paris (France), a MBA from Pace University (NY) and a
B.S. in Electrical Engineering form the Conservatoire des Arts et Mtiers of Paris (France). Dr.
Goedert is an Instrumentation Research Scientist with extensive experience in analytical and
biochemical instrumentation. Dr. Goedert is currently working as a Research Consultant in

14*

microfluidics for the San Jose State University (CA), where he is developing planar columns for
capillary electrokinetic chromatography.
Dr. Goedert has a strong background in the areas of instrumentation miniaturization using microelectro-mechanical-systems (MEMS) and microfluidics that he gained during his employment at
the corporate research centers of Agilent Laboratories (CA) and Perkin-Elmer (CT). Following a
post-doctoral position at Arizona State University from 1972 to 1973, Dr. Goedert was a Principal
Scientist at Perkin-Elmers Corporate Research Laboratory. There, Dr. Goedert held an
engineering management position, where he led a research team through the definition,
development and support of a gas chromatograph product line. In 1989, Dr. Goedert became a
lead research engineer at Agilent Laboratories Corporate Research Center (formerly Hewlett
Packard), Life Science Technologies Lab, Microfluidics and Miniaturization team. Dr. Goedert
led a multidisciplinary team of approximately 10 in the design, development, and testing of a
microelectromechanical system (MEMS) assembly to integrate and interface a miniaturized
contactless mode of sensing compounds fabricated by conventional MEMS techniques to a planar
lab-on-a-chip. Other related projects at Agilent included the design and evaluation of
microfluidics channels of various shapes to provide the layout of more advanced microstructures
for assay miniaturization as well as the design and development of sensor technologies to detect
the presence of living cells and microparticles.
Dr. Goedert has a great deal of experience and expertise in the design and evaluation of complex
physico-chemical systems applicable to gas and liquid chromatography, capillary electrophoresis
instrumentation and lab-on-a-chip technology. He is the author and co-author of several patents
and technical publications, listed below.
Patents Awarded
US 6331678 (2001)
Patent Assignee: Agilent Technologies
Inventor(s): Wang, T.K., Barth, P.W., Goedert, M.G.
Reduction of blistering and delamination of high-temperature devices with metal film.
US 5581028 (1996)
Patent Assignee: Hewlett Packard CO
Inventor(s): Barth, P.W., Goedert, M.G., Littau, E.
Fluid property sensors incorporating plated metal rings for improved packaging.
US 4935040 (1990)
Patent Assignee: Perkin-Elmer Corporation
Inventor(s): Goedert, M.G.
Miniature devices useful for gas chromatography.

Selected Peer-Reviewed Publications (Selected from 23 peer-reviewed publications)

Goedert, M.G., Higdon, W.: Microelectromechanical systems technology applied to the miniaturization of
field instrumentation. Encyclopedia of Analytical Chemistry 5: 3759-3783, 2000.
Goedert, M.G.: Field-portable instrumentation. Encyclopedia of Environmental Analysis and Remediation
3: 1687-1757, 1998.
Vidal-Madjar, C., Gonnard, M.F., Goedert, M.G.: Gas solid chromatographic measurements of the change
in the heat capacity during adsorption on graphitized thermal carbon blocks. J. Phys. Chem. 79 (7): 732-41,
1975.

15*

Goedert, M.G., Guiochon, G.: High precision measurements in gas chromatography. A study of systematic
errors on the determination of retention times. Analytical Chemistry 45 (7): 1188-1196, 1973.
Goedert, M.G., Guiochon, G.: Sources of error in quantitative gas chromatography: reproducibility of the
response of a katharometer. J. Chromatogr. Sci. 7 (6): 323-339, 1969.

