TW200918888A - Plastic microfluidic separation and detection platforms - Google Patents

Abstract

Plastic electrophoresis separation chips are provided comprising a plurality of microfluidic channels and a detection window, where the detection window comprises a thin plastic; and the detection window comprises a detection region of each microfluidic channel. Such chips can be bonded to a support provided an aperture is provided in the support to allow detection of samples in the electrophoresis chip at the thin plastic detection window. Further, methods for electrophoretically separating and detecting a plurality of samples on the plastic electrophoresis separation chip are described.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the field of nucleic acid sorting and fragment size measurement by electrophoresis under laser-induced fluorescence detection. The analysis was performed on a plastic electrophoresis wafer. This application is based on US Provisional Application No. 60/921,802, filed on Apr. 4, 2007, and U.S. Provisional Application No. 60/964,502, filed on August 13, 2007, in The right to file the filing date of U.S. Provisional Application No. 61/028,073, filed on Feb. 12, the entire disclosure of which is hereby incorporated by reference. Two US applications in the same semester application as the present application: the agent name "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS" " U.S. Patent Application Serial No. 07-801-US, entitled "INTEGRATED NUCLEIC ACID ANALYSIS", is incorporated herein by reference in its entirety. (Maxam and Gilbert, 1977, Proc Natl Acad Sci USA 74: 560-564; Sanger et al., 1977, Proc 74: 5463-5467) The widespread use of such technologies has evolved since its inception. The test equipment is used to perform DNA sequencing. For example, in 1986, Applied Biosystems marketed an automated DNA sequencer based on the isolation of DNA fragments generated by the Sanger ranking method; the DNA fragment was used in a 131623.doc 200918888 Groups of four glory dyes are labeled and separated by capillary electrophoresis (Smith et al, 86' 321: 674-679). Therefore, the Sanger ranking method has become the most widely used in the past three decades. Preface technology.

In recent years, a variety of new sorting techniques and related test equipment have been developed and followed by the so-called "next generation" method (Metzker, 2005, reviewed by Gewowe heW 15: 1767·1776). These chemical methods include coke filling-text Sorting method join reaction sorting method and single molecule sorting method. Driven research The main goal of the next-generation sorting technique is generally to perform high-yield genome sequencing, and especially to reduce the cost of obtaining complete genome sequences. Although each of the next generation technologies The cost of the base pair can be comparable to the cost of the sorting method in some cases 2 'but all of these methods (including Sanger) are costly and require a lot of time, labor and laboratory equipment. Seeking a very large number of self-sufficient genomic genomes The emphasis of the sequence data does not = quickly obtain the value of a relatively small number of genomic sequences. For example, many common human diseases can be based on difficult sequences of less than 1 base pair.

(Diagnosing a number of orders of magnitude smaller than the number of bases in which A is produced by the complete human genome). Similarly, the precision of a group of less than 20 specific DNA fragments produced by complex tandem recombination is sufficient to identify a given individual. Pairing of nucleic acid analysis (defined as a rapid identification of a subset of a given human, moving mussel or pathogen genome)

(The precision of nucleic acid sequencing or fragment size determination B "" 疋)) The development of instruments and techniques exists. The focus of nucleic acid analysis will enable the end user to make a few hands of immediate clinical, forensic or other Judging the lack of eight, ~ 矾 application and 疋 'focus nuclear analysis can be performed in a variety of situations, including hospital laboratories, physicians 131623.doc 200918888, bedside or in the field in forensic or environmental applications For nucleic acid (DNA and RNA) sequencing, clinical applications include diagnosing bacterial, fungal, and viral diseases (including measuring drug resistance profiles in organisms), cancer (including measuring responses to chemotherapy), and hereditary and other Common diseases (including determination of response to drugs). Focused nucleic acid sequences are also very suitable for pharmacogenomic analysis and some forensic applications (including, for example, mitochondrial DNA). Ugly for nucleic acid fragment size determination, focused nucleic acid analysis is available Forensic and clinical applications. For example, a class of human identification methods is based on short tandem repeat (STR) analysis. Edwards et al., 1991, — 49(4) 746: 756). In STR analysis, a series of primers are used to amplify certain genomic regions containing a variable number of short tandem repeats. Nucleic acid fragment size determinations (usually using capillary electrophoresis) are performed, and the size of each member of the set of STR alleles uniquely identifies the individual. STR typing has become the global standard for human forensic genetic identification and allows for 铿The only bioassay technique for the individual and the genetic relatives of the individual. In clinical applications, nucleic acid fragment size determination can be used to diagnose a given condition (eg, (eg, Friedrichich Ataxia) By searching for feature deletions or inserting or determining the size of the nucleotide repeat region, KPandolfo, M., 2006, M〇/. 126: 197-216). Fragment size determination can also be used to identify infectious agents; DNA fingerprinting is available. In the pathogen diagnosis. The application of focused nucleic acid analysis is not limited to the applications discussed above. Focused nucleic acid analysis can be used to identify 131623.do by both sorting and fragment size determination. c 200918888 Bed and clothing i-like oral biological weapons (bi〇l〇gical weap〇ns agent). Veterinary and food testing applications also reflect the above applications. Veterinary dry identification applications (such as horse breeding and Tracking, livestock breeding, and pet testing are also within the scope of the invention. The application of focused nucleic acid analysis is numerous. In short, focused nucleic acid analysis has the potential to significantly transform several industries. : The sorting instrument based on the capillary and the next-generation sorter are not capable of performing focused nucleic acid analysis in a timely and cost effective manner. The economies of scale sought by these techniques are driven by the reduced cost of obtaining and analyzing extremely large amounts of sequence data. In order for instruments and systems capable of performing focused nucleic acid analysis to enter conventional use, they should be designed to have certain "ideal" properties and characteristics. In particular, such instruments and systems should produce results quickly (ideally within a few minutes) so that indictable information can be generated as quickly as possible. It should be easy to handle and reagents and consumer products should be inexpensive. Moreover, for some applications, it is useful to perform nucleic acid isolation in a disposable article; this significantly reduces the likelihood of sample contamination. To achieve these properties, polymer-based biochips are suitable as separate substrates than other materials such as glass and germanium. An attempt to achieve DNA fragment size determination on plastic wafers is reported by McCormick (^«a/Chem 69(14): 2626 1997)

