WO2000009757A9 - Polymeres caracterises optiquement - Google Patents

Polymeres caracterises optiquement

Info

Publication number
WO2000009757A9
WO2000009757A9 PCT/US1999/018438 US9918438W WO0009757A9 WO 2000009757 A9 WO2000009757 A9 WO 2000009757A9 US 9918438 W US9918438 W US 9918438W WO 0009757 A9 WO0009757 A9 WO 0009757A9
Authority
WO
WIPO (PCT)
Prior art keywords
polymer
radiation
units
constructed
optical
Prior art date
Application number
PCT/US1999/018438
Other languages
English (en)
Other versions
WO2000009757A1 (fr
Inventor
Jonas O Tegenfeldt
Robert H Austin
Eugene Y Chan
Original Assignee
Us Genomics Inc
Univ Princeton
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Us Genomics Inc, Univ Princeton filed Critical Us Genomics Inc
Priority to JP2000565191A priority Critical patent/JP2002522780A/ja
Priority to CA002340228A priority patent/CA2340228A1/fr
Priority to EP99942167A priority patent/EP1105535A1/fr
Priority to IL14131099A priority patent/IL141310A0/xx
Priority to AU55606/99A priority patent/AU5560699A/en
Publication of WO2000009757A1 publication Critical patent/WO2000009757A1/fr
Publication of WO2000009757A9 publication Critical patent/WO2000009757A9/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/44—Resins; rubber; leather
    • G01N33/442—Resins, plastics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • the present invention is directed to optical systems, methods and products for analyzing polymers, and more particularly to optical systems, methods and products that utilize highly localized optical radiation for characterizing individual units of polymers.
  • Cells have a complex microstructure that determine the functionality of the cell. Much of the diversity associated with cellular structure and function is due to the ability of a cell to assemble various building blocks into diverse chemical compounds. The cell accomplishes this task by assembling polymers from a limited set of building blocks referred to as monomers or units.
  • the key to the diverse functionality of polymers is based in the primary sequence of the monomers within the polymer and is integral to understanding the basis for cellular function, such as why a cell differentiates in a particular manner or how a cell will respond to treatment with a particular drug.
  • the ability to identify the structure of polymers by identifying their sequence of monomers is integral to the understanding of each active component and the role that component plays within a cell.
  • Expression maps relate to determining mRNA expression patterns.
  • the need to identify differentially expressed mRNAs is critical in the understanding of genetic programming, both temporally and spatially. Different genes are turned on and off during the temporal course of an organisms' life development, comprising embryonic, growth, and aging stages. In addition to developmental changes, there are also temporal changes in response to varying stimuli such as injury, drugs, foreign bodies, and stress. The ability to chart expression changes for specific sets of cells in time either in response to stimuli or in growth allows the generation of what are called temporal expression maps. On the other hand, there are also body expression maps, which include knowledge of differentially expressed genes for different tissues and cell types. Since generation of expression maps involve the sequencing and identification of cDNA or mRNA, more rapid sequencing necessarily means more rapid generation of multiple expression maps.
  • DNA sequencing is performed using one of two methods.
  • the first and more popular method is the dideoxy chain termination method described by Sanger et al. ("DNA sequencing with chain-terminating inhibitors," Proc. Natl. Acad. Sci. USA. 74:5463- 7, 1977).
  • This method involves the enzymatic synthesis of DNA molecules terminating in dideoxynucleotides.
  • ddNTPs By using the four ddNTPs, a population of molecules terminating at each position of the target DNA can be synthesized.
  • Subsequent analysis yields information on the length of the DNA molecules and the base at which each molecule terminates (either A, C, G, or T). With this information, the DNA sequence can be determined.
  • the second method is Maxam and Gilbert sequencing (Maxam and Gilbert, "A new method for sequencing DNA,” Proc. Natl. Acad. Sci. USA. 74:560-4, 1977), which uses chemical degradation to generate a population of molecules degraded at certain positions of the target DNA. With knowledge of the cleavage specificities of the chemical reactions and the lengths of the fragments, the DNA sequence is generated. Both methods rely on polyacrylamide gel electrophoresis and photographic visualization of the radioactive DNA fragments. Each process takes about 1-3 days. The Sanger sequencing reactions can only generate 300-800 bases in one run. Sanger-based methods have been proposed to improve the output of sequence information. The Sanger-based methods include multiplex sequencing, capillary gel electrophoresis, and automated gel electrophoresis.
  • Sanger independent methods use a completely different methodology to realize the base information.
  • This category contains the most novel techniques, which include scanning electron microscopy (STM), mass spectrometry, enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA) sequencing, exonuclease sequencing, and sequencing by hybridization.
  • STM scanning electron microscopy
  • ELIDA enzymatic luminometric inorganic pyrophosphate detection assay
  • exonuclease sequencing and sequencing by hybridization.
  • reaction samples are analyzed by small diameter, gel-filled capillaries.
  • the small diameter of the capillaries 50 ⁇ m
  • high field strengths can be used without excessive Joule heating (400 Vim), lowering the separation time to about 20 minutes per reaction run.
  • high field strengths can be used without excessive Joule heating (400 Vim), lowering the separation time to about 20 minutes per reaction run.
  • many capillaries are analyzed in parallel (Wooley and Mathies, "Ultra-high-speed DNA sequencing using capillary electrophoresis chips," Anal. Chem.
  • Multiplex sequencing is a method which more efficiently uses electrophoretic gels (Church and Kieffer-Higgins, "Multiplex DNA sequencing," Science. 240:185-88, 1988).
  • Sanger reaction samples are first tagged with unique oligomers and then up to 20 different samples are run on one lane of the electrophoretic gel. The samples are then blotted onto a membrane. The membrane is then sequentially probed with oligomers that correspond to the tags on the Sanger reaction samples. The membrane is washed and reprobed successively until the sequences of all 20 samples are determined. Even though there is a substantial reduction in the number of gels run, the washing and hybridizing steps are as equally laborious as running electrophoretic gels. The actual sequencing rate is comparable to that of automated gel electrophoresis.
  • Mass spectrometry sequencing first entails creating a population of nested DNA molecules that differ in length by one base. Subsequent analysis of the fragments is performed by mass spectrometry.
  • an exonuclease is used to partially digest a 33-mer (Smirnov, "Sequencing oligonucleotides by exonuclease digestion and delayed extraction matrix- assisted laser desorption ionization time-of-flight mass spectrometry," Anal. Biochem. 238:19- 25, 1996).
  • a population of molecules with similar 5' ends and varying points of 3' termination is generated.
  • the reaction mixture is then analyzed.
  • the mass spectrometer is sensitive enough to distinguish mass differences between successive fragments, allowing sequence information to be generated.
  • Mass spectrometry sequencing is highly accurate, inexpensive, and rapid compared to conventional methods.
  • the major limitation, however, is that the read length is on the order of tens of bases.
  • matrix-assisted laser desorption ionization time-of- flight (MALDI-TOF) mass spectroscopy (Smirnov et al., "Sequencing oligonucleotides by exonuclease digestion and delayed extraction matrix-assisted laser desorption ionization time- of-flight mass spectrometry," Anal. Biochem. 238:19-25, 1996), can only achieve maximum read lengths of 80-90 base pairs. Much longer read lengths are physically impossible due to fragmentation of longer DNA at guanidines during the analysis step. Mass spectrometry sequencing is thus limited to verifying short primer sequences and has no practical application in large-scale sequencing.
  • STM sequencing (Ferrell, “Scanning tunneling microscopy in sequencing of DNA.” In Molecular Biology and Biotechnology, R.A. Meyers, Ed. VCH Publishers, New York, 1997) method was conceived at the time the STM was commercially available.
  • the initial promise of being able to read base-pair information directly from the electron micrographs no longer holds true.
  • DNA molecules must be placed on conducting surfaces, which are usually highly ordered pyrolytic graphite (HOPG) or gold. These lack the binding sites to hold DNA strongly enough to resist removal by the physical and electronic forces exerted by the tunneling tip. With difficulty, DNA molecules can be electrostatically adhered to the surfaces.
  • HOPG pyrolytic graphite
  • purines can be distinguished from pyrimidines, but the individual purines and pyrimidines cannot be identified. The ability to achieve this feat requires electron microscopy to be able to distinguish between aldehyde and amine groups on the purines and the presence or absence of methyl groups on the pyrimidines.
  • Enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA) sequencing uses the detection of pyrophosphate release from DNA polymerization to determine the addition of successive bases.
  • the pyrophosphate released by the DNA polymerization reaction is converted to ATP by ATP sulfurylase and the ATP production is monitored continuously by firefly luciferase.
  • the method uses successive washes of ATP, CTP, GTP, and TTP. If a wash for ATP generates pyrophosphate, one or more adenines are incorporated.
  • the number of incorporated bases is directly proportional to the amount of pyrophosphate generated. Enhancement of generated sequence information can be accomplished with parallel analysis of many ELIDA reactions simultaneously.
