Slug control during thermal cycling

Abstract

The present invention, in one aspect, provides methods and systems for controlling slugs using temperature dependent fluorescent dyes. In some embodiments, the present invention uses one or more techniques to enhance the visibility of slugs, enhance a system's ability to differentiate between slugs, and enhance a system's ability to identify the positions of slugs.

What is claimed is: 
     
         1 . A system for controlling the position of a slug within a microfluidic device, comprising:
 (a) a microfluidic device comprising one or more fluid channels and one or more slugs in said one or more fluid channels, wherein said one or more slugs include one or more fluorescent dyes, and   (b) a slug excitation device configured to excite said one or more fluorescent dyes within said one or more slugs;   (c) a light-sensing unit configured to capture fluorescence from at least a region of interest of the microfluidic device, wherein said region of interest includes one or more of said slugs;   (d) a processing unit configured to receive information from said light-sensing unit;   (e) a position identification unit configured to identify the position of one of said one or more slugs within said region of interest; and   (f) a slug control unit, configured to position the slug within the microfluidic device based upon the identified position.   
     
     
         2 . The system of  claim 1 , wherein said slug control unit comprises a proportional-integral-derivative control unit operatively engaged with a fluid control device configured to move the slug. 
     
     
         3 . The system of  claim 1 , wherein the light-sensing unit comprises a digital single-lens reflex camera. 
     
     
         4 . The system of  claim 1 , wherein said slug excitation device comprises a light source. 
     
     
         5 . A method for controlling the position of a fluid slug during polymerase chain reaction (PCR) amplification, wherein the PCR has a denaturation step, an annealing step and an extension step, comprising:
 exciting a fluorescent dye in said slug;   acquiring images of the fluorescence of said slug;   processing data in said images;   identifying the position of said slug from said processed image data;   controlling the position of said slug based on the detected position;   
       wherein the denaturation step is of shorter duration than the period at which said images are acquired. 
     
     
         6 . The method of  claim 5 , wherein the annealing step is of longer duration than the denaturation step, and at least a portion of the images are acquired during the annealing step. 
     
     
         7 . The method of  claim 6 , wherein about 30% or more of the images are acquired during the annealing step. 
     
     
         8 . The method of  claim 7 , wherein about 50% or more of the images are acquired during the annealing step. 
     
     
         9 . The method of  claim 6 , wherein the annealing step is about seven to about ten seconds in length. 
     
     
         10 . The method of  claim 5 , wherein the step of acquiring images of the fluorescence of said slug occurs at an image acquisition frequency that is greater than or equal to about one image per second. 
     
     
         11 . The method of  claim 10 , wherein the image acquisition frequency is about one to about thirty images per second. 
     
     
         12 . The method of  claim 5 , wherein the extension step is of longer duration than the denaturation step, and at least a portion of the images are acquired during the extension step. 
     
     
         13 . The method of  claim 12 , wherein about 30% or more of the images are acquired during the extension step. 
     
     
         14 . The method of  claim 13 , wherein about 50% or more of the images are acquired during the extension step. 
     
     
         15 . The method of  claim 12 , wherein the extension step is about seven to about ten seconds in length. 
     
     
         16 . The method of  claim 12 , wherein the step of acquiring images of the fluorescence of said slug occurs at an image acquisition frequency that is greater than or equal to about one image per second. 
     
     
         17 . The method of  claim 16 , wherein the image acquisition frequency is about one to about thirty images per second. 
     
     
         18 . The method of  claim 5 , wherein no images are acquired during the denaturation step. 
     
     
         19 . The method of  claim 5 , wherein said denaturation step has a duration of about one second, said annealing step has a duration of about eight seconds, and said extension step has a duration of about one second. 
     
     
         20 . The method of  claim 5 , wherein said denaturation step has a duration of about 2 seconds, said annealing step has a duration of about 1.5 seconds, and said extension step has a duration of about 6.5 seconds. 
     
     
         21 . A method for controlling the position of a slug during a polymerase chain reaction (PCR) thermal cycle, comprising:
 a denaturation step comprising subjecting the slug to a temperature sufficiently high to allow denaturation,   an annealing/extension step comprising subjecting the slug to a temperature lower than the denaturation temperature and which is sufficient to allow primer annealing and extension,   exciting a fluorescent dye in said slug during said thermal cycle;   acquiring images of the fluorescence of said slug;   processing data in said images;   identifying the position of said slug from said processed image data; and   controlling the position of said slug based on the detected position.   
     
     
         22 . The method of  claim 21 , wherein the denaturation step comprises subjecting the slug to a first temperature sufficiently high to allow denaturation and a second temperature lower than the first temperature but still sufficiently high to allow denaturation during subsequent denaturing steps during the PCR thermal cycle. 
     
     
         23 . The method of  claim 22 , wherein the first temperature is about 95° C. or greater, and wherein the second temperature is about 90° C. or lower. 
     
