Re: [GRG] Diagnostics on Demand – DARPA

Nov 15, 2013 (Vol. 33, No. 20)

Modern Microfluidics via Lab-on-a-Chip

From our November 15 issue: Platforms that manage microscale and
nanoscale fluid flows effectively bring the laboratory to the
patient’s bedside (or anywhere else).
Josh P. Roberts

Click Image To Enlarge +

One of the first lab-on-a-chip devices to be developed was the DNA
microarray. These are used in biological research to simultaneously
measure the expression of thousands of genes. [nanela/iStock]

There are two major design subfields in microfluidics—chip-in-a-lab
and lab-on-a-chip. Chip-in-a-lab aims to shrink laboratory
operations down to the microliter (or smaller) scale, mainly for
the cost, time, and reagent savings.

Lab-on-a-chip tries to fit a laboratory benchtop into a handheld
device. Both approaches were in evidence at the recent Gordon
Research Conference, “Microfluidics: Challenges, Advances, and New
Technologies for Diagnostics.”

Although microfluidics, and in particular paper microfluidics, is a
hot, sexy new topic and seems like the latest, greatest thing to
hit the practical sciences, “almost everything in the field is
old,” according to Barry Lutz, Ph.D., research assistant professor
in the University of Washington’s department of bioengineering. The
theories have been around, in some cases, for a few hundred years.
The materials themselves may be advanced, but even they have been
exploited at least since the early 1980s in the form of home
pregnancy tests.

In these tests, fluid moves on its own without the aid of a pump.
There is no need to pipette. All the reagents are stored on the
device. It’s very fast, very easy to use. “The simple lateral flow
test is just an ingenious invention,” Dr. Lutz exclaimed.

The paper may look uniform, “but it’s really a bunch of tortuous
pathways,” Dr. Lutz said. Yet by understanding a few basic physical
principles of how fluids move through paper, which in many ways is
very similar to how pollutants in groundwater move through sand or
rock, a researcher should be able to understand and predict
behavior in a microfluidics device.

The Reynolds number (a relationship between a fluid’s inertia and
its viscosity) helps to determine whether flow is fast or slow,
turbulent or not, for example. The boundary conditions are
different for flow through paper than they are for flow through a
tube. Fluid moves through the entire width of a paper conduit,
while fluid near the wall of a tube is essentially pinned and does
not move. Wicking power, or capillary pressure, propels the fluid
into the paper. The flow due to wicking does not happen
instantaneously because of a countering force—flow resistance. “We
have to drag the fluid through these small pores,” Dr. Lutz said.

There is also dispersion. “We have soluble components moving with
the fluid—proteins, small molecules, salts, maybe nucleic acids,”
said Dr. Lutz. “An important question is: Do they move with the
fluid or does the paper change the way they are distributed in the
fluid?” It turns out that both effects matter—classical
chromatographic effects, where the molecule sticks to the paper for
a few seconds and then unsticks, as well as hydrodynamic effects,
where the fluid wends its way through the paper.

Dr. Lutz discussed his lab’s work using dissolvable sugar as a
programmable flow delay in a multistep microfluidic system. By
applying different concentrations of sucrose to the paper and
allowing it to dry, the group was able exploit the varying time
delays created as the fluid reached the sugar and had to dissolve
it. “We were running an ELISA-like test in which the reagents have
to be run sequentially,” Dr. Lutz said. In this way, fluid could be
added to all the legs of the device at the same time, yet the first
leg would turn itself on while the second waited its turn, and so

Compared with a traditional 96-well-plate-scale ELISA, in which
incubation times are on the order of minutes to hours, one of the
brilliant things about flow tests is that mixing takes place in
seconds by diffusion, noted Dr. Lutz.

Diagnostics on Demand

The University of Washington has been working with GE Global
Research and other collaborators under a DARPA “Diagnostics on
Demand” program to develop technology that will enable low-cost,
high-performance tests to be used in a point-of-care or even a
limited-resource setting.

The paper- or membrane-based handheld device will be user-friendly,
instrument-free, and fully disposable, with power contained within
(so there is no need to plug it into a wall). “We want to be able
to do a test from sample to readout in less than an hour, ideally
30 minutes,” explained David Moore, Ph.D., the project’s co-
principal investigator and manager of GE Global Research’s Membrane
and Separation Technologies Lab.