ΦΧ174 RF DNA //will limit the separation of fragments. While these separations were performed in a single wafer under a single sample, they exhibited poor resolution separation and poor sensitivity. Furthermore, the system is only capable of detecting emissions from a single fluorophore. Although Sassi (JC/zromaiogr 894(1-2): 203 2000) reported an acrylic wafer consisting of 16 flow-separated separation channels for STR 131623.doc 200918888 for size measurement, 'this method also shows poor resolution Ability and low sensitivity. This low system sensitivity prevents detection of the allelic ladder (a sizing standard that is strictly required in forensic analysis) when performing simultaneous 16 separations and detections. Using a 2 Hz scan rate (representing an attempt to increase the system signal to noise ratio) causes degradation in both resolution and accuracy. Ultimately, the system is only capable of detecting emissions from a single fluorophore. ShK£/ec(4)a 24(19-20): 3371 2003 and Shi, 2〇〇6, Electron〇ph〇resis 27(10): 3703) reported 2 and 4 color separation and detection in a single sample single-channel plastic separation device . Although it is reported that the 4.5 cm channel provides a single base resolution, it is actually proved to be poor by the appearance of the alleles separated by a test pair (THOl 9.3 and 10 allele peaks and valleys) The ratio is close to 1). Equipment with longer separation channels (6, 10 and 18 cm) was used in this study to achieve higher resolution compared to 4.5 cm equipment for analysis. When using the best screening matrix composition for resolution, equipment of 1 inch and 18 cm length is limited by equipment stratification. In fact, plastics have been found to present several major obstacles to their use in biochips designed for nucleic acid sequencing and fragment size determination. The self-fluorescence of plastic materials interferes with the detection of wavelengths in the visible range of 4 50 to 800 nm (Puriska, 2005, Lab Chip 5(12): 1348; Wabuyele, 2001 Electrophoresis 22(18): 3939-48; Hawkins and Yager 2003 Ιαό 3(4): 248-52). These wavelengths are used in commercial sets of Sanger sorting and STr sizing. In addition, the existing plastic equipment has a low adhesion strength to the commonly used substrate and a poor performance result under the commonly used screening substrate. Finally, through 131623.doc -10- 200918888 the inner surface of the channel interacts with the screening matrix and the DNA sample, and the poor resolution of the substrate due to electroosmotic flow and interaction of DNA and wall (Kan, 2004, V/ecirop/zorub 25 (2i_22): 3564). Therefore, there is an unmet need for an inexpensive, multi-channel plastic biochip capable of performing focused nucleic acid analysis with high resolution and high signal-to-noise ratio. And [invention content]

SUMMARY OF THE INVENTION The present invention provides an inexpensive, multi-channel plastic biochip capable of performing focused nucleic acid analysis with high resolution and high signal to noise ratio and methods of using such wafers. In a first aspect, the present invention provides a plastic separation wafer and, in particular, an electrophoretic wafer, comprising an anode portion, a cathode portion, and a central portion between the anode portion and the cathode portion, wherein the cathode portion comprises at least one a first through hole; the anode portion includes at least one second through hole; and the central portion includes a plurality of micro flow channels and a detection window, each of the micro flow channels has a separation area and a detection area; wherein each of the micro flow channels and at least a first through hole and at least one second through hole are in fluid communication; wherein the plurality of micro flow channels are in substantially the same plane; the plurality of micro holy motorized channels do not intersect each other in the central portion; the detection window includes莼2, and the detection_ port contains the detection area of each micro flow channel. The portion of the wafer outside the detection zone may have the same thickness or a thickness greater than the thickness of the detection zone. In a second aspect, the present invention provides an apparatus comprising a support having a top surface and a bottom surface, which includes an anode portion, a cathode portion, and a knife between the anode portion and the cathode portion at 131623.doc 200918888 a central portion, wherein the central portion includes a: f| 13⁄4 3 aperture on the detection window, the anode portion includes at least one anode aperture, and the cathode portion is 5 small to the trowel a 3 to the cathode aperture; The device further comprises a wafer according to the first aspect having a top surface and a matte side, wherein the top surface of the wafer is in contact with the bottom surface of the supporting body, and the micro flow channel is connected to the bottom surface. The aperture is in fluid communication with the cathode aperture and the anode aperture; and the wafer is fixedly attached to the support.

In the first, the present invention provides a method for simultaneously separating and detecting a plurality of samples by electrophoresis, comprising providing a plurality of samples to each of a plurality of micro flow channels on a microchip according to the first aspect. Applying a potential across a plurality of microfluidic channels to inject a sample into the separation channel and separating the detectable substance comprising each of the plurality of analytical samples; and detecting each of the plurality of isolated samples that are detectable on the detection window substance. The invention will be apparent from the following detailed description of the preferred embodiments of the invention. [Embodiment] The present invention provides a plastic separation crystal capable of detecting a nucleic acid substance separated by a size of about one test pair and having a DNA template concentration level of at least U ng, which is to be analyzed for the analysis of str before the evening PCR reaction. The content sample contains a nucleic acid template Q having less than _ copy, less than copy, less than 200 copies, ^1 〇 (Μ, less than 50 copies, less than 3 copies, less than _本 or 】 copy nucleic acid template The lowest concentration sample to be analyzed for sorting contains a nucleic acid template having less than 〇·5 pm〇le, less than 〇“less than 〇〇] 131623.doc 200918888 pmole as an input to the Sanger sorting reaction. "Injection channel" means an intersecting channel that allows a sample to be introduced into a microflow channel that intersects it. The channel can be in a single cross channel, a single T-connection, or an offset double-Tau connection configuration. The phrase "fluid communication" means that two chambers or other components or regions containing fluid are connected together so that fluid can flow between the two chambers, components or regions. The two chambers in "fluid communication" can be connected, for example, by a micro flow channel between the two chambers so that fluid can flow freely between the two chambers. The passageway may optionally include one or more valves that may close or close to block and/or otherwise control fluid communication between the chambers. As used herein, the phrase "fluorescent dye" It means that the dye emits light having a wavelength of from 380 to 850 nm after excitation with a light source. Preferably, the dye emits light having a wavelength between about 450 and 800 nm; more preferably, the dye is emitted at about 495- Light at a wavelength between 775 nm. As used herein, the term "self-fluorescence" means fluorescence produced by light other than a related fluorophore. The phrase "substantially not used" as used herein. Fluorescent" means when subjected to light illumination (eg, 'at one or more wavelengths between about 350-500 nm, 400-500 nm, or 450-500 nm; especially at 488 nm; laser illumination) Background fluorescent signals from the target (eg, solid or solution) (eg , at about 380-850 nm, 400-800 nm, 450-800 nm, 500-800 nm, or 495-775 nm) with a conventional glass microfluidic device that is lower than a 0.7 mm thick boro float glass The background level of the fluorescent signal. 131623.doc -13· 200918888 The term "norbornene-based polymer" as used herein means a polymer prepared from at least one monomer comprising a norbornene moiety, The norbornene dilute system is polymerized according to a ring-opening metathesis polymerization according to methods known to those skilled in the art (see, for example, U.S. Patent Nos. 4,945,135, 5,198,511, 5,312,940 and 5,342,909). As used herein, the term "poly(methyl methacrylate or "PMMA" means a synthetic polymer of methyl methacrylate, including but not limited to, under the trade names PlexiglasTM, LimacrylTM, R_CastTM, PerspexTM,

PlazcrylTM > AcrylexTM ^ AcryliteTM > AcrylplastTM > AltuglasTM, P〇lycastTM and the polymers sold by them, as well as U.S. Patent Nos. 5,561,208, 5,462,995 and 5,334,424 (by reference each These polymers are described in the herein). The term "polycarbonate" as used herein means a polyester of carbonic acid with a diol or a divalent phenol. Examples of such diols or divalent phenols are p-xylene diol, 2,2 bis (4-oxygen). Phenyl)propane, bis(4-oxophenyl)methane, ^-bis(4-oxophenyl)ethane, 1,1-bis(oxyphenyl)butane, 1,1-bis(oxyphenyl) Cyclohexane, 2,2-bis(oxygen)butane and mixtures thereof including, but not limited to, under the tradename CalibreTM ' MakrolonTM ' PanliteTM > MakroclearTM CyrolonTM, LexanTM and their sale Glycol or Divalent 0 The term "nucleic acid" as used herein is intended to include both single-stranded and double-stranded DNA & RNA 'and any and all forms of alternative nucleic acids containing modified nucleotides, sugars and backbones. The term "nucleic acid" includes, but is not limited to, single-stranded or double-stranded DNA or RNA (and may be in the form of a partial single-stranded or partially double-bonded form 131623.doc-14.200918888), cDNA, aptamer, peptide nucleic acid ( "PNA"), 2'-5' DNA (a synthetic material with a shortened backbone that matches the A configuration of DNA, the shortened backbone contains one base) 2,-5, DNA will usually not hybridize to DNA in B form, but it will readily hybridize to RNA) and lock nucleic acids ("LNA"). Nucleic acid analogs include similar or improved binding, hybridization Known analogs of natural nucleotides of base pairing properties. "Similar" forms of purines and pyrimidines are well known in the art and include, but are not limited to, aziridinyl cytosine, 4-ethenyl Pyrimidine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminopurine uracil, inosine, N6-isopentenyl gland嗓吟, 1-methyl gland " 吟, 1-methyl pseudourine, 1_曱 guanine, 1-methylinosine, 2,2-dimethylguanine, 2-mercapto Adenine, 2-mercapto-bird, 3-methyl-mole bite, 5-methylcytidine, N6-mercapto adenine, 7-methylguanine, 5-mercaptoamine Methyluracil, 5-methoxyaminomethyl-2-thiouracil, p_D_mannosyl q-nucleoside (beta_D_mannosylqueosine), 5-methoxyuracil, 2-methylthio-N- 6-isopentenyl adenine, uracil-5-oxyacetate, Uracil, q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxy Acetic acid and 2,6-diaminoguanidine. The DNA backbone analogs provided by the present invention include phosphodiesters, phosphorothioates, dithiophosphates, methylphosphonates, aminophosphates, alkyl groups. Phosphate triester, amine sulfonate, 3, thioacetal, fluorenylene (methylimido), 3'-N-carbamate, N-morpholinyl urethane and Peptide nucleic acid (PNA), methylphosphonate bond or alternating methylphosphonate and acid diester bond (Strauss-Soukup, 1997, Biochemistry 36: 8792-8698) and benzoate 131623.doc -15· 200918888 Phosphonate linkages, as discussed in US 6,664,057; see also OLIGONUCLEOTIDES AND ANALOGUES, A PRACITICAL APPROACH, F. Eckstein, ed., IRL Press, Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, 600 volumes, edited by Baserga and Denhardt (NYAS 1992); Milligan, 1993, «/· Met/. C/zem. 36:1923-1937 ; Antisense Research and Appli Cances (1993, CRC Press). Nucleic acids herein can be prepared from cells or synthesized synthetically in any manner known to those skilled in the art; for example, in other sources, 'nucleic acids can be chemically synthesized or transcribed or reverse transcribed from cDNA or mRNA. The term "through hole," as used herein, means a through-hole formed in a solid material to allow a fluid connection between the top and bottom surfaces of the material. An exemplary electrophoretic wafer in accordance with various embodiments of the present invention is shown in FIG. The wafer (100) includes an anode portion (101), a cathode portion (1〇2), and a central portion (i〇3) between the 1% pole portion and the cathode portion. The cathode portion includes at least one first through hole (104), and the anode portion includes at least one second through hole (105). The central portion includes a plurality of micro flow channels (1〇6) and a detection window (1〇7). Each micro flow channel has a separation region and a detection region, wherein each micro flow channel and at least one first through hole and at least A second through hole is in fluid communication. The plurality of micro flow channels are substantially in the same plane and do not intersect each other in the central portion. Each of the micro flow props has a region in which excitation and/or detection of the sample can be performed. The area of the pot containing the excitation and detection zones of the plurality of microfluidic channels is referred to as the detection window and the window contains thin plastic. 131623.doc •16- 200918888 The phrase “thin plastic” as used herein means that the material comprises a thickness (its minimum dimension) of less than 1 mm, less than 750 μηι, less than 650 μπι, less than 50〇μηι, less than 400 μπι, a plastic of less than 3 arrays, less than 200 μm or less than 1 μm; or the material comprising a thickness of 25_2〇〇〇μηη, 25·1〇〇〇μηι, 25-750 μπι, 25-500 μηι, 25 Plastics in the range of -400 μιη, 25-300 μηι or 25-200 μπι. Although the wafer is designed to be thin in the detection window, the portion of the wafer outside the detection zone may have the same thickness or have a thickness greater than the thickness of the detection zone. For the sake of illustration, the wafer shown in Fig. 1 has four micro flow channels. However, the disclosure is not intended to be limiting. In fact, those skilled in the art will readily recognize that wafers may contain interlaced micro flow channels (below). , including a wafer having one channel and a wafer having two or more channels. The term "plurality" as used herein means two or more, four or more, eight or more, 16 or more, 32 or more than 32, 48 or 48. More than 64, 64 or more, 96 or 96, 128 or 128, 256 or 256, 384 or 384, 5 12 or 5 12 or more or 1 024 Or more than 1,024; or 2-4, 2-8, 2-16, 2-32, 2-48, 2-64, 2-96, 2-128, 2-3 84, 2-512, 2- 1024 micro flow channels. As shown in Figure 3, the wafer (250) includes a substrate layer (360) and a cover layer (370). A plurality of trenches (361) are patterned into the substrate layer. Holes (ie, through holes) (371, 372) are formed in the cover layer to allow fluid to enter the micro flow channels and may be located at the ends of the micro flow channels in the anode and cathode portions of the wafer. Alternatively, the vias may be on the substrate layer. In the middle of the non-cover layer, 131623.doc 200918888 is used to achieve the same function. The top surface of the substrate layer is bonded to the bottom surface of the cover layer to form a micro flow channel. The technology of the dynamic system is extensively reviewed by Becker A Gartner (Becker, 2〇〇〇, 5:21:12_26 and 仏^ 2〇〇8, 390(1):89), which is incorporated herein by reference in its entirety. Any number of such methods can be used to fabricate the plastic release wafers described herein. In particular, the plastic release wafers of the present invention can be prepared by means of a negative standard embossed thin thermoplastic film having a structure to be fabricated. Standard molds can be prepared by electroforming a device prepared in a solid substrate. The solid substrate can be a glass sheet that is patterned by standard photolithography and chemical etching methods known to those skilled in the art. The substrate and the cover layer are diffusion bonded by applying heat and pressure. The substrate and cover layer of the wafer can be constructed from a variety of plastic substrates including, but not limited to, polyethylene, poly(acrylate) (eg, poly(methacrylic) Formazan)), poly(carbonate) and unsaturated, partially unsaturated or saturated cyclic olefin polymers (COP) or unsaturated, partially unsaturated or saturated cyclic olefin copolymers (coc) (eg, zeonort) m, ZEONEXTM or TOPAStm). For details of the wafer application of the present invention, C〇p and coc are preferred because they are optically exhibited in the visible wavelength range and are inherently lower than other polymers. Self-fluorescence. The thickness of the plastic substrate and the cover layer utilized in the method of the present invention is maintained to minimize the self-fluorescence from the wafer. The plastic substrate and the cover layer may each independently have a size of less than 2 mm and less than 1 mm, less than 750, less than 650 μηη, less than 5 μπηη, less than 400 μηη, less than 300 131623.doc 200918888 μιη, less than 200 μηη or less than 100 4 claw thickness; or the plastic substrate and the cover layer may each independently comprise Thickness in the range of 25-2000 μηι, 25-1 000 μηη, 25-750 μηη, 25-650 μπι, 25-500 μηη, 25-400 μτη, 25-300 μηι, 25-200 μηι or 2 5-100 μηη Plastic inside. In one embodiment, as illustrated in FIG. 2, the wafer (250) is attached to a support (201) having a top surface and a bottom surface, including an anode portion (2〇2), a cathode portion (203), and A central portion (204) between the anode portion and the cathode portion 'where the central portion includes a detection window (2〇5), the anode portion includes at least one %-electrode hole (2 〇6)' and the cathode portion includes at least one cathode hole ( 207). The top surface of the wafer having the upward via is in contact with the bottom surface of the support, and the wafer is fixedly attached to the support. The wafer can be attached to the support according to methods known to those skilled in the art (e.g., diffusion bonding, solvent bonding, or adhesive bonding). The bulk layer can be constructed from a variety of plastic substrates including, but not limited to, polyethylene, poly(acrylate) (eg, poly(methyl methacrylate)), poly (carbonated vinegar), and unsaturated, partially unsaturated or A saturated cyclic olefin polymer or an unsaturated, partially unsaturated or saturated cyclic olefin copolymer (COC) (for example, ZEONORTM, ZEONEXTM or TOPASMm). The thickness of the plastic support layer utilized in the method of the present invention is sufficiently thick to provide structural rigidity and to provide a sufficient amount of sample and buffer in the reservoir. The thickness of the plastic support body will be in the range of 100-15,000 μηι. Alternatively, the wafer can be fabricated by patterning the trenches on a solid support to form the wafer substrate with the support structure. The cover layer can be bonded to the support to complete the structure. In this configuration, the thickness of the support window for the support body and the wafer, which is consistent with the detection portion of the micro flow pass 131623.doc -19·200918888, is kept thin to minimize self-fluorescence. The thickness of the wafer portion is less than 丨〇〇〇μηι, less than 750 μηη, less than 500 μηι or less than 250 μηι; or in the range of 25-1000 μηη, 25-75 0 μιη or 25-5 00 μιη .

Each of the plurality of micro flow channels may have a depth of at least 丨〇, 5〇μηι, 1 〇〇, 2〇〇μηι, 500 μηι, or 1 mm; or have a density of 1-1000 μηι, 10-100 μηι, 1〇_50 μιη425_5() Depth in the range of μπι. The plurality of micro flow channels may have a width of at least 25 μm, 5 μm, 100 μm, 200 μm, 500 μΓΠ4 mm; or have a width in the range of 25_1000 μηι, 25-200 μηι or 50-200 μηΐ2. The microchannel cross section of each channel can have a substantially square, rectangular, circular, semi-circular elliptical, digonal or trapezoidal cross-face. Those skilled in the art will recognize that the depth, width and cross-section of the micro flow channels may or may not be consistent.

4 of the plurality of micro flow channels (10) 6) include a separation zone separation zone typically having a separation length of about 2- (108) and a detection zone (jog) 50 cm, 10-50 cm, 2-25 cm, defined The injection point and sample detection for the sample is typically less than the cathode and anode storage. 10-25 cm channel. Separate the channel portion between the length points. Separating the total length of the separation channels between the long stacks and analyzing a plurality of samples is abbreviated - " Μ 力 force, engraving, j injection and stacking into any of the separate wafers described herein to perform the technique It is familiar with the presence of an electric field along the separation channel (for example) on the surface of the channel (for example), causing the sample to move along the cathode portion of the channel 隹13I623.doc -20-200918888 toward the anode portion or from the anode portion toward the cathode portion. The movement of the sample through the screening substrate is based on the size of the separation material f. When the separated sample passes through the detection window, the dye attached to the sample can be excited and the resulting fluorescent light can be detected. At the end of the region, the detection window usually overlaps with the detection region of the plurality of microchannels. Typically, the detection regions of the plurality of microfluidic channels are at substantially the same position along the channels, such that the detection window It can be at a single location in the central portion of the support.

Preferably, the wafer is provided with a syringe for simultaneously injecting a plurality of samples into a plurality of samples or buffer wells to enable simultaneous multiple sample separation and detection. The injectors provide, for example, one of a plurality of samples to one of a plurality of microfluidic channels. Injections can be introduced into the channel by any means known to those skilled in the art, for example, by electrical: delivery, gas transmission or liquid transfer via a needle or tube or channel that connects the sample to the separation channel. In some embodiments, the sample can be loaded into the wafer via the cathode reservoir of the wafer. Each sample of the injection amount can be introduced through one of the cathode holes according to a method known to those skilled in the art. For example, the sample and the waste liquid hole can be biased via the parent channel and the waste channel of the separation channel and/or the knife to provide a part of the sample hole (ie, the injection amount) to the separation channel. Inject the sample. After the sample is injected, additional buffer is dissolved in the (4) cathode hole; a sufficient amount can be provided to dilute any remaining sample in the well. For example, it will be about ~5, 1 〇, 25' 5 〇 or 1 〇〇 times the amount of sample injected into the buffer 丨 cathode hole. Alternatively, an amount of buffer in the range of about 5-100 times, 5-50 times, or ι 〇 50 131623.doc • 21 · 200918888 times the amount of sample injected is introduced into the cathode hole. In other embodiments, each of the plurality of microfluidic channels further comprises - an injection channel for introducing a sample. For example, referring to Fig. 4; there is shown a perspective view of the wafer (400) showing the abutting portions of the cathode portion (4 〇) and the center portion (4 〇 3). The cathode portion includes at least one second passage (4〇5)' and the core portion includes a plurality of micro flow passages (4G6). In the cathode portion of the wafer, for each of the micro flow channels, each of the micro flow channels further includes an injection channel (408) including a sample well (4〇9) and a waste liquid (41〇). The main shot channel can be in a single intersection (as illustrated in Figure 4), a single τ connection or an offset dual T connection configuration. In some embodiments, the injection channel is an offset dual T connection configuration, It minimizes sample injection, thereby improving separation resolution. Injection of the sample from the injection channel to the microfluidic channel can be accomplished according to methods known to those skilled in the art, including electrophoretic injection via application of an appropriate potential in the sample, waste, anode, and cathode wells. An alternative embodiment of a microfluidic separation and detection wafer is illustrated in FIG. The wafer (500) includes an anode portion (5〇1), a cathode portion (5〇2), and a central portion (503). For each of the micro flow channels (5〇6), the cathode portion includes a first through hole (504)' and for each of the micro flow channels (5〇6), the anode split includes at least one second through hole (5〇5) . The central portion includes a plurality of micro flow channels (506) and a detection window (5〇7), each of the micro flow channels has a separation area and a detection area; wherein each of the micro flow channels and a first through hole and a second pass The holes are in fluid communication. A plurality of micro flow channels 131623.doc -22· 200918888 f are in the same plane and do not intersect each other in the center portion. The detection window contains thin plastic and overlaps the detection zone of each micro flow channel. In this case, the injection passage is omitted to facilitate the anode (second) and cathode (first) through holes of each of the micro flow passages. Each sample of the injection amount is introduced via one of the cathode through holes according to a method known to those skilled in the art (above). After the sample is injected, an additional buffer solution is introduced into each of the cathode buffer wells; preferably, a sufficient amount is provided to dilute any remaining sample in the well, thereby modulating any background signal from the long-term sample injection and improving the detection window The observed signal noise ratio. For example, a buffer of about at least 5, , ^, or 100 times the amount of sample injected is introduced into the cathode well. Alternatively, a buffer of about 5 to 100 times, 5 to 5 times, or 1 to 5 times the sample injection amount is introduced into the anode buffer hole. Electrophoretic doping of the sample within the microfluidic channel is provided by applying a potential difference across the microchannels on the microchip. .s # π L λ 通十 By placing the cathode and anode in the cathode and anode holes, respectively, an electric field is established along the separated portion of the micro flow channel, and the sample is sampled (for example, & lack, A ^ t 1 hi Moving from the end of the cathode via the separation portion: the detection portion: and finally to the anode, the high-speed effective knife can be applied across the end of the microchannel to remove the required lightning error, s A 士 Λ Λ Λ 50 50 50 50 50 50 50 50 50 50 50 The voltage of V/cm is applied from the voltage of the power source via the electrodes, and buffer is used in the anode and cathode reservoirs to connect Til ^ /, the electrical contact between the electrodes and the screening polymer. The sample is separated and added to the separation channel using an electrode in contact with the buffer of > T in the cathode and anode wells. Hydrolysis by t = L on the electrode in contact with the buffer 'causes the -mine and ^ formation causes the buffer value of the buffer to change with time, and 131623.doc -23. 200918888 The buffer of the anode in the polar reservoir The change in value can be attenuated by the anode and the cathode... enough to buffer the solution (for example, 1X TTE, · a.) into bubbles and have 'electrodes and! The contact between the substrates is selected. Shaped in the buffer? The tendency to move into the screening matrix, blocking the channel, leading to poor nucleic acid isolation. The following steps can be used to prevent the formation of the ::1 channel on the electrode by using the following method. First, the electrodes in the reservoir can be raised to move the source of the emulsion (electrode) away from the manhole in the channel. Second, a frit, polymer frit or polymer film or polymer filter can be inserted between the cathode inlet and the electrode tip. In particular, a polymeric frit (e.g., polyethyl ketone (PEEK)) can be inserted between the cathode inlet and the electrode tip. A glass frit, film or filter that does not have electricity and has an aperture that blocks the bubble enthalpy formed on the electrode is selected. Since a glass frit, a polymer film or a filter is interposed between the electrode and the screening substrate, bubbles formed from the electrolysis process can be prevented from entering the channel. This implementation reduces and/or eliminates failures caused by air clogging of the passage. Electrically connected separate devices for simultaneous analysis consisting of a plurality of samples so that a common power source can be used to simultaneously bias a plurality of channels. In addition, physical limitations of wafers and instruments will generally not allow all channels to have the same physical layout in terms of length, depth and width. To achieve substantially the same electrophoretic injection and separation conditions for each of the plurality of microfluidic channels, each channel segment of the individual device should have substantially the same electrical resistance and thus have substantially the same electric field. The substantially identical electric field (i.e., the 131623.doc 24·200918888 electric field crossing each of the plurality of micro flow channels does not exceed about +/- 5%) can be adjusted by simultaneously adjusting a plurality of micro flow channels The length, width and depth of each are established by adjusting the resistance of each section of the channel. The resistance 各 of each segment can be described by the following relationship: where is the resistivity, / is the length and J is the cross-sectional area of the channel. : The surface charge present on the channel walls of the separation wafer can cause electro-osmosis and the interaction of the ρ sample with the walls. This effect can be minimized by applying a surface coating to the inner wall of the micro flow channel. Such surface coatings and modifications can be accomplished by methods known to those skilled in the art (e.g., Ludwig and Beider, 2003 24(15): 2481-6). A large number of candidates for surface modification can be utilized, including hydroxypropyl decyl cellulose (HPMC), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), polydimethyl methacrylate ( PDMA), poly(vinylpyrrolidone), dimercaptopropenylamine (DMA), diethyl acrylamide (DEA), poly(diethyl propylene fluorene U amine) (PDEA), and mixtures thereof, such as PDMA: PDEA. Moreover, for use in electrophoretic applications, each of the plurality of microfluidic channels is preferably filled with a screening matrix. In a non-limiting example, the screening matrices may comprise linear polyacrylamide (PAA), polydimethyl methacrylate (PDMA), polydiethyl acrylamide (PDEA), polyvinyl pyrrolidine. Ketones (PVP) and combinations thereof, including, for example, PVP: PAA, PDMA: PAA, PDEA: PAA, PDEA: PDMA: PAA. In certain embodiments, the screening matrix comprises 0.1-50% by weight of polyacrylamide. A large number of these screening matrices are also 131623.doc -25- 200918888 with dynamic self-coating capabilities. As embodied in such embodiments using the electrophoretic separation wafer of the present invention, the nucleic acid is electrophoretically moved from the anode to the cathode end via a screening substrate and is separated therein by size. As noted above, the inside of the channel = can be coated to minimize the effects of electroosmosis and interaction of the nucleic acid with the wall. The resolution (herein specified as electrophoretic resolution) is the force (or by base size) that clearly distinguishes between two timely separated peaks. The resolution is defined by Λ = (21η2), /2 - '广' 丨^b{hwx +hw2) where / is the movement time of the ηth peak, h is the full width and half maximum of the ηth peak: And 铋 is the difference in the number of bases between the two peaks. The single base pair resolution is defined at the point of RA_·4. Visually, the two peaks can be distinguished from each other when the peak-to-valley ratio is greater than 0.7. R and peak: The valley requires that both must be met to have a high resolution of 1 and the resolution can be considered as a feature of a series of segment sizes. The fragment size of the alleles in the STM knife ranged from 90 to 400 bp' and the single-sequence pair resolution across the range was STR. The fragment size range of up to 1200 bp was analyzed. . The ability to achieve long read lengths and data yields is determined, in part, by the range of wafer susceptibility and soap base pair resolution. ^ The detection limit of the detection system is defined by the signal to noise ratio (S N R). This is the ratio of the power of the power to the power of the signal and the noise power of the signal (the noise of the noise). A high SNR indicates a higher certainty of the presence of the signal. 3 The noise ratio is generally defined as the acceptable for the identification signal. 131623.doc -26· 200918888 Ί LV sfl ratio (Gilder, 2007, J Forew z'c Sc/. 52(1): 97) .

When analyzing and detecting a plurality of nucleic acid species in a nucleic acid sample, the self-fluorescence of the plastic from the wafer detection window has a strong influence on the fluorescent background. An advantageous feature of the electrophoretic separation wafer of the present invention is the use of a thin detection window to minimize background fluorescence from the plastic. This background level is compared to borofloat®, which is a common substrate used to fabricate micro flow separation wafers. By using a thin plastic window, a minimum of 1000 copies, 3 copies, 100 copies, 30 copies, and 1 copy of the fragments that produce the fluorescent markers for analysis can be detected in the PCR method, A copy of the template nucleic acid. A nucleic acid template for a minimum of 〇5 pm〇ie, 〇1 pmole, 0.01 pm〇le or 〇.001 pm〇le for sorting reactions can also be detected. Separating and testing wafer applications does not appreciate the various. Examples for human identification include criminal forensics and national security, such as identification at military checkpoints, borders and ports, airports, and large-scale disaster locations. Veterinary identification applications (including, #马养殖 breeding and tracing, house breeding and pet identification) are also within the scope of the present invention. < Uses of electricity, water, and daily use. In addition, the apparatus of the present invention can be reinforced and used in the field of the results of (4). The instruments can be used for military inspections at ports, airports and mass casualties. The application of mediation and nucleic acid sequencing technology can be divided into a variety of studies, including (for example, bacterial infection and clinical diagnosis of drug-sensitive H-members and drug resistance profile analysis), hereditary disease axious infection (identification is a miscellaneous condition (asthma, heart 131623) .doc 200918888 Dirty diseases, diabetes and pharmacogenomics; veterinary clinical diagnostics; research nine sorts, including reordering and completion; identification of biological weapons, including, for example, Bacillus anthracis (anti-four) and Ebola virus o/a testing, and food safety. Some examples are as follows. A patient with HIV requires a drug resistance test. Currently, it can take several weeks to establish resistance. Drug resistant strains can be controlled during this time. There is an unmet need for instruments and systems that provide answers within 2 hours of waiting in the clinic. The use of electrophoretic separation wafers according to the present invention allows for frequent drug resistance monitoring, clinical and cost-effective use of antiviral agents and more Good patient outcome. Patients with bacteremia are in shock. Currently, it can take several days to determine whether the disease is resistant to antibiotics. Sex and its identity. During this period, broad-spectrum antibiotics must be used to treat patients, and broad-spectrum antibiotics can cause serious side effects to patients and promote the current prevalence of antibiotic resistance. In addition, these treatments It can be sub-optimal. The use of electrophoretic separation of wafers in accordance with the present invention allows identification of pathogen antibiotic resistance profiles in 丨_2 hours, resulting in more effective targeted therapies, reduced antibiotic toxicity, and better patient outcomes. The benefits of public health are praised. Patients with cancer are undergoing surgery. Currently, when the patient is on the operating table, the tumor sample is taken to the pathological examination. Based on the results of the simple histopathological strain, how the surgeon should make progress The decision to use the electrophoretic separation wafer according to the present invention can be used to replace the histopathology with a clear nucleic acid diagnosis of cancer within less than one hour, allowing for a surgical decision based on better information. 131623.doc -28-200918888. The following examples illustrate the invention. Specific embodiments and their various uses. The purpose of the invention is not to be construed as limiting the invention. EXAMPLES Example 1 Japanese Film Design and Electrophoresis Example\People: Wafer Design A schematic diagram of a particular embodiment of the apparatus of the present invention is illustrated in Figure 6. This microfluidic device consists of 16 Composed of microchannels, each microchannel has a double-crossing syringe and a syringe. The cross-sectional dimensions of the channel (9 〇 wide and 4 〇 ^ claw depth) and the length of the channel between the anode and the syringe (25 cm) for all channels The words are equal. The separation length of each channel (the distance between the intersection point and the excitation/detection window) is in the range of 16 to 20 cm long. The channel cross-sectional area between the cathode hole and the syringe is adjusted to the cathode. All the resistances between the intersections and thus the electric field are substantially (4) under bias. This ensures that the electric field experienced by the sample is the same regardless of the separation channel loaded into the sample. The intersection voltage of all channels is basically the same. The sample inlet and sample waste branch for the injection of the sample are both 2.5 mm long. The offset between the two channels is 5〇〇. f example \ it ·, wafer and support manufacturing by heat (four), drilling to form a manhole and diffusion minus the sealing channel, = film W case. A master is produced in a glass cap by light using a chemical wet wire engraving method. This glass master is then used to make a squeezing fixture from electroforming to produce a negative replica of the glass master. Zed 1 fiber film (size 5, x2, and thickness i88 (4) is used as the base plate 131623.doc •29· 200918888. On these plates, the cathode, anode, sample and waste liquid inlet are drilled. Holes are formed. Thereafter, the wafer design features are hot stamped onto the substrate on the embossing tool. As illustrated in 囷7, the stack is placed at 135 ° C and at a compression pressure of 1 250 psi. Embossing was carried out in a heated water press for 5 minutes. The stack was held at a compression pressure of 1250 psi and allowed to cool to 38 ° C prior to release. Fabrication of this wafer with a thin thermoplastic polymer containing norbornene monomer resulted in Low background fluorescence is produced at the excitation and detection windows. Achieving high bond strength diffusion bonding allows the use of high viscosity screening substrates.

The diffusion bonding of the substrate is achieved by aligning a ZenorTM-1420R film sheet (size 5 ι, χ2 ιι and 1 88 μπι thickness) on the substrate and subjecting the stack to heat and pressure. No adhesive is applied between the film sheets; the bonding is achieved entirely by heat and pressure. The final thickness of the wafer is about 376 μηη. The discrete wafers fabricated by this method were tested and demonstrated to withstand a pressure of at least 830 psi prior to damage. Figure 8 illustrates a wafer support made from a 3/8, thick acrylic plate by CNC milling. The wafer support consists of three main parts: a cathode plate, a center portion, and an anode plate. The cathode plate contains cathode holes, sample and waste holes and alignment. The anode plate contains anode holes and alignment & Both the cathode and anode plates are 3/8, and the thickness is read for a sufficient sample volume for sample injection and sufficient buffer volume for electrophoresis. The central portion is 〇〇4, thick and has an opening for the "detection window" for laser induced fluorescence detection in the microchannel. The factory, ~ " skill rewards are controlled by a substrate of approximately 376 μηη thick. The separation is attached to the sheet support using a double-sided pressure-sensitive adhesive. Choose to be inert to the separation buffer and screening matrix. 131623.doc •30- 200918888 Primer. The support is attached to the separation wafer using a pressure sensitive epoxy resin. The thickness of the plastic in the excitation and detection zone is minimized by making slits in the carrier in this zone.

The optical emission spectrum of Zen〇rTM-1420R has a Raman emission peak at 57 〇 nmT, which limits the fluorescent detection of fluorescent dyes. Circle 9 s is compared to a typical glass-separated wafer (glass 14 mm and glass crucible 7), the plastic wafer (Pchipl and PChp2) has a low self-fluorescence. By selecting the cop polymer and reducing the thickness of the device in the detection zone Minimize and achieve low self-fluorescence of plastic wafers by fabricating the device with a film. Example 1C: 4 face rose: lean and select shame f by initially pretreating the microchannel surface with deionized water, followed by 1 M The surface of the microchannel was treated with NaOH to effect surface modification. The fluid was removed from the channel using a nitrogen flush. Following surface treatment, 0.1 °/〇 (w/v) hydroxypropyl methylcellulose (HPMC) was applied. The solution flows through the channel and is then incubated overnight at room temperature. High purity nitrogen is used to flow through the channel to remove fluid inside the channel. The screening matrix used in this special experiment is 7 guanidine and IX TTE (Amresco) buffer. 4% linear polyacrylamide (lpa). f Example VD.. Electrophoresis STR size determination Electrophoretic separation and analysis of nucleic acid analysis was performed on a Genebench-FXTM Series 1 (Network Biosystems, Inc., Woburn, MA). The instrument is configured to accept plastic separation wafers In consideration of the wafer support member between the wafer and excellent optical instruments, electrical and thermal coupling. In the entire operation, the chamber temperature was maintained at 50 ° C. 131623.doc 31 200918888

For DNA size determination experiments, human genomic DNA is used in ABI

AmpFISTR# .^(Applied Biosystems Inc., Foster City, CA) Amplification. The PCR product (2.7 pL) was mixed with 0.3 pL size measurement standard and i〇 called methotrexate and loaded into the sample well for analysis. The assay consisted of performing pre-electrophoresis at 156 V/cm for 6 minutes, then introducing the sample by applying a potential difference of 3900 V to the anode aperture and grounding the cathode aperture. An electric field of 350 V/cm was applied over 18 seconds, followed by applying an electric field of 350 V/cm across the sample and waste holes while applying an electric field of 15.6 V/cm across the cathode and anode holes. Load to introduce DNA samples. After the sample was injected, the electrophoresis D N A separation was performed by applying an electric field of 156 v/cm across the cathode and the anode aperture while maintaining a rejection voltage of 800 V for a minute. For DNA sequencing experiments, Mn plastids were circulated with GE a(R) (7) DYEnamicTM ET dye terminator cycle sequencing kit (GE Healthcare), ethanol killed and resuspended in 10 μί deionized water. The separation test consisted of performing pre-electrophoresis at 156 v/cm for 6 minutes and then introducing a sample by applying a potential difference of 3 Torr to the anode hole and grounding the cathode hole. II introduces a sample by applying an electric field of 35 历 over 60 seconds. mouth. After the sample was injected, the electrophoresis βΝΑ separation was performed by applying the i56 of the i56 across the cathode and the anode opening, and holding the blocking voltage of 400 V for 6 minutes. The DNA separation resolution is calculated by extracting peak information (peak interval and peak width) from Peakfit®. Successful separation is achieved simultaneously in 16 lanes in the plastic wafer. Figure Contours Alleles from the 5-color marker set _ AmpF1STR Identifiler set 131623.doc -32· 200918888 Gene ladder allele call overview. These results demonstrate that the apparatus of the present invention is capable of separating and clearly interpreting alleles in five colors in a plastic wafer, including alleles spaced apart by a distance of only a single base pair (th〇, allele 9.3 and 10). Figure u shows an overview of the STR of the 9947 eight human genome 〇1^8 alleles, showing a complete overview under the 丨.〇 ng 1)1^8 template. Circle 12 shows that for up to 48〇 卟, the resolution of R>〇4 is at most bp. Figure u illustrates this resolution by showing that the two alleles separated by one nucleotide can be clearly and unambiguously resolved. Figures 14 and 15 show a DNA ranking overview demonstrating the resolution of a single base pair. Example 2 Electric Injection Plastic Wafer f Example 2A. Wafer Design Another configuration of the electrophoretic separation wafer of the present invention was separated using a single channel. Each sample was introduced into the separation channel by injection of a motorized sample. This alternative approach allows the use of small sample volumes and significantly simplifies the separation process. A schematic of the wafer design for motorized sample injection is shown in the separation diagram of Figure 16, showing the baffle body and the separated wafer portion. The device consists of 16 microchannels separated by a length of 20 cm. Each channel has an access hole at each end. These channels are 90 μηι wide and 40 μιη deep. Example 2: The equipment for forming the circle 16 was fabricated in accordance with the procedure described in the section above. In summary, an entrance hole (1 mm in diameter) was formed in a 188 pn^COP film (ZeonorTM-1420R). Then, the COP cover (ZeonorTM-1420R) was diffusion bonded to the I31623.doc-33.200918888 substrate by hot stamping to form a channel pattern (9〇μηι width and 40 μηι deep) to seal the channel. Example 2C: Tasting by using Part of the modification is applied to the channel to prepare the device for separation. Thereafter, the channel is filled with the screening matrix. The sample is loaded into the sample/cathode reservoir. The injection field is applied to the sample via the electrode. The negatively charged DNA is injected into the separation channel. After the DNA is injected into the channel, a buffer (IX TTE; Ameresco) is added to the sample/cathode reservoir in an amount 10 times the amount of the sample. An electric field is applied to the anode to separate the i% DNA from the injection plug down the separation channel. The sample is added to dilute the sample/cathode and the sample does not have to be removed prior to loading the buffer. Separation and detection in the Genebench-FXTM Series 100 instrument Execution was performed and data analysis was performed using the software described in the previous example. Example 3 DNA Sequencing For DNA sequencing analysis, DNA template was amplified in a reaction mixture consisting of: PCR SpeedSTAR HS (Takara, Madison, WI) (U/pL): 0.025, Fast buffer l: lx, dNTPs: 0.25 mM, primer (forward): 250 nM and primer (reverse): 250 nM. The required amount of template DNA is added to the mixture. DI water or TE buffer (Tris 10 mM or EDTA 0.1 mM) is added to the reaction mixture to a total amount of 10 pL. According to the manufacturer's recommended protocol, the PCR reaction mixture The thermal cycle consists of 60 seconds of hot initiation activation at 60 ° C, 30 denaturation cycles, annealing and expansion (5 seconds at 98 ° C, 10-15 seconds at 55 ° C and 5-10 at 72 ° C). Seconds/kbp) and finally extended for 60 seconds at 72 ° C. 131623.doc -34- 200918888 According to the protocol of Weishang, the entire PCR product was purified by using a 30K MWCO UF transition (pall, East mUs, Νγ). The purified product consisting of DNA in 〇1 is diluted or used as a template for the sequencing reaction. The DYEnamicTM ET Terminator Cycle Sorting Set (ge Amersham B1〇Sciences) was used to perform PCR using the following reaction mixture under a half intensity reaction. Cycle sorting of templates. Sort premix: * pL, dilution buffer · 4 μί ' Sub (1 μ μΜ) : 5 pm 〇 1. Add the DNA template to the sequencing reaction mixture. Add (1) water to the reaction mixture to 2 〇. According to the manufacturer's recommended circulation scheme, the cycle conditions used consisted of (95. 20 seconds at 20 minutes, 15 seconds at 50 °C, 60 seconds (60 seconds)). The sorted reaction mixture was purified by ethanol precipitation. The precipitated product was resuspended in 13 3 L of DI water and used as a sample for separation and detection.

For the STR analysis, the amplification line was carried out in a 10 μΐ reaction with the following reaction mixture consisting of the following: PCR enzyme SpeedSTAR HS (Takara, Madison, WI) (U/VL): 0.0315, Fast buffer

1: lx ′ primer setting: 2 μΐ, Fast buffer 1:1X, dNTPs: 200 μΜ ‘introduction (forward/reverse): 2 0 from AmpFISTR

ProfilerTM, C0FilerTM 4identifilerTM (Applied Biosystems, Foster City, CA). The cycling protocol consisted of the following conditions: 95. 6 seconds of hot start activation, followed by 28 denaturation cycles, annealing and expansion (at 98 ° C 4

s, 15 s at 59 ° C for 5 s at 72 ° C and finally at 72 °. (: The next extension is 60 seconds. The PCR product is used as a sample for separation and detection. Alternatively, the PCR product can also be purified and used as a sample for separation and detection. 131623.doc -35- 200918888 It should be understood that the above disclosure highlights the present invention. The particular embodiments of the invention, and all modifications or alternative equivalents thereof, are within the spirit and scope of the invention as set forth in the appended claims. One of the embodiments is a microfluidic separation and detection wafer. Figure 2 illustrates a separate support and wafer layer that can be used to construct a microfluidic separation and detection wafer in accordance with various embodiments of the present invention. Figure 3 illustrates that it can be used to construct a method in accordance with the present invention. One of the various embodiments of the microfluidic separation and detection of the separation device layer of the wafer. Figure 4 illustrates a perspective view of an anode portion of a microfluidic separation and detection wafer in accordance with various embodiments of the present invention. Figure 5 illustrates various embodiments in accordance with the present invention. A micro flow separation and detection wafer having an injection channel. Figure 6 is a micro flow separation and inspection according to various embodiments of the present invention. Schematic diagram of the wafer. Figure 7 illustrates the stack for imprinting. Figure 8 illustrates a wafer support made from 3/8, thick acrylic sheet by CNC milling; (top) top view; bottom (side Figure 9. Figure 9 shows the fluorescence spectrum of a plastic wafer with low self-fluorescence compared to a typical glass-separated wafer; (a) assembled plastic wafer (pchip2); (b) assembled plastic wafer (Pchipl) (c) Plastic cover only; glass wafer '1.4 mm thick; (e) glass wafer, 〇.7 mm thick; (f) plastic substrate only. 131623.doc -36· 200918888 The picture shows the 5-color mark set (aBI AmpFISTR Identifier kit) Allele ladder allele call profile (aUele_called profile); top to bottom: blue, green, yellow, red, orange detector signals. • Figure 11 is 9947A human genomic DNA Alleles call the STR profile 'top to bottom: blue, green, yellow, red, orange detector signals, all profiles are implemented on 1. 〇ng DNA template. Figure 12 shows R> for up to 480 bp _4 resolution, proof of single test ( Resolution up to 480 bp; top to bottom: blue, green, yellow, red, orange detector signals. Figure 3 shows two alleles separated by one nucleotide (thoi 9.3 and 10) Figure 14 is a DNA sequencing analysis of pGEM fragments; top to bottom: blue, green, yellow, and red detector signals. Figure 15 is a composite four-base pair map showing DNA sequencing analysis of pGEM fragments. Figure 16 is a schematic exploded view of a wafer design for direct motor sample injection showing the support (top) and wafer (bottom) layers. : [Main component symbol description] 100 wafer 101 anode portion 102 cathode portion 103 a central portion between the anode portion and the cathode portion 104 at least one first through hole 131623.doc • 37- 200918888 f- ί.

J 105 at least one second through hole 106 a plurality of micro flow channels 107 detection window 108 separation region 109 detection region 201 having a top surface and a bottom surface support 202 anode portion 203 cathode portion 204 at the anode portion and cathode portion 205 detecting window 206 at least one Anode hole 207 at least one cathode hole 250 wafer 360 substrate layer 361 a plurality of trenches 370 cover layer 371 through L 372 through hole 400 wafer 401 cathode portion 403 central portion 405 at least one second channel 406 a plurality of micro flow channels 408 containing samples Hole and waste core portion 131623.doc 38- 200918888 409 Sample hole 410 Waste hole 500 Wafer 501 Anode portion 502 Cathode portion 503 Center portion 504 First through hole 505 Second through hole 506 Multiple micro flow channels 507 Detection Window 131623.doc -39-

Claims (1)

200918888 X. Patent Application Scope · An electrophoretic separation wafer comprising an anode portion, a cathode portion and a central portion between the anode portion and the cathode portion, wherein the cathode portion comprises at least one first through hole; The anode portion includes at least one second through hole; and the central portion includes one or more micro flow channels and a detection window' each micro flow channel has a separation region and a detection region; wherein each micro flow channel and at least one first The through hole and the at least one second through hole are in fluid communication; the plurality of micro flow channels are in substantially the same plane; the plurality of micro flow channels are not intersected with each other in the central portion; Plastic; and the detection window overlaps the detection zone of each micro flow channel. The wafer of claim 1, wherein the thin plastic region comprises polyethylene, poly(acrylate), poly(carbonate), unsaturated, partially unsaturated or saturated cyclic olefin polymer (COP), unsaturated, partially non-saturated A saturated or saturated cyclic olefin copolymer (COC) or a norbornene thermal polymer. The wafer of claim 1 wherein the thin plastic comprises ze〇N〇rtm, ZEONEXTM or t〇pastm polymer. The wafer of claim 1, wherein the thin plastic has a thickness of less than about 300 μη. The day of the month 1 item, wherein the thin plastic has a thickness of less than about 500 μη. ', 6. The wafer of claim 1, wherein the thin plastic has a thickness of 1 mm or less of 131623.doc 200918888. 7_如5青求曰曰η, 甘士&amp; $曰曰 The micro flow channels of each of the micro flow channels have a separation length of 2 cm to 50 cm. 8. For example, ^ 求 y, -rf· ί. . y , 曰曰 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中9. The wafer of claim 1, wherein the germanium plastic substantially does not emit fluorescence having a wavelength between 500 and 800 nm when excited at a wavelength between about 450 and 500 nm. V 1 〇 如 吻 吻 求 9 , , , , , , , , , , , , 9 9 9 9 9 9 9 9 9 9 9 9 激发 激发 激发 激发 激发 激发 激发 激发 激发 激发 激发 激发 激发 激发 激发 激发 激发 激发At 1Q, a signal to noise ratio greater than 3 can be used to detect a plurality of nucleic acid species in a nucleic acid sample produced for a fragment size determination application. 12. A wafer of the claimed item, wherein a single copy of the dna template for Pcr amplification, can be used to detect a plurality of nucleic acid species in a nucleic acid sample produced for DNA sequencing applications using a signal to noise ratio greater than three. C) 13. The wafer of claim 1 wherein each of the plurality of microfluidic channels further comprises a surface coating. 14. The wafer of claim 13 wherein the surface coating is hydroxypropyl decyl cellulose (HPMA), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(dimethyl Acrylamide (PDMA), poly(vinyl, piroxicam), dimercaptopropeneamine (DMA), diethyl acrylamide (DEA), poly(diethyl acrylamide) And mixtures thereof. 15. The wafer of claim 13 wherein each of the plurality of microfluidic channels further comprises a screening matrix. 16. The wafer of claim 15 wherein the screening matrix comprises linear or cross-linked poly(N,N-dialkyl acrylate), linear polyacrylamide, polydidecyl acrylamide, polyvinyl pyrrolidine Ketone or a combination thereof. 17. The wafer of claim 16, wherein the screening matrix comprises i_5〇 of polyacrylamide. 18. The wafer of claim </ RTI> further comprising a porous layer between each of the cathode apertures and each of the micro flow channels, wherein the porous layer is capable of substantially blocking the passage of bubbles from the cathode apertures into the respective micro flow channels. 1 9. The wafer of claim 18 wherein the porous layer comprises a frit, a polymer frit, a polymer film or a polymer filter. 20. As requested! A wafer in which a plurality of nucleic acid substances in a nucleic acid sample can be detected at a single base resolution after electrophoretic analysis of the nucleic acid sample. 21. The wafer of claim 1 further comprising a syringe for simultaneously injecting at least two samples into the plurality of microfluidic channels. 22. A device comprising a support having a top surface and a bottom surface, comprising an anode portion, a cathode portion, and a central portion between the anode portion and the cathode portion, wherein the central portion is The detection window includes a hole, the anode portion includes at least one anode hole, and the cathode portion includes at least one cathode hole; the device further comprises a wafer of the first item 1 having a top surface and a bottom surface, wherein The top surface of the sheet is in contact with the bottom surface of the support, 131623.doc 200918888 the micro flow channels are in fluid communication with the cathode holes and the anode holes via the through holes; and the wafer is fixedly attached to The support. The device of claim 22, wherein the separation wafer comprises a substrate layer and a cover layer each having a top surface and a bottom surface, wherein the substrate layer includes a plurality of micro flow channels in a top surface thereof; The cover layer includes a through hole for each of the anode and cathode holes of the wafer support, wherein a top surface of the substrate layer is in contact with a bottom surface of the cover layer, thereby forming a plurality of micro flow channels in the separation wafer. 24. The device of claim 23, wherein the substrate layer and the cover layer are thermally bonded. 25. A method of simultaneously separating and detecting a plurality of samples by electrophoresis, comprising providing a plurality of samples to each of a plurality of microfluidic channels on a microchip as claimed; A potential is applied to separate a detectable substance comprising each of the plurality of analytical samples; each of the detectable substances comprising the plurality of separated samples is detected at the detection window. 26. The method of claim 25, further comprising the step of maintaining substantially the same electric field across each of the plurality of micro flow channels. 27. The method of claim 26, wherein the substantially identical electric field is maintained across each of the plurality of microfluidic channels by balancing the electrical resistance of each of the plurality of microfluidic channels . The method of claim 25, wherein the detectable substance comprises a nucleic acid. 29. The method of claim 28, wherein the detectable substances comprise a dye functionally linked to the nucleic acids. 131623.doc