  • Exonuclease sequencing involves a fluorescently labeled, single-stranded DNA molecule which is suspended in a flowing stream and sequentially cleaved by an exonuclease. Individual fluorescent bases are then released and passed through a single molecule detection system.
  • the temporal sequence of labeled nucieotide detection corresponds to the sequence of the DNA (Ambrose et al., "Application of single molecule detection to DNA sequencing and sizing," Eer. Bunsenges. Phys. Chem. 97:1535-1542, 1993; Davis et al, "Rapid DNA sequencing based on single-molecule detection,” Los Alamos Science.
  • a target DNA is sequentially probed with a set of oligomers consisting of all the possible oligomer sequences.
  • the sequence of the target DNA is generated with knowledge of the hybridization patterns between the oligomers and the target (Bains, "Hybridization methods for DNA sequencing,” Genomics. 11 :294-301, 1991 ; Cantor et al., “Reporting on the sequencing by hybridization workshop,” Genomics. 13: 1378-1383, 1992; Drmanac et al., "Sequencing by hybridization.” In Automated DNA Sequencing and Analysis Techniques, J. Craig Ventor, Ed. Academic Press, London, 1994). There are two possible methods of probing target DNA.
  • the “Probe Up” method includes immobilizing the target DNA on a substrate and probing successively with a set of oligomers.
  • "Probe Down” on the other hand requires that a set of oligomers be immobilized on a substrate and hybridized with the target DNA.
  • Probe Up methods would require, for an 8-mer, 65,536 successive probes and washings, which would take an enormous amount of time.
  • Probe Down hybridization generates data in a few seconds. With perfect hybridization, 65,536 octamer probes would determine a maximum of 170 bases. With 65,536 "mixed" 1 1-mers, 700 bases can be generated.
  • a short read length means that additional genetic sequence information needs to be sequenced before the linear order of a target DNA can be deciphered.
  • the short fragments have to be bridged together with additional overlapping fragments.
  • a minimum of 9 x 10 9 bases need to be sequenced before the linear sequence of all 3 x 10 9 bases of the human genome are properly ordered.
  • the number of bases needed to generate a believable genome is approximately 2 x 10 10 bases.
  • Protein sequencing generally involves chemically induced sequential removal and identification of the terminal amino acid residue, e.g., by Edman degradation. See Stryer, L., Biochemistry, W. H. Freeman and Co., San Francisco (1981) pp. 24-27. Edman degradation requires that the polypeptide have a free amino group which is reacted with an isothiocyanate.
  • the isothiocyanate is typically phenyl isothiocyanate. The adduct intramolecularly reacts with the nearest backbone amide group of the polymer thereby forming a five membered ring.
  • polypeptide sequencing has been described by preparing a nested set (sequence defining set) of polymer fragments followed by mass analysis. See Chait, B. T. et al., Science 257:1885-94 (1992). Sequence is determined by comparing the relative mass difference between fragments with the known masses of the amino acid residues. Though formation of a nested (sequence defining) set of polymer fragments is a requirement of DNA sequencing, this method differs substantially from the conventional protein sequencing method consisting of sequential removal and identification of each residue. Although this method has potential in practice it has encountered several problems and has not been demonstrated to be an effective method.
  • the rate of sequencing has limited the capability to generate multiple body and temporal expression maps which would undoubtedly aid the rapid determination of complex genetic function.
  • the invention relates to new systems, methods and products for analyzing polymers and in particular new systems, methods and products useful for determining the sequence of polymers.
  • the invention has numerous advantages over prior art systems and methods used to sequence polymers. Using the methods of the invention the entire human genome could be sequenced several orders of magnitude faster than could be accomplished using conventional technology.
  • the systems, methods and products of the invention can be used to create comprehensive and multiple expression maps for developmental and disease processes. The ability to sequence an individual's genome and to generate multiple expression maps will greatly enhance the ability to determine the genetic basis of any phenotypic trait or disease process.
  • a system for optically analyzing a polymer of linked units includes an optical source, an interaction station, an optical detector, and a processor.
  • the optical source is constructed to emit radiation of a selected wavelength.
  • the interaction station is constructed to receive the emitted radiation and produce a localized radiation spot from the radiation emitted from the optical source.
  • the interaction station is also constructed to sequentially receive units of the polymer and arranged to irradiate sequentially the units at the localized radiation spot.
  • the optical detector is constructed to detect radiation including characteristic signals resulting from interaction of the localized radiation spot with the units.
  • the processor is constructed and arranged to analyze the polymer based on the detected radiation. Preferred embodiments of this aspect include one or more of the following features:
  • the interaction station is constructed to sequentially receive the units being selectively labeled with a radiation sensitive label and the interaction includes interaction of the localized radiation with the radiation sensitive label.
  • the radiation sensitive label includes a fluorophore.
  • the interaction station includes a waveguide constructed to receive the emitted radiation and provide the evanescent radiation in response thereto.
  • the interaction station includes a slit having a width in the range of 1 nm to 500 nm, wherein the slit produces the localized radiation spot.
  • the interaction station includes a microchannel and a slit having a submicron width arranged to produce the localized radiation spot.
  • the microchannel is constructed to receive and advance the polymer units through the localized radiation spot.
  • the width of the slit is in the range of 10 nm to 100 nm.
  • the system may include a polarizer and the optical source is a laser constructed to emit a beam of radiation and the polarizer is arranged to polarize the laser beam prior to reaching the slit.
  • the polarizer may be arranged to polarize the laser beam in parallel to the width of the slit, or perpendicular to the width of the slit.
  • the interaction station may include several slits located perpendicular to the microchannel that is arranged to receive the polymer in a straightened form.
  • the interaction station may include a set of electrodes constructed and arranged to provide electric field for advancing the units of the polymer through the microchannel.
  • the system may further include an alignment station constructed and arranged to straighten the polymer and provide the straightened polymer to the interaction station.
  • a method for optically analyzing a polymer of linked units comprising: labeling selected units of the polymer with radiation sensitive labels; sequentially passing the units of the polymer through a microchannel; generating radiation of a selected wavelength to produce therefrom a localized radiation spot; irradiating sequentially the labeled units of the polymer at the localized radiation spot; detecting sequentially radiation providing characteristic signals resulting from interaction of the localized radiation spot with the labels or the units; and analyzing the polymer based on the detected radiation.
  • an article of manufacture used for optically analyzing a polymer of linked units comprising an interaction station fabricated on a substrate and constructed to receive radiation and produce therefrom a localized radiation spot.
  • the interaction station is further constructed to sequentially receive units of the polymer and arranged to irradiate sequentially the units at the localized radiation spot to generate characteristic signals of radiation.
  • a system for optically analyzing a polymer of linked units includes an optical source, an interaction station, an optical detector, and a processor.
  • the optical source is constructed to emit radiation of a selected wavelength.
  • the interaction station is constructed to receive the emitted radiation and constructed to sequentially receive units of the polymer and arranged to irradiate sequentially the units of the polymer with evanescent radiation excited by the radiation emitted from the source.
  • the optical detector is constructed to detect radiation including characteristic signals resulting from interaction of the evanescent radiation with the units.
  • the processor is constructed and arranged to analyze the polymer based on the detected radiation.
  • the interaction station is constructed to sequentially receive the units being selectively labeled with a radiation sensitive label and the interaction includes interaction of the evanescent radiation with the radiation sensitive label.
  • the radiation sensitive label includes a fluorophore.
  • the interaction station includes a waveguide constructed to receive the emitted radiation and provide the evanescent radiation in response thereto.
  • the waveguide is a dielectric waveguide constructed to achieve total internal reflection of introduced light.
  • the waveguide is a rectangular mirror waveguide with a dielectric surrounded by metallic mirror layers constructed to have a low loss of introduced light.
  • the waveguide includes a tip including an aperture in the metallic mirror layers and arranged to emit the evanescent radiation.
  • the waveguide includes a tip constructed to emit the evanescent radiation.
  • the interaction station includes a nanochannel located at the tip of the waveguide and arranged to receive the polymer in a straightened form.
