Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2000-06-22
2003-09-30
Nguyen, Nam (Department: 1753)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403060, C435S287200
Reexamination Certificate
active
06627067
ABSTRACT:
BACKGROUND OF THE INVENTION
Rapid, reliable and inexpensive characterization of polymers, particularly nucleic acids, has become increasingly important. A high-throughput device that can probe and directly read, at the single-molecule level, hybridization state, base stacking, and sequence of a cell's key biopolymers such as DNA, RNA and even proteins, will dramatically alter the pace of biological development.
Church et al. in U.S. Pat. No. 5,795,782 recently reported that a voltage bias could drive single-stranded charged polynucleotides through a 1-2 nanometer transmembrane channel in a lipid bilayer. Data in the form of variations in channel ionic current provide insight into the characterization and structure of biopolymers at the molecular and atomic levels. The passage of an individual strand through the channel could be observed as a transient decrease in ionic current. Experiments using biological membranes and pores have demonstrated extraordinary electronic sensitivity to the structure of translocating molecules. See, U.S. Pat. No. 5,795,782 and Kasianowicz et al. (“Characterization of individual polynucleotide molecules using a membrane channel”,
Proc. Natl. Acad. Sci
. 93:13770 (November 1996)).
This is demonstrated in
FIG. 1
, in which a lipid bilayer
10
having a &agr;-hemolsin channel
12
therein is shown. A
Staphylococcus aureus
&agr;-hemolsin channel is used because its inner diameter has a limiting aperture of 1.5 nm, which is adequate to admit single-stranded DNA. The layer separates two solution-filled compartments
14
,
16
in which ions are free to migrate through the channel
12
in response to an applied voltage. The unobstructed ionic current
18
is illustrated in the upper channel
12
of FIG.
1
. If negatively charged molecules, such as DNA, are placed in compartment
14
and a negative bias is applied, the molecules are pulled one at a time into, and through, the channel. The ionic current is reduced as a polymeric molecule
17
traverses the channel from the cis to the trans compartment, as is illustrated in the lower channel
19
of the figure. The number of transient decreases of ionic current per unit time (the blockade rate) is proportional to the concentration of polymer in the source solution. Furthermore, the duration of each blockade is proportional to polymer length.
FIG. 2
is an example of actual current traces obtained using a lipid membrane containing an
S. aureus
&agr;-hemolsin channel. The voltage applied across the bilayer (−120 mV) produces a current of ions that flow through the channel. After adding DNA, transient reductions in current are evident in the trace (FIG.
2
A). The time it takes for the DNA to be drawn through the channel (FIG.
2
B), effectively measures the length of a DNA molecule (here, 1300 &mgr;s corresponding to a 1,060 nt polymer). The extent to which ionic flow is reduced (here, from about 120 &rgr;A to 15 &rgr;A) reflects the physical properties of the nucleotides in the polymer.
While a protein channel has demonstrated the ability to identify characteristics of polynucleotides, attaining the resolution and precision needed to achieve error-free sequencing of individual monomers has proved to be a challenge. For example, it has been demonstrated that detection sensitivity extends along the entire length of the &agr;-hemolsin protein channel, and this despite the sharp limiting asperity of 1.5 nm at its neck. The interactions of multiple monomer units along its entire length contribute to the blocked current magnitude, thereby making it difficult to obtain unambiguous resolution of individual monomers characteristics.
The currently available biological pore membrane system suffers from a number of additional disadvantages, including limited temperature and bias voltage operating ranges, limited chemical environment accommodation, limited device lifetime due to pore diffusion in the membrane, high electronic noise levels associated with large membrane capacitance, and limited availability of pores with the desired diameter and lengths on the 1-10 nm scale. In order to maximize the capabilities of the present technology, certain advances in the technology are required.
SUMMARY OF THE INVENTION
The present invention provides methods and apparatuses based upon solid-state materials for molecular detection. In addition to providing remedies for the above problems associated with biological pores, a solid-state system for molecular detection offers the ability to provide and accommodate local, embedded conducting electrodes and “on chip” integrated electronics that can extend the capabilities of “ionic” current measurements and also offer the prospect of local and very sensitive electronic sensing by mechanisms such as injection tunneling spectroscopy.
In general, the method and apparatus of the invention provide for the traverse of individual monomers of DNA or any other linear polymer molecule across or through a limited volume in space in sequential order, preferably on the nanoscale range, e.g., a volume on a scale which accommodates a single monomer for interacting with a detector such as 1-10,000 nm
3
, and preferably 1-1000 nm
3
. The limited space reduces background noise associated with polymer detection, so that subtle differences in structure may be observed. The use of a limited volume also ensures that the monomers move in single file order.
In one aspect of the invention, evaluation of a polymer molecule including linearly connected monomer residues is accomplished by contacting a polymer-containing liquid with an insulating solid-state substrate having a detector capable of detecting polymer molecule characteristics, and causing the polymer molecule to traverse a limited volume on the solid-state substrate so that monomers of the polymer molecule traverse the limited volume in sequential order, whereby the polymer molecule interacts linearly with the detector and data suitable to determine polymer molecule characteristics are obtained.
In another aspect of the invention, evaluation of a polymer molecule including linearly connected monomer residues is accomplished by contacting a polymer-containing liquid with an insulating solid-state membrane having an aperture therein, wherein the aperture includes an entry port and an exit port defining a channel there between, and causing the candidate polymer molecule to traverse the aperture of the membrane, whereby the polymer molecule interacts linearly with the aperture and data suitable to determine polymer molecule characteristics are obtained.
“Solid-state” is used herein to refer to materials that are not of biological origin. By biological origin is meant derived from or isolated from a biological environment such as an organism or cell, or a synthetically manufactured version of a biologically available structure. Solid-state encompasses both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as Si
3
N
4
, Al
2
O
3
, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses, although there is no specific limitation to the materials that may be used according to the invention.
A “solid-state substrate” of the invention is an insulating material, which is integratable with the electronic devices, e.g., electrodes, necessary to monitor and detect polymer interactions at the solid-state substrate surface. A solid-state substrate is not required to have an aperture.
A “membrane” is a layer prepared from solid-state materials, in which one or more apertures is formed. The membrane may be a layer, such as a coating or film on a supporting substrate, or it may be a free-standing element. Alternatively, it may be a composite of various materials in a sandwich configuration. The thickness of the membrane may vary, and in particular, the membrane may be considerably thinner in the region containing the aperture. In embodiments, in which the membrane is a layer on a suppor
Branton Daniel
Denison Timothy J.
Golovchenko Jene A.
Hale and Dorr LLP
Nguyen Nam
President and Fellows of Harvard College
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