Chuck Witkowski, M.B.A., is the founder, Chief Executive Officer, and President of QGENICS. In
addition to an economics degree with honors, Mr. Witkowski has earned a Masters of Business
Administration degree (M.B.A.) from the University of Tennessee, Knoxville, with a concentration in
new venture development. Mr. Witkowski has received numerous leadership and entrepreneurial
recognitions. Mr. Witkowski began his entrepreneurial career at the age of 17 by obtaining a
commercial bank loan and launching a successful medical equipment leasing business, through which
he financed his undergraduate college education. In 2000, Mr. Witkowski worked as a Project Manager
at Motorola Corporation, Semiconductor Sector, in Austin, Texas. Mr. Witkowski has valuable
experience developing and managing all aspects of company development.
Justin Treadwell, P.E., M.B.A., is the Chief Operating Officer of QGENICS. Mr. Treadwell has an
undergraduate engineering degree from Cornell University and Master of Engineering and M.B.A.
degrees from the University of Tennessee, Knoxville. While at Cornell, Mr. Treadwell was also an All
Ivy League basketball player. In addition to his two years of experience with QGENICS, Mr. Treadwell
has over five years of engineering consulting experience with Williams Environmental Services in
Birmingham, Alabama and with CH2M Hill in Oak Ridge, Tennessee. Mr. Treadwell is a licensed
Professional Engineer in the State of Tennessee and has successfully managed over 10 contracts
ranging in value from $70,000 to $800,000, including service as a Principal Investigator for a Phase I
Small Business Innovation Research contract for the development of MEMS technology. Clients for
these projects have included Southwire Company, T.R. Miller, Inc. NASA, the National Science
Foundation, the Department of Defense, and the Department of Energy. Mr. Treadwells blend of
engineering, business, and government contracting experience is extremely valuable to the company.
Karolyn Hansen, Ph.D. is currently a Principal Scientist for QGENICS. Dr. Hansen received a
B.S. in Biology from Pennsylvania State University in 1980, a M.S. in Environmental Toxicology
from Drexel University in 1984, and a Ph.D. in Marine Biology and Biochemistry from the
University of Delaware in 1990. Following the attainment of these degrees, she served as a postdoctoral researcher for three years at the University of Delaware performing research in protein
biochemistry. In 1995, she joined Geo-Centers, Inc. working on surface chemistry at the U.S.
Naval Research Laboratory. In 1999, she began consulting with Dr. Thomas Thundats Nanoscale
Sciences and Devices group at ORNL as a subcontractor. Through work with Dr. Thundats group
at ORNL, Dr. Hansen has gained extensive experience with the design and development of
microscale sensors.
Jay Harkins is a Research Associate for QGENICS. Mr. Harkins has worked as a technician in
the Chemical Technology Division of the Oak Ridge National Laboratory in the Bioprocessing
Research and Development Group (1994-1997), and as a research associate for Genase, LLC
(Knoxville, TN) and Photogen, Inc. (Knoxville, TN) (1997-2002). At Genase, he was responsible
for conducting experiments to study the effect of enzymes and was intimately involved with
bench-scale assays for the production testing of cellulase products. Furthermore, he worked
directly with sales staff and end users to optimize product performance. As a Research Associate
for Photogen, he was responsible for the preparation, oversight, and completion of a variety of
laboratory experiments designed to research the effectiveness of chemical compounds and
devices for use in photodynamic therapy (use of light-activated drugs in medicine).

16*

Item 8. Consultants and Collaborators

Georges A. Guiochon, Ph.D. graduated with a MS degree in engineering at Ecole Polytechnique
(Paris, France) and received a Ph.D. in chemistry from the University of Paris. He was a
Professor of chemistry at Ecole Polytechnique (until 1985) and at the University of Paris VI or
Universite Pierre et Marie Curie (until 1984), then at Georgetown University (1984-87). He was
appointed a UTK/ORNL Distinguished Scientist in June 1987 and is now a Distinguished
Professor at the University of Tennessee (Department of Chemistry) and a Senior Scientist at the
Oak Ridge National Laboratory (Division of Chemical and Analytical Sciences).
Dr. Guiochons research interests include all aspects of gas and liquid chromatography, theory,
instrumentation and applications, and the problems of physical chemistry related to
chromatography. These include solution and adsorption thermodynamics, mass and energy
transfers, detector principles, and the consolidation of beds of fine particles. His current work is
in the theory of nonlinear chromatography, the development of separation processes based on
chromatography (including SMB), and the preparation of efficient columns. He has nearly 800
scientific publications. His most recent books are "Quantitative Gas Chromatography, written in
cooperation with C. Guillemin and published by Elsevier (1988) and "Fundamentals of
Preparative and Nonlinear Chromatography, written in cooperation with S. G. Shirazi and A. M.
Katti and published by Academic Press (1994).
Dr. Guiochon organized four major International Symposia on Chromatography (ISC in 1974 and
1980, HPLC in 1981 and 1988) and fifteen Symposia on Preparative Liquid Chromatography
(between 1985 and 2002). He is a member of the editorial boards of Chromatographia, the
Journal of Chromatography, the Journal of Liquid Chromatography and the Journal of
Chromatographic Sciences. He was the first foreign member of the Editorial Advisory Board of
Analytical Chemistry and the Associate Editor of this journal for the Separation Sciences in 19851993.
Dr. Guiochon is an Honorary Member of the Chromatographic Society of the UK, of GAMS (the
French Society of analytical chemistry), and of the Spanish Royal Society of Physics and
Chemistry. He was awarded the 1978 Silver Medal of C.N.R.S. (the French NSF), the 1978 Dal
Nogare Award of the Chromatography Forum of the Delaware Valley, the Tswett Medal of
Advances in Chromatography, the Tsvet Medal of the Russian Academy of Sciences, the A.J.P.
Martin Award of the Chromatographic Society, Honorary Doctorates by the Technical University
of Budapest (Hungary) in 1991 and by the University of Pardubice (Czech Republic) in 1999, the
1991 Separation Sciences Award and the 1998 Chromatography Award of the American Chemical
Society, and the Eastern Analytical Symposium Award in Separation Sciences in 2001. In 1994
he received an Alexander von Humboldt Award as a Senior American Scientist.
David L. Hachey, Ph.D. is currently the Mass Spectrometry Director, Mass Spectrometry
Facilities Core, and a Professor of Biochemistry at Vanderbilt University (Nashville, TN). Dr.
Hachey has a great deal of expertise relating to all aspects of protein chemistry, separations, and
mass spectrometry. Dr. Hachey received a B.A. in Chemistry from Oakland University
(Rochester, MI) in 1967 and Ph.D. in Chemistry from the University of California, Santa Barbara
(Santa Barbara, CA) in 1972. Dr. Hachey completed a postdoctoral position in the field of
Chemistry at the Argonne National Laboratory (Argonne, IL) from 1971 to 1974. Subsequently,
Dr. Hachey held was an Assistant Chemist and then a Chemist at Argonne National Laboratory
from 1974 to 1980. From 1976 to 1980, Dr. Hachey was also a Research Associate (Assistant