  • the interaction station includes a set of electrodes constructed and arranged to provide electric field for advancing the units of the polymer through the nanochannel.
  • the electrodes are internal electrodes.
  • the electrodes are external electrodes.
  • the nanochannel is between 2 and 50 nanometers.
  • the waveguide is further constructed and arranged to receive the radiation including the characteristic signals and optically couple the received radiation to the optical detector.
  • the interaction station includes another waveguide constructed and arranged to receive the radiation including the characteristic signals and optically couple the received radiation to the optical detector.
  • the system further includes an alignment station constructed and arranged to straighten the polymer and provide the straightened polymer to the interaction station.
  • the invention is a system for optically analyzing a polymer utilizing confocal fluorescence illumination of linked units.
  • the system includes an optical source constructed to emit optical radiation; a filter constructed to receive and filter said optical radiation to a known wavelength; a dichroic mirror constructed to receive said filtered optical radiation; an interaction station constructed to receive said filtered optical radiation and produce a localized radiation spot from said filtered optical radiation, said interaction station being also constructed to sequentially receive units of said polymer and arranged to irradiate sequentially said units at said localized radiation spot; an optical detector constructed to detect radiation including characteristic signals resulting from interaction of said units at said localized radiation spot; and a processor constructed and arranged to analyze said polymer based on said detected radiation including said characteristic signals.
  • the interaction station is constructed to sequentially receive said units being selectively labeled with a radiation sensitive label producing said characteristic signals at said localized radiation spot.
  • the radiation sensitive label includes a fluorophore.
  • the filter is a laser line filter.
  • the system may also include an objective, wherein the objective focuses said filtered optical radiation.
  • the proposed system and method for analyzing polymers is particularly useful for determining the sequence of units within a DNA molecule and can eliminate the need for generating genomic libraries, cloning, and colony picking, all of which constitute lengthy pre- sequencing steps that are major limitations in current genomic-scale sequencing protocols.
  • the methods disclosed herein provide much longer read lengths than achieved by the prior art and a million-fold faster sequence reading.
  • the proposed read length is on the order of several hundred thousand nucleotides. This translates into significantly less need for overlapping and redundant sequences, lowering the real amount of DNA that needs to be sequenced before genome reconstruction is possible.
  • FIG. 1 illustrates diagrammatically a system for characterizing polymers.
  • Fig. 2 illustrates an alignment and a first interaction station used in the system of Fig. 1.
  • Fig. 3 is a cross-sectional view of the alignment and the first interaction station along lines 3-3 shown in Fig. 2.
  • Fig. 4 is a top view of a portion of the alignment and the first interaction station shown in Fig. 2.
  • Fig. 4A illustrates the arrangement of a nanoslit located in the first interaction station shown in Fig 4.
  • Fig. 4B illustrates an optical system for characterizing polymer units labeled by a fluorophore.
  • Figs. 5 and 5 A illustrate a second interaction station used in the system of Fig. 1.
  • Figs. 6 through 7B illustrate the fabrication of the alignment and first interaction station shown in Fig 4.
  • Fig. 8 is an SEM micrograph of the fabricated alignment and first interaction stations.
  • Figs. 9, 10A, 10B, and IOC show results of a test measurement of the alignment and interaction station of Fig. 8.
  • Fig. 11 is a cross-sectional view of the central line of optical waveguides according to another embodiment of the first interaction station.
  • Fig. 11 A is a perspective view of the optical waveguides shown in Fig. 11
  • Figs. 1 IB and 11C illustrate the interaction of with a linearized polymer with evanescent radiation emitted from the optical waveguide.
  • Fig. 12 illustrates optical systems for near-field and far-field detection as used with the optical waveguide of Fig. 11.
  • Figs. 13, 13A and 13B illustrate coupling of electromagnetic radiation into the optical waveguide of Fig. 11.
  • Figs. 14A through 16G illustrate the fabrication of the optical waveguides shown in Fig. 1 1.
  • Fig. 17 is a schematic of an optical apparatus which utilizes confocal fluorescence illumination and detection for linear analysis of polymers.
  • Fig. 18 is a top view of another embodiment of the alignment station for aligning and stretching polymer.
  • an interactive system for characterizing individual units of a polymer includes a system controller 10, a polymer supply 20, a microfluidic pump 25, a polymer alignment station 30, a first interaction station 40, and a second interaction station 50.
  • System controller 10 may be a general purpose computer.
  • Microfluidic pump 25 supplies selected amounts of polymer 27 from polymer supply 20 to polymer alignment station 30.
  • the first interaction station 40 uses an optical system for characterizing individual units of the polymer passing through.
  • the optical system includes an optical source 42. an optical filter 45, an optical detector 46 and other optical elements and electronic elements associated with the source and detector.
  • the optical system is controlled by an optical controller 48. As the individual units of the polymer pass through interaction station 40, optical source
  • the optical component 42 emits radiation directed to an optical component of interaction station 40.
  • the optical component produces a localized radiation spot that interacts directly with polymer units, or interacts with labels selectively attached to the polymer units, or interact with both the polymer units and the labels.
  • the localized radiation spot includes non-radiating near field or an evanescent wave, localized in at least one dimension.
  • the localized radiation spot provides a much higher resolution than the diffraction-limited resolution used in conventional optics.
  • interaction station 40 uses unique arrangements and geometries that allow the localized radiation spot to interact with one or several polymer units or attached labels that are on the order of nanometers or smaller.
  • Optical detector 46 detects light modified by the interaction and provides a detection signal to optical controller 48.
  • Second interaction station 50 uses electric or electromagnetic field.
  • polymer alignment station 30 and first interaction station 40 include a substrate 92. a quartz wafer 60. and a glass cover 90, which is optional.
  • Substrate 92 is machined from a non-conducting, chemically inert material, such as Teflon® or Delrinl. to facilitate a flow of conducting fluid 96 (for example, agarose gel) and the examined polymer.
  • Substrate 92 includes trenches 94A and 94B machined to receive gold wires 98A and 98B. respectively, which have a selected shape in accordance with the shape of the electric field used for advancing polymer molecules 39 across first interaction station 40. Quartz wafer 60 is sealed onto substrate 92 around regions 91.
  • trenches 94A and 94B and wires 98A and 98B may be replaced by metallic regions located directly on quartz wafer 60, or may be replaced by external electrodes for creating the electric field.
  • the electrodes are spaced apart over a distance in the range of about millimeter to 5 centimeters, and preferably 2 centimeters and provide typically field strengths of about 20 V/cm.
  • Figs. 4 and 4A show a presently preferred embodiment of alignment station 30 and first
  • Fig. 4 is a top view of a portion of alignment station 30 and first interaction station 40 (also shown in Fig. 2). which are fabricated on quartz wafer 60.
  • Quartz wafer 60 includes a quartz substrate covered with a metal layer 62 (e.g. aluminum, gold, silver) and having a microchannel 41 fabricated on the surface.
  • a metal layer 62 e.g. aluminum, gold, silver
  • slits 36A. 36B and 36C Fabricated through metal layer 62 are slits 36A. 36B and 36C. which form the optical elements that provide the localized radiation spot.
  • Slits 36A. 36B and 36C have a selected width in the range between 1 nm and 5000 nm. and preferably in the range between 10 nm and 1000 nm.
  • Slits 36A. 36B and 36C are located across microchannel 41 , which has a width in the range of 1 micrometer to 50 micrometers and a length of several hundred micrometers.
  • polymer alignment station 30 includes several alignment posts 32 located in regions 3 1 . Regions 31 are connected via transition regions 34 to microchannel 41. Alignment posts 32 have a circular cross-section and are about 1 micron in diameter. Alignment posts 32 are spaced about 1.5 microns apart and located about 5 ⁇ m to 500 ⁇ m (and preferably about 10 ⁇ m to 200 ⁇ m) from microchannel 41 depending on the length of the examined polymer. For example, when the polymer is bacteriophage T4 DNA, which has about 167 000 base pairs, alignment posts 32 are located about 30 ⁇ m from nanoslit 36A. In general, the distance from nanoslit 36A is about one half of the expected length of polymer 39.
  • Fig. 4A illustrates interaction of a light beam 65. emitted from optical source 42. with a nanoslit 36. formed in metal layer 62, to produce a localized radiation spot 67.
  • Laser beam 65 which has a size many times larger that the width of nanoslit 36, irradiates the back side of quartz wafer 60, propagates through quartz wafer 60 and interacts with nanoslit 36.
  • Localized radiation spot 67 which is a non-radiating near field, irradiates sequentially the units of polymer chain 39 as polymer chain 39 is pulled through microchannel 41. Localized radiation spot 67 may be understood as an evanescent wave emitted from nanoslit 36. Because the width of nanoslit 36 is smaller than the wavelength of light beam 65 the radiation is in the Fresnel mode.