According to Dr. Moore, “membranes and papers are what make up the
porous channels that allow for the transport of the biospecimens
that you’re collecting, processing, amplifying, and then
detecting.” The steps will all be carried out within modules of the
microfluidic device with no further intervention necessary,
producing a visual result.

The first test will be for detection of MRSA DNA from a nasal swab.
MRSA is problematic in institutional settings such as military
bases (thus the interest of DARPA), prisons, and hospitals where
many physicians who will later practice in remote settings do their

The groups are also looking at using blood as a sample fluid, and
at identifying DNA or RNA of other pathogens such as STDs and
viruses. Ultimately, they hope to develop a device that could be
used in the field to detect a panel of infectious diseases, for
example, so that a soldier’s health could be monitored in a war

The readout might be a series of lines or a pattern of dots “that
could then be imaged or captured using a cell phone,” Dr. Moore
explained. “The phone would have an algorithm associated with it to
capture, process, store, and transmit that information to the
healthcare provider of interest.”

For now, they’re in the process of doing multiple iterative loops
of developing what a prototype will look like, with a variety of
different pack-of-cards-sized designs being considered. But
although the form factor has not yet been decided upon, “I can say
we’ve made very nice progress integrating the modules of the
technology into something that works from sample all the way to
result,” Dr. Moore noted.

DARPA is not the only agency looking to develop rapid diagnostics
for use in remote locations. Wave 80 Biosciences is working under
NIH contract to develop rapid quantification of HIV from a single
drop of blood. The company is designing an enclosed microfluidic
cartridge to directly target the HIV virion RNA molecule.

Patients sometimes present at clinics in places like rural Africa
with flu-like symptoms and very large levels of virus in their
blood before anti-HIV antibodies can be detected. The
infrastructure doesn’t exist to send them home, run a sophisticated
diagnostic overnight, and expect them to come back the following
day for a result. They may be highly infectious, so it’s important
to have a test to determine the presence of HIV RNA on the spot.

The device’s 10-cm-long cartridge is designed to fit inside a
handheld processing unit containing electronics for operating
fluidic actuators and incubating the sample at a specified
temperature. Once the incubation is complete, “you stick it in the
analyzer unit, which has the user interface as well as the optics
for reading out the luminescent signal,” explained Wave 80
scientist William Behnke-Parks, Ph.D. Up to 10 samples can be
processed in parallel.

“A lot of steps have to occur in a small amount of real estate,”
Dr. Behnke-Parks said. The process begins by placing a drop of
blood onto one side of the disposable cartridge, and then the
company’s proprietary slit-capillary array fluidic actuators
(SCAFAs) act like pumps to move the blood through. The blood is
lysed to release the HIV RNA. It’s passed through a solid-phase
extraction to clean up the sample, and the RNA is amplified
isothermally. HIV RNA can then be optically detected following an
“ultrasensitive bipartite signal amplification assay.”

The process is even more complex than it sounds because “you’re
working with a limited number of molecules and have to push through
very small channels to control it—the physics is very different at
that length scale,” pointed out Dr. Behnke-Parks. For example, when
mixing on scales that we’re used to—centimeters or meters—there is
a large component of the flow that is inertial, and this causes
turbulence. But microliter-size samples and submillimeter
dimensions operate under a very low Reynolds number regime where
inertia, and therefore turbulence, is almost negligible.

The team at Wave 80 used a pulsatile flow to rapidly and
successively interleave two fluids such that each pulse produces a
plug so small that they can mix by diffusion to produce a
homogenous distribution within seconds.

Smooth Operator

Click Image To Enlarge +

Fluigent’s Microfluidic Flow Control System applies pressure to
independently drive liquid through multiple microfluidic channels.
Because no mechanical or moving parts are involved, flows are
pulseless and highly stable, and pressures may be adjusted to
control the flow rate.

Some issues in microfluidics—mixing, for example—are easier to deal
with in a laboratory setting with “big, powerful pumps to pump
fluids through small mixing geometries that then have very high
backpressures,” Dr. Behnke-Parks said.

Yet using syringe pumps or peristaltic pumps to control flow has
its own drawbacks, noted Anne Le Nel, Ph.D., R&D manager at
Fluigent. Such solutions in the nanoliter range can lead to poor
temporal response times and, because of the rotating motors,
pulsing and instabilities in flow delivery.

