Abstract:
The invention relates to new systems, methods and products for analyzing polymers and in particular new systems, methods and products useful for obtaining sequence information from polymers. The invention has numerous advantages over prior art systems and methods used to obtain sequence-related information. Using the methods of the invention the entire human genome could be analyzed several orders of magnitude faster than could be accomplished using conventional technology. In addition to obtaining sequencing information for the entire genome, 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 analyze an individual&#39;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.

Description:
This patent application claims priority from U.S. Provisional Application No. 60/096,544 filed on Aug. 13, 1998, and U.S. Provisional Application No. 60/120,414 filed on Feb. 14, 1999, both of which are incorporated by reference. 
    
    
     FIELD OF THE INVENTION 
     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. 
     BACKGROUND 
     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 identifying their sequence of monomers is integral to the understanding of each active component and the role that component plays within a cell. By determining the sequences of polymers it is possible to generate expression maps, to determine what proteins are expressed, to understand where mutations occur in a disease state, and to determine whether a polysaccharide has better function or loses function when a particular monomer is absent or mutated. 
     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&#39; 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. 
     Currently, only 1% of the human genome and an even smaller amount of other genomes have been sequenced. In addition, only one very incomplete human body expression map using expressed sequence tags has been achieved (Adams et al., 1995). Current protocols for genomic sequencing are slow and involve laborious steps such as cloning, generation of genomic libraries, colony picking, and sequencing. The time to create even one partial genomic library is on the order of several months. Even after the establishment of libraries, there are time lags in the preparation of DNA for sequencing and the running of actual sequencing steps. Given the multiplicative effect of these unfavorable facts, it is evident that the sequencing of even one genome requires an enormous investment of money, time, and effort. 
     In general, 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. 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. Recently, there has also been increasing interest in developing Sanger independent methods as well. 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. 
     Currently, automated gel electrophoresis is the most widely used method of large-scale sequencing. Automation requires reading of fluorescently labeled Sanger fragments in real time with a charge coupled device (CCD) detector. The four different dideoxy chain termination reactions are run with different labeled primers. The reaction mixtures are combined and co-electrophoresed down a slab of polyacrylamide. Using laser excitation at the end of the gel, the separated DNA fragments are resolved and the sequence determined by computer. Many automated machines are available commercially, each employing different detection methods and labeling schemes. The most efficient of these is the Applied Biosystems Model 377XL, which generates a maximum actual rate of 115,200 bases per day. 
     In the method of capillary gel-electrophoresis, reaction samples are analyzed by small diameter, gel-filled capillaries. The small diameter of the capillaries (50 μm) allows for efficient dissipation of heat generated during electrophoresis. Thus, high field strengths can be used without excessive Joule heating (400 V/m), lowering the separation time to about 20 minutes per reaction run. Not only are the bases separated more rapidly, there is also increased resolution over conventional gel electrophoresis. Furthermore, many capillaries are analyzed in parallel (Wooley and Mathies, “Ultra-high-speed DNA sequencing using capillary electrophoresis chips,”  Anal. Chem.  67:3676-3680, 1995), allowing amplification of base information generated (actual rate is equal to 200,000 bases/day). The main drawback is that there is not continuous loading of the capillaries since a new gel-filled capillary tube must be prepared for each reaction. Capillary gel electrophoresis machines have recently been commercialized. 
     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. 
     Sequencing by mass spectrometry was first introduced in the late 80&#39;s. Recent developments in the field have allowed for better sequence determination (Crain,  MassSpectrom. Rev.  9:505-54, 1990; Little et al.,  J. Am. Chem. Soc.  116:4893-4897, 1994; Keough et al.,  Rapid Commun. Mass Spectrom.  7:195-200,1993; Smirnov et al., 1996). 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. In one example, 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. Even the best method, 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. 
