Abstract:
Methods and systems of performing multiple reactions in a high throughput format by utilizing interfacial mixing of adjacently positioned reagent slugs in a fluid conduit. Preferred applications of the methods and systems are in performing biochemical analyses, including genotyping experiments for multiple different loci on multiple different patient samples. Microfluidic systems are provided that increase throughput, automation and integration of the overall reactions to be carried out.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Patent Application No. 60/298,058, filed Jun. 13, 2001, which is incorporated herein by reference in its entirety for all purposes. 
    
    
     BACKGROUND OF THE INVENTION 
     Higher throughput experimentation is a consistent goal for high-technology industries that depend upon research and development for growth, e.g., pharmaceutical, biotechnology and chemical industries. In the case of biological and chemical research, microfluidic technology has attempted to address this need by miniaturizing, automating and multiplexing experiments so that more experiments can be carried out faster and in a less expensive fashion. However, even these advances have highlighted the need and/or desire for even higher throughput experimentation within these industries. In particular, as with every other type of fluid based experimentation, microfluidic technology is limited by the fact that analyzing a given reaction requires mixing the reagents together in isolation and analyzing the results. Typically, such analysis has required a separate reaction vessel into which the different reagents must be pipetted. Higher throughput has then been achieved by increasing the number of reaction vessels, e.g., through the use of multiwell plate formats, increasing the complexity of pipetting systems, or in some rare cases, by carrying out multiple reactions in a single mixture. As can be readily appreciated, when one wishes to perform a matrixed experiment, e.g., testing each of a first library of reagents against each of a second library of experiments, the number of different reactions can potentially be staggering. 
     One example of such a matrixed experiment that is of considerable interest is that involved in genotyping experiments, e.g., SNP genotyping. In particular, it has been hypothesized that there is a correlation between the genetic footprint of a patient, e.g., as represented by the pattern of different genetic markers, e.g., SNPs, and that patient&#39;s response to different pharmaceutical treatments, susceptibility to disease, etc. In order to identify such a pattern, a large number of different patients need to be genotyped as to a large number of different genetic marker loci, in order to identify such correlations, so that they can be later used as diagnostic or therapeutic aids. 
     Microfluidic systems have addressed the throughput need for analytical operations, including genetic analysis, by providing very small fluidic channels coupled to an external fluid sipping element, e.g., a sampling capillary, through which reagents are drawn into the fluidic channel, where different reactions are carried out (See commonly owned U.S. Pat. No. 5,942,443). By serially drawing different samples into flowing reagent streams, such systems are capable of analyzing large numbers of different reactions in a relatively short amount of time. Further, by providing multiple parallel sipping and channel systems, one can further increase the number of experiments that are carried out. 
     While these systems have proven highly effective, each channel network has typically only been used to perform a single assay against a battery of test compounds or reagents. For example, in a particular channel, a given enzyme or target system is screened against a large number of potential inhibitors or test compounds. In the case of a matrixed experiment, e.g., screening a large number of enzymes or targets against a large number of potential inhibitors or test compounds, this particular operation would amount to one column of the matrix. Different columns of the matrix would be performed by other channel systems that are either within the same body or device, or are alternatively, completely separate. For example, one channel may be used to screen compounds for an effect on one enzyme system, while another channel in the same device, would be used to screen those compounds for an effect on a different enzyme system. 
     By way of example, in previously described operations, a first reagent is resident within the microfluidic device and is continuously introduced into the channels of the device. A large number of different second reagents are then serially introduced into the channel system to be reacted with (or interrogated against) the first reagent. Other reaction channel networks in the same device then optionally include different first reagents to perform other columns of the matrix. However, complexities of fixed sampling element positioning in microfluidic devices make such experiments difficult to configure, as different channel systems would not visit all of the same external sample sources, e.g., certain channels would not be able to access all of the test sample wells in a multiwell plate. 
     The present invention addresses the needs of higher throughput, matrixed experimentation, while taking advantage of the benefits of microfluidic technology in miniaturization, integration and automation. 
     SUMMARY OF THE INVENTION 
     The present invention generally provides methods and systems that utilize interfacial mixing of adjacent fluid plugs within a fluid conduit to perform multiple different analytical reactions. In at least one aspect, the invention provides a method of analyzing a plurality of reactions. The method comprises serially introducing plugs of first, second and third fluid borne reagents into a first fluid conduit under conditions suitable for performing the plurality of reactions whereby the plug of the first fluid borne reagent is abutted by the plug of the second fluid borne reagent at a first interface, and the plug of the second fluid borne reagent is abutted by the plug of the third fluid borne reagent at a second interface. The reagents are allowed a sufficient time for diffusion of effective amounts of the first and second reagents across the first interface whereupon the first and second reagents mix and react in a first reaction mixture, as well as sufficient time for diffusion of effective amounts of the second and third reagents across the second interface, whereupon the second and third reagents react in a second reaction. The results of the first and second reactions are then analyzed. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  schematically illustrates interfacial matrixed reactions in accordance with the present invention. 
         FIG. 2  schematically illustrates the interactions at an interface between fluid slugs in a microfluidic channel. 
         FIG. 3  schematically illustrates a channel network for use in carrying out the methods of the invention. 
         FIG. 4  schematically illustrates an overall system for use in carrying out the interfacial reactions of the present invention. 
         FIG. 5  shows results of PCR amplification reactions that are carried out in an interfacial format, in accordance with the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides methods of rapidly performing a large number of reactions on one or more different materials of interest in a single fluidic system, and with extremely small quantities of reagents. The methods of the invention are particularly suited to performing matrixed experiments, e.g., experiments that are performed using a range of first reactants separately reacted with each of a range of second reactants. In particular, the present invention takes advantage of interfacial diffusion/dispersion in reagent plugs that are serially introduced into a fluid conduit to process, in series, different columns of the matrixed reaction. This is illustrated in  FIG. 1  for a matrixed reaction of a library of first reagents A, B and C (shown as hatched slugs-///) against a library of second reagents  1 ,  2  and  3  (shown as hatched slugs-\\\). Although described with reference to three-member reagent libraries, it will be appreciated that each library will range in size from, e.g., about 5 different reagent members to up to thousands of reagent members, e.g., 10,000, 100,000 or more, and typically, from about 10 to about 5000 different reagent library members. 
     As shown in  FIGS. 1A–C  a conduit is provided. A reagent from the first reagent library, e.g., reagent A, is introduced as a plug into the conduit followed by a plug of first reagent from the second library, e.g., reagent  1  ( FIG. 1A ). Another plug of reagent A is then introduced into the conduit following and adjoining or abutting the plug of reagent  1 . A plug of a second reagent from the second library, e.g., reagent  2 , is introduced following and abutting reagent A. Again, a plug of reagent A follows reagent  2 , which is in turn followed by reagent  3 . Subsequent to, and optionally abutting reagent  3 , a plug of a second reagent from the first library is introduced into the conduit. This is then followed by reagent  1 , reagent B and reagent  2 , and optionally reagent B followed by reagent  3  (although this is not necessary, as reagent B already abuts reagent  3 ). This is further repeated with reagent C. The resulting conduit (as shown in  FIG. 1C ) includes all of the various reagents of the first library abutting each of the reagents in the second library at one or more fluid interfaces. 
     In the conduit, each of the different reagent plugs then diffuses and/or disperses into the adjoining reagent slugs. In each of these resulting reaction mixtures, the reactions of interest are carried out, and the results are determined/measured and recorded as the material moves through the conduit past a detection point. As can be seen, a simple organization of reagent plugs dictates the reactions that occur. As is also apparent from  FIG. 1 , the organization of the fluid plugs can result in certain reactions being duplicated, which in some cases, may not be desirable, e.g., where maximum throughput is desired, and where there is a substantial number of duplicated reactions, e.g., where the number of reagents in each library are similar. In such cases, algorithms may be used which minimize the duplication of reactions. By way of example, in the simple illustration shown in  FIGS. 1A–C , one could rearrange the order in which reagents are introduced in order to eliminate any duplicated interfaces.  FIG. 1D  illustrates just such an arrangement. As shown, reagent one is followed by reagent A which is followed by reagent  2 . To avoid duplication of the 2+B interface, reagent B is then introduced followed by reagent  3 , and reagent C. This is then followed by reagent  1 , followed by reagent B and reagent  3 , which is followed by reagent A. In this fashion, all possible combinations of interfaces have been represented with only one duplicated reaction, as opposed to  3  in the previous example. For other types of reactions, e.g., where a large number of different first reagents are screened against a small number of second reagents, the effect of duplications on throughput is relatively minor and could be ignored in favor of simpler sampling strategies. 
     The specific interfacial interactions are schematically illustrated in  FIGS. 2A and 2B . As shown, a first reagent plug  202  is introduced into the conduit  200 . A second reagent  204  is introduced immediately adjacent to and bounded by the first reagent  202 . A third reagent  206  is then introduced after and adjacent to the second reagent  204 . The third reagent may be the same as or different from the first reagent  202 . As shown in  FIG. 2B , after a short period, the reagents  202 ,  204  and  206  diffuse and/or disperse into each other at their respective interfaces to form areas of reagent mixtures  208  and  210 , respectively (illustrated by cross hatching). Thus, with the three reagent slugs  202 ,  204  and  206 , potentially two different reactions are being carried out, e.g., within mixtures  208  and  210 , if the first and third reagents represented two different reagents, or different concentrations of the same reagent. Further, as can be seen, the system includes a built in spacing fluid region in the form of the slug of the second reagent  204 . By lining up slugs of different reagents, one can rapidly carry out a large number of different reactions, and particularly matrixed reactions, in a serial format. 
     The methods of the present invention are particularly useful in performing genotyping reactions on a relatively large number of patients with respect to a relatively large number of different genetic loci. By way of example, the first library of reagents consists of “patient specific” reagents, e.g., the genomic DNA from a number of different patients who are to be genotyped. The second library of reagents then consists of the “locus-specific” reagents, e.g., amplification primers for the subsequence that contains the particular locus of interest, as well as any other reagents specific to and necessary for discriminating the nature of the polymorphism at the locus, e.g., locus specific probes, i.e., nucleic acid or analog probes. Other reagents that are generic to the whole process are then included as part of one of the reagent libraries or are included in the system buffers, e.g., as part of each different library reagent plug, or are separately and continuously flowed into the conduit along with all of the different library reagents. 
     In the genotyping example, and with reference to  FIG. 2 , a first locus specific reagent mixture, e.g., primers and probes for amplifying a particular locus containing region of a patient&#39;s DNA, may be represented by reagent slug  202 , while the template or patient DNA is contained in reagent slug  204 . A second mixture of different locus specific reagents (specific for a different locus on the patient&#39;s DNA) would be represented by reagent slug  206 . As each of the two different locus specific reagent mixtures diffuses and/or disperses into the patient specific reagent slug, e.g., containing the template DNA, the regions of overlap will be capable of supporting amplification of each of the two loci. Specifically, reagent mixture  208  would include the template DNA from slug  204 , as well as the primers for amplifying the first locus containing region from slug  202 , and any other locus specific reagents, e.g., locus specific probes that would be used for discrimination in certain processes. The second reagent mixture  210  would include the same template DNA, but primers (and optionally discrimination reagents, e.g., probes) that would be specific for the second locus. When the entire train of reagents, e.g., as shown in  FIG. 2B  is subjected to thermal cycling in the presence of a DNA polymerase and dNTPs, only the complete mixtures  208  and  210  would support amplification. Further, the amplified products would be distinguishable from each other by virtue of their physical isolation from each other. 
     Although described in terms of using discrimination reagents, e.g., nucleic acid probes that are specific for one variant or the other at a given locus, e.g., Molecular beacons or other signal generating probes, i.e., TaqMan probes, in certain preferred aspects, the discrimination is carried out by virtue of the use of an allele specific primer sequence used during amplification. A variety of different discrimination techniques are generally described in U.S. Patent Application No. 60/283,527, filed Apr. 12, 2001, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. Specifically, one of the primers is made to be sufficiently complementary to one variant of the polymorphic position in the template sequence, whereby the presence of the other variant will prevent hybridization and, consequently, amplification. In such case, no additional discrimination reagents are required, and detection is carried out by detecting whether amplification has occurred in the first instance. Such allele specific amplification is well known and is described in e.g., U.S. Pat. Nos. 5,525,494, 5,866,366, 6,090,552, and 6,117,635. 
     The interfacial mixing method allows all, or virtually all, of the reaction steps involved in performing the particular experiment, e.g., SNP genotyping, to be carried out in a single conduit for a large number of different patients and different loci. In particular, reagent mixing, amplification, discrimination and detection can all be carried out in this conduit while also including a built-in separation between the various experiments by virtue of the slugs of different reagents through which the other reagents have not completely diffused and/or dispersed. Stated in an alternative manner, one can screen an entire battery of reagents, e.g. locus specific reagents in a first reagent train where each of the locus specific reagent slugs is bounded by one patient specific reagent plug. The same battery can then be screened against another patient&#39;s DNA, by substituting a second patient specific reagent plug as the spacing reagent between the locus specific reagents. 
     The interfacial mixing methods of the present invention were demonstrated using two reagent slugs repeatedly and alternately introduced into a capillary channel. One of the reagent slugs included primers designed for amplification of a specific region of a template nucleic acid, a DNA polymerase, the four naturally occurring dNTPs, and a TaqMan probe that gave increasing fluorescence upon amplification of the specific region of the template. The other fluid contained the template nucleic acid. The contents of the capillary were subjected to thermal cycling through a temperature profile that supported melting of the template, annealing of the primers to the template and extension of those primers along the template. 
       FIG. 3  schematically illustrates a microfluidic channel network useful in carrying out the methods of the invention. As shown, the channel network is disposed in a body structure of a microfluidic device  300 . A sampling capillary (not shown) is attached to the body structure and used to sample reagents into the main channel  302  of the network, via port  304 . The sampling capillary is placed into fluid communication with each of the different reagent source, in series, in order to serially introduce the different reagent slugs. Optional additional reagent sources  306  and  308  may be provided in the body structure  300 , and in fluid communication with main channel  302 , e.g., via channels  310  and  312 , respectively. In the case of SNP genotyping experiments, it is generally desirable to include a heating zone  320  in main channel  302 . The heating zone may be provided by placing an external or integral heating element, i.e., a resistive heater or peltier device, adjacent to or within the heating zone. Alternatively, electrical or “Joule heating” may be used to control the temperature of the heating zone  320 . Controlled Joule heating is described in detail in U.S. Pat. No. 6,174,675. In the case of electrical heating, electrodes  322  and  324  are placed so as to be able to pass electrical current through the fluid in the heating zone  320  of main channel  302 . As shown, such electrodes are placed in wells  326  and  328 , respectively that are fluidly connected to main channel  302  at opposite ends of the heating zone  320 . As will be appreciated, different applications may require multiple different heating zones, e.g., to heat to different temperatures, or for different uses, e.g., for generating thermal melting curves, etc. The electrodes are in turn, typically coupled to an appropriate electrical controller for providing current through the fluid in the heating zone in response to measured temperatures and desired temperature profiles. A detection zone  330  is also provided, which typically comprises a transparent region of the main channel  304 , through which optical signals can be passed. 
       FIG. 4  schematically illustrates an overall system  400  for carrying out the methods of the invention. The system includes a microfluidic device  300  (shown as including external capillary element  350 ). The reagents are accessed through the capillary element  350  from source plates, e.g., multiwell plate  402 . A flow controller  404  is also provided operably coupled to the microfluidic device  300 , e.g., by a vacuum line, in the case of vacuum based flow, for driving fluid movement into and through the channels of device  300 . Also shown is a temperature controller  406  operably coupled to the heating zone, for controlling and monitoring (either through an included sensor or via the monitoring of fluidic resistance, in the case of certain Joule heating embodiments) the temperature of the heating zone(s) in response to preprogrammed instructions from the user. As shown, the temperature controller is connected to the reservoirs of the device, as would be the case in Joule heating applications, although connection is similarly made to resistive heating elements attached to or disposed adjacent to the heating zone of the device. A detection system  408  is also typically included disposed within sensory communication of the main channel or channels of the microfluidic device, in order to detect the signal that is ultimately produced in the discrimination analysis. Typically, such detection systems include optical, and preferably, fluorescence detection systems that are well known in the art. In particular, in the case of genotyping experiments, a number of different discrimination techniques have been developed that produce a fluorescent signal that is indicative of one variant allele or the other, thus requiring fluorescence detection. Although illustrated as different units, it will be appreciated that the flow controller, temperature controller and detector may be integrated into a single instrument, for ease of use. A computer or other processor  410  is also typically included operably coupled to the various controllers and detectors of the system, in order to receive information from these system components, and instruct their operation in accordance with pre-programmed instructions. 
       FIG. 5  is a plot of the fluorescent signal received from the capillary as the reagent slugs passed the detection point. The areas of increased fluorescence result from the fluorescent signal of a TaqMan probe that indicates amplification. As can be seen, amplification occurs only in regular spaced intervals that correspond to the regions surrounding the interface of the slugs of the two different reagents that were repeatedly interspersed into the capillary. Notably, the regions indicating amplification are separated by regions where no apparent amplification is taking place. 
     Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. All publications and patent applications referenced herein are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.