References, Patents & News for Nano-Scale,
Single Nucleic Acid Amplification and Digital PCR™

Controlled Formation of Low-Volume Liquid Pillars between Plates
with a Lattice of Wetting Patches by Use of a Second Immiscible Fluid

Jonathan Silver,*,2 Zihou Mi,* Keiji Takamoto,* Peter Bungay,Ý James Brown,ÝÝ and Adam Powellý

*Laboratory of Molecular Microbiology, Building 4/338, NIAID, National Institutes of Health, Bethesda, Maryland 20892; ÝBioengineering and Physical
Science Program, Building 13/3N17, MSC 5766, National Institutes of Health, Bethesda, Maryland 20892; ÝÝCytonix Corporation, Beltsville, Maryland
20705; and ýMetallurgy Division (855) MATLS B164, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
E-mail: jsilver@nih.gov, bungay@box-b.nih.gov, jfbrown@cytonix.com, adam.powell@nist.gov.

Journal of Colloid and Interface Science 219, 81&shyp;89 (1999) Article ID jcis.1999.6422, available online at http://www.idealibrary.com


Abstract: Method for forming an array of microdropletsbetween two plates, at least one of which is patterned with a lattice
of wetting patches, using a second immiscible fluid to control droplet formation. The method may be useful for performing
multiple, small-volume biochemical reactions in parallel. We analyze the forces responsible for droplet formation, describe results
of a computer simulation using Surface Evolver, and derive an analytic criterion for droplet formation in terms of the contact
angles of the droplet:second fluid interface on the wetting patches and surrounding surface, the diameter of the wetting patches, the
distance between wetting patches, and the distance between the plates. © 1999 Academic Press


FIG. 1. (A) Schematic diagram of nano droplet forming device. (B) Device being filled with 40 µl of 1% food coloring in water (to visualize the
aqueous phase) followed by 100 µl of displacing fluid. Shadow at upper left is from loading pipette, and ruler units are cm. (C) and (D), 53
photomicrographs of aqueous-displacing fluid interface before and just after isolation of droplet.

Nanoliter scale PCR with TaqMan detection

Olga Kalinina, Irina Lebedeva, James Brown* and Jonathan Silver

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda MD 20892, USA and *Cytonix Corporation, Beltsville, MD, USA


ABSTRACT: We monitored PCR in volumes of the order of 10 nl in glass microcapillaries using a
fluorescence energy transfer assay in which fluorescence increases if product is made due to
template-dependent nucleolytic degradation of an internally quenched probe (TaqMan assay).
This assay detected single starting template molecules in dilutions of genomic DNA.
The results suggest that it may be feasible to determine the number of template
molecules in a sample by counting the number of positive PCRs in a set
of replicate reactions using terminally diluted sample. Since the assay system
is closed and potentially automatable, it has promise for clinical applications.

Nucleac Acid Research, Volume 25: January - December 1997, Pages 1999-2004, Oxford University Press

Issued Patents:

6,391,559 Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase
chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly

6,143,496 Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase
chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly

Published Applications, Others Pending:

20040171055 Method for detecting the presence of a single target nucleic acid in a sample

20020164820 Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase
chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly

News November 20, 2006 -- Cytonix Corporation receives exclusive license from the National Institutes of Health for single molecule DNA and RNA detection.

The Cytonix Corporation announced today that it has signed an exclusive license with the National Institutes of Health (NIH) for a novel method of sampling, amplifying and quantifying nucleic acids and for the production of nano-scale devices needed for the application of this technology. This technology allows Cytonix to detect a single nucleic acid molecule in a nanoarray, vastly facilitating identification of signature DNA or RNA molecules. Nano devices from Cytonix will make it possible to count the number of individual DNA or RNA molecules as the unit of absolute quantity.

James F. Brown of Cytonix is a co-inventor of this technology in U.S. Patent No. 6,143,496 (filed April 17, 1997, entitled "Methods of Sampling, Amplifying and Quantifying Segment of Nucleic Acid") and Divisional Application No. 20020164820 and is inventor in divisional Patent No. 6,391,559 (entitled "Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly") and Divisional Application No. 20040171055. According to Mr. Brown, "this technology provides Cytonix Corporation the unique ability to identify single molecules of contaminating DNA or RNA in food, water or blood. By simply counting the number of fluorescent wells in a nano array, it provides for the first time a true quantitative procedure for measuring nucleic acids. This technology offers an alternative to traditional rt-PCR as a direct means of quantifying nucleic acids by treating every nucleic acid molecule as an individual, overcoming a major limitation of current PCR procedures in detecting rare sequence differences."

Detection and quantification methods based on single molecule detection have exquisite sensitivity, immense dynamic range, and unprecedented quantitative precision. Cytonix's new ultra-sensitive technology is easier to use, considerably faster, more accurate and less expensive than current PCR methods, accelarating the development of applications in diagnosis of human disease, forensics, environmental monitoring and detection of pathogens during an emerging infectious disease or a deliberate biological attack.

Since 1985, Cytonix Corporation technologies have enabled the development of innovative products for life sciences, medical, aerospace and communications industries worldwide.

Cytonix Corporation, 8000 Virginia Manor Road,
Beltsville, MD 20705, phone 301-470-6267 or 888-CYTONIX