     The Scanning tunneling microscope (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. Even with successful immobilization of the DNA, it is difficult to distinguish base information because of the extremely high resolutions needed. With current technology, 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. To determine base specificity, 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 nucleotide detection corresponds to the sequence of the DNA (Ambrose et al., “Application of single molecule detection to DNA sequencing and sizing,”  Ber. Bunsenges. Phys. Chem.  97:1535-1542, 1993; Davis et al., “Rapid DNA sequencing based on single-molecule detection,”  Los Alamos Science.  20:280-6, 1992; Jett et al., “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,”  J. Of Bio. Structure  &amp;  Dynamics.  7:301-9, 1989). Using a processive exonuclease, it theoretically is possible to sequence 10,000 bp or larger fragments at a rate of 10 bases per second. 
     In the sequencing by hybridization method, 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. With the advent of the “DNA chip,” which applies microchip synthesis techniques to DNA probes, arrays of thousands of different DNA, probes can be generated on a 1 cm 2  area, making Probe Down methods more practical. Probe Up methods would require, for an 8-mer, 65,536 successive probes and washings, which would take an enormous amount of time. On the other hand, Probe Down hybridizations generates data in a few seconds. With perfect hybridization, 65,536 October probes would determine a maximum of 170 bases. With 65,536 “mixed” 11-mers, 700 bases can be generated. 
     The most common limitation of most of these techniques is a short read length. In practice 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. Theoretically, with a 500 base read length, a minimum of 9×10 9  bases need to be sequenced before the linear sequence of all 3×10 9  bases of the human genome are properly ordered. In reality, the number of bases needed to generate a believable genome is approximately 2×10 10  bases. Comparisons of the different techniques show that only the impractical exonuclease sequencing has the theoretical capability of long read lengths. The other methods have short theoretical read lengths and even shorter realistic read lengths. To reduce the number of bases that need to be sequenced, it is clear that the read length must be improved. 
     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 flee 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. This adduct rearranges and the terminal amino acid residue is then cleaved using strong acid. The released phenylthiohydantoin (PTH) of the amino acid is identified and the shortened polymer can undergo repeated cycles of degradation and analysis. 
     Further, several new methods have been described for carboxy terminal sequencing of polypeptides. See Inglis, A. S., Anal. Biochem. 195:183-96 (1991). Carboxy terminal sequencing methods mimic Edman degradation but involve sequential degradation from the opposite end of the polymer. See Inglis, A. S., Anal. Biochem. 195:183-96 (1991). Like Edman degradation, the carboxy-terminal sequencing methods involve chemically induced sequential removal and identification of the terminal amino acid residue. 
     More recently, 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. 
     Each of the known methods for sequencing polymers has drawbacks. For instance most of the methods are slow and labor intensive. The gel based DNA sequencing methods require approximately 1 to 3 days to identify the sequence of 300-800 units of a polymer. Methods such as mass spectroscopy and ELIDA sequencing can only be performed on very short polymers. 
     A need exists for de noveau polymer sequence determination. 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. A need also exists for improved systems and methods for analyzing polymers in order to speed up the rate at which diagnosis of diseases and preparation of new medicines is carried out. 
     SUMMARY OF THE INVENTION 
     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. In addition to sequencing the entire genome, 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&#39;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. 
     According to one aspect, 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 andy 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 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. 
     In another embodiment 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 interactions of the localized radiation spot with the labels or the units; and 
     analyzing the polymer based on the detected radiation. 
     In another embodiment, 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. 
     According to another aspect, 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. 
     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 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. 
     In yet another aspect 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. 
