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  Integration of biochemical protocols in a continuous flow microfluidic deviceUSPTO Application #: 20060011478Title: Integration of biochemical protocols in a continuous flow microfluidic device Abstract: Provided is a microfluidic device comprising a microfluidic substrate comprising at least one pathway for sample flow; and at least one thermal transfer member which is capable of cycling between at least two temperatures. The thermal transfer member is adapted to heat at least a portion of the sample pathway while a sample is flowing along said at least a portion of said sample pathway. Provided also are methods of carrying out biochemical protocols using such a device. (end of abstract) Agent: Saliwanchik Lloyd & Saliwanchik A Professional Association - Gainesville, FL, US Inventors: Yves Fouillet, Claude Vauchier, Jean-Frederic Clerc, Christine Peponnet USPTO Applicaton #: 20060011478 - Class: 204451000 (USPTO) Related Patent Categories: Chemistry: Electrical And Wave Energy, Non-distilling Bottoms Treatment, Electrophoresis Or Electro-osmosis Processes And Electrolyte Compositions Therefor When Not Provided For Elsewhere, Capillary Electrophoresis The Patent Description & Claims data below is from USPTO Patent Application 20060011478. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] The present application is a continuation of U.S. application Ser. No. 09/627,647, filed Jul. 28, 2000, which claims priority to French patent application serial No. 99/09806, filed Jul. 28, 1999; French patent application serial No. 99/11652, filed Sep. 17, 1999; and French patent application serial No. 99/12317, filed Oct. 1, 1999, the disclosures of all of which are incorporated herein by reference in their entireties. BACKGROUND [0002] Microfluidics consist of using microchannels instead of test tubes or microplates to carry out analyses and reactions. These microchannels or microcircuits are etches into silicon, quartz, glass, ceramics or plastic. The size of these channels is on the order of micrometers, while the reaction volumes are on the order of nonoliters or microliters. The principle of a microfluidic device is to guide reaction media containing reagents and samples, over zones which correspond to the different steps of the protocol. The integration of reactors, chromatographic columns, capillary electrophoresis systems and miniature detection systems into these microfluidic systems allows the automation of complex protocols by integrating them into a single system. These "laboratories on chips" have made it possible to obtain results which are efficient in terms of reaction speed, in terms of product economy and in terms of miniaturization which allows the development of portable devices. Complex protocols have been integrated and automated, including biochemical or molecular biology protocols which often require extensive manipulation. These manipulations include mixing reagents and samples, controlling the reaction temperature, carrying out thermal cycling, separation by electrophoresis, and detection of reaction products. [0003] Wolley et al. (Anal. Chem. 68: 4081-4086 (1996), the contents of which is incorporated herein by reference in its entirety) discloses the integration of a PCR microreactor, a capillary electrophoresis system and a detector in a single device. The PCR reaction, separation of PCR products by electrophoresis, and detection of PCR products are carried out automatically. This device does not, however, integrate the mixing of reagents, and it does not allow large scale protocols to be performed. [0004] A device or substrate allowing integration of the steps of reagent mixing and enzymatic reaction has been described by Hadd et al. (Anal. Chem. 69, 3407-3412, (1997), the contents of which is incorporated herein by reference in its entirety). This device provides a microcircuit of channels and reservoirs etched into a glass substrate. The moving and mixing of the fluids takes place by electrokinetics. [0005] Microfluidic systems for the integration of protocols and of analyses have been described in international patent application WO 98/45481. One of the difficulties in implementing these devices resides in the movement of the fluids. The fluids are generally moved by electroosmosis or by electrokinetics, which requires a network of electrodes. Other systems use micropumps and microvalves which are integrated in the microfluidic substrate. In the majority of cases the reactions are carried out while stationary in a microreactor and then the fluids are thus moved from one reactor to another at each step of the protocol. These systems which integrate electrodes, microvalves or micropumps are very costly and their complexity does not allow large scale applications for simultaneously treating a very large number of samples. One of the major difficulties is the distribution, mixing and transport of a very large number of products in parallel or in series. [0006] Thus, there exists a need to develop a device comprising a microfluidic substrate allowing the manipulation of a large number of fluids and/or allowing a large number of complex protocols, particularly protocols involving temperature treatment, to be carried out at a low cost. SUMMARY OF THE INVENTION [0007] The present invention provides a device comprising a microfluidic substrate which comprises at least one microchannel in which reactions or sequences of reactions, which make up a protocol, are carried out. [0008] The present invention provides a microfluidic substrate which comprises at least one microchannel in which reactions or sequences of reactions, which make up a protocol, are carried out, where the channel is fed in continuous flow. [0009] Combining the microfluidic substrate with a thermal support makes it possible to control the reaction temperature in the different zones of the channel corresponding to the various steps of the protocol. The invention relates to advantageous devices and processes for carrying out thermal cycling in continuous flow on thermal cycling zones. [0010] The device is based on a system for distributing and moving the fluids by hydrostatic pressure. All steps of a protocol are carried out in continuous flow; wherein sequential injections of samples and of reagents make it possible to carry out a large number of reactions one after the other in the same channel. Reagents can be injected successively at different stages of the protocol. By arranging several channels in parallel, it is possible to carry out the same protocol in series in the same channel and in parallel in various channels. Synchronizing the reactions in the channels arranged in parallel makes it possible to distribute the reagents simultaneously into the various channels. This arrangement has a particularly advantageous application in improving the throughput and reducing the number of distributions to be carried out. [0011] The microfluidic substrate of the present invention is preferably semi-disposable (used for a few hundred reactions or some tens of hours) and is added on, in a removable fashion, to the thermal support, the fluid feed devices and the detection means. The control of the temperature, the movement of the fluids, the injection of the reagents, the mixing of the solutions in continuous flow and the detection are entirely automated. In addition, the combination of a permanent device and a disposable but relatively inexpensive microfluidic substrate allows a considerable reduction in costs relative to systems in which everything is integrated on the same microfluidic device. [0012] One embodiment of the present invention is a device comprising a microfluidic substrate comprising at least one pathway for sample flow and at least one thermal transfer member which is capable of cycling between at least two temperatures, said at least one thermal transfer member being adapted to bring at least a portion of said sample pathway to said at least two temperatures while a sample is continuously flowing along said at least a portion of said sample pathway. In some aspects of this embodiment, the device further comprises a force supplying member operably linked to said at least one pathway for sample flow wherein said force supplying member applies a force to said sample such that said sample travels along said at least one pathway. The device may further comprise a sample supplier which supplies a sample to said at least one pathway. The device may also further comprise at least one inlet basin positioned at a first end of said at least one pathway such that said sample supplier supplies said sample to said inlet basin and said sample travels from said inlet basin to said at least one pathway. The device of may also further comprise at least one outlet basin positioned at a second end of said pathway. In some aspects of the present invention, the device further comprises at least one reagent supplier positioned between said inlet basin and said outlet basin. In other aspects of the present invention, the device comprises a plurality of said pathways. The pathways may comprise channels arranged in parallel. The force generated by said force supplying member may be pressure. The microfluidic substrate may consist essentially of silicon. The device may further comprise a detector for measuring a physicochemical property of said biological sample. The thermal transfer member may comprise a metal bar in fluid communication with a plurality of water sources containing water at said at least two temperatures, said metal bar being in thermal communication with said at least a portion of said sample pathway. [0013] Another embodiment of the present invention is a method for conducting a biochemical or chemical process comprising cycling at least a portion of at least one sample flow pathway between at least two temperatures while a sample comprising the reagents for said biochemical or chemical process is flowing through said at least a portion of said at least one sample flow pathway. The sample flow pathway may be located on a microfluidic substrate. The sample flow pathway may be in thermal communication with at least one thermal transfer member which cycles between said at least two temperatures while said sample is continuously flowing through said at least a portion of said at least one sample flow pathway. The thermal transfer member may cycle through said at least two temperatures a plurality of times while said sample is continuously flowing through said at least a portion of said at least one sample flow pathway. The thermal transfer member may cycle through said at least two temperatures from about 2 to about 35 times while said sample is continuously flowing through said at least a portion of said at least one sample flow pathway. In some aspects of this embodiment, at least a portion of a plurality of sample flow pathways are simultaneously cycled between said at least two temperatures while a plurality of samples are simultaneously flowing through said sample flow pathways. The biochemical or chemical reaction may comprise a nucleic acid amplification procedure. [0014] The nucleic acid amplification procedure may comprise polymerase chain reaction. The method may further comprise determining the identity of at least one polymorphic nucleotide in the product of said nucleic acid amplification procedure. [0015] Another embodiment of the present invention is a process for carrying out biochemical protocols on at least one sample, comprising feeding at least one channel with a continuous flow of a solution containing at least one sample, injecting at least one reagent from a reagent reservoir into said channel, thereby mixing said sample and said reagent, and transferring heat between at least one thermal support and at least one temperature regulated portion of said at least one channel. The feeding step may comprise applying a pressure difference between a feed basin of said at least one channel and an outlet basin of said at least one channel. The process may further comprise detecting at least one physicochemical parameter of said sample in said at least one channel. In some aspects of the process, a temperature of said solution is adjusted to a predetermined level when said solution runs through said at least one temperature regulated portion of said at least one channel. The process may further comprise cycling said at least one thermal support through at least two different temperatures. The cycling may be repeated 1 to 35 times while solution is running through said at least one portion of said at least one channel. In other aspects of the process, a plurality of samples separated by separators are sequentially introduced into said at least one channel. In some aspects of the process, said feeding, said injecting, and said transferring are carried out simultaneously on a plurality of channels arranged in parallel. [0016] Another embodiment of the present invention is a process for carrying out in continuous flow at least one temperature cycle on a solution containing at least one sample comprising feeding at least one channel with a continuous flow of said solution, running said solution through at least one temperature regulated zone, and cycling said at least one temperature regulated zone successively through a temperature cycle of at least two temperatures in a predetermined temporal series, such that the solution undergoes said temperature cycle at least once in running through the at least one temperature regulated zone once. The process may further comprise detecting at least one physicochemical parameter of said sample in said channel. The feeding may comprise applying a pressure difference between a feed basin of said at least one channel and an outlet basin of said at least one channel. The feeding may be sequentially repeated with a plurality of samples separated by separators. In some aspects of the process, said feeding, said running and said cycling are carried out simultaneously on a plurality of channels arranged in parallel. [0017] Another embodiment of the present invention is a process for amplifying nucleic acids, comprising: a) mixing at least one sample comprising said nucleic acids with reagents which are suitable for amplifying nucleic acids to form at least one reaction mixture, b) feeding at least one channel with a continuous flow of said at least one reaction mixture, c) running said at least one reaction mixture through at least one temperature regulated zone, and d) cycling said temperature regulated zone through a temperature cycle of at least two temperatures in a predetermined temporal series, wherein the at least two temperatures, a duration of the temperature cycle, and a rate of said running are preselected such that said at least one nucleic acid sample undergoes a denaturation-hybridization-elongation cycle one or more times while flowing through said at least one temperature regulated zone. The feeding may comprise applying a pressure difference between a feed basin of said at least one channel and an outlet basin of said at least one channel. The channel may be formed in a microfluidic substrate. The microfluidic substrate may consist essentially of silicon. In some aspects of the process, said feeding is sequentially repeated with a plurality of nucleic acid samples separated by separators. In other aspects of the process, steps a), b), c) and d) are carried out simultaneously on a plurality of channels arranged in parallel. [0018] Another embodiment of the present invention is a process for identifying in continuous flow at least one nucleotide in at least one target nucleic acid, comprising: a) feeding a channel with a continuous flow of a solution comprising said at least one target nucleic acid, b) injecting a microsequencing reagent comprising a microsequencing buffer, at least one microsequencing primer, at least one ddNTP and a polymerase into said channel, thereby mixing said nucleic acid solution and said reagent, c) running the solution through at least one temperature regulated zone in such a way as to produce at least one cycle comprising denaturation of said at least one target nucleic acid, hybridization of said nucleic acid with said at least one microsequencing primer, and incorporation of a ddNTP which is complementary to the nucleotide to be identified at a 3' end of said primer, and d) identifying the nucleotide which has been incorporated at the 3' end of the microsequencing primer. The feeding may comprise applying a pressure difference between a feed basin of said channel and an outlet basin of said channel. The process may further comprise amplifying said at least one target nucleic acid using the method above prior to performing said method for identifying at least one nucleotide. The ddNTPs may be labelled with fluorophores and the fluorescence of the incorporated ddNTP may be detected. The feeding, said injecting and said running may be carried out simultaneously on a plurality of channels arranged in parallel. [0019] Another embodiment of the present invention is a process for detecting in continuous flow at least one nucleotide in at least one target nucleic acid, comprising: a) feeding a channel with a continuous flow of a solution containing at least one target nucleic acid, b) injecting the reagent for amplifying a region of the at least one target nucleic acid which carries at least one nucleotide to be detected into said channel from a first reagent reservoir, c) running the solution through at least one temperature regulated zone in such a way that the nucleic acid undergoes a denaturation-hybridization-elongation cycle one or more times, d) injecting the reagent for purifying the amplification product into said channel from a second reagent reservoir, e) running the solution through at least one temperature regulated zone to carry out a purification reaction, f) injecting the microsequencing reagent comprising the microsequencing buffer, at least one microsequencing primer, at least one ddNTP and a polymerase into said channel from a third reagent reservoir, g) running the reaction mixture through at least one temperature regulated zone in such a way as to produce at least one cycle comprising the denaturation of the target nucleic acid, the hybridization of said nucleic acid with the at least one microsequencing primer, and the incorporation of the ddNTP which is complementary to the nucleotide to be detected, at the 3' end of said primer, and h) detecting at least one ddNTP which is incorporated at the 3' end of the microsequencing primer. The feeding may comprise applying a pressure difference between a feed basin of said channel and an outlet basin of said channel. In some aspects of the process, in steps c) and e), the temperature regulated zone is brought successively to at least two temperatures in a temporal series which forms at least one cycle. The ddNTPs may be labelled with fluorophores, and wherein in step h) the fluoresence of the incorporated ddNTP is detected. The reagent for the purification may comprise an exonuclease and an alkaline phosphatase. In some aspects of the process, steps a), b), c), d), e), f), g) and h) are carried out simultaneously on a plurality of channels arranged in parallel. BRIEF DESCRIPTION OF THE DRAWINGS [0020] FIG. 1 is a schematic view of one embodiment of the invention; Continue reading... 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