TWI647439B - Integrated microfluidic systems, biochips and methods for the detection of nucleic acids and biological molecules by electrophoresis - Google Patents

Abstract

The present disclosure provides a fully integrated microfluidic system for nucleic acid analysis. These processes include sample collection, nucleic acid extraction and purification, amplification, sequencing, and separation and detection. The present disclosure also provides an optical detection system and method for separating and detecting biomolecules. In particular, aspects of the invention enable simultaneous separation and detection of a plurality of biomolecules (typically fluorescent dye-labeled nucleic acids) in one or more microfluidic chambers or channels. The nucleic acids can be labeled with at least six dyes, each having a unique peak emission wavelength. The systems and methods of the present invention are particularly useful for applications that measure DNA fragment size (such as human recognition by genetic fingerprinting) and DNA sequencing applications (such as clinical diagnostics).

Description

Integrated microfluidic system, biochip and method for detecting nucleic acid and biomolecule by electrophoresis

This invention relates to the field of microfluidics for nucleic acid analysis.

This application claims the right to file the filing date of the following application in accordance with 35 USC § 119(e): U.S. Provisional Application No. 60/921,802, filed on Apr. 4, 2007; U.S. Provisional Application No. 60/964, 502, and U.S. Provisional Application No. 61/028, 073, filed on Feb. 12, 2008, the entire contents of each of which is incorporated herein by reference. The entire contents of the following two U.S. patent applications are hereby incorporated by reference in their entirety in the application in the the the the the the the the the the the the File number 08-318-US; and second file name "Plastic Microfluidic separation and detection platforms", agent file number 07-865-US.

A centralized nucleic acid analysis that allows complete integration (ie, sample entry into the resulting output) (defined as rapid identification of a subset of a given human, animal, plant, or pathogen genome (by nucleic acid sequencing or fragment size determination)) There is an unmet need for the development of instruments and technologies. Centralized nucleic acid sequencing will enable end users to make immediate clinical decisions, forensic decisions, or other decisions. For example, many common human diseases can be based on less than 1000 DNA sequences. Base pairs (less than the order of magnitude required to produce the entire human genome) are diagnosed. Similarly, an accurate determination of the size of a collection of less than 20 specific DNA fragments produced by short tandem repeat sequence analysis is sufficient to identify a given individual. Depending on the application, centralized nucleic acid analysis can be performed in the field in a variety of configurations, including hospital laboratories, physician offices, bedsides, or in forensic or environmental applications.

There are several unmet needs for improved DNA sequencing and fragment size determination systems. There is an unmet need for an instrument that is easy to use and does not require highly trained operators for DNA sequencing and fragment size determination. Second, there is an unmet need for systems that eliminate all manual processing. Therefore, only minimal operator training is required and the system should be susceptible to individual operations limited by challenging environments such as, for example, a first responder wearing a haz-mat suit.

Third, there is an unmet need for ultra-fast analysis that does not sacrifice the need for complete, accurate, and reliable data. For human identification applications, the appropriate time to produce results is 45 minutes or less, which is much less than the days to weeks required to use conventional techniques. For clinical applications, such as sequencing infectious agents to determine appropriate therapeutic therapies, 90 minutes or less is a reasonable response time so that treatment with antibacterial and antiviral drugs can begin as soon as the patient arrives in the emergency room. Regardless of the application, there is an unmet need to produce instant and useful data. A shorter response time also allows for an accompanying increase in sample throughput.

Fourth, there is an unmet need for miniaturization. Many DNA analysis systems require the entire laboratory and related support. For example, the high throughput Genome Sequencer FLX (Roche Diagnostics Corp, Indianapolis, IN) DNA sequencing system requires only a bench for installation, but requires a large laboratory for the required library construction. Miniaturization is important for laboratory and on-site point-of-care and field operations. It is also important to reduce the cost of each sample.

Fifth, there is an unmet need for durability. For many applications, especially For applications in forensic, military, and defense applications, DNA analytical instruments must be operational in the field. Accordingly, the instrument must be capable of being transported whether it is carried by a soldier, transported by a police car, or dropped by a helicopter to the battlefield. Similarly, the instrument must be able to withstand extreme temperatures including temperature, humidity, and airborne particles (eg, grit) and be able to operate in such extreme environments.

Sixth, there is an unmet need for systems that can accept multiple sample types and can perform highly multiplexed analysis in parallel. For most applications, the ability to analyze DNA from a single sample type in a single reaction is unacceptable for meaningful DNA analysis.

Seeking microfluidics (also known as micro-total analysis systems (μTAS) or lab-on-a-chip) for concentrating complex laboratory operations on biochips, see Manz et al., Sens. Actuators B 1990, 1, 244-248) Developers have clearly recognized these unmet needs, but have not yet been able to design biochemical and physical technologies that are necessary or desirable for microfluidic nucleic acid analysis to meet these needs. Process integration of biochips and instruments. Therefore, concentrated nucleic acid analysis has not yet entered widespread use in today's society.

The development of microfluidic systems involves the integration of micro-assembly components (such as micro-separation, reactions, microvalves and pumps) and various detection mechanisms into full-featured devices (see, for example, Pal et al, Lab Chip 2005, 5, 1024-1032). ). Since Manz et al. (supra) demonstrated the phenomenon of capillary electrophoresis on wafers in the early 1990s, others have sought to improve it. Several groups have demonstrated integration of DNA processing functions with biochip separation and detection. An integrated device for the hybrid structure of glass-PDMS (polydimethylmethoxane) has been reported (Blazej et al, Proc Natl Acad Sci USA 2006, 103, 7240-5; Easley et al, Proc. Natl. Acad. Sci. USA 2006, 103, 19272-7; and Liu et al., Anal. Chem. 2007, 79, 1881-9). Liu combines multiple polymerase chain reaction (PCR), separation, and four-dye detection for human recognition by short tandem repeat (STR) size determination. Blazej combines Sanger sequencing reaction, Sanger reaction purification, electrophoretic separation and four-dye detection for DNA sequencing of the pUC18 amplicon. Easley combines solid phase extraction of DNA, PCR, electrophoretic separation, and monochrome detection to identify the presence of bacterial infections in the blood. An integrated glass-lined device incorporating PCR, electrophoretic separation, and monochrome detection is shown by Burns (Pal, 2005, Id. ). Huang reported combining a glass-PDMS fraction of PCR with a poly(methyl methacrylate) (PMMA) fraction separated by electrophoresis and a single color detection to identify a hybrid device for the presence of bacterial DNA (Huang et al., Electrophoresis 2006, 27). , 3297-305).

Koh et al. reported combining PCR with biochip electrophoresis separation and single color detection for a plastic device for identifying the presence of bacterial DNA (Koh et al., Anal. Chem. 2003, 75, 4591-8). Asogawa reports on the integration of DNA extraction, PCR amplification, biochip electrophoresis separation and monochrome detection (Asogowa M, Development of portable and rapid human DNA Analysis System Aiming on-site Screening, 18th International Symposium on Human Identification, Poster , October 1-4, 2007, Hollywood, CA, USA). U.S. Patent No. 7,332,126 (Tooke et al.) describes the use of centrifugal force to perform the microfluidic operations required for nucleic acid isolation and cycle sequencing. However, this method is based on small sample volumes (about one to several μL of them). Therefore, the device is not suitable for processing a large number of samples for separation and analysis of nucleic acids, especially in a highly parallel manner, since the fluid sample must be applied to the device at rest, ie the disk must be capable of containing the operating device prior to centrifugation. All fluids required (up to hundreds of mL for highly parallel devices). Second, the device is limited to bacterial preparation and cycle sequencing (eg, plastid DNA).

There are several drawbacks in their attempts to separate DNA processing from biochip electrophoresis. First, the detection is limited by the amount of information (mostly using a monochrome detector, but some have a four-color detection system) or throughput (single sample or two sample processing capabilities). Second, these devices do not reflect complete sample-response integration. For example, Blazej's device requires off-board amplification of template DNA prior to cycle sequencing, while others use samples that require some pretreatment (eg, Easley and Tooke need to dissolve the sample before it is added). Third, certain processing options for such devices have a negative impact on time-response: for example, the hybridization-based method of Blazej requires more than 20 minutes to purify the cycle sequencing product. Fourth, many of these devices are made, in part or entirely, of glass or enamel. The use of such substrates and related manufacturing techniques makes them inherently more costly (Gardeniers et al, Lab-on-a-Chip (Oosterbroeck RE, van den Berg A, ed.), Elsevier: London, pp. 37-64 (2003). )) and it is limited to applications where re-use of such devices is necessary; for many applications, such as human IDs, this poses a risk of sample contamination. Finally, the techniques demonstrated are not suitable for both applications, namely human identification via STR analysis and sequencing. For example, both Easley and Pal devices suffer from poor resolution, which is much worse than single base resolution. Fragment size determination applications (eg, human recognition by short tandem repeat profiling) and sequencing require a single base analysis.

In addition to the limitations of prior art in terms of microfluidic integration, the issue of fluorescence detection also limits the wide range of applications of nucleic acid analysis beyond the well-known laboratory studies. The most widely used of a commercial sequencing kit (BigDye ^TM v3.1 [Applied Biosystems] and ^{DYEnamic TM ET [GE Healthcare Biosciences Corp} , Piscataway, NJ]) is based on the presence of two decades four-color detection methods (see, e.g. U.S. Patent Nos. 4,855,225; 5,332,666; 5,800,996; 5,847,162; 5,847,162). This method is based on the resolution of the emission signal of the dye-labeled nucleotide into four different colors, each color representing each of the four bases. These four-color dye systems have several disadvantages, including inefficient excitation of fluorescent dyes, significant spectral overlap, and inefficient collection of emission signals. These four-color dye systems are particularly problematic because they limit the amount of information that can be obtained from a given electrophoresis (or other) separation of the sequenced product.

There is an unmet need for a system capable of separating and detecting DNA fragments based on fragment size and obtaining high information content by color (dye wavelength) in an electrophoresis system. The maximum number of DNA fragments that can be discerned by electrophoresis is determined by the length of reads and resolution of the device. The maximum number of detectable colors is determined in part by the fluorescent dye. The resolution of the wavelength of the use and detection system. Although four-color detection has been reported, existing bio-disc detection systems are generally limited to monochrome.

STR analysis for human recognition is an example of DNA fragment size based on color multiplicity and allows simultaneous analysis of up to 16 sites (AmpFlSTR Identifiler kit, Applied Biosystems, Foster City, CA; and PowerPlex 16 kit, Promega Corporation, Madison) , WI). Using four or five fluorescent dyes, a single separation channel can identify the size of many of the dual gene variants at each point. Several sample size applications will require separation and detection of more than 16 segments on a single lane. For example, fingerprint recognition of pathogens (ie, isolation and detection of a large number of unique DNA fragments) and diagnosis of aneuploidy by measuring the entire human genome can be achieved by focusing on dozens or hundreds of loci, respectively.

One way to increase the number of sites that can be detected in a single separation channel is to broaden the resulting fragment size range in part by increasing the fragment size of the extra sites. However, the use of longer fragments for additional sites is non-ideal because the amplification of larger fragments is more sensitive to inhibitors and DNA degradation, resulting in lower yields of longer fragments relative to shorter fragments. In addition, the generation of longer segments also requires an increase in the expansion time and thus an increase in the total verification time. There is an unmet need to increase the number of detectable sites in a given separation channel by increasing the number of dye colors that can be simultaneously detected.

There is an unmet need to increase the Sanger sequencing separation ability (and thus the cost, labor, and space of the method) by increasing the number of DNA sequences that can be analyzed in a single separation channel. In addition, in some applications, sequencing of multiple DNA fragments results in "mixed sequence" data that is difficult to read; a method for correctly interpreting the mixed sequences needs to be developed.

One way to increase the capacity of the Sanger separation channel and develop the ability to interpret the mixed sequence is to increase the number of dye colors used in the sequencing reaction. In DNA sequencing and fragment size determination, multiple fragments labeled with different dyes can be detected simultaneously. In general, the separation between the peak emission wavelengths of adjacent dyes must be sufficiently large relative to the peak width of the dye. Thus, the throughput of each separation channel can be used, for example, by using two in two separate sequencing reactions. The four dyes were combined and the products were combined and doubled on a single channel. This method requires the use of a total of 8 dye colors, where the first sequencing reaction uses one set of four dye colors suitable for labeling the dideoxynucleotide terminator, and the second reaction uses another set for labeling the terminators. Four dye colors; each set of dye colors is independent so that there may be no overlap in the interpretation of the two sequences. Using this same method, a set of 12 dyes can be used to allow simultaneous analysis of the sequence of three DNA fragments in a single channel, a set of 16 dyes allowing analysis of four sequences, etc., which significantly increases the amount of information for the Sanger separation. .

The novel instruments and biochips of the present application meet a number of unmet needs, including those listed above.

The present invention provides a fully integrated microfluidic system for nucleic acid analysis. Such processes include sample collection, DNA extraction and purification, amplification (which can be highly multiplexed), sequencing, and separation and detection of DNA products.

The separation and detection module of the present invention is rugged and better than single base analysis. It is capable of detecting six or more colors and, as such, is suitable for self-sequencing and fragment size applications to produce high information content.