     In one embodiment 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. In another embodiment the radiation sensitive label includes a fluorophore. In some embodiments 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. The actual time taken to read a given number of units of a polymer is a million-fold more rapid than current methods because of the tremendous parallel amplification supplied by a novel apparatus also claimed herein, which is referred to as a nanochannel plate or a microchannel plate. The combination of all these factors translates into a method of polymer analysis including sequencing that will provide enormous advances in the field of molecular and cell biology. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     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 5A 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,  10 A,  10 B, and  10 C 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. 11A is a perspective view of the optical waveguides shown in FIG.  11 . 
     FIGS. 11B 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,  13 A and  13 B 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.  11 . 
     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. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Referring to FIG. 1, 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 . Polymer alignment station  30 , controlled by system controller  10 , straightens and aligns individual polymers using force field and mechanical obstacles, and dispenses the polymers to first interaction station  40 . 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 in the polymer pass through interaction station  40 , optical source  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. 
     Furthermore, 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, X-ray radiation, or visible or infrared radiation for characterizing the polymer passing from first interaction station  40  through second interaction station  50 . A controller  56  controls the operation of second interaction station  50 . Both controllers  48  and  56  are connected to system controller  10 . 
     Referring to FIGS. 2 and 3, 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 Delrin®, to facilitate a flow of conducting fluid  96  (for example, agarose gel) and the examined polymer. Substrate  92  includes trenches  94 A and  94 B machined to receive gold wires  98 A and  98 B, 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 . 
     Alternatively, trenches  94 A and  94 B and wires  98 A and  98 B may be replaced by metallic regions located directly on quartz wafer  60 , or may be replaced by external electrodes for creating the electric field. In general, 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 interaction station  40 . 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 . Of course, a single quartz wafer  60  may include hundreds or thousands of the alignment and first interaction stations. Quartz wafer  60  included a quartz substrate covered with a metal layer  62  (e.g., aluminum, gold, silver) and having a microchannel  41  fabricated on the surface. Fabricated through metal layer  62  are slits  36 A,  36 B and  36 C, which form the optical elements that provide the localized radiation spot. Slits  36 A,  36 B and  36 C have a selected width in the range between 1 nm and 5000 nm, and preferably in the range between 10 nm and 1000 nm, and more preferably in the range between 10 nm and 100 nm. Slits  36 A,  36 B and  36 C 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. The electric field, created by gold wires  98 A and  98 B, pulls a polymer chain  39  (such as a DNA molecule) through microchannel  41  past slits  36 A,  36 B and  36 C. 
     As shown in FIG. 4, polymer alignment station  30  includes several alignment posts  32  located in regions  31 . 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 μm to 500 μm (and preferably about 10 μm to 200 μm) 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 μm from nanoslit  36 A. In general, the distance from nanoslit  36 A 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 than 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  46 . When 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. When 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 . By selectively polarizing the incident beam  65 , 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 the light emitted from laser source  80 . The excitation beam  65  interacts with nanoslit  36  (shown in FIG.  4 A and designated here as region  40 ) to create the non-radiating near-field  67 . The electric field between gold wires  98 A and  98 B (FIGS. 2 and 3) pulls polymer chain  39  at a known rate causing interaction of each labeled unit with radiation  67 . As fluorophore  68  moves pass slits  36 A,  36 B and  36 C (shown FIG.  4 ), emitted radiation  67  excites fluorophore  68  that re-emits fluorescent radiation  72 . Notch filter  86  passes the fluorescent wavelength  72  of radiation  70  and attenuates the excitation wavelength to increase the signal to noise resolution, as is known in the art. CCD detector  88  located few millimeters to few centimeters above quartz wafer  60  detects fluorescent radiation  72 . CCD detector  88  can detect separately for each nanoslits  36 A,  36 B and  36 C fluorescent radiation  72  as the fluorophore moves across. This process occurs at a large number of nanoslits located on quartz wafer  60 . 