  • the optical system may also include a polarizer 43 placed between optical source 42 and quartz wafer 60. and a notch filter 45, placed between quartz wafer 60 and optical detector
  • the polarizer orients light beam 65 with the E vector parallel to the length of nanoslit 36, there is near-field radiation emitted from nanoslit 36 and no far field radiation.
  • the polarizer orients light beam 65 with the E vector perpendicular to nanoslit 36 (which is many wavelengths long), there is far-field emission from nanoslit 36.
  • the optical system can switch between the near- field and far-field emissions.
  • Fig. 4B illustrates an optical system for characterizing polymer units labeled by a fluorophore.
  • the optical system includes a laser source 80. an acousto-optic tunable filter 82. a polarizer 84, a notch filter 86. an intensifier and a CCD detector 88, and a video monitor 87 connected to a video recorder VCR 89.
  • the individual units of polymer chain 39 are selectively labeled by a fluorophore 68 sensitive to a selected excitation wavelength.
  • Acousto-optic tunable filter 82 is used to select the excitation wavelength of light emitted from laser source 80.
  • the excitation beam 65 interacts with nanoslit 36 (shown in Fig.
  • Electric field may be used to position polymer 39 close to nanoslit 36.
  • Nanoslit 36 "emits" the non-radiating field 67, which is attenuated over a distance of only one or two wavelengths.
  • polymer 39 may need to be pulled closer to nanoslit 36 (and metal film 62) and thus closer to metal layer 62.
  • Polymer 39 is pulled closer to nanoslit 36 using dielectric forces created by applying AC field to metal layer 62. See, e.g., "Trapping of DNA in Nonuniform Oscillating Electric Fields," by Charles L.
  • second interaction station 50 measures ionic current across a nanochannei as linearized polymer molecules approach the nanochannel and pass through.
  • the detected blockages of the ionic current are used to characterize the length of the polymer molecules and other characteristics of the polymer.
  • Interaction station 50 receives linearized polymer 39 from first interaction region 40 and applies transchannel voltage using electrodes 52 and 53 in a direction perpendicular to electrodes 54 and 55 to draw the polymer molecules through a channel 51.
  • Electrodes 54 and 55 are connected to a microampere meter 56A, located in controller 56, to measure the ionic current across nanochannel 51.
  • a microampere meter 56A located in controller 56, to measure the ionic current across nanochannel 51.
  • the microampere meter is replaced by a bridge 56B, which compares the impedance of channel 51 without polymer 39 (Z,) with the instantaneous impedance of (Z . Without polymer 39 present in channel 51. the voltmeter measures 0 V. As the extended, nearly linear string 39 passes through channel 51 , its presence detectably reduces, or completely blocks, the normal ionic flow from electrode 54 to electrode 55.
  • Electrodes 54 and 55 are fabricated using submicron lithography and are connected to the bridge to detect changes in the impedance or the microampere meter to measure the ionic current.
  • the measured data across the channel are amplified, and the amplified signal is filtered (e.g.. 64,000 samples per second) using a low pass filter, and the data is digitized at a selected sampling rate by an analog-to-digital converter.
  • System controller 10 correlates the transient decrease in the ionic current with the speed of the polymer units and determines the length of the polymer, for example the length of a DNA or RNA molecule.
  • the optical system includes an ultra fast, highly sensitive spectrophotometer capable of detecting fluorescence from a single fluorophore.
  • Optical source 42 is a mode-locked Nd:YAG laser emitting radiation of an excitation wavelength.
  • the system uses a splitter providing a reference beam to a photodiode and a discriminator (e.g., Tennelec TC454) that provides the start pulse to a time-to-amplitude converter (e.g., Tennelec 863).
  • the primary beam 65 is directed through a neutral density filter that adjusts the power level.
  • fluorophore 68 interacting with non-radiation near-field 67 excites fluorescent light 72, which is collected by detector 46 after being spectrally filtered by an
  • RECTIFIED SHEET (RULE 91) /EP interference filter (e.g.. made by Omega Optics) and detected by an avalanche photodiode or a photomultiplier (e.g.. Hamamatsu R1562UMCP microchannel photomultiplier).
  • the microchannel photomultiplier signal is amplified by an amplifier and shaped by a discriminator (for example. Tennelec C4534 discriminator).
  • the signal having appropriate time delays are provided to the time-to-amplitude converter (TAC).
  • TAC time-to-amplitude converter
  • the time-gated TAC output is counted by a multiscaler and interfaced via a VME interface to system controller 10.
  • System controller 10 provides, for the signal from each detector, a time-delay histogram that is characteristic for each type of the fluorescing fluorophore coupled to a unit of polymer 39.
  • Fluorescent lifetime is useful for identification of the fluorophore.
  • the system can use related dyes with similar spectra but different lifetimes thus employing only one laser source emitting the excitation wavelength and one detector detecting the fluorescent radiation.
  • the optical system uses modulated radiation (e.g., single side band or double side band modulation) at frequencies in the range of 10 MHz to 1 GHz using phase modulation techniques to characterize fluorescence of a single fluorophore located next to a polymer unit.
  • modulated radiation e.g., single side band or double side band modulation
  • a laser source emits a light beam 65, which is intensity modulated using a sinusoidal signal at a frequency of 100 MHz.
  • the excited fluorescent radiation 72 is detected using a photomultiplier.
  • the corresponding signal is homodyne or heterodyne detected to resolve the characteristic signal from the fluorophore. e.g.. fluorescent lifetime.
  • Figs. 6 through 7B illustrate the fabrication of alignment region 30, microchannel 41 and slits 36A, 36B and 36C, shown in Fig. 4.
  • Fig. 6 is a side view of quartz wafer 60, which is about 400 microns thick and polished on both sides.
  • HMDS hexamethyldisiloxane
  • a photoresist Shipley 1813 was spun onto the wafers at 4000 rpm 60 sec, and the wafer was baked on a hotplate at 115°C to harden the resist (Fig. 6 A). The wafer was exposed, and the photoresist developed in 1 : 1 MF 312 developer and water for 60 seconds.
  • RECTIFIED SHEET (RULE 91) SA/EP
  • the coarse aluminum pattern was etched using a Cl reactive ion etcher PK 1250 for 1.5 mm.
  • Fig. 6B Fig 6C shows an overview of the wafer with the devices shown as squares and alignment marks as crosses. All resist residues were removed using the resist descum process in the Branson barrel etcher at 1000 RF power for 10 minutes (Fig. 6D).
  • Fig. 6E the PMMA resist (4% 950 K in MIBK) was spun onto the wafers at
  • PECVD plasma enhanced chemical vapor deposition
  • the SiO- layer was planarized by chemical mechanical polishing (CMP).
  • Figs. 7 through 7B are side views of the wafer along one of the nanochanels.
  • alignment region 30 and microchannel 41 were defined by first spinning photoresist Shipley 1813 onto the wafers at 1800 rpm for 60 sec. and baking the resist on a hotplate at 1 15°C for 60 sec.
  • the resist as exposed in a high resolution mask aligner, such as a 5x g-line stepper, and developed in 1 1 MF 312 and water for 60 sec.
  • the SiO- la er was etched (Fig 7 A) using reactive ion etching (RIE) in CHF, (50 seem) + O, (2 seem) to define the pattern in the SiO- laver as shown in Fig 4.
  • RIE reactive ion etching
  • the photoresist was removed using the Branson barrel etcher at 1000 W RF power for 10 minutes.
  • a protective SiO, layer of 10 nm to 100 nm was deposited deposited PECVD (Fig. 7B).
  • Glass cover 90 (shown in Fig. 2) may be anodically bonded to quartz wafer 60, or may be attached to chip 60 using a thin layer of RTV.
  • Fig. 8 shows an SEM micrograph with two fabricated alignment regions 30 and two interaction regions 40.
  • Each alignment region 30 includes microposts 32, and each interaction regions 40 includes microchannel 41 and nanoslits 36A, 36B, and 36C, as drawn in Fig. 4.
  • Figs. 9 through 10C the fabricated alignment regions 30 and interaction regions 40 (shown in Fig. 8) were tested in the following experiment. CW laser light from a collimated Ar:Kr ion laser was focused onto the back side of wafer 60
  • RECTIFIED SHEET (RULE 91) ISA/EP as shown in Fig. 4A.
  • Laser beam 65. having excitation wavelength of 488 nm. created a non- radiating near field on the other side film 62 near a fluorophore 68.
  • a microscope objective captured the fluorescent far-field radiation of 560 nm. which was recorded in a time- dependent manner by a photomultiplier. This time-dependent signal then gave a record of the passage of the object over the slit with a spatial resolution roughly equal to the width of the slit 36.
  • Fig. 9 shows a response of the photomultiplier for 0.5 micron balls passing a 2.0 micron wide slit (curve 94A) and 0.1 micron wide slit (curve 94B).
  • Curves 94A and 94B represent the voltage of the photomultiplier as a function of time. As expected, the smaller slit produces the narrower curve 94B, which is the minimum response of this setup.
  • Figs. 10A through 10C show the imposition of fluorescent beads and yoyo- 1 stained T4 DNA simultaneously passing through two nanoslits which are spaced lO ⁇ m apart.
  • Fig. 10A shows two intensity peaks of a bead passing through the first slit and then through the second slit.
  • Fig. 10B shows a partly uncoiled strand of DNA passing through the delivery channel. Broader peaks 99A and 99B are due to the geometry of the DNA coil. The passage of the fluorescent bead is superimposed of the DNA signal.