In 2005, the Paris-based company introduced the Microfluidic Flow
Control System (MFCS), a pressure-driven flow controller with no
mechanical parts that can be used to independently drive liquid
through multiple microfluidic channels. To do so, it applies
pressure from a source such as bottled gas or a compressor onto
liquid reservoirs. “The use of pressure to move liquid can have the
consequence of having very stable flows and also short settling and
response times,” said Dr. Le Nel.

Yet customers began asking to know the flow rate. So Fluigent
integrated precise flow sensors to monitor the velocity of the
liquids inside the microfluidic systems.

Still not content, some customers wanted to be able to control the
flow rate as well. “So we developed a specific algorithm in which
the pressure pump adjusts its pressure to be able to control the
flow rate,” Dr. Le Nel explained.

It is possible to order flow rates in a syringe pump system, for
example. “But then you don’t know if there is some delay in the
response, and you also don’t know if there is stability or
instability in the flow,” Dr. Le Nel said. “Because you don’t have
a sensor, you are only pushing liquids.”

About Johnny Adams

My full-time commitment is to slow and ultimately reverse biological aging and age related decline for more years of healthy living. I’ve been active in this area since the 1970s, steadily building skills and accomplishments. I have a good basic understanding of the science of aging, and have many skills that complement those of scientists so they can focus on science to advance our shared mission. Broad experience Top skills: administration, management, information technology (data and programming), communications, writing, marketing, market research and analysis, public speaking, forging ethical win-win outcomes among stakeholders (i.e. high level "selling"). Knowledge in grant writing, fundraising, finance. Like most skilled professionals, I’m best described as a guy who defines an end point, then figures out how to get there. I enjoy the conception, design, execution and successful completion of a grand plan. Executive Director Gerontology Research Group (GRG). Manages Email discussion forum, web site, meetings and oversees supercentenarian (oldest humans, 110+ years) research. CEO / Executive Director Aging Intervention Foundation (dba for Carl I. Bourhenne Medical Research Foundation), an IRS approved 501(c)(3) nonprofit. Early contributor to Supercentenarian Research Foundation. Co-Founder Geroscience Healthspan Forum. Active contributor to numerous initiatives to increase healthy years of life. Co-authored book on conventional, practical methods available today to slow the processes of aging – nutrition, exercise, behavior modification and motivation, stress reduction, proper supplementation, damage caused by improper programs, risk reduction and others. Fundamental understanding of, and experience in the genomics of longevity (internship analyzing and curating longevity gene papers). Biological and technical includes information technology, software development and computer programming, bioinformatics and protein informatics, online education, training programs, regulatory, clinical trials software, medical devices (CAT scanners and related), hospital electrical equipment testing program. Interpersonal skills – good communication, honest, well liked, works well in teams or alone. Real world experience collaborating in interdisciplinary teams in fast paced organizations. Uses technology to advance our shared mission. Education: MBA 1985 University of Southern California -- Deans List, Albert Quon Community Service Award (for volunteering with the American Longevity Association and helping an elderly lady every other week), George S. May Scholarship, CA State Fellowship. BA psychology, psychobiology emphasis 1983 California State University Fullerton Physiological courses as well as core courses (developmental, abnormal etc). UCLA Psychobiology 1978, one brief but fast moving and fulfilling quarter. Main interest was the electrochemical basis of consciousness. Also seminars at the NeuroPsychiatric Institute. Other: Ongoing conferences, meetings and continuing education. Aging, computer software and information technology. Some molecular biology, biotech, bio and protein informatics, computer aided drug design, clinical medical devices, electronics, HIPAA, fundraising through the Assoc. of Fundraising Professionals. Previous careers include: Marketing Increasing skill set and successes in virtually all phases, with valuable experience in locating people and companies with the greatest need and interest in a product or service, and sitting across the table with decision makers and working out agreements favorable to all. Information Technology: Management, data analysis and programming in commercial and clinical trials systems, and bioinformatics and protein informatics. As IT Director at Newport Beach, CA based technology organization Success Family of Continuing Education Companies, provided online software solutions for insurance and financial professionals in small to Fortune 500 size companies. We were successful with lean team organization (the slower moving competition was unable to create similar software systems). Medical devices: At Omnimedical in Paramount CA developed and managed quality assurance dept. and training depts. for engineers, physicians and technicians. Designed hospital equipment testing program for hospital services division. In my early 20’s I was a musician, and studied psychology and music. Interned with the intention of becoming a music therapist. These experiences helped develop valuable skills used today to advance our shared mission of creating aging solutions.
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