     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. To position fluorophore  68  within the range of the non-radiating field  67 , 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. Ashbury and Ger van den Engh, Biophysical Journal Vol 74, pp 1024-1030 (1998), “Molecular Dielectrophoresis of Biopolymers,” by M. Washizu, S. Suzuki, O. Kurosawa, T. Nishizaka, and T. Shinohara, in IEEE Transactions on Industry Applications, Vol 30, No 4, pp. 835-843 (1994), and “Electrostatic Manipulation of DNA in Microfabricated Structures,” by M. Washizu, and O. Kurosawa, in IEEE Transactions on Industry Applications, Vol 26, No 6, pp. 1165-1172 (1990). In general, see “Dielectrophoresis: The Behavior of Neutral Matter in Nonuniform Electric Fields,” by Pohl, H. A., Cambridge University Press, Cambridge, UK, 1978. The inhomogeneous field will attract polarized units of polymer  39  (e.g., DNA molecule) to metal layer  62 . 
     Referring to FIG. 5 second interaction station  50  measures ionic current across a nanochannel 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 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  56 A, located in controller  56 , to measure the ionic current across nanochannel  51 . Alternatively, referring to FIG. 5A, the microampere meter is replaced by a bridge  56 B, which compares the impedance of channel  51  without polymer  39  (Z l ) with the instantaneous impedance of (Z x ). 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. 
     In another embodiment, 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., Tenneled TC454) that provides the start pulse to a time-to-amplitude converter (e.g., Tunnelec 863). The primary beam  65  is directed through a neutral density filter that adjusts the power level. As described above, 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 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, Tunnelec C4534 discriminator). The signal having appropriate time delays are provided to the time-to-amplitude converter (TAC). 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 . 
     Different fluorophores have different fluorescent lifetimes (i.e., the average amount of time that the molecule remains excited before returning to the ground electronic state through the emission of a fluorescent photon) that usually have an exponential probability distribution. Fluorescent lifetime is useful for identification of the fluorophore. In rapid sequencing, 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. 
     In another embodiment, 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. For example, 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. (See, for example, Lackowicz, J. R., “Gigahertz Frequency-Domain Fluorometry: Resolution of Complex Intensity Decays, Picosecond Processes and Future Developments,” Photon Migration in Tissues, Academic Press, NY, pp.169-186, 1989; see also other references cited therein) 
     FIGS. 6 through 7B illustrate the fabrication of alignment region  30 , microchannel  41  and slits  36 A,  36 B and  36 C, 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. First a 300 nm thick aluminum film  62  is evaporated on the wafer and primed in hexamethyldisiloxane (HMDS) for 35 minutes (FIG.  6 ). Then, 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. The coarse aluminum pattern was etched using a Cl reactive ion etcher PK 1250 for 1.5 min. (FIG.  6 B). 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 W RF power for 10 minutes (FIG.  6 D). 
     Referring to FIG. 6E, the PMMA resist (4% 950 K in MIBK) was spun onto the wafers at 3000 rpm for 60 seconds and the wafer was baked on a hotplate at 180° C. for 30 min. Then a 100 Å layer of gold was evaporated onto the PMMA photoresist to avoid a charge build-up. The PMMA photoresist was exposed in an e-beam system to define the nanoslits. The exposed PMMA resist was developed in IPA:MIBK 3:1 for 1 min, and the 100 Å layer of gold metal was etched (FIG.  6 F). Next, the nanoslit patterns were defined by etching aluminum using the Cl reactive ion etch PK 1250 for 1.5 min (FIG.  6 G). The photoresist was removed using the Branson barrel etcher at 1000 W RF power for 10 minutes (FIG.  6 H). To create alignment region  30  and microchannel  41 , a one micron layer of SiO 2  was deposited using plasmna enhanced chemical vapor deposition (PECVD) at T=240 C., 450 mTorr, 50 W RF power using 15 sccm silane, 50 sccm N 2 O (FIG.  6 I). The SiO 2  layer was planarized by chemical mechanical polishing (CMP). 
     FIGS. 7 through 7B are side views of the wafer along one of the nanochanels. Referring to FIG. 7, 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 115° C. for 60 sec. The resist was exposed in a high resolution mask aligner, such as a 5×g-line stepper, and developed in 1:1 MF 312 and water for 60 sec. The SiO 2  layer was etched (FIG. 7A) using reactive ion etching (RIE) in CHF 3  (50 sccm)+O 2  (2 sccm) to define the pattern in the SiO 2  layer as shown in FIG.  4 . The photoresist was removed using the Branson barrel etcher at 1000 W RF power for 10 minutes. Next, a protective SiO 2  layer of 10 nm to 100 nm was deposited deposited PECVD (FIG.  7 B). 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  36 A,  36 B, and  36 C, as drawn in FIG.  4 . 