17*

Professor) in the Department of Medicine at The University of Chicago (Chicago, IL). In 1978,
Dr. Hachey received the University of Chicago Award for Distinguished Performance at Argonne
National Laboratory. From 1980 to 1993, Dr. Hachey held the position of Assistant Professor of
Pediatrics at Baylor College of Medicine (Houston, TX). From 1993 to 1998, Dr. Hachey was an
Associate Professor of Pediatrics at the Baylor College of Medicine. Since that time, Dr. Hachey
has held the positions of Professor of Pharmacology and Professor of Biochemistry at Vanderbilt
University. Dr. Hachey brings a wealth of relevant experience to this proposed project.
Alfred L. Yergey, Ph.D. received a B.S. in Chemistry, Magna cum laude in 1963 from
Muhlenberg College and a Ph.D in Chemistry (Physical) in 1967 under the direction of F.W.
Lampe at Rice University, where he completed a postdoctoral position with J.L. Franklin from
1967-69. He has published widely in a number of areas involving the use of mass spectrometry
ranging from thermodynamics, quantitative analysis, high precision isotope ratiometry and most
recently protein characterization. Dr. Yergey's current research interests lie in the areas of 1)
problems of protein identification with emphasis on using affinity interactions for isolation prior
to mass spectrometry and 2) equilibrium ion-molecule chemistry applied to hydration
thermodynamics. Particular interests in the area of mass spectrometric protein characterization are
in the areas of sample preparation methods to increase sensitivity, de novo sequencing of
peptides, improvements in protein identification algorithms and mathematical approaches for
resolving mixtures of intact proteins. Dr. Yergey is currently the Chief at the Section on Metabolic
Analysis and Mass Spectrometry, National Institute of Child Health and Human Development,
National Institutes of Health. Dr. Yergey will serve as a collaborator on the proposed project.

Item 9. Facilities and Equipment

QGENICS is located in the Knoxville-Oak Ridge, Tennessee area and has close collaborations
with scientists at the Oak Ridge National Laboratory (ORNL), The University of Tennessee, the
University of Tennessee Medical Center, St. Judes Research Hospital, and the Vanderbilt Ingram
Cancer Center. QGENICS laboratory facilities are located at 11020 Solway School Road, Suite
108 in the Fairview Technology Center, which is an incubator for early stage technology
companies. The laboratory space is approximately 700 square feet in space and houses the
following equipment: an Atomic Force Microscope (AFM) head, an AFM read-out/control box, a
data logger and module, a spectrum analyzer, a digitizing oscilloscope, an analytical fume hood,
computer systems, a freezer, refrigerators, a dry bath, chemical mixers, focusing optics, galvos,
galvo drivers, a power source with voltage programmer, a potentiostat, a lock-in amplifier, and
other standard biological and chemical laboratory equipment. QGENICS also has established a
Cooperative Research and Development Agreement (CRADA) with ORNL, which allows the
company full access to the state-of-the-are resources available at the laboratory and grants
QGENICS full rights to any intellectual property generated while at the facility (supporting
documentation available upon request).

E. PRIOR, CURRENT, OR PENDING SUPPORT OF SIMILAR PROPOSALS

An SBIR proposal of equivalent work is also being submitted to the National Institutes of Health,
National Institute on Drug Abuse (NIDA). The appropriated information is disclosed below.
1. NIDA
2. SBIR contract proposal

18*

3. True Two-Dimensional Liquid Chromatography on a Chip

4. Dr. Michel Goedert
5. It is estimated that Dr. Goedert will provide at least 20 hours per week on either this
proposal work or the NIDA proposed work. Since the proposals are essentially the same,
only one contract would be initiated.
6. $100,000 proposed
7. Estimated period of support: March 1, 2003 through September 1, 2003
8. Date of proposal submission: November 8, 2002
9. PHS 2003-1 Small Business Innovative Research Contract Proposals
10. Solicitation number and title: 049 Technologies for Proteomic Analysis in the Nervous
System

ITEM F. PRIOR SBIR PHASE II AWARDS

N/A

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19*

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2