     
         24 . A system for controlling the position of a slug within a microfluidic device, comprising:
 (a) a microfluidic device comprising one or more fluid channels, one or more thermal elements in communication with said one or more channels, wherein said one or more fluid channels are configured to receive one or more slugs and wherein said one or more slugs include one or more fluorescent dyes,   (b) a slug excitation device configured to excite said one or more fluorescent dyes within said one or more slugs;   (c) a light-sensing unit configured to capture fluorescence at an acquisition rate from at least a region of interest of the microfluidic device, wherein said region of interest is configured to include one or more of said slugs;   (d) a temperature control unit configured to control the temperature of said slugs using said thermal elements of the microfluidic device, wherein said temperature control unit is configured to subject said slug to (i) a denaturation step at a first temperature for a first duration of time, (ii) an annealing step at a second temperature for a second duration of time, and (iii) an extension step at a third temperature for a third duration of time;   (e) a processing unit configured to receive information from said light-sensing unit;   (f) a position identification unit configured to identify the position of one of said one or more slugs within said region of interest; and   (g) a slug control unit, configured to position the slug within the microfluidic device based upon the identified position, and   
       wherein the duration of the denaturation step is of a shorter duration than the period between image acquisitions. 
     
     
         25 . The system of  claim 24 , wherein the duration of the annealing step is longer than the duration of the denaturation step, and at least a portion of the images are acquired during the annealing step. 
     
     
         26 . The system of  claim 24 , wherein the duration of the extension step is longer than the duration of the denaturation step, and at least a portion of the images are acquired during the extension step. 
     
     
         27 . The system of  claim 24 , wherein said denaturation step has a duration of about one second, said annealing step has a duration of about eight seconds, and said extension step has a duration of about one second. 
     
     
         28 . The method of  claim 24 , wherein said denaturation step has a duration of about 2 seconds, said annealing step has a duration of about 1.5 seconds, and said extension step has a duration of about 6.5 seconds. 
     
     
         29 . A method for controlling the position of a fluid slug during polymerase chain reaction (PCR) amplification, wherein the PCR has a denaturation step, an annealing step and an extension step, comprising:
 exciting a fluorescent dye in said slug;   acquiring images of the fluorescence of said slug;   processing data in said images;   identifying the position of said slug;   controlling the position of said slug based on the detected position;   
       wherein the annealing step is of longer duration than the denaturation period, such that more than 30% of the images collected are obtained during the annealing step. 
     
     
         30 . The method of  claim 29 , wherein more than 50% of the images collected are obtained during the annealing step. 
     
     
         31 . A method for controlling the position of a fluid slug during polymerase chain reaction (PCR) amplification, wherein the PCR has a denaturation step, an annealing step and an extension step, comprising:
 exciting a fluorescent dye in said slug;   acquiring images of the fluorescence of said slug;   processing data in said images;   identifying the position of said slug;   controlling the position of said slug based on the detected position;   
       wherein the extension step is of longer duration than the denaturation period, such that more than 30% of the images collected are obtained during the extension step. 
     
     
         32 . The method of  claim 31 , wherein more than 50% of the images collected are obtained during the annealing step. 
     
     
         33 . A method for controlling the position of a fluid slug during a polymerase chain reaction (PCR) amplification,
 wherein the PCR comprises:
 a first denaturation step comprising subjecting the slug to a temperature sufficiently high to allow denaturation, and 
 a second annealing and extension step comprising subjecting the slug to a temperature lower than the denaturation temperature to allow primer annealing and extension, 
   
       comprising:
 exciting a fluorescent dye in said slug; 
 acquiring images of the fluorescence of said slug; 
 processing data in said images; 
 identifying the position of said slug; and 
 controlling the position of said slug based on the detected position. 
 
     
     
         34 . The method of  claim 33 , wherein no images are obtained during the denaturation step. 
     
     
         35 . A system for controlling the position of a slug within a microfluidic device, comprising:
 (a) a microfluidic device comprising one or more fluid channels, one or more thermal elements in communication with said one or more channels, wherein said one or more fluid channels are configured to receive one or more slugs and wherein said one or more slugs include one or more fluorescent dyes,   (b) a slug excitation device configured to excite said one or more fluorescent dyes within said one or more slugs;   (c) a light-sensing unit configured to capture fluorescence at an acquisition rate from at least a region of interest of the microfluidic device, wherein said region of interest is configured to include one or more of said slugs;   (d) a temperature control unit configured to control the temperature of said slugs using said thermal elements of the microfluidic device, wherein said temperature control unit is configured to subject said slug to (i) a denaturation step at a first temperature for a first duration of time, and (ii) an annealing/extension step at a second temperature for a second duration of time.   (e) a processing unit configured to receive information from said light-sensing unit;   (f) a position identification unit configured to identify the position of one of said one or more slugs within said region of interest; and   (g) a slug control unit, configured to position the slug within the microfluidic device based upon the identified position.