Rapid Detection of Waterborne Pathogens in Drinking Water
Rheonix, Inc.
22 Thornwood Drive
Ithaca, NY 14850
Telephone: 607-257-1242
http://www.rheonix.com
Environmental Problem
Drinking water in the United States is among the
safest in the world. Despite that, undetected micro-
bial contamination can lead to serious illness and
death. One such pathogen, Cryptosporidium
parvum, can cause debilitating diarrhea leading to
serious illness. A number of outbreaks of crypto-
sporidiosis have been reported in the United States,
including a major outbreak in Milwaukee in 1993
that killed more than 100 people,. To monitor drinking
water for the presence of C. parvum oocysts, water
utilities often rely on collecting water samples that
then are submitted to an outside reference labora-
tory for testing. Besides the expense of $350-$650
per analysis, public health is jeopardized by a delay
of up to 10 days in obtaining the final results.
Another problem inherent in current testing methods
is that determination of the viability status of the
oocysts requires additional complicated tests. As
effective methods to inactivate C. parvum are imple-
mented on a widespread basis in water utilities, it
will be even more important to determine the
viability of microbes that manage to enter the
plant's distribution system, whether in an active
or inactive state,.
SBIR Technology Solution
With support from EPAs SBIR Program, Rheonix
has developed a fully automated and rapid molecu-
lar diagnostic system that is able to detect single
oocysts of C. parvum in drinking water and distin-
guish viable from nonviable oocysts. Moreover, its
patented Chemistry And Reagent Device (CARD™)
is able to automatically perform all sample prepara-
tion, analysis, and readout without user interven-
tion. A bench-top assay was originally developed by
Innovative Biotechnologies International, Inc. (IBI),
prior to its acquisition by Rheonix in 2008, that
could be completed within 4-6 hours. Considerable
direct intervention, however, was required. Those
steps included: (1) immunomagnetic separation
and washing of oocysts; (2) heat-shock induction of
the hsp70 rnRNA response to differentiate viable
from nonviable oocysts; (3) lysis and purification of
oocysts; (4) extraction and purification of rnRNA;
(5) nucleic acid sequence-based amplification
(NASBA) gene amplification of the target gene
sequences; and (6) detection of the NASBA ampli-
cons on a lateral flow system utilizing liposomes,
conjugated to molecular probes, that also encapsu-
late signal-generating molecules to provide an inex-
pensive method to detect the amplicons.
The bench-top assay was adapted to Rheonix's fully
integrated CARD™ platform, which analyzes clinical
specimens automatically. Once a "raw" water sam-
ple is applied to the C/ypfoDetect CARD™,
required steps are performed seamlessly and auto-
matically. The ease-of-performance reduces the
currently high costs associated with monitoring
drinking water for the presence of C. parvum and
SBIR Success Stories
significantly reduces the level of training required,
providing time and cost benefits in water treatment
plants' testing of drinking water for microbial safety.
Commercialization Information
As a result of EPAs SBIR funding, Rheonix currently
is preparing GypfoDetect CARD™ devices and the
software-interfaced control system for evaluation by
Battelle Memorial Institute as part of EPA's Environ-
mental Technology Verification (ETV) testing program.
Top: Schematic of Card™ showing 6 pumps and 25
valves and general locations of the various functional
components of the assay. Bottom: Photograph of fully
integrated CrypfoDetect CARD™.
1. Immunomagnetic
Separation
6. End detection (LFA) 5. NA SBA reaction 4. Oiigo dT RNA isolation
2. Heat Shock
3. Chemical lysis and
purification

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Simultaneously, Rheonix is pursuing strategic rela-
tionships with companies that actively sell to and
service the drinking water industry The ideal part-
ner for Rheonix is a company that not only main-
tains a dominant presence in the marketplace but
also has complementary products whose sales can
be leveraged by the availability of the unique
Crypto Detect CARD™. The Company will contin-
ue to collaborate with EPA to achieve the necessary
regulatory approvals to permit the C/yptoDetect
CARD™ to be implemented on a nationwide basis,
thereby further improving the safety of the U.S.
drinking water supply.
Company History
Rheonix began its microfluidic efforts approximately
6 years ago as the microfluidic division of KIONIX,
Inc. (Ithaca, NY), a global leader in the design and
fabrication of high-performance, silicon-microma-
chined Micro-Electro-Mechanical Systems (MEMS)
inertial sensors. After spinning out of KIONIX and
acquiring IBI in December 2008, Rheonix has con-
tinued its development of the fully integrated
CARD™ platform for molecular diagnostics.
Numerous CARD™ devices have been developed
for use in both human in vitro diagnostics markets,
as well as the drinking and recreational water test-
ing markets. An existing bench-top assay generally
can be migrated and prototyped on a CARD™
device within 6-12 weeks. Having recently raised
equity capital, the company has adequate financial
resources to complete the necessary development
and regulatory approval processes to permit it to
offer revolutionary products in multiple market
niches. In addition to EPA funding, the company
also is the recipient of grants from the National
Institutes of Health (NIH), National Science
Foundation (NSF), and New York State Energy
Research and Development Authority (NYSERDA).
SBIR Impact
Waterborne pathogens, such as C. parvum, that enter the U.S.
drinking water supply pose significant hazards to public health.
Rheonix developed the fully automated and customizable CARD™
platform for the rapid detection of C. parvum oocysts.
The microfluidic platform incorporates a patented, low-cost, disposable
CARD™ that can analyze single or multiple raw clinical samples and provide
multiplexed endpoint analysis.
SBIR funding assisted IBI to develop its bench-top assay and attract
the attention of and acquisition by Rheonix, which improved and
commercialized the technology with additional EPA SBIR support.
SBIR Success Stories

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