  • Fig. 10C shows a highly extended DNA in transit through three slits, 36A. 36B and 36C. Again, for reference, the signal from a fluorescent bead is superimposed on the DNA signal.
  • Fig. 1 1 is a cross-sectional view of quartz wafer 150 with waveguide 160 taken along a central axis of the waveguide.
  • Waveguide 160 includes and two waveguides 166 A and 166B with a rectangular cross-section fabricated on quartz wafer 150.
  • Rectangular waveguides 166A and 166B may be rectangular dielectric waveguides that use two dielectric materials with different refractive indexes and confine light in a core material with a larger refractive index (n : ) than the refractive index (n,) of the surrounding dielectric material (n, > n,).
  • rectangular waveguides 166A and 166B may be rectangular mirror waveguides that use a dielectric core material surrounded by a metallic material, or waveguides 166A and 166B by be formed by a combination of the two types of waveguides.
  • the rectangular dielectric waveguides ideally achieve the total internal reflection of light propagation, where the incident angle ⁇ ,> ⁇ c .
  • interaction station 40 uses a triangular waveguide with a very small angle at the tip.
  • Rectangular mirror waveguides usually exhibit a higher loss depending on the quality of the metallic mirrors. Rectangular mirror waveguides convey light up to a
  • ⁇ 2 ⁇ h.
  • Nanochannel 171 has a width in the range of 2 nm to 100 nm, and preferably in the range of 5 nm to 50 nm.
  • Gold wires 98 A and 98B (shown in Fig. 1 I B) are spaced about 3 to 25 millimeters from nanochannel 171.
  • the two waveguide arrangement may be replaced by a single waveguide with an opposite electrode forming a wider channel in the range of 100 nm to 1 ⁇ m.
  • Triangular waveguides 166A and 166B shown in Figs. 1 1 and 1 1A are about 10 ⁇ m wide. 5000 ⁇ m long, and over 1 ⁇ m high and are made of Si0 2 .
  • Waveguides 166A and 166B are isolated from substrate 162 by metallic layers 164A and 164B and from a glass cover 152 by metallic layers 174 A and 174B. respectively.
  • metallic layers 164A and 174A for waveguide 166A, or metallic layers 164B and 174B waveguide 166B may be replaced by dielectric layers with a lower refractive index.
  • the introduced plane wave 176 is coupled into triangular waveguide 166 A at an input side 168 A and undergoes internal reflection at waveguide sides 172A and 173 A as it is transmitted toward waveguide tip 170A.
  • Waveguide tip 170 A emits waves of evanescent radiation (illustrated in Fig. 1 IB) into nanochannel 171.
  • the evanescent radiation interacts with individual units of polymer 39 producing radiation with a characteristic signal.
  • the evanescent radiation interacts with a fluorophore located next to a specific unit of polymer 39.
  • Triangular waveguide 166B collects the radiation including the characteristic signal (e.g., fluorescent radiation) from nanochannel 171 and transmits this radiation toward coupling region 168B.
  • the characteristic signal e.g., fluorescent radiation
  • the collected radiation propagates inside waveguide 166B.
  • the radiation may undergo the total internal reflection at the triangular sides 172B and 173B.
  • the output side 168B. providing radiation 188 is optically coupled to optical detector 46 (Fig. 1).
  • the radiation from nanochannel 171 is also emitted in the direction 189, through glass cover 152.
  • Another, external optical detector, located few millimeters to few centimeters above nanochannel 171 detects far-field radiation 189, as shown in Fig. 12.
  • Fig. 1 IB is a cross-sectional view of two triangular waveguides 166 A and 166B surrounded by metal layers on each side, wherein the cross-hatched pattern denotes a metal
  • Waveguide 166A conveys introduced light beam 1 76 to tip 170A by confining substantially the entire wave inside the SiO : volume. At tip 170A.
  • evanescent wave is attenuated over a distance of only one or two wavelengths for the total internal reflection ( ⁇ ,> ⁇ c ).
  • Waves of evanescent radiation 177 interact with the units of polymer 39 passing through nanochannel 171.
  • evanescent waves 177 interact with a fluorophore 178 selectively attached to a selected unit of polymer 39.
  • Fluorophore 178 emits fluorescent radiation 179 propagating in all directions.
  • Fluorescent radiation 179 is collected by waveguide 166B and conveyed to detector 46 (Fig. 1).
  • Fig. 1 1 C is a cross-sectional view of another embodiment using a single triangular waveguide 166 and a metal electrode 185.
  • a channel 171 A formed between waveguide 166 and metal electrode 185 is about 0.5 ⁇ m. which is significantly larger than nanochannel 171.
  • Triangular waveguide 166 is surrounded by metal layers on all sides and is fabricated similarly as waveguides 166A and 166B (Fig.
  • tip 170A emits evanescent waves 177. which are attenuated over a distance of only one or two wavelengths. Therefore, polymer 39 has to be pulled closer to tip 170 than electrode 185 to irradiate fluorophore 1 78 with evanescent waves 177.
  • Polymer 39 is pulled closer to tip 170 using dielectric forces created by applying AC field to electrode 185 and waveguide 166, i.e., metal layers 164 and 174, in addition to the DC field applied across wires 98A and 98B.
  • the AC field applied capacitively with respect to the DC field generates inhomogeneous field in nanochannel 171 A as described above in connection with Fig. 4A.
  • Fig. 12 illustrates an optical system 100 for detecting near field and far field radiation emitted from nanochannel 171.
  • Optical source 44 emits light beam 176, which is focused onto input side 168 A of waveguide 166 A using techniques described in connection with Figs 13 through 13B. After the interaction of evanescent waves 176 with polymer 39, the near field radiation is collected by waveguide 166B and optically coupled to optical detector 46 from
  • RECTIFIED SHEET (RULE 91) ISA/EP output side 168B
  • the far field 100. emitted in direction 189. is collected by a lens 102. filtered by a tunable filter 104 and provided to a PMT detector 106
  • Optical source 42. such as an LED or a laser diode may be incorporated onto quartz wafer 1 0. This arrangement would eliminate the need for an external optical source which as to be aligned with in put side 168A
  • the optical sources are made using a direct bandgap material, for example GA ' for generating UV radiation, or GaP.N for generating radiation of a green wa elength
  • Quartz wafer 150 may also include an integrated optical detector 46 in order to avoid external setup for detection and filtering
  • Various integrated optical elements are described in "Integrated
  • a corrugated waveguide is used as a contradirectional coupler so that light within a narrow frequency band will be reflected back resulting in a filtering action.
  • Another filter is made using two wa eguides with different dispersion relations in close proximity Light from one wa eguide will be coupled into the other for wavelengths for which there is a match in the index of refraction Bv applying a oltage to the waveguides, the dispersion curve is shifted and the spectrum of the resulting filter is altered providing a tunable filter.
  • the optical system is an ultra fast, highly sensitive spectrophotometer capable of detecting fluorescence from a single fluorophore as desc ⁇ bed above.
  • the optical system uses radiation modulated at frequencies in the range of 10MHz to 1GHz as desc ⁇ bed above.
  • Figs. 13 through 13B show different ty pes of coupling of light from an external optical source into a waveguide.
  • lights source 42 emits light beam 176. which is focused onto the input side 168 A of triangular waveguide 166A using a focusing lens 180.
  • a prism 182 is used to couple light beam 176 into triangular waveguide 166A.
  • Light beam 176 is diffracted bv pnsm 182 and undergoes inside the total internal reflection.
  • Prism 182 is located on the surface of SiO, volume 166A and is arranged to optically couple beam 176 across a layer 184 into waveguide 166A. Referring to Fig.
  • a diffraction grating 186 is used to couple light beam 176 into triangular waveguide 166A.
  • Grating 186 is fabricated on waveguide 166A so that it diffracts light beam 176 toward tip 170 A.
  • an optical fiber couples light beam 176 to
  • Waveguides 166 A and 166B are fabricated on quartz or another insulating material to avoid electrical currents in substrate 150.
  • the fabrication process uses UV lithography alone or in combination with deep UV lithography, e-beam lithography or X-ray lithography.
  • the contiguous waveguide is first defined using standard UV lithography, and then nanochannel (or microchannel 171 A described in connection with Fig. 1 I C) is defined in separate e-beam or X-ray lithography steps.
  • the slit (or a hole) is fabricated by creating a concave shape of the photoresist (i.e., an undercut) at the very tips 170A and 170B of waveguides 166A and 166B. and by creating a convex shape of the photoresist at the sides 172A, 173 A, 172B and 173B before evaporating the metal.
  • the convex sides will be covered by the evaporated metal, but not the concave tip.
  • the small tip (the small hole) is fabricated by first creating a very thin wall and then using lift-off or etching to create a metal film with the small slit over the wall.
  • metal hard masks are used to keep the resist thickness down and the resolution high, as is known in the art.
  • Figs. 14A through 14K that are side views along the central line of waveguides 166 A and 166B are fabricated as follows: To improve adhesion of the resist to the wafers, the wafers are primed in hexamethyldisiloxane (HMDS) for 34 minutes (Fig. 14A). Then, a photoresist Shipley 1830 is spun onto the wafers at 4000 rpm 60 sec to achieve a 1.3 micron thick resist and the wafers are baked on a hotplate at 1 15 C for 60 sec to harden the resist (Fig. 14B). The photoresist is exposed in a high resolution mask aligner such as a 5x g-line stepper and baked in a pressurized NH 3 oven.
  • HMDS hexamethyldisiloxane
  • the wafer is flood exposed for 1 min in the HTG/contact aligner with 405 nm light and developed with Microposit 321 for lmin.