Highly multiplexed rapid PCR on a biochip is the subject of a U.S. patent application filed on the same day, having the assignee file number MBHB 08-318-US and entitled "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS", the patent The entire disclosure of the application is hereby expressly incorporated by reference. In addition, the PCR product can be isolated and detected in a biochip as described in U.S. Patent Application Serial No. U.S. Patent Application Serial No. 07-865-US, the entire disclosure of which is incorporated herein by reference. The manner of citation is expressly incorporated into the present application.

Therefore, in a first aspect, the present invention provides an optical detector comprising one or more light sources positioned to illuminate one or a plurality of detection locations on a substrate; one or more a first optical component positioned to collect and direct light emitted from the detection locations on the substrate; and a light detector positioned to receive light from the first optical component, wherein the light The detector includes a wavelength dispersive element for separating light from the first optical elements according to a wavelength of light and positioning to provide a portion of the separated light to the detecting element, wherein each of the detecting elements And communicating with a first control element for simultaneously collecting detection information from each of the detection elements, wherein the photodetector detects fluorescence from at least 6 dyes marking one or more biomolecules Each dye has a unique peak emission wavelength.

In a second aspect, the invention provides a system for isolating and detecting biomolecules comprising: an assembly for simultaneously separating a plurality of biomolecules in one or a plurality of channels on a substrate, wherein each The channel includes a detection location; one or more light sources positioned to illuminate the detected locations on the substrate; and one or more plurality of light positioned to collect and direct light emitted from the detection locations An optical component; and a photodetector positioned to receive light from the first optical component, wherein the photodetector includes a wavelength dispersive component for separating from the first optics according to a wavelength of light The light of the component is positioned to provide a portion of the separated light to the detecting component, wherein each of the detecting components and a first control for collecting detection information from each of the detecting components simultaneously The components are connected, and wherein the photodetector detects fluorescence from at least six dyes labeling one or more biomolecules, each dye having a unique peak wavelength.

In a third aspect, the present invention provides a method for isolating and detecting a plurality of biomolecules, comprising: providing one or more analytical samples on one of a substrate or a plurality of microfluidic channels, wherein each The microfluidic channel comprises a detection location, and each of the analysis samples independently comprises a plurality of biomolecules, each biomolecule being independently labeled by one of at least six dyes, each dye having a unique peak wavelength; and simultaneously in each microfluidic channel Separating the plurality of labeled biomolecules; and detecting the plurality of separated target analytes in each microfluidic channel by: illuminating each detection location with a light source; Light emitted by each detection location; directing the collected light to a photodetector; and (i) separating the collected light according to the wavelength of the light; and (ii) simultaneously detecting one or more biomolecules from the marker Fluorescence of at least 6 dyes, each dye having a unique peak wavelength.

In a fourth aspect, the present invention provides an integrated biochip system comprising (a) a biochip comprising one or more microfluidic systems, wherein each microfluidic system comprises a microfluidic communication with a separation chamber a first reaction chamber, wherein the first reaction chamber is adapted for nucleic acid extraction, nucleic acid purification, purification before nucleic acid amplification, nucleic acid amplification, purification after nucleic acid amplification, purification before nucleic acid sequencing, nucleic acid sequencing, purification after nucleic acid sequencing, Reverse transcription, pre-transcriptional purification, post-transcriptional purification, nucleic acid ligation, nucleic acid hybridization or quantification, and the separation chamber comprises a detection site; and (b) a separation and detection system comprising (i) one for Separating a plurality of discrete analytes of the target analyte simultaneously in the separation chamber; (ii) one or more light sources positioned to illuminate the detection locations on the biochip; (iii) one or more Positioning a first optical component for collecting and directing light emitted from the detection locations; and (iv) a light detector positioned to receive light from the first optical component, wherein the light detection Contains a wavelength of color a dispersing element for separating light from the first optical elements according to a wavelength of light and positioned to provide a portion of the separated light to at least six detecting elements, wherein each of the detecting elements is used The first control element is configured to simultaneously collect detection information from each of the detection elements, and wherein the photodetector detects fluorescence from at least six dyes marking one or more biomolecules, each dye Has a unique peak wavelength.

In a fifth aspect, the present invention provides an integrated biochip system comprising (a) a biochip comprising one or more microfluidic systems, wherein each microfluidic system comprises a microfluidic communication with a separation chamber a first reaction chamber, wherein the first reaction chamber is adapted for nucleic acid extraction, nucleic acid purification, purification before nucleic acid amplification, nucleic acid amplification, purification after nucleic acid amplification, purification before nucleic acid sequencing, nucleic acid sequencing, purification after nucleic acid sequencing, Reverse transcription, pre-transcriptional purification, post-transcriptional purification, nucleic acid ligation, nucleic acid hybridization or quantification, and the separation chamber package a detection and detection system; and (b) a separation and detection system comprising (i) a separation element for simultaneously separating a plurality of biomolecules comprising a DNA sequence in the separation chambers; (ii) one or a plurality of light sources positioned to illuminate the detected locations on the biochip; (iii) one or a plurality of first optical components positioned to collect and direct light emitted from the detection locations; (iv) a photodetector positioned to receive light from the first optical element, wherein the photodetector includes a wavelength dispersive element for separating the first optical element from the wavelength of the light Light and positioned to provide a portion of the separated light to at least six detection elements, wherein each of the detection elements and a first one for collecting detection information from each of the detection elements simultaneously The control element is in communication, and wherein the photodetector detects fluorescence from at least eight dyes marking one or more DNA sequences, each dye having a unique peak wavelength, the dyes being at least two children containing four dyes a member of the set to enable the collection of such dyes to The number of DNA sequences in a single channel detector at least two DNA sequences, wherein the number of dye is a multiple of four, and to be equal to the multiple of detection, so that each of these different dyes are present in only a subset.

40‧‧‧Fluorescence excitation and detection assembly

42‧‧‧ openings

50‧‧‧Protective layer

55‧‧‧Test module

60‧‧‧Laser

62‧‧‧Scanner/Scanner System

64‧‧‧Photodetector / Multi-Component PMT

68‧‧‧Mirror

72‧‧‧ lens

75‧‧‧埠

Room 101‧‧

104‧‧‧埠

105‧‧‧埠

106‧‧‧埠

107‧‧‧埠

108‧‧‧埠

109‧‧‧埠

202‧‧‧through hole

203‧‧‧through hole

204‧‧‧ sample room

205‧‧‧First hybrid joint

208‧‧‧ distribution channel

209‧‧ ‧ measuring room

210‧‧‧Capillary valve

211‧‧‧Capillary valve

212‧‧‧Mixed spheres

213‧‧‧ Contraction

214‧‧‧ mixed channel

215‧‧‧through hole

216‧‧‧through hole

217‧‧‧through hole

218‧‧‧cycle sequencing reagent metering room

219‧‧‧Capillary valve

220‧‧‧Capillary valve

221‧‧‧Capillary valve

227‧‧‧through hole

303‧‧‧sample channel

304‧‧‧through hole/channel

305‧‧‧through hole

306‧‧‧through hole

Room 307‧‧

308‧‧‧through hole

Room 309‧‧

310‧‧‧ channel

311‧‧‧through hole

314‧‧‧through hole

315‧‧‧through hole

316‧‧‧through hole

317‧‧‧through hole

320‧‧‧through hole

336‧‧‧through hole

402‧‧‧through hole

403‧‧‧through hole

404‧‧‧through hole

502‧‧‧PCR room

503‧‧‧Cycle sequencing room

1104‧‧‧埠

1105‧‧‧ channel

Room 1106‧‧

1108‧‧‧Capillary valve

1110‧‧‧through hole capillary valve

1111‧‧‧through hole

1112‧‧‧UF input room

1113‧‧‧Capillary valve

1115‧‧‧Filter room

1116‧‧‧Ultrafiltration (UF) filter

1119‧‧‧埠

1120‧‧‧埠

1121‧‧‧Reservoir/room

1122‧‧‧ channel

1123‧‧‧ overflow chamber

1124‧‧‧埠

1301‧‧‧Integrated biochip

1302‧‧16-sample biochip/subassembly

1303‧‧‧16-channel plastic separation biochip/separation subassembly

1304‧‧‧ delivery point

1305‧‧‧Input hole

1306‧‧‧Separation channel

1307‧‧‧Detection area

1308‧‧‧ recess

1401‧‧‧Liquid receiving hole/hole reservoir

1402‧‧‧Main separation electrode

1403‧‧‧Counter electrode

Figure 1 is a graphical representation of an embodiment of an integrated biochip for dissolution and template amplification of four individual samples.

2 is an illustration of an embodiment of a first layer of the bio-wafer of FIG. 1.

3 is an illustration of an embodiment of a second layer of the biowafer of FIG. 1.

4 is an illustration of an embodiment of a third layer of the biowafer of FIG. 1.

Figure 5 is an illustration of an embodiment of a fourth layer of the biochip of Figure 1.

6 is a diagram of an embodiment of the assembly and connection of the biochip of FIG. 1.

Figure 7 is a graph illustrating capillary valve pressure as a function of reverse hydraulic diameter for valves for deionized water and cycle sequencing reagents for both valves (in-plane valves and through-hole valves).

8 is a graphical representation showing an embodiment of a fluid step of the biowafer of FIG. 1 for PCR template amplification.

Figure 8a is a graphical representation showing the loading of a sample and PCR reagent into a biochip of the present invention.

Figure 8b is a diagram showing the transfer of a sample through a channel to a sample chamber (which is shown at different locations along the sample channel to illustrate the flow path).

Figure 8c is a graphical representation showing a sample in a sample chamber.

Figure 8d is a graphical representation showing the delivery of a PCR reagent to a reagent chamber.

Figure 8e is a graphical representation showing the extraction of excess PCR reagents.

Figures 8f and 8g are graphical representations showing the initial mixing step and retention of the liquid by the first set of capillary valves.

Figures 8h through 8j are diagrams showing the transfer of mixed liquid to a PCR chamber where thermal cycling begins.

9 is a diagram showing an embodiment of a fluid step of integrating a biochip.

Figures 9a through 9e are graphical representations showing the delivery of a circulating sequencing reagent to a metering chamber in layer 1 and removal of excess reagent from the vicinity of the chambers.

Figures 9f and 9g are graphical representations showing the introduction of PCR products into the Sanger reaction chamber.

Figures 9h-9k are graphical representations showing the mixing of a Sanger reagent with a PCR product by reciprocating motion.

Figure 119 is a graphical representation showing that the recycled product can be removed for analysis.

Figure 10 is a sequencing trace (electropherogram) of sequencing the product produced in the biochip of Figure 1.

11 is a graphical representation showing an embodiment of an integrated biochip for ultrafiltration performance of cycle sequencing products. The wafer assembly is similar to the assembly of the biochip 1 except that an ultrafiltration (UF) filter 1116 is added between layers 3 and 4.

Figure 12 is a graphical representation showing the fluid steps of the biochip of Figure 11 during purification of the sequencing product.

Figures 12a and 12b are diagrams showing the transfer of the Sanger product to the UF input chamber.

Figure 12c is a graphical representation showing that the sequencing product has been delivered to the filtration chamber.

Figure 12d is a graphical representation showing the almost complete filtration of the sequencing product.

Figures 12e through 12g are diagrams showing the delivery of detergent to the UF input chamber followed by removal of excess detergent from the transfer channel.

Figure 12h is a graphical representation showing the beginning of the first wash cycle; followed by filtration as in Figure 12d and subsequent wash cycles.

Figures 12i and 12j are diagrams showing the dissolved liquid (the same liquid as the detergent) delivered to the UF input chamber.

Figures 12k through 12m are illustrations showing a single cycle of pressurizing the UF input chamber and closing the output port and then releasing the pressure resulting in a reciprocating motion.

Figure 12n is a graphical representation showing the purified product prepared for further processing or removal.

13 is a graphical representation showing an embodiment of an integrated biochip for template amplification, cycle sequencing, sequencing product purification, separation by electrophoresis, and efficacy by laser induced fluorescence detection.

Figure 14 is a graphical representation showing the concentration of labeled nucleic acid fragments by a counter electrode and injection into a separation channel.

15 is a diagram showing an embodiment of an excitation and detection system.

16 is a diagram showing an embodiment of an excitation and detection system.

Figure 17 is an electropherogram generated by separating and detecting a 6-dye sample. The lines in the figure represent the signals from each of the 32 elements of the 32-anode photomultiplier tube (PMT). The lines are offset relative to one another to allow the material to be easily viewed.

Figure 18 is a graph showing the dye spectra of each of the six dyes extracted from the electropherogram; also showing the background fluorescence spectrum.

Figure 19 is a graph showing the dye emission spectra of 6-FAM, VIC, NED, PET, and LIZ dyes.

Figure 20 is a graph showing the dye emission spectrum of 5-FAM, JOE, NED and ROX dyes. Figure.

Figure 21 is an electropherogram generated by separating and detecting four dye samples. The traces in the figure represent the signals from each of the 32 elements of the 32-anode PMT. The traces are offset relative to each other to allow the material to be easily viewed.