     Referring to 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  as shown in FIG.  4 A. Laser beam  65 , having excitation wavelength of 488 nm, created a nonradiating 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  94 A) and 0.1 micron wide slit (curve  94 B). Curves  94 A and  94 B represent the voltage of the photomultiplier as a function of time. As expected, the smaller slit produces the narrower curve  94 B, 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 10 μm 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  99 A and  99 B are due to the geometry of the DNA coil. The passage of the fluorescent bead is superimposed on the DNA signal. FIG. 10C shows a highly extended DNA in transit through three slits,  36 A,  36 B and  36 C. Again, for reference, the signal from a fluorescent bead is superimposed of the DNA signal. Broader peaks  97 A,  97 B and  97 C are due to the geometry of the DNA coil. 
     FIG. 11 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  166 B with a rectangular cross-section fabricated on quartz wafer  150 . Rectangular waveguides  166 A and  166 B 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 2 ) than the refractive index (n 1 ) of the surrounding dielectric material (n 2 &gt;n 1 ). Alternatively, rectangular waveguides  166 A and  166 B may be rectangular mirror waveguides that use a dielectric core material surrounded by a metallic material, or waveguides  166 A and  166 B 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 θ 1 &gt;θ c . To confine the introduced light using total internal reflection, 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 wavelength (λ) equal twice the height (h) of the waveguide (λ=2·h). Thus these waveguides have a height designed for propagation of light in a selected range of wavelengths useful for polymer examination. For further details see “Fundamentals of Photonics,” by Bahaa E. A. Saleh and Malvin Carl Teich, John Wiley &amp; Sons, 1991. 
     As shown in a perspective view in FIG. 11A, waveguides  166 A and  166 B are located symmetrically with their tips  170 A and  170 B aligned along the symmetry axis defining a nanochannel  171  (shown in FIG.  11 B). Nanochannel  171  has a width in the range of 2 nm to 100 nm, and preferably in the range of 5 nm to 50 mm. Gold wires  98 A and  98 B (shown in FIG. 11B) are spaced about 3 to 25 millimeters from nanochannel  171 . Alternatively, as shown in FIG. 11C, 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 μm. 
     Triangular waveguides  166 A and  166 B shown in FIGS. 11 and 11A are about 10 μm wide, 5000 μm long, and over 1 μm high and are made of SiO 2 . Waveguides  166 A and  166 B are isolated from substrate  162  by metallic layers  164 A and  164 B and from a glass cover  152  by metallic layers  174 A and  174 B, respectively. (Alternatively, metallic layers  164 A and  174 A for waveguide  166 A, or metallic layers  164 B and  174 B waveguide  166 B, 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  172 A and  173 A as it is transmitted toward waveguide tip  170 A. Waveguide tip  170 A emits waves of evanescent radiation (illustrated in FIG. 11B) into nanochannel  171 . In nanochannel  171 , the evanescent radiation interacts with individual units of polymer  39  producing radiation with a characteristic signal. For example, the evanescent radiation interacts with a fluorophore located next to a specific unit of polymer  39 . Triangular waveguide  166 B collects the radiation including the characteristic signal (e.g., fluorescent radiation) from nanochannel  171  and transmits this radiation toward coupling region  168 B. As the collected radiation propagates inside waveguide  166 B, the radiation nay undergo the total internal reflection at the triangular sides  172 B and  173 B. The output side  168 B, providing radiation  188 , is optically coupled to optical detector  46  (FIG.  1 ). Furthermore, 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. 11B is a cross-sectional view of two triangular waveguides  166 A and  166 B surrounded by metal layers on each side, wherein the cross-hatched pattern denotes a metal layer on waveguide sides  172 A,  172 B,  173 A, and  173 B. However, the metal layer does not cover completely the apex of tips  170 A and  170 B of triangular waveguides  166 A and  166 B. The metal layer at tips  170 A and  170 B my be removed during the etching or milling process that is used to create nanochannel  171 , as described below. Waveguide  166 A conveys introduced light beam  176  to tip  170 A by confining substantially the entire wave inside the SiO 2  volume. At tip  170 A, waveguide  166 A emits evanescent waves  177 , which are attenuated as q −1  wherein q=n 1,2  ω/c[(sin θ 1 /sin θ c ) 2 −1] 1/2  in a dielectric waveguide (see, e.g., “Optical Waves in Layered Media” by P. Yeh , John Wiley &amp; Sons, 1988). Thus the evanescent wave is attenuated over a distance of only one or two wavelengths for the total internal reflection (θ 1 &gt;θ c ). Waves of evanescent radiation  177  interact with the units of polymer  39  passing through nanochannel  171 . For example, 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  166 B and conveyed to detector  46  (FIG.  1 ). 
     FIG. 11C 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 μm, which is significantly larger than nanochannel  171 . Triangular waveguide  166  is surrounded by metal layers on all sides and is fabricated similarly as waveguides  166 A and  166 B (FIG.  11 A), wherein the cross-hatched pattern denotes a metal layer on waveguide sides  172  and  173 . Similarly as for waveguide  166 A, tip  170 A 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  178  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  98 A and  98 B. 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.  4 A. 