  • Fig. 14D a 1000 Angstrom Al layer is deposited and the lift-off is performed using Microposit 1 165 resist remover or acetone at room temperature (Fig. 14E). All resist residues are removed using the resist descum process in the Branson Barrel etcher, 0.6 Torr O, at 150 W RF power.
  • the SiO, waveguide is created as follows: A 1
  • the SiO, layer is planarized by chemical mechanical polishing (CMP), as shown in Fig. 14G.
  • CMP chemical mechanical polishing
  • the top metal mask is defined by spinig photoresist Shipley 1830 onto the wafers at 4000 ⁇ m for 60 sec to achieve a 1.3 micron thick resist and baking it on a hotplate at 1 15°C for 60 sec.
  • the resist is exposed in a high resolution mask aligner, such as a 5x g-line stepper, and baked in a pressurized NH 3 oven. This reverses the positive tone of the photoresist and provides the necessary backward leaning profile (i.e.. the undercut) for the subsequent lift-off process, as shown in Fig. 141.
  • the resist is flood exposed for 1 min in the HTG/contact aligner by 405 nm light and developed in Microposit 321 for lmin.
  • a layer of 1000 A Al metal is deposited. The excess metal is removed by a lift-off using the Microposit 1165 resist remover or acetone at room temperature.
  • Figs. 15A through 15G are side views along the central line and Figs. 16A through 16G are side views along a line pe ⁇ endicular to the central line.
  • the PMMA resist 496K is spun onto the wafers at 2500 ⁇ m to achieve a 200 nm thick resist and bakes on a hotplate at 180°C for 60 min. to harden the resist.
  • the PMMA is exposed by the e-beam system to create the pattern in the nanochannel region.
  • the exposed PMMA resist is developed in IPA:MIBK 3: 1 for lmin and a 1000 A layer of Al metal is deposited as shown in Fig. 15C.
  • the waveguide is etched, but without the microchannel pattern, in the Plasma Therm 72 etcher using reactive ion etching (RIE) in CHF 3 (50 seem) + O- (2 seem) at 200 W RF power and 40 mTorr, > 1 micron to create a wall shown in Fig. 15B.
  • the bottom metal is wet etched in the solution of 16 :H-P0 4 : 1 : HN0 3 ; 1 :acetic acid; 2 : water; wetting agent, or dry etched in Cl.
  • the remaining resist is removed in a Branson Barrel O, plasma etcher at 1000 W RF power for 15 min.
  • the aluminum is removed in a wet etch using 16 : H,P0 4 ; 1 : HNO-; 1 : acetic acid; 2 : water; wetting agent.
  • a photoresist Shipley 1830 is spun onto the wafers at 4000 ⁇ m for 60 sec to achieve a 1.3 micron thick resist and baked on a hotplate at 1 15°C for 60 sec. to harden the resist.
  • the resist is exposed in a high resolution mask aligner, such as a 5x g-line stepper, and baked in a pressurized NH, oven. This reverses the positive tone of the photoresist and provides the necessary backward leaning profile (i.e., the undercut) for the subsequent lift-off process.
  • the resist is flood exposed for 1 min in the
  • RECTIFIED SHEET (RULE 91) ISA/EP HTG/contact aligner 405 nm light and developed in Microposit 321 for lmin.
  • a 1000 A Al layer is deposited as shown in Figs. 15F and 16F. The excess metal is lifted-off using the Microposit 1165 resist remover or acetone at room temperature.
  • a layer of Cr metal is deposited on the top of the device as follows. First, a mask for the nanochannel was etched and then the Shipley 1830 resist was spun onto the wafers at 4000 m for 60 sec to achieve a 1.3 micron thick resist and baked on a hotplate at 115 ° C for 60 sec to harden the resist. The resist was exposed in a high resolution mask aligner, such as a 5x g-line stepper, and baked in a pressurized NH 3 oven. This process reverses the positive tone of the photoresist and provides the necessary backward leaning profile (i.e., the undercut) for the subsequent lift-off process.
  • a high resolution mask aligner such as a 5x g-line stepper
  • the resist was flood exposed for 1 min in the HTG/contact aligner using 405 nm light and developed in Microposit 321 for lmin. Then, a 1000 A Cr layer was deposited and a lift-off of excess metal was performed in the Microposit 1165 resist remover or acetone at room temperature.
  • a PMMA 496K resist was spun onto the wafers at 2500 m to achieve a 200 nm thick resist and baked on a hotplate at 180° C for 60 min. to harden the resist.
  • the resist was exposed in the e-beam system to define the desired pattern, and the wafer was developed in IPA:MIBK 3:1 for lmin. Then, a 1000 A Cr layer was deposited and the lift-off of excess metal was performed in the Microposit 1165 resist remover or acetone at room temperature.
  • Nanochannel 171 was crated by etching the first metal layer (i.e., the Al layer) in a Cl based dry etch, wherein Cr acts as an etch mask. Then, the SiO 2 was etched in Plasma Therm 72 using reactive ion etching (RIE) in CHF3 (50 seem) + O 2 (2 seem) at 200 W RF power and 40 mTorr, > 1 micron to create a wall. The bottom metal layer was etched in a Cl based dry etch and the remaining Cr was removed using a wet etch.
  • nanochannel 171 can be fabricated by focussed ion beam milling to define the gap and the aperture in the tip.
  • the individual molecules can be selectively labeled as described in the PCT application PCT/US98/03024 filed on Feb. 11, 1998, which is incorporated by reference.
  • the sequencing is done using a combination of single-stranded DNA molecules (ssDNA), which have been hybridized with fluorescently tagged oligonucleotides of test sequences.
  • ssDNA single-stranded DNA molecules
  • the tagged sequence is now at a fixed position on the DNA molecule.
  • the process can use three tags: "start” and “stop” tags, which signal the 3' and 5' beginning and end of the ssDNA, and the tagged oligo which is used for sequencing.
  • the system By observing a large population of these tagged molecules using a spectrum of oligonucleotide sequences as they pass through the microchannel and recording the position of the oligonucleotide labels, the system obtains the sequence of the molecule at an unprecedented level of speed, accuracy and low molecule concentration.
  • An optical apparatus 200 utilizes confocal fluorescence illumination and detection. Confocal illumination allows a small optical volume (on the order of picoliters) to be illuminated. Both Raleigh and Raman scattering are minimized using a small probe olume.
  • Optical apparatus 200 includes a light source 202. a filter 204, a dichroic mirror 206. an objective 208, a narrow ⁇ band pass filter 210, a pinhole 212. a lens 214, and a detector 216.
  • Light source 202 which is a 1 mW argon ion laser, emits a laser beam 201, which passes through filter 204.
  • Filter 204 is a laser line filter that provides a focused beam of a wavelength of about 514 nm.
  • the filtered beam 205 is reflected by dichroic mirror 206 and is focussed by objective 208 onto a region of a DNA sample or another polymer.
  • Objective 208 is a lOOx 1.2 NA oil immersion objective.
  • the DNA sample is a straightened DNA molecule with one or several units tagged by a fluorescent tag.
  • the fluorescent tag on the DNA can be one of several dyes including Cy-3, tetramethylrhodamine. rhodamine 6G, and Alexa 546.
  • intercalator dyes can be used such as TOTO-3 (Molecular Probes).
  • the excited tag provides a fluorescence emission that is passed through dichroic mirror 206, narrow bandpass filter 210 (e.g., manufactured by Omega Optical) and is focused onto a 100 ⁇ m pinhole 212.
  • the fluorescent light 213 is focussed by aspheric lens 214 onto detector 216, which is an avalanche photodiode (e.g., manufactured by EG&G Canada) operating in the photon counting mode.
  • the output signal from the photodiode is collected by a multichannel scalar (EG&G) and analyzed using a general pu ⁇ os computer.
  • EG&G multichannel scalar
  • the confocal apparatus is appropriate for quantitative applications involving time-of- flight. Such applications include measuring distances on the DNA, detecting tagged sequences, and determining degrees of stretching in the DNA. Single fluorescent molecules can be detected using the apparatus.
  • an imaging apparatus uses an intensified CCD (ICCD, Princeton Instruments) mounted on a microscope.
  • Fig. 18 shows a presently preferred embodiment of alignment station 220 for aligning and stretching polymers before they reach an interaction station 231 , where they interact with optical radiation.
  • Alignment station 220 is fabricated on a quartz wafer, which may be covered with a metal layer 222 (e.g., aluminum, gold, silver).
  • Alignment station 220 includes a triangular microchannel 224, micropost region 228, and an entrance region 230, all fabricated on the surface.
  • ISA/EP Entrance region 230 is about 50 micron wide and is in communication with micropost region 228.
  • Micropost region 228 includes several alignment posts 226. Alignment posts 226 have a circular cross-section and are about 1 micron in diameter. Alignment microposts 226 are spaced about 1.5 microns apart in 12 to 15 rows. Micropost region 228 is canted at about 26.6 degrees.
  • Microposts 226 are located about 100 ⁇ m to 5,000 ⁇ m (and preferably about 1,000 ⁇ m to 3,000 ⁇ m) from the interaction station, where the units of the polymer (e.g. DNA) interact with optical radiation.
  • MicroChannel 224 is a region of constant x-direction shear that maintains the polymer in extended conformation after release from microposts 226. The electric field pulls the examined polymer through microchannel 224.
  • a very effective technique of stretching a polymer (e.g., DNA) uniformly is to have an obstacle field inside the tapered microchannel 224, followed by a constant-shear section to maintain the stretching obtained and straighten out any remaining coiling in the polymer.
  • the preferred embodiment is a structure that combines microposts with two regions of different funnel designs as shown in Fig. 18. Pressure flow is the preferred driving force because of the predictable behavior of fluid bulk flow.
  • a constant shear rate, or change in average velocity with distance in the channel is defined as S:
  • x is the distance down a substantially rectangular channel
  • u is the average fluid velocity, which is computed from the overall fluid flow (Q) and the cross-sectional area (A) of the channel as follows:

Abstract

L'invention concerne un système servant à caractériser optiquement des polymères. De préférence, le système est utilisé pour mettre en oeuvre une analyse linéaire de polymères.
PCT/US1999/018438 1998-08-13 1999-08-13 Polymeres caracterises optiquement WO2000009757A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2000565191A JP2002522780A (ja) 1998-08-13 1999-08-13 ポリマーの光学的な特性決定
CA002340228A CA2340228A1 (fr) 1998-08-13 1999-08-13 Polymeres caracterises optiquement
EP99942167A EP1105535A1 (fr) 1998-08-13 1999-08-13 Polymeres caracterises optiquement
IL14131099A IL141310A0 (en) 1998-08-13 1999-08-13 Optically characterizing polymers
AU55606/99A AU5560699A (en) 1998-08-13 1999-08-13 Optically characterizing polymers

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US9654498P 1998-08-13 1998-08-13
US12041499P 1999-02-14 1999-02-14
US60/096,544 1999-02-14
US60/120,414 1999-02-14

Publications (2)

Publication Number Publication Date
WO2000009757A1 WO2000009757A1 (fr) 2000-02-24
WO2000009757A9 true WO2000009757A9 (fr) 2000-10-12

Family

ID=26791804

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/018438 WO2000009757A1 (fr) 1998-08-13 1999-08-13 Polymeres caracterises optiquement

Country Status (7)

Country Link
EP (1) EP1105535A1 (fr)
JP (1) JP2002522780A (fr)
CN (1) CN1323355A (fr)
AU (1) AU5560699A (fr)
CA (1) CA2340228A1 (fr)
IL (1) IL141310A0 (fr)
WO (1) WO2000009757A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007010428A3 (fr) * 2005-07-18 2007-04-05 Koninkl Philips Electronics Nv Capteur de luminescence faisant appel a une structure de substrat multicouche
US8859201B2 (en) 2010-11-16 2014-10-14 Nabsys, Inc. Methods for sequencing a biomolecule by detecting relative positions of hybridized probes
US8882980B2 (en) 2008-09-03 2014-11-11 Nabsys, Inc. Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US9588051B2 (en) 2004-09-17 2017-03-07 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis

Families Citing this family (66)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120160687A1 (en) 1995-03-17 2012-06-28 President And Fellows Of Harvard College Characterization of individual polymer molecules based on monomer-interface interactions
US6362002B1 (en) 1995-03-17 2002-03-26 President And Fellows Of Harvard College Characterization of individual polymer molecules based on monomer-interface interactions
US8142708B2 (en) 1995-04-03 2012-03-27 Wisconsin Alumni Research Foundation Micro fluidic system for single molecule imaging
US7775368B2 (en) 1995-04-03 2010-08-17 Wisconsin Alumni Research Foundation Micro-channel long molecule manipulation system
DE69825601T2 (de) 1997-02-12 2005-04-28 Chan, Eugene Y, Brookline Verfahren zur analyse von polymeren
US6263286B1 (en) 1998-08-13 2001-07-17 U.S. Genomics, Inc. Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer
US7056661B2 (en) 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US6627067B1 (en) 1999-06-22 2003-09-30 President And Fellows Of Harvard College Molecular and atomic scale evaluation of biopolymers
US6762059B2 (en) 1999-08-13 2004-07-13 U.S. Genomics, Inc. Methods and apparatuses for characterization of single polymers
US6927065B2 (en) 1999-08-13 2005-08-09 U.S. Genomics, Inc. Methods and apparatus for characterization of single polymers
CA2381361A1 (fr) * 1999-08-13 2001-02-22 U.S. Genomics, Inc. Procede et appareil d'etirage de polymeres
US6696022B1 (en) 1999-08-13 2004-02-24 U.S. Genomics, Inc. Methods and apparatuses for stretching polymers
US6528258B1 (en) 1999-09-03 2003-03-04 Lifebeam Technologies, Inc. Nucleic acid sequencing using an optically labeled pore
US6908736B1 (en) 1999-10-06 2005-06-21 Medical Biosystems, Ltd. DNA sequencing method
GB9923644D0 (en) 1999-10-06 1999-12-08 Medical Biosystems Ltd DNA sequencing
EP1285290A1 (fr) 2000-04-28 2003-02-26 Edgelight Biosciences, Inc. Dispositif a microreseau destine a detecter la fluorescence d'ondes evanescentes
JP2002048765A (ja) * 2000-08-03 2002-02-15 Masabumi Youda 水平面型ゲル電気泳動装置用試料導入装置、水平面型ゲル電気泳動システム、その試料導入方法およびその試料採取方法
EP2465943A3 (fr) 2001-03-16 2012-10-03 Kalim Mir Affichage de polymère linéaire
SG187992A1 (en) 2001-07-25 2013-03-28 Univ Princeton Nanochannel arrays and their preparation and use for high throughput macromolecular analysis
US9678038B2 (en) 2001-07-25 2017-06-13 The Trustees Of Princeton University Nanochannel arrays and their preparation and use for high throughput macromolecular analysis
US8423294B2 (en) 2001-09-18 2013-04-16 Pathogenetix, Inc. High resolution linear analysis of polymers
JP2005289809A (ja) 2001-10-24 2005-10-20 Vlaams Interuniversitair Inst Voor Biotechnologie Vzw (Vib Vzw) 突然変異重鎖抗体
EP1572860B1 (fr) 2002-04-16 2018-12-05 Princeton University Structures de gradient interfa ant des elements microfluidiques et des elements nanofluidiques, leurs procedes de fabrication et d'utilisation
EP1546380A4 (fr) 2002-05-28 2007-02-14 Us Genomics Inc Procedes et appareils d'analyse de polymeres simples
US7282330B2 (en) 2002-05-28 2007-10-16 U.S. Genomics, Inc. Methods and apparati using single polymer analysis
WO2004007692A2 (fr) * 2002-07-17 2004-01-22 U.S.Genomics, Inc. Procedes et compositions d'analyse de polymeres au moyen de marqueurs chimeres
WO2004035212A1 (fr) * 2002-10-18 2004-04-29 Wisconsin Alumni Research Foundation Systeme de manipulation de molecules a chaine longue a micro-canaux
AU2002363822A1 (en) * 2002-11-20 2004-06-15 Richard Fritz Sauter Method for analyzing molecules for molecule sequencing and spectrometer therefor
US7141801B2 (en) * 2002-12-26 2006-11-28 Applied Precision, Llc System and method of illuminating living cells for imaging
US20040214211A1 (en) * 2003-01-23 2004-10-28 U.S. Genomics, Inc. Methods for analyzing polymer populations
JP2006522940A (ja) * 2003-04-10 2006-10-05 ユー．エス． ジェノミクス， インコーポレイテッド マイクロチャネルにおけるポリマーの操作
US7846738B2 (en) 2003-08-15 2010-12-07 President And Fellows Of Harvard College Study of polymer molecules and conformations with a nanopore
WO2005085849A2 (fr) * 2003-10-24 2005-09-15 U S. Genomics, Inc Identification de risque biologique par dosage immunologique par fluorescence et par detection de molecule simple
US7977048B2 (en) 2004-01-13 2011-07-12 Pathogenetix, Inc. Detection and quantification of analytes in solution using polymers
US7595160B2 (en) 2004-01-13 2009-09-29 U.S. Genomics, Inc. Analyte detection using barcoded polymers
US7238485B2 (en) 2004-03-23 2007-07-03 President And Fellows Of Harvard College Methods and apparatus for characterizing polynucleotides
EP1786932A4 (fr) 2004-08-23 2010-10-27 Us Genomics Inc Systemes et procedes de detection et d'analyse de polymeres
CA2579150C (fr) 2004-09-17 2014-11-25 Pacific Biosciences Of California, Inc. Appareil et procede d'analyse de molecules
WO2006098772A2 (fr) 2004-10-13 2006-09-21 U.S. Genomics, Inc. Systemes et procedes d'optimisation de mesures
WO2006044728A2 (fr) 2004-10-18 2006-04-27 U.S.Genomics, Inc. Procedes d'isolation d'acides nucleiques de spores procaryotiques
JP2006313151A (ja) * 2005-04-07 2006-11-16 Sysmex Corp 血液分析装置、試料分析装置及びフローサイトメータ
PL1888641T3 (pl) 2005-05-18 2012-05-31 Ablynx Nv Białka wiążące albuminę surowicy
DE102005023617A1 (de) 2005-05-21 2006-11-23 Aspre Ag Verfahren zum Mischen von Farben in einem Display
US9410889B2 (en) * 2005-06-10 2016-08-09 Applied Biosystem, Llc Method and system for multiplex genetic analysis
SE529254C2 (sv) * 2005-06-17 2007-06-12 Aamic Ab Optiskt testsystem
US20100019155A1 (en) * 2005-06-23 2010-01-28 Koninklijke Philips Electronics, N.V. Luminescence sensors using sub-wavelength apertures or slits
US8075841B2 (en) * 2005-12-20 2011-12-13 Koninklijke Philips Electronics N.V. Biosensor with one-dimensional sub-diffraction-limited apertures composed of a grid and a polarizer
CN101346621B (zh) * 2005-12-22 2010-12-29 皇家飞利浦电子股份有限公司 在反射模式下工作的发光传感器
WO2008078263A1 (fr) 2006-12-21 2008-07-03 Koninklijke Philips Electronics N.V. Biocapteur d'ouverture à tranchées
EP1936340A1 (fr) * 2006-12-21 2008-06-25 Koninklijke Philips Electronics N.V. Ouverture inferieure aux longueurs d`ondes
AU2008232616B2 (en) 2007-03-28 2014-08-07 Bionano Genomics, Inc. Methods of macromolecular analysis using nanochannel arrays
US8278047B2 (en) 2007-10-01 2012-10-02 Nabsys, Inc. Biopolymer sequencing by hybridization of probes to form ternary complexes and variable range alignment
CN104862383B (zh) 2008-03-28 2019-05-28 加利福尼亚太平洋生物科学股份有限公司 用于核酸测序的组合物和方法
US8262879B2 (en) 2008-09-03 2012-09-11 Nabsys, Inc. Devices and methods for determining the length of biopolymers and distances between probes bound thereto
US9650668B2 (en) 2008-09-03 2017-05-16 Nabsys 2.0 Llc Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels
EP2580588B1 (fr) 2010-06-08 2014-09-24 President and Fellows of Harvard College Dispositif à nanopores avec membrane lipidique artificielle sur support de graphène
US8715933B2 (en) 2010-09-27 2014-05-06 Nabsys, Inc. Assay methods using nicking endonucleases
WO2012109574A2 (fr) 2011-02-11 2012-08-16 Nabsys, Inc. Procédés de dosage à l'aide de protéines de liaison à l'adn
EP2574923A1 (fr) 2011-09-28 2013-04-03 Koninklijke Philips Electronics N.V. Appareil pour le traitement de molécules uniques
WO2013158860A1 (fr) * 2012-04-18 2013-10-24 Pathogenetix, Inc. Dispositifs et méthodes de préparation et d'analyse d'acides nucléiques
US8685708B2 (en) 2012-04-18 2014-04-01 Pathogenetix, Inc. Device for preparing a sample
US9028776B2 (en) 2012-04-18 2015-05-12 Toxic Report Llc Device for stretching a polymer in a fluid sample
CN102923635B (zh) * 2012-10-26 2015-06-03 中国科学院苏州纳米技术与纳米仿生研究所 纳米流体二极管及其制造方法
US9914966B1 (en) 2012-12-20 2018-03-13 Nabsys 2.0 Llc Apparatus and methods for analysis of biomolecules using high frequency alternating current excitation
US10294516B2 (en) 2013-01-18 2019-05-21 Nabsys 2.0 Llc Enhanced probe binding
LU100392B1 (en) 2017-07-27 2019-02-12 Wisconsin Alumni Res Foundation Warf A method to determine the throughput speed of a pore