Figure 22 is a sequencing trace.

I. Integration and integration system A. General description of integration

The use of microfluidics allows the fabrication of features that perform more than one function on a single biochip. Two or more of these functions may be in microfluidic relationship to enable continuous processing of the sample; this combination is referred to as integration.

Although not all processes must be implemented for any given application, there are a number of possible functions or constituent processes that must be integrated to achieve any given application. Therefore, the chosen integration method must be suitable for effectively linking several different constituent processes in different orders. Processes that can be integrated include, but are not limited to, the following: 1. sample insertion; 2. removal of foreign matter (eg, large particles such as dust, fibers); 3. cell separation (ie, removal except for analysis) Cells other than the cells of the nucleic acid, such as human cells removed from the clinical sample containing the microbial nucleic acid to be analyzed (and correspondingly removing human genomic DNA); 4. Concentrating the cells containing the nucleic acid of interest; Dissolving the cells and extracting the nucleic acid; 6. Purifying the nucleic acid from the lysate; at the same time, it is possible to concentrate the nucleic acid to a smaller volume; 7. Purifying the nucleic acid before amplification; 8. Purifying after amplification; 9. Purifying before sequencing; 10. Sequencing; 11. Purification after sequencing (eg, removal of uncombined dye-labeled terminators and ions that interfere with electrophoresis); 12. Nucleic acid isolation; 13. Nucleic acid detection; 14. Reverse transcription of RNA; 15. Pre-transcriptional purification; 16. Post-transcriptional purification; 17. Nucleic acid ligation; 18. Nucleic acid quantification; 19. Nucleic acid hybridization; and 20. Nucleic acid amplification (eg, PCR, rolling circle amplification, strand displacement amplification, and multiples) Replacement amplification).

One of many methods that can combine some of these processes is an integrated system for human identification by STR analysis. Such systems may require DNA extraction, human-specific DNA quantification, addition of quantitative DNA to PCR reactions, multiplex PCR amplification, and separation and detection (and, where appropriate, purification steps to remove reaction components or primers) ). One or more samples of whole blood, dried blood, cheek inner surface, fingerprints, sexual assault, contact or other forensic related samples may be collected by techniques such as wiping (see Sweet et al, J. Forensic Sci. 1997, 42,320-2). Exposure to lysate (as appropriate in the presence of agitation) releases DNA from the swab into the tube.

B. General description of integrated components and their uses 1. Sample collection and initial processing

For many applications, the following discrete components are advantageously integrated into a biochip: sample insertion, removal of foreign matter, removal of interfering nucleic acids, and concentration of cells of interest. In general, the biochip pretreatment assembly accepts samples for granules and contains The initial removal of the cells from the nucleic acid and concentration of the cells of interest to a smaller volume. One method is to perform a dissolution and extraction step using a sample tube that can hold a swab (eg, like a "Q-tip") and is filled with a dissolution solution. The swab can be placed in contact with several cell-containing sites (including blood, fingerprints, water, air filters) or clinical sites (eg, buccal swabs, wound swabs, nasal swabs). The interface of such tubes to other components of the biochip can include filters for removing foreign matter. Another method is to use a large volume of blood or environmental sample collection cartridge that processes 1-100 mL of sample. In the case of blood, the white blood cell reducing medium can remove human leukocytes and interfere with DNA when passing through a microorganism containing the nucleic acid of interest. For environmental samples, large mesh filters can be used to remove dust and stains, while small mesh filters (eg <20μm, <10μm, <5μm, <2.5μm, <1μm, <0.5μm, < A 0.2 μm, <0.1 μm filter can be used to trap microorganisms and concentrate them into small volumes. These pre-treatment components can be separate consumables or attached to the integrated wafer at the time of manufacture. Alternatively, the biowafer can be designed for differential solubilization to separate cells according to type (eg, sperm from vaginal epithelial cells or red blood cells from bacteria).

2. Dissolution and extraction

Various dissolution and extraction methods can be used. For example, one typical procedure involves applying heat after mixing the sample with a small amount of a degrading enzyme, such as Protease-K, which breaks down the cell wall and releases the nucleic acid. Other methods available are sonication and ultrasonic treatment, either or both of which are sometimes performed in the presence of beads.

For example, a sample containing 10 ⁶ cells or 10 ⁶ or less cells can be dissolved and extracted. Depending on the application, a smaller number of starting cells can be used in the biochip and method of the invention, less than 10 ⁵ , less than 10 ⁴ , less than 10 ³ , less than 10 ² , less than 10, and in the case where multiple copies of the sequence are to be analyzed, less than one.

3. Purification of nucleic acids

One form of nucleic acid purification can be obtained by inserting a purification medium between an input channel and an output channel. This purification medium can be based on cerium oxide fibers and uses a chaotropic-salt reagent (chaotropic-salt reagent) to dissolve a biological sample, expose DNA (and RNA) and bind DNA (and RNA) to the purification medium. The lysate is then passed through a purification medium via an input channel to bind the nucleic acid. The bound nucleic acid is washed by an ethanol based buffer to remove contaminants. This can be accomplished by flowing the wash reagent through the purification membrane through the input channel. The bound nucleic acid is then eluted from the membrane by flow of a suitable low salt buffer (e.g., Boom U.S. 5,234,809). A variation of this method involves the use of solid phases of different configurations. For example, silicone can be used to bind nucleic acids. Paramagnetic ceria beads can be used and fixed to the channel or chamber wall by their magnetic properties during the bonding, washing and dissolving steps. Non-magnetized ceria beads can also be used, which are packed in a dense 'column' where it is held in a frit (usually made in the plastic of the device, but these can also be Inserted during assembly, or "free" during a particular phase of its operation. The free beads can be mixed with the nucleic acid and then flowed in the device relative to the frit or crucible to capture it so that it does not interfere with downstream processes. Other forms include sol-gels having cerium oxide particles distributed in a gel medium and polymer monomers having particles comprising cerium oxide, wherein the carrier is crosslinked for greater mechanical stability. Basically, any nucleic acid purification method that functions under conventional configurations can be applied to the integrated biochip of the present invention.

4. Nucleic acid amplification

Various nucleic acid amplification methods can be used, such as PCR and reverse transcription PCR, which require thermal cycling between at least two temperatures and more typically three temperatures. Isothermal methods such as strand displacement amplification can be used, and multiple displacement amplification can be used for whole genome amplification. The teachings of the U.S. Patent Application Serial No. 08-318-US, entitled "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS", filed on the same day, is hereby incorporated by reference herein in ).

5. Nucleic acid quantitative

One method of quantification in the microfluidic format is based on real-time PCR. In this quantitative method, a reaction chamber is fabricated between the input channel and the output channel. The reaction chamber is coupled to a thermal cycler and an optical excitation and detection system is coupled to the reaction chamber to allow fluorescence from the reaction solution to be measured. The amount of DNA in the sample is related to the intensity of the fluorescence from the reaction chamber of each cycle. See, for example, Heid et al, Genome Research 1996, 6, 986-994. Other methods of quantification include the use of intercalating dyes such as picoGreen, SYBR or ethidium bromide before or after amplification, which can then be detected using fluorescence or absorbance.

6. Secondary purification

For STR analysis, multiplex amplified and labeled PCR products can be used directly for analysis. However, electrophoretic separation performance can be greatly improved by purifying the product to remove ions necessary for PCR but that interfere with separation or other subsequent steps. Similarly, purification following cycle sequencing or other nucleic acid processing may be suitable. In general, any purification step following the initial extraction or purification of the nucleic acid can be considered as secondary purification. Various methods can be used, including supersonic processing, in which a small ion/introduction/unincorporated dye label is driven through the filter to leave the desired product on the filter, which product can then be lysed and applied directly to the separation or subsequent In the module. Ultrafiltration media include polyether oxime and regenerated cellulose "woven" filters, as well as trajectory erosive films (where highly uniform sized pores are formed in very thin (1-10 μm) films). The latter has the advantage of collecting products of a size larger than the size of the pores on the surface of the filter rather than capturing some of the product under the surface. The amplified nucleic acid can also be purified using the same method as described above (i.e., typical cerium oxide solid phase purification). Other methods include the use of hydrogels, crosslinked polymers having the property of pore size variability, i.e., the size of the pores varies in response to environmental variables such as heat and pH. In one case, the pores are dense and the PCR product cannot pass. As the pores expand, the hydrodynamic or electrophoretic flow of the product may pass through the pores. Another method is to use hybridization, the product non-specifically hybridizing to random DNA or specific hybridization immobilized on a surface (such as the surface of the beads) (wherein the complement of the sequence tag on the product is on a solid surface). In this method, the product of interest is immobilized via hybridization and the unwanted material is removed by washing; the heated duplex is then heated to melt and the purified product is released.

7. Cycle sequencing reaction

Typical cycle sequencing requires thermal cycling, almost identical to PCR. A preferred method is to use a dye-labeled terminator such that each extension product carries a single fluorescent label corresponding to the final base of the extension reaction.

8. Injection, separation and detection

The injection, separation and detection of the labeled nucleic acid fragments in the electrophoresis channel can be performed in various ways, which has been filed on the same day entitled "PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS" (given agent file number 07-865- The disclosure of U.S. Patent Application Serial No. is hereby incorporated by reference in its entirety in its entirety in its entirety in its entirety in its entirety in First, a cross-injector as discussed therein can be used to inject a portion of the sample. In an alternate embodiment, electric injection ("EKI") can be used. In either case, further concentration of the sequencing product near the open end of the loading channel (in the case of cross-injection) or the separation channel (in the case of EKI) can be performed by electrostatically concentrating the product in the vicinity of the electrode. A two electrode sample well on the electrophoresis portion of the wafer is shown in FIG. Both electrodes are coated with a permeable layer that prevents DNA from contacting the electrode metal but allows ions and water to enter between the sample well and the electrode. The permeable layers may be formed from crosslinked polyacrylamide (see U.S. Patent Application Publication No. US 2003-146145-A1). The electrode furthest from the channel opening is the separation electrode, and the electrode closest to the channel opening is the counter electrode. By positively charging the counter electrode relative to the separation electrode, the DNA is drawn to the counter electrode and concentrated near the opening of the separation channel. The concentrated product is electrically injected by floating the counter electrode and using a separate electrode and anode injection at the distal end of the separation channel.

C. Integration method

Biochips also contain several different components for integrating functional modules. Such components involve the transport of liquid from one point to another on the biochip, the flow rate dependent process for flow rate dependent processes (eg, certain washing steps, particle separation and dissolution), and the time of fluid movement on the gated biochip. And spacing (eg, via the use of some form of valve), and mixing of fluids.

Various methods can be used for fluid transport and controlled fluid flow. One method is positive displacement pumping in which the plunger driving fluid in contact with the fluid or intervening gas or fluid moves a precise distance during movement based on the volume displaced by the plunger. An example of such a method is a syringe pump. Another method is to use an integrated elastic membrane that is pneumatically, magnetically actuated, or otherwise actuated. Separately, such membranes can be used as valves to accommodate fluids in defined spaces and/or to prevent premature mixing or transfer of fluids. However, when used in series, such membranes can form a pump similar to a peristaltic pump. By simultaneous, continuous actuation of the membrane, the fluid can be "pushed out" from its rear side because the membrane on the front side is opened to accept the moving fluid (and any displacement air in the passage of the evacuation device). A preferred method for actuating such membranes is pneumatic actuation. In such devices, the biochip is comprised of a fluid layer, at least one of which has a membrane, one side of which is exposed to the fluid passages and chambers of the device. The other side of the membrane is exposed to a pneumatic manifold layer perpendicular to the pressure source. The membranes are opened or closed by application of pressure or vacuum. The valve can be changed under pressure or vacuum application using a valve that is normally open or normally closed. It is noted that any gas can be used for actuation because the gas is not in contact with the fluid under analysis.

Another method of driving the fluid and controlling the flow rate is to apply a vacuum or pressure directly on the fluid itself by changing the pressure at the meniscus, the meniscus, or the two meniscuses before the fluid. Appropriate pressure is applied (typically in the range of 0.05-3 psig). The flow rate can also be controlled by adapting the size of the fluid passage, since the flow rate is proportional to the pressure difference across the fluid and the fourth power of the hydraulic diameter, and inversely proportional to the length of the passage or liquid plug and its viscosity. .

Fluid gates can be achieved using a variety of active valves. The foregoing may include a piezoelectric valve or solenoid valve that may be incorporated directly into the wafer, or applied to the biochip to communicate the helium on the main wafer body with the valves, direct fluid into the valves, and then return to the wafer. . One disadvantage of these types of valves is that for many applications it can be difficult to manufacture and too expensive for incorporation into disposable integrated devices. As mentioned above, the preferred method is to use a membrane as a valve. E.g A membrane actuated by 10 psig can be used to successfully accommodate the fluid subjected to PCR.

In some applications, a capillary microvalve (which is a passive valve) may be preferred. Basically, the microvalve is tightened in the flow path. In microvalves, surface energy and/or geometric features such as sharp edges can be used to prevent flow when the pressure applied to the fluid is below a critical value known as burst pressure, which is often given by the following relationship: P _valve α(γ/d _H )*sin(θ _c ), where γ is the surface tension of the liquid, d _H is the hydraulic diameter of the valve (defined as 4×(sectional area)/section perimeter), and θ _c is The contact angle of the liquid to the valve surface.