     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  166 B and optically coupled to optical detector  46  from output side  168 B. 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  150 . This arrangement would eliminate the need for an external optical source which is to be aligned with input side  168 A. The optical sources are made using a direct bandgap material, for example GaN for generating UV radiation, or GaP:N for generating radiation of a green wavelength. 
     Quartz wafer  150  may also include an integrated optical detector  46  in order to avoid external setup for detection and filtering. An integrated avalanche photodiode or a PIN photodiode, together with an insitu filter for filtering out the excitation wavelength, receive light beam  188 . Various integrated optical elements are described in “Integrated Optoelectronics—Waveguide Optics, Photonics, Semiconductors,” by Karl Joachim Ebeling, Springer-Verlag, 1992. For example, 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 waveguides with different dispersion relations in close proximity. Light from one waveguide will be coupled into the other for wavelengths for which there is a match in the index of refraction. By applying a voltage to the waveguides, the dispersion curve is shifted and the spectrum of the resulting filter is altered providing a tunable filter. 
     In another embodiment, the optical system  110  is an ultra fast, highly sensitive spectrophotometer capable of detecting fluorescence from a single fluorophore as described above. 
     In another embodiment, the optical system  120  uses radiation modulated at frequencies in the range of 10 MHz to 1 GHz as described above. 
     FIGS. 13 through 13B show different types of coupling of light from an external optical source into a waveguide. Referring to FIG. 13, lights source  42  emits light beam  176 , which is focused onto the input side  168 A of triangular waveguide  166 A using a focusing lens  180 . Alternatively, referring to FIG. 13A, a prism  182  is used to couple light beam  176  into triangular waveguide  166 A. Light beam  176  is diffracted by prism  182  and undergoes inside the total internal reflection. Prism  182  is located on the surface of SiO 2  volume  166 A and is arranged to optically couple beam  176  across a layer  184  into waveguide  166 A. Referring to FIG. 13B, alternatively, a diffraction grating  186  is used to couple light beam  176  into triangular waveguide  166 A. Grating  186  is fabricated on waveguide  166 A so that it diffracts light beam  176  toward tip  170 A. Alternatively, an optical fiber couples light beam  176  to triangular waveguide  166 A. Different ways to couple light into a waveguide are described in Fundamentals of Optics, by Clifford R. Pollock, Richard D. Irwin Inc., 1995. 
     Waveguides  166 A and  166 B are fabricated on quartz or another insulating material to avoid electrical currents in substrate  150 . To achieve the required high definition in the nanochannel region (i.e., 10 nm resolution), 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. 11C) is defined in separate e-beam or X-ray lithography steps. In waveguide embodiments that include a radiation slit at tips  170 A and  170 B, the slit (or a hole) is fabricated by creating a concave shape of the photoresist (i.e., an undercut) at the very tips  170 A and  170 B of waveguides  166 A and  166 B, and by creating a convex shape of the photoresist at the sides  172 A,  173 A,  172 B and  173 B before evaporating the metal. Thus, the convex sides will be covered by the evaporated metal, but not the concave tip. Alternatively, 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. When using e-beam lithography, metal hard masks are used to keep the resist thickness down and the resolution high, as is known in the art. 
     Referring to FIGS. 14A through 14K that are side views along the central line of waveguides  166 A and  166 B are fabricated as follows: To improve adhesion of the resist to the wafers, the wafers are primed in hexamethyldisiloxane (HMDS) for 34 minutes (FIG.  14 A). 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 115 C. for 60 sec to harden the resist (FIG.  14 B). The photoresist is exposed in a high resolution mask aligner such as a 5×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 shown in FIG. 14C) for the subsequent lift-off process. The wafer is flood exposed for 1 min in the HTG/contact aligner with 405 nm light and developed with Microposit 321 for 1 min. Referring to FIG. 14D, a 1000 Angstrom Al layer is deposited and the lift-off is performed using Microposit 1165 resist remover or acetone at room temperature (FIG.  14 E). All resist residues are removed using the resist descum process in the Branson Barrel etcher, 0.6 Torr O 2  at 150 W RF power. 