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6150089A (en) * 1988-09-15 2000-11-21 New York University Method and characterizing polymer molecules or the like
US5624845A (en) * 1995-03-16 1997-04-29 International Business Machines Corporation Assembly and a method suitable for identifying a code
US5538898A (en) * 1995-03-16 1996-07-23 International Business Machines Corporation Method suitable for identifying a code sequence of a biomolecule
WO1996030508A1 (fr) * 1995-03-24 1996-10-03 Ely Michael Rabani Assemblage d'objets moleculaires et supramoleculaires complexes, dispositifs ainsi obtenus et leurs utilisations
EP0871640A4 (fr) * 1995-04-03 2002-04-17 Univ New York Procede permettant de mesurer des caracteristiques physiques d'acides nucleiques par imagerie microscopique
US5699157A (en) * 1996-07-16 1997-12-16 Caliper Technologies Corp. Fourier detection of species migrating in a microchannel
DE69825601T2 (de) * 1997-02-12 2005-04-28 Chan, Eugene Y, Brookline Verfahren zur analyse von polymeren

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US9588051B2 (en) 2004-09-17 2017-03-07 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
WO2007010428A3 (fr) * 2005-07-18 2007-04-05 Koninkl Philips Electronics Nv Capteur de luminescence faisant appel a une structure de substrat multicouche
US8882980B2 (en) 2008-09-03 2014-11-11 Nabsys, Inc. Use of longitudinally displaced nanoscale electrodes for voltage sensing of biomolecules and other analytes in fluidic channels
US8859201B2 (en) 2010-11-16 2014-10-14 Nabsys, Inc. Methods for sequencing a biomolecule by detecting relative positions of hybridized probes

Also Published As

Publication number Publication date
CA2340228A1 (fr) 2000-02-24
WO2000009757A1 (fr) 2000-02-24
EP1105535A1 (fr) 2001-06-13
CN1323355A (zh) 2001-11-21
AU5560699A (en) 2000-03-06
IL141310A0 (en) 2002-03-10
JP2002522780A (ja) 2002-07-23

Similar Documents

Publication Publication Date Title
US6790671B1 (en) Optically characterizing polymers
WO2000009757A9 (fr) Polymeres caracterises optiquement
US7013054B2 (en) Waveguides for performing spectroscopy with confined effective observation volumes
US8865459B2 (en) Nucleic acid analysis device and nucleic acid analyzer using the same
US6376177B1 (en) Apparatus and method for the analysis of nucleic acids hybridization on high density NA chips
US6210896B1 (en) Molecular motors
US7444053B2 (en) Integrated electrical and optical sensor for biomolecule analysis with single molecule sensitivity
US7248771B2 (en) Integrated sensor with electrical and optical single molecule sensitivity
JP3935509B2 (ja) ポリマー分析のための方法および製品
US5538898A (en) Method suitable for identifying a code sequence of a biomolecule
JP2005504275A (ja) 高分解能線形解析用のポリマーの差示的タグ付け
US20050186576A1 (en) Polymer sequencing using selectively labeled monomers and data integration
Hubarevich et al. λ-DNA through porous materials—surface-enhanced raman scattering in a simple plasmonic nanopore
US5624845A (en) Assembly and a method suitable for identifying a code
KR20050010946A (ko) 신호 확장 및 데이터 집적화에 의한 핵산의 서열화
JPH11507514A (ja) 薄膜の化学的、生化学的ならびに生物学的加工方法
JP2005503269A (ja) 分子アレイの製造
KR100590546B1 (ko) 마이크로 ｐｃｒ 장치, 이를 이용한 핵산의 증폭방법 및ｐｃｒ 증폭산물의 농도를 측정하는 방법
US5607568A (en) Assembly suitable for identifying a code sequence of a biomolecule in a free-solution embodiment
US5609744A (en) Assembly suitable for identifying a code sequence of a biomolecule in a gel embodiment
EP0732584A2 (fr) Procédé et assemblage pour identifier une séquence de codes d'une biomolécule
Kitamori et al. Subsingle-molecule determination of nonfluorescent species by scanning thermal lens microscope and its application to single-cell measurement

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 99811963.6

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WD Withdrawal of designations after international publication

Free format text: US

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: C2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 13-24 AND 26, DESCRIPTION, REPLACED BY NEW PAGES 13-24 AND 26; PAGES 5/20, 6/20, 9/20, 11/20-15/20 AND 20/20, DRAWINGS, REPLACED BY NEW PAGES 5/20, 6/20, 9/20, 11/20-15/20 AND 20/20; PAGES 2/20, 3/20 AND 4/20 RENUMBERED AS PAGES 3/20, 4/20 AND 2/20; AFTER RECTIFICATION OF OBVIOUS ERRORS AS AUTHORIZED BY THE INTERNATIONAL SEARCHING AUTHORITY

WWE Wipo information: entry into national phase

Ref document number: 55606/99

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1999942167

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 141310

Country of ref document: IL

ENP Entry into the national phase

Ref document number: 2340228

Country of ref document: CA

Ref document number: 2340228

Country of ref document: CA

Kind code of ref document: A

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1999942167

Country of ref document: EP