Preferred properties for passive valves for certain applications include: very low dead volume (usually in the picoliter range) and small physical extent (each is only slightly larger than the access valve and the exit valve) Channel). The small physical range allows for a high density valve on a given surface of the biochip. In addition, some capillary valves are very easy to manufacture and consist essentially of small holes in a surface treated or untreated plastic sheet. Proper use of capillary valves reduces the total number of membrane valves required, simplifies overall manufacturing and creates a robust system.

There are two types of capillary valves constructed in the device of the present invention: in-plane valves, wherein the small passages and sharp corners of the valve are formed by forming a "groove" in one layer and joining the layer to a featureless cover (usually a device) Formed by another layer; and a via valve in which a small (typically 250 μm or less) hole is fabricated in the intermediate layer between the two fluid carrying layers of the device. In both cases, fluoropolymer treatment can be used to increase the contact angle of the fluid to the valve contact.

Figure 7 shows the valve modulating efficiency of these valves as a function of valve size for the liquid of interest (i.e., deionized water and cycle sequencing reagent) in the case of fluoropolymer treatment. In both cases, the expected dependence of the valve pressure on the valve size (pressure about 1/diameter) was observed. Through-hole valves have the advantage of being significantly superior to in-plane valves. First, it is easy to manufacture because the small through holes can be easily formed in the plastic sheet by molding around post, perforating, die cutting, drilling or laser drilling after the valve layer is fabricated. In-plane valves require fairly precise manufacturing, and extremely fine valves (with high valve pressure) must use lithography to create the required Type or embossing tool. Secondly, the through-hole valve can be coated more completely with fluoropolymer on the "all sides". Application of a low surface tension fluoropolymer solution to the pores results in complete coating of the inner wall of the pore by capillary action. All sides of the in-plane valve need to be applied to the valve and to the area of the matching layer on the valve. Thus, a typical in-plane valve is formed without the "top" of the valve being coated.

In machined prototypes, the through-hole valve is both easy to implement and exhibits a large valve-regulating pressure, as shown in FIG.

Mixing can be achieved in a variety of ways. First, the fluid can be mixed by diffusion by co-injecting two fluids into a single channel, wherein the channel typically has a small lateral dimension and a sufficient length to satisfy the diffusion time at a given flow rate: t _D = (width ) ² / (2 × diffusion constant)

Unfortunately, such mixing is often insufficient for fast mixing of large volumes because the diffusion or mixing time is proportional to the square of the channel width. Mixing can be enhanced in a variety of ways, such as lamination, where the fluid stream is separated and recombined (Campbell and Grzybowski Phil. Trans.R. Soc. Lond. A 2004, 362, 1069-1086); or by using fine microstructures in the flow A disordered advection is formed in the channel (Stroock et al., Anal. Chem. 2002, 74, 5306-5312). In systems using active pumps and valves, mixing can be achieved by multiple cycles of fluid between the two points on the device. Finally, the latter can also be achieved in systems using capillary valves. A capillary valve disposed between two channels or chambers serves as a pivot for fluid flow; when fluid flows from one channel to another via a capillary, if a sufficiently low pressure is used to draw fluid, the rear meniscus is intercepted. The reversal of pressure drives the fluid back into the first passage and again causes it to stop at the capillary. Multiple cycles can be used to effectively mix the ingredients.

The method of separation and detection in a microfluidic format is described in the U.S. Patent Application entitled "PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS" (Attorney Docket No. MBHB 07-865-US), filed on the same day, the entire The manner of reference is incorporated herein (see, for example, paragraph 68- 79, 94-98).

The top half of Figure 13 shows the construction of an integrated biochip ( 1301 ) from two components that are joined during manufacture or during manufacture. The first component is a 16-sample biochip ( 1302 ) that combines the dissolution, amplification, and sequencing features of the biochip of Figure 1 with the sequencing product purification features of the biochip of Figure 11, and the second component is a 16-channel plastic separation. Biochip ( 1303 ). The purified sequencing product can also be electroporated prior to separation.

D. Manufacturing method

The device of the present invention can be constructed primarily of plastic. Types of plastics that may be used include, but are not limited to, cyclic olefin polymers (COP); cyclic olefin copolymers (COC); (when having sufficient molecular weight, both have excellent optical properties, low hygroscopicity, and high handling Temperature); poly(methyl methacrylate) (PMMA) (easy to process and obtain excellent optical properties); and polycarbonate (PC) (highly formable with good impact resistance and high operating temperature) . Further information on materials and methods of manufacture is contained in U.S. Patent Application entitled "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS" (Attorney Docket No. 08-318-US), the entire disclosure of which is incorporated by reference. Incorporated herein (as described above).

Various methods can be used to fabricate individual parts of the biochip and assemble them into the final device. Because the biowafer can be constructed from one or more types of plastic, it is possible to include an insert assembly, the method of interest being related to the manufacture of individual parts, followed by post-part processing and assembly.

Plastic components can be fabricated in a number of ways, including injection molding, hot stamping, and machining. The injection-molded part can be constructed from general features such as fluid reservoirs and fine features such as capillary valves. In some cases, it is preferred to make fine features on one set of parts and to make larger features on the other set, as the method of injection forming of such differently sized features may vary. For large reservoirs (measured on one side (approximately 1-50 mm) mm and depths of several millimeters (approximately 1-10 mm) and capable of accommodating hundreds of microliters) A conventional injection molding tool or a tool manufactured by burning a graphite electrode into steel or other metal using a conventional electrode formed into a negative electrode of the tool.

For fine features, tool manufacturing and forming processes can vary. Tools are typically fabricated on a substrate of interest using a lithography process (eg, isotropic etching of glass, or deep reactive ion etching or other processes on the crucible). The substrate can then be plated with nickel (typically after deposition of the chrome layer to promote adhesion) and the substrate removed, for example by etching in an acid. This nickel "child" coating is an injection molding tool. The forming process can also be slightly different from the above. For fine, narrow features, it has been found that compression injection molding (where the mold is slightly physically compressed after the plastic is injected into the cavity) is better than standard injection molding in terms of fidelity, precision and reproducibility.

For hot stamping, similar issues regarding the overall and fine features described above are controlled and the tool can be fabricated as described above. In hot stamping, the plastic resin may be applied to the tool surface or a flat substrate in the form of pellets or as preforms formed by forming or stamping. The second tool can then be contacted at a precisely controlled temperature and pressure to raise the plastic temperature above its glass transition temperature and cause the material to flow to fill the cavity of the tool. Imprinting under vacuum avoids the problem of air trapping between the tool and the plastic.

Machining can also be used to make parts. Many individual parts can be manufactured daily from self-forming, extrusion or solvent-cast plastic using a high-speed computer numerical control (CNC) machine. Reasonable selection of milling machines, operating parameters and cutting tools for high surface quality (50nm surface roughness at high speed milling of COC (Bundgaard et al., Proceedings of IMechE Part C: J.Mech.Eng.Sci. 2006) , 220, 1625-1632)). Milling can also be used to create geometries that may be difficult to shape or imprint and to easily mix feature sizes on a single part (eg, large reservoirs and fine capillary valves can be machined into the same substrate). Another advantage of milling over forming or stamping is the removal of the finished part from the forming tool without the use of a release agent.

Post processing of individual parts includes optical inspection (which can be automated), cleaning operations to remove defects such as burrs or hanging plastics, and surface treatment. If an optical quality surface is required in the processed plastic, solvent vapor polishing suitable for plastics can be utilized. For example, for PMMA, dichloromethane can be used, and for COC and COP, cyclohexane or toluene can be used.

Surface treatment can be applied prior to assembly. Surface treatment can be performed to promote or reduce wetting (i.e., to alter the hydrophilicity/hydrophobicity of the part); to inhibit the formation of bubbles within the microfluidic structure; to increase the valve pressure of the capillary valve; and/or to inhibit protein adsorption to the surface. The coating that reduces wettability includes a fluoropolymer and/or a molecule having a fluorine moiety, wherein the fluorine moiety can be exposed to the liquid when the molecule is adsorbed or bound to the surface of the device. The coating can be adsorbed or deposited, or it can be covalently bonded to the surface. Methods that can be used to make such coatings include dip coating, transfer of coating reagents through channels of the assembled device, inking, chemical vapor deposition, and inkjet deposition. The covalent bond between the coated molecule and the surface can be formed by treatment with oxygen or other plasma or UV-ozone to form an active surface, and then the surface treated molecules are deposited or co-deposited onto the surface (see Lee et al. Human, Electrophoresis 2005, 26, 1800-1806; and Cheng et al, Sensors and Actuators B 2004, 99, 186-196).

Assembly of component parts in the final device can be done in a variety of ways. An insertion device such as a filter can be die cut and then placed with a picker.

Thermal diffusion bonding can be used, for example, for joining two or more layers of the same material, with each layer being of uniform thickness. In general, the parts can be stacked and placed in a hot press where the temperature can be increased to near the glass transition temperature of the material containing the parts to cause interfacial fusion between the parts. One of the advantages of this method is that the joint is "general", that is, regardless of the internal structure of the layer, any two stacks of layers of substantially the same size can be joined because the heat and pressure can be uniformly Applied to the layers.

Thermal diffusion bonding can also be used to join more complex parts by using specially fabricated splice brackets, such as those that are not planar on the joint or opposite surface. The brackets and desires The surface of the joined layer is uniform.

Other bonding variants include solvent assisted thermal bonding in which a solvent such as methanol partially dissolves the plastic surface to enhance bonding strength at lower bonding temperatures. Another variation is the use of a spin coating layer of a low molecular weight material. For example, a polymer having the same chemical structure as the substrate composition but having a lower molecular weight may be spin-coated on at least one of the layers to be joined, the components are assembled, and the resulting stack is joined by diffusion bonding. During the thermal diffusion bonding process, the low molecular weight component can undergo a glass transition temperature at a temperature lower than the components and diffuse into the substrate plastic.

Adhesives and epoxies can be used to join different materials and adhesives and epoxies are likely to be used when manufacturing the joint components in different ways. The adhesive film can be die cut and placed on the assembly. The liquid adhesive can also be applied via spin coating. Adhesives can be successfully applied to the structured part by ink (such as in nanocontact printing) to apply the adhesive to the structured surface without the need to "guide" the adhesive to a particular area.

In one example, the biowafer of the present invention can be assembled as shown in FIG. Layers 1 and 2 can be aligned by included features such as pins and sockets; respectively, layers 3 and 4 can be similarly aligned by the included features. The stack of layer 1 plus layer 2 can be inverted and applied to the stack of layer 3 plus layer 4 and can then be combined into a whole stack.

E. Example Example 1 Integrated biochip for nucleic acid extraction and amplification

An integrated biochip for DNA extraction and PCR amplification is shown in FIG. The 4-sample device incorporates the following functions: reagent dispensing and metering; mixing of reagents and samples; transfer of sample to thermal cycling of the wafer; and thermal cycling. The same biochip was used in Example 2 below and had additional structure for cycle sequencing performance.

The biochip was constructed from 4 layers of thermoplastic as shown in Figures 2-5. The four layers are processed PMMA and have layer thicknesses of 0.76 mm, 1.9 mm, 0.38 mm, and 0.76 mm, respectively. Degree, and the side dimension of the biochip is 124 mm x 60 mm. In general, three or more layers of biochips allow the use of an unlimited number of common reagents dispensed between multiple assays: two fluid layers and a layer containing at least a via such that the fluid channels in the outer layer can each other 'cross'. (It should be recognized that there are special circumstances - such as the use of only one common reagent in multiple samples - making a three layer construction not necessary). Four layers were chosen to be compatible and fully integrated with wafer construction for other functions such as ultrasonic processing, Example 3 (Example 4).

The cross-sectional dimension of the channel of the biochip is in the range of 127 μm × 127 μm to 400 μm × 400 μm, and the section of the reservoir is in the range of 400 μm × 400 μm to 1.9 × 1.6 mm; the distance between the channel and the reservoir is 0.5 mm to several tens Mm is short. The capillary valve used in the biochip is that the "in-plane" valve has a size of 127 μm × 127 μm and the through-hole capillary valve has a diameter of 100 μm.

Some of the four processing layers, reservoirs, and capillary valves were treated with a hydrophobic/oleophobic material PFC 502A (Cytonix, Beltsville, MD). The surface treatment was carried out by coating with a wet Q-tip and then by air drying at room temperature. The dried fluoropolymer layer was found to have a thickness of less than 10 μm by optical microscopy. Surface treatment is used for two purposes: to prevent the formation of bubbles in the liquid, especially in low surface tension liquids such as cycle sequencing reagents (this may occur when the liquid rapidly wets the channel or the wall of the chamber (and can be replaced in air) The front "closed" bubble)); and enhances the capillary burst pressure at the capillary valve against liquid flow. The untreated area is a thermal cycling chamber for PCR and cycle sequencing.

After the surface treatment, the layers are joined as shown in FIG. The connection is made using a thermal diffusion connection in which the stack of components is heated under pressure to a temperature close to the glass transition temperature ( _Tg ) of the plastic. Applying 45 pounds (lbs) of force over the entire 11.5 square inch biowafer during a thermal connection profile consisting of increasing ambient temperature to 130 ° C, holding at 1300 ° C for 7.5 minutes, and rapidly cooling to room temperature 15 minutes.

Pneumatic instruments are developed for driving fluids within the biochip of the present invention. Two small peristaltic pumps provide pressure and vacuum. The positive pressure output is distributed between three regulators having a range of about 0.05-3 psig. The vacuum was transferred to a regulator having an output vacuum of about (-0.1)-(-3) psig. Transfer the fourth, higher pressure from the N ₂ cylinder or from the high capacity pump to the other regulator. Apply positive or negative pressure to a series of 8 pressure selector modules. Each module is equipped with a solenoid valve that selects the output pressure to be transmitted to the biochip from the five inputs. The output pressure line terminates at at least one pneumatic interface. The interface is held on the wafer by an O-ring on the wafer cassette on the input side of the wafer (the ridge is along the top of the features).

Additional solenoid valves (ie, gate valves; 8 per interface) from the output pressure line of the pressure selector module are just on the biochip. These valves, which are very close to the wafer, provide a low dead volume interface (about 13 [mu]L) between the pressure line and the wafer. When a pressure is applied to move other liquids, the low dead volume interface prevents unintentional movement of certain fluids on the biochip (eg, small gas volume between the liquid plug and the shut-off valve due to gas compression when pressure is applied) Determine the maximum amount of pluggable.) All pressure selector valve and the valve train based LabView ^TM instruction of the program operated under computer control. An important feature of this system is that short pressure cycle times are possible. Some fluid control events can be performed that require pressure pulses as short as 30 milliseconds, and/or complex pressure profiles can be used, where pressure can be quickly changed from one value to another (ie, one regulator to another) (That is, the time lag does not exceed 10-20 milliseconds).

Samples of about ¹⁰⁶ cells / mL pGEM sequencing by mass of insertion (target pUC18 sequencing) Transformation of Escherichia coli (E.coli) DH5 bacteria suspension composition. The PCR reagent consisted of dNTP KOD Taq polymerase (Novagen, Madison, WI) (concentration 0.1 μM).

A sample of 1.23 μL of the bacterial suspension was added to each of the four crucibles 104 , each of which formed vias 202 and 336 in layers 1 and 2, respectively. The sample is then present in sample channel 303 in layer 2. Next, 10 μL of the PCR reagent was added to the crucible 105 , which constitutes the vias 217 and 306 in the layers 1 and 2. The PCR reagent is then present in chamber 307 in layer 2 (see Figure 8a). The enthalpy for venting the replacement air for the PCR reagent is 埠107, which constitutes 109 and the via 203 + 305 .

In operation, air displaced by the sample and downstream processes (such as metering of reagents, mixing of fluids) is discharged through a crucible 108 on the output end of the wafer, which is formed by vias 227 . The final volume of the PCR reaction can be increased or decreased as desired.

The biochip is placed in the aerodynamic manifold described above. Perform the following automated pressure profile with no delay between steps. Unless otherwise stated, the pneumatic interface valve (corresponding to the enthalpy along the input side of the wafer) is closed during all steps.

A pressure of 0.12 psig was applied to the crucible 104 for 15 seconds to drive the sample along the channel 303 to the via 304 . The sample passes through the via 304 and appears on the other side of layer 2 in the sample chamber 204 of layer 1 and is driven to the first hybrid junction 205 . At the first mixing junction, the sample is held by capillary valve 210 (see Figures 8b-c).

A pressure of 0.12 psig was applied to 埠105 for 10 seconds to drive the PCR reagent through the via 320 . The PCR reagent appears on the other side of layer 2 in the distribution channel 208 and moves into the metering chamber 209 , which defines a reagent volume equal to the sample volume, wherein it is retained by the capillary valve 211 at the mixing junction 205 . (See Figure 8d).

A pressure of 0.12 psig was applied to crucible 107 (consisting of vias 203 and 305 ) (where 埠105 is open to the atmosphere) for 3 seconds to evacuate channel 208 (see Figure 8e).

A pressure of 0.8 psig was applied to 埠107 and 105 for 0.03 seconds while a pressure of 0.7 psig was applied to 埠104 for 0.03 seconds to allow sample and PCR to pass through the capillary valves 210 and 211 and through the valves. Initial mixing of reagents (see Figure 8f).

A pressure of 0.12 psig was applied to helium 104 and 107 for 10 seconds to pump the sample and PCR reagent into mixing channel 214 and retained at capillary valves 210 and 211 . The incorporation of the conical portion 212 into the constricted portion 213 constitutes additional hydraulic resistance to the flow, thereby reducing the high velocity imparted by the high pressure pulses described above.

A pressure of 0.7 psig was applied to the crucibles 104 and 107 for 0.03 seconds to separate the liquid from the capillary valves 210 and 211 (see Figure 8g).

A pressure of 0.12 psig was applied to the crucibles 104 and 107 for 3 seconds to pump liquid through the mixing channel 214 to the capillary valve 219 where the liquid was retained (see Figure 8h).

A pressure of 0.7 psig was applied to 埠104 and 107 for 0.1 second to drive the mixture of sample and PCR reagent through vias 315 and 402 and through the bodies of layers 2 and 3 into PCR chamber 502 (see Figure 8i).

A pressure of 0.12 psig was applied to helium 104 and 107 for 3 seconds to effect pumping of the mixture of sample and PCR reagent into chamber 502 . The leading edge of the mixture of sample and PCR reagent is then passed through the vias 403 and 316 , appearing in layer 1 and blocked at capillary valve 220 (see Figure 8j).

The biowafer was then pressurized to 30 psig N ₂ and subjected to thermal cycling using a gas bladder compression mechanism via a Peltier effect for PCR amplification, as claimed in the present application entitled "METHODS FOR RAPID" US Patent Application for MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS" (Attorney Profile Number 08-318-US) and International Patent entitled "DEVICES AND METHODS FOR THE PERFORMANCE OF MINIATURIZED IN VITRO ASSAYS", filed on February 6, 2008 The entire disclosure of each of these applications is hereby incorporated by reference in its entirety in its entirety in its entirety in its entirety in the the the the the the the the the the the

The selected sample, reagent volume, and PCR chamber size are such that the liquid fills the area between valve 219 and valve 220 . Thus, the liquid/vapor interface of a small cross-sectional area (typically 127 [mu]m x 127 [mu]m) is located about 3 mm from the bottom surface of the thermal cycle of layer 4. The application of pressure during the thermal cycle inhibits the degassing of dissolved oxygen in the sample. The small cross-sectional area of the liquid/vapor interface and the distance from the Peltier surface inhibit evaporation.

The temperature at the top of the biowafer observed during the cycle never exceeds 60 ° C, and thus the vapor pressure at the liquid/vapor interface is significantly lower than if the interface would have a vapor pressure in the PCR chamber. For a 2 [mu]L sample, where 1.4 [mu]L was in chamber 502 and the remainder in vias and capillary valves, the amount of evaporation observed in 40 cycles of PCR was less than 0.2 [mu]L. The uncirculated fluid volume (0.6 μL in this case) can be reduced by selecting a smaller diameter through hole.

Perform PCR using the following temperature profiles:

○ Heat the bacteria for 3 minutes at 98 ° C

○ 40 cycles of the following items:

○ Denaturation at 98 ° C for 5 seconds

○ Anneal at 65 ° C for 15 seconds

○ Extend at 72 ° C for 4 seconds

○ Final extension at 72 ° C (2 minutes)

The PCR product was recovered by flushing chamber 502 with about 5 [mu]L of deionized water and analyzed by slab gel electrophoresis. The PCR yield is as high as 40 ng/reaction, much more than the amount required for subsequent sequencing reactions. In the present application, bacterial nucleic acids are produced only by lysing bacteria. The nucleic acid can be subjected to the desired purification, which can also improve the efficiency of amplification, sequencing and other reactions.

Example 2

Integrated biochip for cyclic sequencing reagent dispensing, mixing with PCR products, and cycle sequencing

A biochip as described in Example 1 was used. The PCR product produced in the test tube using the protocol described in Example 1 was added to the sample of the biochip as described above and the PCR reagent. The 50μL cycle sequencing reagents ^{(BigDye TM 3.1 / BDX64, MCLab} , San Francisco) was added to port 106 (composed of a through hole 308, and 215), and chamber 309. After installing two pneumatic interfaces (one for the wafer input and one for the wafer output), the PCR product was processed as described in Example 1 up to the PCR chamber, but without the PCR thermal cycling step. The treatment of the fluid in the wafer is shown in Figure 9a.

The following pressure profiles were performed using a pneumatic system software; all solenoid valves corresponding to the wafer cassette were closed unless otherwise stated:

1. Apply a pressure of 0.1 psig to 埠106 (where 埠109 is open to the atmosphere) for 10 seconds to pump the cycle sequencing reagent into channel 310 (see Figure 9b).

2. Apply a pressure of 0.7 psig to 埠106 and 108 for 0.2 seconds (consisting of vias 216 and 314 ) to drive the cycle sequencing reagent from channel 304 via via 311 , via the body of layer 2, and onto layer 1. The cycle sequencing reagent is metered into chamber 218 (see Figure 9c).

3. Apply a pressure of 0.1 psig to 埠106 (where 埠109 is open to the atmosphere), drive the cycle sequencing reagent to capillary valve 221 , and hold the sequencing reagent in it (see Figure 9d).

4. Apply a pressure of 0.1 psig to 埠108 (where 埠106 is open to the atmosphere) for 1 second to drive the excess cycle sequencing reagent back into chamber 101 , leaving channel 310 empty (see Figure 9e).

5. Apply a pressure of 0.7 psig to 埠104 and 107 for 0.1 seconds (where 埠109 is open to the atmosphere) to drive the PCR product through capillary valve 220 and into via 317 through the body of layer 2 and the layer 3 The well 404 enters the loop sequencing chamber 503 of layer 4 (see Figure 9f).

6. Apply a pressure of 0.1 psig to 埠109 (where 埠104 and 107 are open to the atmosphere) for 5 seconds to drive the PCR sample back to the via. Capillary action holds the liquid at the entrance to the through hole, preventing trapped air bubbles from occurring between the PCR product and chamber 503 (see Figure 9g).

7. Apply a pressure of 0.7 psig to 埠108 (where 埠109 is open to the atmosphere) for 0.2 seconds to drive the cycle sequencing reagent into chamber 503 while applying 0.1 psig to 埠104 and 107 to allow PCR product Contact with the sequencing reagent (see Figure 9h).

8. Apply a pressure of 0.1 psig to 埠104 , 107, and 108 for 10 seconds (where 埠109 is open to the atmosphere) to drive the PCR product and the Sanger reagent into the chamber. The meniscus after the PCR product and the meniscus after the sequencing reagent are blocked by capillary valves 220 and 221 (see Figure 9i).

9. Apply a vacuum of 0.25 psig and five vacuum pulses of 0.1 second duration to 埠108 (where 埠109 is open to the atmosphere) to draw the two liquid portions back into the reagent metering chamber 218 (see Figure 9j).

10. Apply a pressure of 0.1 psig to 埠104 , 107, and 108 (where 埠109 is open to the atmosphere) for 10 seconds to pump the mixture back to chamber 503 , where the back meniscus is blocked from the capillary valve as in step 8. (See Figure 9k).

Additional steps 9-10 were repeated an additional step to effect mixing of the sequencing reagent with the PCR product.

The biochip is then pressurized to 30psig N ₂ and performing a thermal cycle using the following temperature profile:

● 95 ° C / 1 minute initial denaturation

○ 30 cycles of the following items

○ Denaturation at 95 ° C for 5 seconds

○ Annealing at 50 ° C for 10 seconds

○ Extend at 60 ° C for 1 minute

Sample (see FIG. 9L) system by ethanol precipitation and recovered and purified as described by (part II, Example 5) to said instrument Genebench ^TM electrophoresis and laser-induced fluorescence detection analysis. Phred quality analysis yielded 408 +/- 57 QV 20 bases/sample.

Example 3 Ultrafiltration in 4-sample biochips

Four layers of 4-sample biochips suitable for sequencing product purification were constructed as described in Example 1 and are shown in FIG. One additional component in the construction is an ultrafiltration (UF) filter 1116 that is cut to size and placed between layers 3 and 4 prior to thermal bonding. Layer 3 is necessary for the formation of a good connection around the UF filter. Layers 3 and 4 form an uninterrupted perimeter around the filter, since all channels leading to and exiting the filter are at the bottom of layer 2 (for example, in the case where the channel passes over the filter, directly in layer 2 with The connection between 4 results in a bad connection to the filter). In this example, a regenerated cellulose (RC) filter (Sartorius, Goettingen, Germany) with a molecular weight cut off (MWCO) of 30 kD was used. A variety of other MWCOs (10 kD, 50 kD, and 100 kD) have been tested when having the alternative material polyether oxime (Pall Corporation, East Hills, NY).

1. Add four samples of 10 [mu]L of cycle sequencing product produced in a tube reaction using the pUC18 template and KOD enzyme to the crucible 1104 in the first layer and drive it through the channel 1105 in the second layer to the chamber in the second layer. 1106 . 200 μL of deionized water was added to the crucible 1120 (through hole in the first layer) to the reservoir 1121 in the second layer. The biochip is then mounted between two pneumatic interfaces.

Use the pneumatic system software to perform the following pressure profiles. All solenoid valves corresponding to the biochip are closed unless otherwise stated.

2. Apply a pressure of 0.09 psig to 埠1104 (where 埠1119 is open to the atmosphere) for 5 seconds to drive the sequencing product to capillary valve 1108 in layer 1, where the sequencing product is retained.

3. Apply a pressure of 0.6 psig to 埠1104 (where 埠1119 is open to the atmosphere) for 0.1 second to expand the sample through capillary valve 1108 in layer 1 and pass it through layer 1111 in layer 2 into layer 2. UF input chamber 1112 .

4. A pressure of 0.09 psig was applied to 埠1104 (where 埠1119 was open to the atmosphere) for 10-30 seconds (different times were used in different experiments) to complete the transfer of the sequencing product to chamber 1112 . The sequencing product is retained by capillary valve 1113 in layer 2 (see Figures 12a and 12b).

5. Apply a pressure of 0.8 psig to 埠1124 (where 埠1119 and 1104 are open to the atmosphere) for 0.5 seconds to drive the sequencing product into valve chamber 1115 via valve 1113 . This also removes the input capillary valve 1108 that holds the liquid.

6. Apply a pressure of 0.09 psig to 埠1124 (where 埠1119 is open to the atmosphere) for 10-30 seconds to complete the transfer of the sequencing product to chamber 1115 . The sequencing product is maintained at valve 1113 (see Figure 12c).

7. Slowly apply a pressure of 7.5 psig to all of the crucibles used for ultrafiltration. During ultrafiltration, when the liquid is driven via filter 1116 , the sequencing product meniscus remains blocked at 1113 and the liquid leading edge "retracts". 10 μL of the sequencing product took about 120 seconds for filtration. The pressure is released after filtration (see Figures 12c and 12d).

8. Apply a pressure of 0.09 psig to 埠1120 (where 埠1124 is open to the atmosphere) for 3 seconds to drive water into channel 1122 (in layer 4) and partially fill overflow chamber 1123 (see Figure 12e).

9. Apply a pressure of 0.8 psig to 埠1120 and 1124 (where 埠1119 is open to the atmosphere) to drive water into chamber 1112 via via capillary valve 1110 in channel 1122 .

10. Apply a pressure of 0.09 psig to 埠1120 (where 埠1119 is open to the atmosphere) for 10-30 seconds to complete the transfer of liquid to chamber 1112 , which is held therein by valve 1113 (see Figure 12f).

11. Apply a pressure of 0.09 psig to the crucible 1124 (where the crucible 1120 is open) to drive the water in chamber 1123 and channel 1122 back to chamber 1121 (see Figure 12g).

12. Apply a pressure of 0.8 psig to 埠1124 (where 埠1119 and 1104 are open to the atmosphere) for 0.5 seconds to drive water through valve 1113 into filter chamber 1115 . This also removes the input capillary valve 1108 that holds the liquid.

13. Apply a pressure of 0.09 psig to 埠1124 (where 埠1119 is open to the atmosphere) for 10-30 seconds to complete the transfer of water to chamber 1115 . The sequencing product is maintained at valve 1113 (see Figure 12h).

As in step 6 above, the water is driven through the UF filter to complete the first wash. Repeat steps 8-13 for an additional iteration.

Steps 8-12 are repeated to partially fill chamber 1115 where the final volume of water is used for dissolution (see Figure 12k).

A vacuum of 1.6 psig was applied to crucible 1104 where all other crucibles were closed for 1 second and some water was drawn from chamber 1115 into chamber 1112 (maximum motion by the meniscus with liquid and the solenoid valve corresponding to 埠1119) The same order of magnitude of vacuum is determined by the space) (see Figure 12l).

The crucible 1104 is allowed to open to the atmosphere for 1 second due to the partial vacuum created between the liquid and the valve corresponding to the crucible 1119 , causing the liquid to move back to the chamber 1115 (see Figure 12m).

16-17 was repeated 50 times to produce 50 dissolution cycles.

A pressure of 0.09 psig was applied to 埠1124 for 10 seconds (where 埠1119 was open to the atmosphere) to drive the liquid such that the rear meniscus was blocked at 1113 .

A pressure of 0.7 psig/0.05 seconds was applied to 埠1124 (where 埠1119 was open to the atmosphere) to separate the lysate (see Figure 12n).

Samples were recovered and directly used as the operation of Genebench ^TM, generating up to 479 QV20 bases.

Example 4 Fully integrated biochip for nucleic acid extraction, template amplification, cycle sequencing, sequencing product purification, and electrophoretic separation and detection of purified products

Figure 13 illustrates an embodiment of a 16-sample biochip 1301 incorporating the dissolution and extraction, template amplification, and cycle sequencing functions of the biochip of Figure 1; ultrafiltration of the wafer of Figure 11; and electrophoretic separation and detection. The ultrafiltration process is performed by subassembly 1302 and can be performed as described in Examples 1, 2, and 3; the transfer point 1304 on the bottom surface of 1302 is aligned with the input aperture 1305 on the separation subassembly 1303 .

The injection is electrically performed using a counter electrode in a pre-concentration step. The input hole 1305 illustrated in FIG. 14 is composed of a liquid receiving hole 1401 , a main separating electrode 1402, and a counter electrode 1403 . The separation channel 1306 leads to the bottom of the hole reservoir 1401 . The separation electrode is typically coated with platinum or gold, and is preferably a planar gold plated electrode that generally covers 1, 3 or 4 of the inner surface of 1401 . The counter electrode is a thin gold, steel or platinum wire (typically 0.25 mm in diameter) which has been coated with a thin layer (about 10 [mu]m) of crosslinked polyacrylamide. This forms a hydrogel protective layer on the electrodes. On panel d, the purified sequencing product (black spots in 1401 ) can be transferred to the wells. A positive potential is applied between 1402 and 1403 , and the negatively charged sequencing product is directed to 1403 , as in panel cd. The hydrogel layer on 1403 prevents the sequencing product from contacting the metal electrode and thus preventing the electrochemical and damage of the sequencing product. The counter electrode 1403 is then floated relative to 1402 . A positive potential is then applied between the primary separation electrode 1402 and the anode (not shown) at the distal end of the separation channel 1306 . This allows product injection (panel e) and electrophoresis along 1306 for separation and detection (panel f). As shown in Figure 14, this flow allows the concentration of the sequencing product near the end of channel 1306 to be significantly increased relative to the sequencing product delivered from the ultrafiltration. While this concentration is ideal for some applications, it is not necessary in all cases. In such cases, the EKI can be directly performed using the holes of Figure 14 without the counter electrode 1403 . Alternatively, the single electrode in the loading well can be one-half of the cross-T or double-T injector (see, for example, "PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS", filed at the same time as the application. U.S. Patent Application Serial No. 07-865-US).

Separation occurs in the separation channel 1306 , and detection of laser-induced fluorescence occurs in the detection zone 1307 . In this biowafer, a recess 1308 is provided to match, for example, a Peltier block (not shown) to the low side of 1301 to provide thermal cycling for PCR and cycle sequencing. A pneumatic interface (not shown) within the instrument is clamped to the end of the wafer to provide microfluidic control.

II. DEPARATION and detection system A. Detailed description of the separation and detection components and their uses Separation instrument

DNA isolation is performed using a biochip and apparatus as described in U.S. Patent Application Publication No. US 2006-0260941-A1. The separation wafer may be glass (see U.S. Patent Application Publication No. US 2006-0260941-A1) or plastic (available at the same time as the present application entitled "Plastic Microfluidic separation and detection platforms", attorney file number 07-865-US U.S. Patent Application, the entire disclosure of which is incorporated herein by reference.

2. Excitation and detection instruments

The instrument includes an excitation and detection subsystem for interacting with the sample and interrogating the sample. Samples typically include one or more biomolecules (including but not limited to DNA, RNA, and proteins) labeled with a dye (eg, a fluorescent dye). The excitation subsystem includes an excitation source and an excitation beam path, wherein the optical element includes a lens, a pinhole, a mirror, and an objective lens to adjust and focus the excitation source in the excitation/detection window. Optical excitation of the sample can be accomplished by a series of laser types in which the emission wavelength is in the visible region between 400 and 650 nm. Solid state lasers provide emission wavelengths of approximately 460 nm, 488 nm, and 532 nm. Such lasers include, for example, Compass, Sapphire and Verdi products from Coherent (Santa Clara, CA). Gas lasers include emission at approximately 488 nm, 514 nm, 543 nm, 595 nm, and 632 Argon ions and helium lasers of visible wavelengths of nm. Other lasers having a visible region emission wavelength are commercially available from Crysta Laser (Reno, NV). In one embodiment, a 488 nm solid state laser Sapphire 488-200 (Coherent, Santa Clara, CA) can be used. In another embodiment, a source having a wavelength in excess of the visible range can be used to excite a dye (eg, an infrared or ultraviolet emitting dye) having an absorption and/or emission spectrum that exceeds the visible range. Alternatively, optical excitation can be obtained by using a non-laser source (including a light-emitting diode and a lamp) having an emission wavelength suitable for dye excitation.

The detection subsystem includes one or more optical detectors, a wavelength dispersion device (which performs wavelength separation), and one or a series of optical components, including but not limited to lenses, pinholes, mirrors, and The objective lens collects the emitted fluorescence from the fluorophore-labeled DNA fragments present at the free excitation/detection window. The emitted fluorescence can be from a single dye or combination of dyes. To discern the signal to determine its contribution from the emissive dye, wavelength separation of the fluorescence can be used. This can be achieved by using a dichroic mirror and a belt pass filter element (available from a number of suppliers including Chroma, Rockingham, VT and Omega Optical, Brattleboro, VT). In this configuration, the emitted fluorescence passes through a series of dichroic mirrors in which one of the wavelengths will be mirrored to continue along the path while the other portions will pass. A series of discrete photodetectors (each positioned at the end of the dichroic mirror) will detect light of a specific range of wavelengths. A band pass filter can be positioned between the dichroic mirror and the photodetector to further narrow the wavelength range before detection. Optical detectors that can be used to detect wavelength-separated signals include photodiodes, crash photodiodes, photomultiplier modules, and CCD cameras. Such optical detectors are commercially available from suppliers such as Hamamatsu (Bridgewater, NJ).

In one embodiment, the wavelength components are separated by using a dichroic mirror and a belt pass filter, and the wavelength components are detected by a photomultiplier tube (PMT) detector (H7732-10, Hamamatsu). The dichroic mirror and the bandpass assembly can be selected such that the incident light on each of the PMTs consists of a narrow wavelength band corresponding to the emission wavelength of the fluorescent dye. The band pass is typically centered around a fluorescent emission peak having a bandpass having a wavelength range between 1 and 50 nm. The system can enter Eight color detections are available and can be designed with eight PMTs and a corresponding set of dichroic mirrors and belt pass filters to split the emitted fluorescence into eight different colors. More than eight dyes can be detected by applying additional dichroic mirrors, belt pass filters and PMT. Figure 15 shows the beam path of a discrete band pass filter and dichroic filter embodiment. One integrated form of this wavelength discrimination and detection configuration is H9797R, Hamamatsu, Bridgewater, NJ.

Another method of identifying dyes that constitute fluorescent signals includes the use of, for example, ruthenium, diffraction gratings, transmission gratings (available from ThorLabs, Newton, NJ; and Newport, Irvine, CA); and Wavelength dispersive elements and systems (available from a number of suppliers including Horiba Jobin-Yvon, Edison, NJ). In this mode of operation, the wavelength components of the fluorescence are dispersed in the physical space. The detector elements placed along the physical space detect light and correlate the physical location of the detector elements to the wavelength. Detectors suitable for this function are array-based and include multi-element photodiodes, CCD cameras, back-end thin CCD cameras, multi-anode PMTs. Those skilled in the art will be able to apply a combination of wavelength dispersive elements and optical detector elements to produce a system that is capable of discerning wavelengths of dyes used in the system.

In another embodiment, a spectrograph is used in place of the dichroic and band pass filter to separate the wavelength components from the self-excited fluorescence. Details of the spectrograph design are available in John James, Spectrograph Design Fundamental , Cambridge, UK: Cambridge University Press, 2007. A spectrograph P/N MF-34 (P/N 532.00.570) having a concave hologram grating with a spectral range of 505-670 nm (HORIBA Jobin Yvon Inc, Edison, NJ) was used in the present application. Detection can be done with a linear 32-element PMT detector array (H7260-20, Hamamatsu, Bridgewater, NJ). The collected fluorescence is imaged, reflected, dispersive, and imaged by a concave holographic image on a pinhole, mounted on a linear PMT detector mounted at the output port of the spectrograph. The use of a PMT-based detector utilizes the low dark noise, high sensitivity, high dynamic range, and fast response characteristics of the PMT detector. The use of a spectrograph for detecting fluorescence and a multi-component PMT detector allows for the flexibility of the number of dyes and the emission wavelength of the dye that can be applied to these systems, without the need for physical reconfiguration instruments. System (dichroic, bandpass and detector). The data collected from this configuration is a wavelength dependent spectrum that traverses the visible wavelength range for each scan of each track. The complete spectrum produced by each scan provides dye elasticity based on the wavelength of the dye emission and the number of dyes that may be present in the sample. In addition, the use of spectrometers and linear multi-element PMT detectors allows for extremely fast read rates when all PMT elements in the array are read in parallel. Figure 16 shows the beam path of a multi-element PMT and spectrograph embodiment.

The instrument can use the start mode of operation to simultaneously detect multiple channels and simultaneously detect multiple wavelengths. In one configuration, the excitation beam impinges on all tracks simultaneously. Fluorescence from this is collected by a two-dimensional detector such as a CCD camera or array. In the initial mode of this collection, a wavelength dispersive element is used. One dimension of the detector represents physical wavelength separation, while another dimension represents spatial or track-channel separation.

For simultaneous excitation and detection of multiple samples, a scanning mirror system (62) (P/N 6240HA, 67124-H-0 and 6M2420X40S100S1, Cambridge technology, Cambridge MA) is used to control the excitation and detection of the beam path to make the organism Each of the wafer tracks is imaged. In this mode of operation, the scanning mirror controls the beam path, continuously scanning from the first track to the last track from the track to the track, and repeats the process again from the first track to the last track. The position of the track is identified using a search algorithm such as U.S. Patent Application Publication No. US 2006-0260941-A1.

An embodiment of an optical detection system for simultaneous multi-channel and multiple dye detection is shown in FIG. The fluorescence excitation and detection system 40 stimulates electrophoretic separation by DNA samples (eg, DNA fragments amplified at a set of STR positions) by scanning energy sources (eg, laser beams) through each of a portion of the microchannels The components are simultaneously collected and delivered from the dye-induced fluorescing to one or more photodetectors for recording and final analysis.

In one embodiment, the fluorescence excitation and detection assembly 40 includes a laser 60 , a scanner 62 , one or more photodetectors 64 and individual mirrors 68 , a spectrograph, and for opening via an opening 42. The laser beam emitted from the laser 60 is transmitted to the test module 55 and returned to the lens 72 of the photodetector 64 . Scanner 62 moves the incident laser beam to various scanning positions relative to test module 55 . In particular, scanner 62 moves the laser beam to the relevant portion of each of the microchannels in test module 55 to detect individual discrete components. The multi-component PMT 64 collects data from the test module 55 (eg, a fluorescent signal from a DNA fragment of varying length) and provides the data to the data acquisition and storage system located outside of the protective layer 50 via cable electronics connected to the 埠75 . . In one embodiment, the data acquisition and storage system may include a reinforced computer available from Option Industrial Computers (13 audreuil-Dorion, Quebec, Canada).

In another embodiment ("start mode"), the excitation source is simultaneously incident on all of the detection points, and the fluorescence from all of the detection points is simultaneously collected. At the same time, spectral dispersion (detection of the wavelength spectrum of the fluorescence) and spatial dispersion (detection point) can be performed via the two-dimensional detector array. In this configuration, the 2-dimensional detector array is positioned in the system such that the spectral components are imaged and detected across one dimension (column) of the array detector, while the spatial components traverse the array detector. One-dimensional imaging and detection.

The preferred instrument uses the scan mode of operation instead of the "start" mode. In scan mode, the signals of each channel need to be collected, integrated, and read out before the scanner matches the path to be interrogated and before it is incident on another channel. A fast readout detector allows for optimal light collection and integration and translates into higher signal-noise performance. Ideally, the readout time of the detector should be significantly less than the total time the scanner and channel meet. The multi-element PMT can be read in less than 0.7 ms and this readout time is much smaller than the integration time of the detection of the individual channels.

The fluorescent incident light on the pinhole can be dispersed by the grating according to its wavelength and focused on the linear multi-anode PMT detector array. The detector provides 32 current outputs, one of each element in the array corresponding to the number of photons incident on the element. In the multi-sample (or track) detection process, when the laser fires the selected path at the appropriate position, the integration circuit will be integrated The PMT outputs current to produce an output voltage that is proportional to the integrated PMT current. At the same time, the Analog Devices (Norwood, MA) differential driver IC (P/N SSM2142) was used to convert the single-ended output voltage into a differential mode. At the end of the integration time (determined by the scan rate and the number of tracks), the data acquisition system will read the differential signal and save the data in its buffer. When the data is saved, the system will move the scanner to transfer the laser beam to another selection and reset the integration circuit.

Each single component PMT module has its own integration circuit. For the 8 color detection system, there are 8 PMT modules and 8 integration circuits. Additional colors can be added using the corresponding number of PMT modules and integration circuits.

Since each of the PMT components (H77260-20, Hamamatsu, Japan) has a similar or faster signal reflection than a single PMT tube (H7732-10, Hamamatsu, Japan) and is read in parallel, it is possible to operate this Detect very quickly. Detector. When coupled to a spectrometer, the spectrometer and multi-anode detector system provides a full spectral scan across the visible spectrum (450 nm to 650 nm) with a readout time of less than 0.1 ms.

The ability to provide a fast update rate allows this spectrometer/detector system to be applied to scan mode embodiments that continuously detect multiple tracks in a single run. The use of PMT-based detectors provides low noise, high sensitivity, high dynamic range and fast response. A 140 mm spectrometer with a concave hologram grating (Horiba Jobin-Yvon) and a multi-anode PMT detector is an H7260-20 detector (Hamamatsu, Japan). Other spectrometer configurations and multi-anode PMT detectors can also be used in this application.

The signal processing algorithm is used to modify, filter, and analyze the data to obtain the nucleotide bases for determining the electropherogram. This process consists of locating a callable signal, correcting the signal baseline, filtering noise, removing color cross-talk, identifying signal peaks, and determining related bases. Positioning the callable signal to remove extraneous data from the beginning and end of the signal is accomplished by using a threshold. The background is then removed from the signal, so the signal has a common baseline for all detected colors. Finally, a low pass filter is applied to remove high frequency noise from the signal.

To eliminate color ambiguity in detection, a weighting matrix is calculated and applied to the signal to amplify the color-space of the nucleotide-dye spectrum. The calculation of this color separation matrix was carried out by the method of Li et al., Electrophoresis 1999, 20, 1433-1442. In this adaptation, the number of dyes used in the autocorrelation assay "m" and the number of detector elements "n" calculate the "m x n" color separation matrix. The signal from the detector space (PMT element) is converted into a dye space by a matrix operation as follows: D = CSM × PMT, where D is the signal in the dye space of each of the m dyes, CSM is The color separation matrix, and the PMT is a matrix of signals with each of the n elements from the detector.

Second, a combination of a zero crossing filter and a frequency analysis is used to identify the peaks in the color separation signal. Finally, for the application of the slice size, the modified trace is allele-called to identify each segment and assign the segment size based on the size criteria. For DNA sequencing applications, the modified trace is base-called to associate one of the four nucleotides with each peak in the trace. A detailed description of base correspondence can be found in Ewing et al., Genome Research , 1998, 8, 175-185, and Ewing et al., Genome Research , 1998, 8, 186-194, the disclosure of which is incorporated herein in its entirety by reference.

3. Dye marking

The dye label attached to the oligonucleotide and the modified oligonucleotide can be synthetic or purchased (e.g., Operon Biotechnologies, Huntsville, Alabama). A large number of dyes (greater than 50) can be used in fluorescent excitation applications. These dyes include those from the luciferin, rhodamine AlexaFluor, Biodipy, Coumarin and Cyanine dye families. In addition, inhibitors can also be used to label oligo sequences to minimize background fluorescence. Dyes having an emission maximum of 410 nm (Cascade Blue) to 775 nm (Alexa Fluor 750) are commercially available and can be used. Dyes in the range between 500 nm and 700 nm have the advantage of being in the visible spectrum and detectable using conventional photomultiplier tubes. A wide range of commercially available dyes allows the selection of dye sets having emission wavelengths throughout the detection range. Detection systems capable of discriminating many dyes have been reported for flow cytometry applications (see Perfetto et al, Nat. Rev. Immunol . 2004, 4 , 648-55; and Robinson et al, Proc of SPIE 2005, 5692 , 359-365). ).

Fluorescent dyes have a peak excitation wavelength that is blue shifted from their peak emission wavelength, typically 20 to 50 nm. Thus, the use of dyes with a wide range of emission wavelengths may require the use of multiple excitation sources in which the excitation wavelength is achieved over the range of emission wavelengths to achieve efficient excitation of the dye. Alternatively, an energy transfer dye can be used to apply a single laser having a single emission wavelength to excite all of the dyes of interest. This is achieved by attaching the energy transfer moiety to the dye label. This portion is typically another fluorescent dye having an absorption wavelength that is compatible with the excitation wavelength of a light source, such as a laser. The placement of this absorber, which is very close to the emitter, allows the absorption of energy from the absorber to the emitter, allowing for more efficient excitation of long wavelength dyes (Ju et al, Proc Natl Acad Sci USA 1995, 92, 4347-51).

Dye-labeled dideoxynucleotides are available from Perkin Elmer, (Waltham, MA).

B. Examples Example 5. Six-color separation and detection of nucleic acid

The following examples illustrate the separation and detection of nucleic acid fragments labeled with six fluorescent dyes and demonstrate the color resolution capabilities of the spectrometer/multi-element excitation/detection system. DNA fragments were labeled with 6-FAM, VIC, NED, PET dyes by applying fluorescently labeled primers in a multiplex PCR amplification reaction. In this reaction, 1 ng of human gene DNA (9947A) was amplified in a 25 μL reaction under the conditions recommended by the manufacturer (AmpFlSTR Identifiler, Applied Biosystems). 2.7 μL of PCR product was removed and mixed with 0.3 μL of GS500-LIZ size standard (Applied Biosystems) and 0.3 μL of HD400-ROX size standard. HiDi (Applied Biosystems) was added to a total of 13 μL and the sample was inserted into the sample well of the isolated biochip and subjected to electrophoresis.

Electrophoresis of DNA using a Genebench consisting of a series of four operations: Pre- Electrophoresis, loading, injecting and separation. These operations were performed on a microfluidic biochip that was heated to a uniform temperature of 50 °C. The biochip contains a 16-channel system for multiple separations and detections, each consisting of an injector channel and a separation channel. The DNA used for analysis is separated along the separation channel via a screening matrix by electrophoretic delivery of DNA. The separation length of the biochip is in the range of 160 to 180 mm.

The first step is pre-electrophoresis, which is achieved by applying a 160 V/cm field along the length of the channel for six (6) minutes. The separation buffer (TTE1X) was injected into the anode, cathode and waste wells. A sample for analysis was injected into the sample well and 175 V was applied from the sample well to the waste well for 18 seconds, then 175 V was applied across the sample and waste wells, and 390 V was applied to the cathode for 72 seconds to load the sample into the separation channel. Injection of the sample was done by applying a field of 160 V/cm along the length of the separation channel while applying fields of 50 V/cm and 40 V/cm across the sample and waste wells, respectively. The separation was continued with an injection voltage parameter for 30 minutes, during which the optical system detected the separation band of DNA. This data collection rate is 5 Hz and the PMT gain is set to -800V.

Sixteen samples containing amplified DNA were loaded for simultaneous isolation and detection. The signals from each of the 32-element PMTs are collected as a function of time to generate an electropherogram. The resulting electropherogram (Figure 17) shows the peak corresponding to the presence of the DNA fragment at the excitation/detection window of one of the 16 lanes. Furthermore, for each peak, the relative signal intensity of each element of the 32-element PMT corresponds to the spectral content of the dye associated with the DNA fragment (or dye, if more than one dye is present in the detection window). Figure 18 shows the emission spectrum of the detected dye and the background spectrum of the substrate. The substrate background spectrum is subtracted from the spectrum to obtain each of the peaks. Performing this practice led to the identification of six different dye spectra. The spectra of the six dyes are superimposed on the same curve. A comparison of this data with the actual published dye spectrum reveals that the relative values of the dyes are similar to those disclosed. This example shows that the system can detect and resolve the six dyes in the reaction solution. This spectral output is used to generate a color correction matrix and convert the signal from the detector space to the dye space representation (Figures 19 and 20).

Example 6. Eight color separation and detection of nucleic acid

In this example, eight dye separations and detections of fluorescent dye-labeled acids are shown. For the 8-site pre-introduction and reverse-introduction pairs, the sequence is selected from the published sequences (Butler et al, J Forensic Sci 2003, 48, 1054-64).

Although any of the sites described in the document and hence the primer pairs can be used in this example, the selected sites are CSF1P0, FGA, THO1, TPOX, vWA, D3S1358, D5S818, and D7S820. For the primer pair, each of the forward primers was labeled with an independent fluorescent dye (Operon Biotechnologies, Huntsville, Alabama). The dyes selected for attachment to the primers include Alexa Fluor Dyes 488, 430, 555, 568, 594, 633, 647 and Tamra. Numerous other dyes are commercially available and can also be used as labels. Each point was independently amplified according to the PCR reaction protocol of (Butler, 2003, Id. ) to produce a reaction solution having a fragment labeled with the corresponding dye. The template for the PCR reaction was 1 ng of human genetic DNA (type 9947A from Promega, Madison WI).

Each PCR reaction was purified by purification by PCR purification column with primers (labeled and dye-labeled primers) and enzyme removed and the PCR buffer exchanged by DI eliminator. The resulting product obtained by purification is a solution of the labeled DNA fragment in DI water. Using MinElute ^TM column (Qiagen, Valencia, CA) for the dye-labeled product was purified according to Smith's scheme. A total of eight reactions were performed. The eight purified PCR reactions were mixed together at a ratio that produced peaks of equal signal intensity to produce a mixture containing fragments labeled with 8 different dyes. Alternatively, primers of 8 sites can be mixed together to form a mixture of major primers for multiplex amplification.

This solution was isolated and detected using the apparatus and protocol as described in Example 1. The grating of the spectrograph is adjusted so that the emission of the eight dyes falls within the 32 pixel range of the detector element. The number of samples loaded for analysis is adjusted so that the detected signal is within the dynamic range of the detection system.

Example 7. Spectrometer / multi-component PMT system

The following examples illustrate the separation/detection of labeled DNA fragments, specifically the sequences used to identify DNA templates, using the spectrometer/multi-component PMT system of Figure 16. In this reaction, depending on the reaction conditions recommended by 0.1pmol DNA template M13 M13 sequencing primers and amplification GE Amersham BigDye ^TM sequencing kit. The reaction mixture was purified by ethanol precipitation and resuspended in 13 μL of DI water. The samples were separated under electrophoretic separation conditions as described in Example 5. Sample loading conditions were varied and applied by applying 175 V across the sample well to the waste well for 105 seconds. Figure 21 shows an electropherogram of a DNA sequence in which colored traces indicate detector elements corresponding to the spectral maximum of each of the four dyes used. The sequence obtained was 519 bases corresponding to a base of Phred quality score > 20 and QV30 of 435 bases (Fig. 22).

Example 8. Simultaneous separation and detection of two sequencing reaction products

In this example, separation and detection of fragments from two DNA template cycle sequencing were performed simultaneously in a single separation channel. The cycle sequencing reaction can be prepared by dye-labeled terminator reaction or dye-labeled primer reaction as follows:

For dye-labeled terminator reactions:

Preparing a cycle sequencing reaction for each template fragment, wherein the template fragment consists of: sequencing primers suitable for the template sequence of interest; and reagents for DNA sequencing, including cycle sequencing buffers, polymerases, oligonucleosides Acid, dideoxynucleotide and labeled dideoxynucleotide. Eight different dyes were used for labeling. In the first cycle sequencing reaction, a set of four deoxynucleotides labeled with four dyes was used. In the second cycle sequencing reaction, another set of four dye-labeled dideoxynucleotides (wherein the emission wavelengths are different from the four dyes used in the first cycle sequencing reaction) are used. Each cycle sequencing reaction was carried out independently according to a scheme in which multiple cycles of each reaction were performed. Each thermal cycle includes denaturation, annealing, and extension steps in which the temperature and number of times follow the Sanger protocol (see, Sanger et al, Proc Natl Acad Sci USA 1977, 74, 5463-7). The cycle sequencing products from the two reactions were pooled to form a sample consisting of a total of eight unique dye-labeled DNA fragments from each of the two DNA templates.

For dye-labeled primer reactions:

Alternatively, samples for separation and detection can be made by cyclic sequencing using primer labeling. Four cycles of sequencing reactions were performed for each DNA template. Each reaction is a cyclic sequencing reaction consisting of a labeled sequencing primer and reagents for DNA sequencing, including cycle sequencing buffers, polymerases, oligonucleotides. In addition, each reaction will include a dideoxynucleotide (ddATP, ddTTP, ddCTP or ddGTP) and one of the labeled primers. Each dye associated with the primer has a unique emission wavelength and is associated with the type of dideoxynucleotide (ddATP, ddTTP, ddCTP or ddGTP) in the circulating sequencing solution. Each cycle sequencing reaction was carried out independently according to a scheme in which multiple cycles of each reaction were performed. Each thermal cycle includes denaturation, annealing, and extension steps in which the temperature and number of times follow the Sanger scheme (see, Sanger, 1977, Id. ). For cycle sequencing of the second DNA template, another set of 4 dyes (where the emission wavelength is different from the four dyes used in the first cycle sequencing reaction) is used. The products of all eight reactions (each having a different dye) were mixed together to form a sample consisting of DNA fragments from each of the two DNA templates.

Samples for separation and detection:

Each of the sequencing reactions was purified by ethanol precipitation. The separation and detection of the samples followed the protocol of Example 8. The result of isolation and detection is the generation of two different DNA sequences corresponding to each of the two template DNA fragments.

The method of the present example can be modified to allow detection of multiples of DNA sequences in a single separation channel using a multiple of four dyes (eg, 12 dyes for simultaneous detection of 3 sequences, for simultaneous detection of 4 species) 16 dyes in the sequence, 20 dyes for simultaneous detection of 5 sequences, etc.). Ultimately, the separation of labeled fragments need not be limited to electrophoresis.

Example 9

Separation and detection of 500 or more sites in a single channel

There are several applications for nucleic acid analysis that can be applied to clinical diagnosis, including DNA and RNA sequencing and fragment size determination. In this example, the simultaneous detection of 10 colors allows interrogation of up to 500 sites. For example, size analysis of a large number of fragments can be used to identify pathogens or to characterize many sites within an individual's genome. In the context of prenatal and preimplantation genetic diagnosis, aneuploidy is currently diagnosed by karyotype and by fluorescence in situ hybridization (FISH). In the FISH study, the presence of two signals per cell indicates the presence of two copies of the site in the cell, one signal indicating monomeric or partial monomericity, and three signals indicating trisomy or partial trisomies . FISH typically uses about 10 probes to analyze whether a cell contains normal chromosome complement. However, this method does not allow for a detailed examination of the entire genome, and cells that are known to be normal by FISH are likely to have major abnormalities that cannot be detected by the technique.

The teachings of the present invention use multicolor separation and detection to allow approximately 500 chromosomal loci to be widely dispersed throughout all chromosomes to be analyzed, allowing for a more detailed analysis of the chromosome structure. In this example, a primer pair sequence of about 500 loci is selected from the published sequences, with each locus present as a single copy of each haploid genome. In addition, 10 sets of 50 primer pairs were selected such that each group defined a corresponding set of DNA fragments such that none of the fragments were of the same size. For each group, the primer pair was previously labeled with a fluorescent dye, and no two groups shared the same dye. The dyes selected for attachment to the primers were Alexa Fluor Dyes 488, 430, 555, 568, 594, 633, 647, 680, 700 and Tamra. Numerous other dyes are commercially available and can also be used as labels. The sites can be amplified in one or several parallel PCR reactions as described in "METHODS FOR RAPID MULTIPLEXED AMPLIFICATION OF TARGET NUCLEIC ACIDS" above. The amplified primers are isolated and detected using the methods described herein. In a single separation channel, all 500 segments can be accurately identified by size, and 50 segments are identified for every ten dyes.

The number of sites, dyes, and separation channels can vary based on the desired application. If necessary, a smaller number of fragments can be detected by using a smaller number of dye labels or generating fewer DNA fragments per label; therefore, less than 500, less than 400, less than 300, less than 200, small A segment of 100, less than 75, less than 50, less than 40, less than 30, or less than 20 can be detected as desired. The maximum number of each identifiable site is based on the read length of the separation system and resolution (eg, a single base pair resolution of a DNA fragment in the range of 20 to 1500 base pairs results in hundreds of fragments) multiplied by detectable The number of different dyes (as described above, several dozens can be obtained). Thus, thousands of sites can be identified in a single separation channel, and this number will increase as additional dyes are developed.

Claims (9)

A system for detecting components separated by electrophoresis, comprising: (i) an integrated biochip comprising a thermal cycling chamber adapted for multiplex amplification in a polymerase chain reaction (PCR), The thermal cycling chamber is in fluid communication with at least one channel on the biochip, wherein each channel includes a detection location; (ii) an indicator that marks six or more fluorescent dyes for generating the six or six a plurality of fluorescent dye-labeled amplified nucleic acid fragments; (iii) at most one light source positioned to illuminate a plurality of detection locations on a physical space on the biochip; (iv) one or more selected from a first optical component of the group consisting of: a lens, a pinhole, a mirror, and an objective lens positioned in the physical space for collecting and directing light emitted from the plurality of detection locations on the biochip; a scanning mirror galvanometer for continuously illuminating and collecting light emitted by the light source from each of the plurality of detection locations; and (vi) a wavelength dispersive element selected from the group consisting of: Diffractive grating, transmission grating, holographic diffracted light And a spectrograph positioned to disperse the fluorescent wavelength component in the physical space in accordance with the wavelength of the light and to reflect substantially the corresponding fluorescence emission of the fluorescent dye present at the at least one detection location by reflection Light of a predetermined wavelength, providing at least a portion of the dispersive wavelength element to at least six detectors or a multi-element photomultiplier tube (PMT), the detector or multi-element PMT being positioned to detect from the Fluorescence of the labeled amplified nucleic acid fragment. A system for simultaneously detecting fragments from at least two DNA templates in a single separation channel, comprising: (i) an integrated biochip comprising at least one thermal cycling chamber adapted for use in Multiple amplification in polymerase chain reaction (PCR), the thermal cycle chamber being in fluid communication with at least one channel on the biochip, wherein each channel comprises a detection location; (ii) marking 8 or more types of firefly a primer for a light dye for producing two sets of nucleic acid fragments labeled with the eight or more fluorescent dyes, the dye being a member of at least two sets of four dyes, such that the at least two sets are The dye emits at a different wavelength; (iii) at most one light source positioned to illuminate a plurality of detected locations on a physical space on the biochip; (iv) one or more selected from the group consisting of a first optical component: a lens, a pinhole, a mirror, and an objective lens positioned on the physical space for collecting and directing light emitted from the plurality of detection locations on the biochip; (v) a scanning microscopy a flow meter for continuously illuminating and collecting light emitted by the light source from each of the plurality of detection locations; and (vi) a wavelength dispersive element selected from the group consisting of: 稜鏡, diffraction grating, transmission Grating, holographic diffraction grating and spectrograph Locating to disperse the fluorescent wavelength element in the physical space according to the wavelength of the light, and providing at least a portion of the dispersive wavelength element to at least eight detectors or a multi-element photomultiplier tube (PMT), The detector or multi-element PMT is positioned to detect fluorescence from the labeled amplified nucleic acid fragment. The system of claim 2, wherein the number of simultaneously detected fragments is at least three, the number of the four dye sets is at least three, and the number of fluorescent dyes is at least 12. The system of claim 2, wherein the number of simultaneously detected fragments is at least four, the number of the four dye sets is at least four, and the number of fluorescent dyes is at least 16 The system of claim 2, wherein the number of simultaneously detected fragments is at least five, the number of the four dye sets is at least five, and the number of fluorescent dyes is at least 20. A system for detecting components separated by electrophoresis, comprising: (i) a biochip comprising a thermal cycling chamber adapted for multiplex amplification in a polymerase chain reaction (PCR); Marking 6 or more fluorescent dye primers for producing amplified nucleic acid fragments labeled with the 6 or more fluorescent dyes; (iii) second chamber adapted for additional processing, The treatment is selected from the group consisting of: nucleic acid extraction; nucleic acid purification; purification before nucleic acid amplification; purification after nucleic acid amplification; purification before nucleic acid sequencing; nucleic acid sequencing; purification after nucleic acid sequencing; reverse transcription; pre-transcription purification; Post-transcriptional purification; nucleic acid ligation; nucleic acid hybridization; and quantification; (iv) at most one light source positioned to illuminate a plurality of detected locations on a physical space on the biochip; (v) one or more first optical components selected from the group consisting of: lenses, pins a hole, a mirror and an objective lens positioned in the physical space for collecting and directing light from the plurality of detection locations on the biochip; (vi) a scanning mirror ammeter for continuous illumination And light emitted from a source of each of the plurality of detection locations; and (vii) a wavelength dispersive element selected from the group consisting of: a 稜鏡, a diffraction grating, a transmission grating, a holographic diffraction grating, and a spectrum An apparatus positioned to disperse a fluorescent wavelength element in the physical space according to a wavelength of light and to provide at least a portion of the dispersive wavelength element to at least six detectors or a multi-element photomultiplier tube (PMT) The detector or multi-element PMT is positioned to detect fluorescence from the labeled amplified nucleic acid fragment. The system of claim 1 or 6, wherein the light source is a laser. The system of claim 1 or 6, wherein each of the detecting elements is capable of detecting light selected from the group consisting of: ultraviolet light, visible light, infrared light, and combinations thereof. The system of any of claims 1, 2, and 6, wherein the physical space on the biochip is plastic.