     Referring to FIGS. 14F through 14K, the SiO 2  waveguide is created as follows: A 1 micron SiO 2  is deposited using plasma enhanced chemical vapor deposition (PECVD) at T=240 C., 450 mTorr, 50 W RF power using 15 sccm silane, 50 sccm N 2 O. The SiO 2  layer is planarized by chemical mechanical polishing (CMP), as shown in FIG.  14 G. The top metal mask is defined by spinig photoresist Shipley 1830 onto the wafers at 4000 rpm for 60 sec to achieve a 1.3 micron thick resist and baking it on a hotplate at 115° C. for 60 sec. The resist is exposed in a high resolution mask aligner, such as a 5×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.  14 I. The resist is flood exposed for 1 min in the HTG/contact aligner by 405 nm light and developed in Microposit 321 for 1 min. As shown in FIG. 14J, 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 perpendicular to the central line. The PMMA resist 496K is spun onto the wafers at 2500 rpm 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 1 min and a 1000 A layer of Al metal is deposited as shown in FIG.  15 C. After performing the lift-off of the excess metal in acetone, the waveguide is etched, but without the microchannel pattern, in the Plasma Therm 72 etcher using reactive ion etching (RIE) in CHF 3  (50 sccm)+O 2  (2 sccm) at 200 W RF power and 40 mTorr, &gt;1 micron to create a wall shown in FIG.  15 B. The bottom metal is wet etched in the solution of 16:H 2 PO 4 ; 1:HNO 3 ; 1:acetic acid; 2:water; wetting agent, or dry etched in Cl. The remaining resist is removed in a Branson Barrel O 2  plasma etcher at 1000 W RF power for 15 min. The aluminum is removed in a wet etch using 16:H 2 PO 4 ; 1:HNO 3 ; 1:acetic acid; 2:water; wetting agent. 
     The deposition of the top Al layer over the waveguide is shown in FIGS. 15E through 15G and  16 D through  16 G. Referring to FIGS. 15E and 16D, a photoresist Shipley 1830 is spun onto the wafers at 4000 rpm 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 is exposed in a high resolution mask aligner, such as a 5×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. The resist is flood exposed for 1 min in the HTG/contact aligner 405 nm light and developed in Microposit 321 for 1 min. 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 rpm 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 5×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. The resist was flood exposed for 1 min in the HTG/contact aligner using 405 nm light and developed in Microposit 321 for 1 min. Then, a 1000 Å 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 rpm 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 1 min. Then, a 1000 Å 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 sccm)+O 2  (2 sccm) at 200 W RF power and 40 mTorr, &gt;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. Alternatively, nanochannel  171  can be fabricated by focussed ion beam milling to define the gap and the aperture in the tip. 
     For DNA sequencing, 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. When hybridization occurs, 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. 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. 
     Another embodiment of the present invention is shown in FIG.  17 . 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 volume. 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 100×1.2 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. In addition, 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 μm 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&amp;G Canada) operating in the photon counting mode. The output signal from the photodiode is collected by a multichannel scalar (EG&amp;G) and analyzed using a general purpose computer. 
     The confocal apparatus is appropriate for quantitative applications involving time-off-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. Alternatively, 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 , microspot region  228 , and an entrance region  230 , all fabricated on the surface. 
     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 μm to 5,000 μm (and preferably about 1,000 μm to 3,000 μm) 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: 
     
       
         
           u/x=S 
         
       
     
     where x is the distance down a substantially rectangular channel, and 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: 
     
       
         
           u=Q/A 
         
       
     
     In one embodiment where the channel cross-section is rectangular, the channel may be defined by a constant height, H and width, W such that the cross-sectional area A=HW, and the average fluid velocity is given by: 
     
       
         u=Q/HW 
       
     
     Applying the boundary condition that the fluid flow must be continuous, Q is constant Hence, u is inversely proportional to W. This relationship can be substituted into the original expression for S to determine a relationship between the shear rate and the width: 
     
       
           S=u/x=Q/H/x (1 /W )=(− Q/HW   2 )( dW/dx ) 
       
     
     
       
           dW/dx =(− SH/Q )( W   2 ) 
       
     
     Integrating this expression, it is found that: 
     
       
           W =( SHx/Q+C )−1 
       
     
     where C is a constant of integration determined by the original width of the channel (boundary condition). This equation for the width of the channel is used to define a channel beyond a post structure. 
     Other embodiments are within the following claims: