Patent Publication Number: US-2015064706-A1

Title: Detection and Mixing in a Conduit in Integrated Bioanalysis Systems

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims a priority benefit under 35 U.S.C. §119(e) from U.S. Patent Application No. 60/946,950, filed Jun. 28, 2007, which is incorporated herein by reference. 
    
    
     FIELD 
     The field of the present disclosure relates to apparatuses and methods for high-throughput detection in integrated bioanalysis systems. 
     BACKGROUND 
     Generally, in bioanalysis, liquid processing is essential for the many process steps involved in obtaining a result. Additionally, many analysis steps, such as sample preparation, reaction, separation, detection, and data processing involved in a broad range of bioanalyses usually require a variety of devices and instrumentation. 
     For many types of bioanalyses, there is desire to reduce the physical complexity of the biotechnology laboratory and at the same time increase throughput. Therefore, there is a need in the art for bioanalysis systems that can integrate analysis steps such as sample preparation, reaction, separation, detection, and data processing into a single footprint, and at the same time have the flexibility to scale throughput. 
     All patents, applications, and publications mentioned here and throughout the application are incorporated in their entireties by reference herein and form a part of the present application. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  and  FIG. 1B  depict variations of liquid processing manifolds for use in embodiments of integrated bioanalysis systems. 
         FIG. 2A  is a perspective view depicting an integrated bioanalysis system illustrative of the present teachings, and  FIG. 2B  is a cross-section of a side view depicting a subassembly of  FIG. 2A . 
         FIG. 3A  and  FIG. 3B  are perspective views that depict variations of integrated bioanalysis systems illustrative of the present teachings. 
         FIG. 4A  is a perspective view depicting an integrated bioanalysis system illustrative of the present teachings, and  FIG. 4B  is a cross-section of a side view depicting a subassembly of  FIG. 4A . 
         FIG. 5  depicts a variation of a scanning detection device for use in conjunction with embodiments of liquid processing manifolds. 
         FIGS. 6A-6C  depict a method for mixing two liquids using various embodiments of liquid processing manifolds, illustrative of the present teachings. 
         FIGS. 7A-7C  depict a method for mixing two liquids using various embodiments of liquid processing manifolds illustrative of the present teachings. 
     
    
    
     It is to be understood that the figures are not drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. 
     DETAILED DESCRIPTION 
     What is disclosed herein are various embodiments of apparatuses and methods in which luminescent detection is integrated with various analysis steps that are practiced in a range of biological analyses. In bioanalysis, functions such as sample preparation, reaction, and separation require the processing of fluids, such as, for example, the dispensing, mixing, and transport of liquids. Additionally, control of environmental conditions that impact analysis, such as, for example, temperature, pH, and ionic strength is frequently required. In the various embodiments of apparatuses and methods disclosed herein, detection is integrated with various liquid processing and environmental control functions to create integrated bioanalysis systems thereby. Though the various embodiments of integrated bioanalysis systems are useful for any number of analysis formats, they are adaptable to high-throughput processing of samples. 
     In disclosed embodiments of apparatuses and methods for integrated bioanalysis systems, liquid processing, environmental control and detection are integrated functions that can be performed in individual conduits. In various embodiments, a plurality of conduits comprises a liquid processing manifold. 
     The term “conduit” as used herein is any number of liquid processing components known in the art of bioanalysis, such as, but not limited by, tubing, piping, needle, pipette, and pipette tip. Such conduits are useful in a variety of manipulations of samples and reagents for a variety of bioanalyses. 
     The term “luminescent detection” as used herein includes photoluminescent detection, such as fluorescence and phosphorescence, as well as chemiluminescent detection, including bioluminescent detection. These types of luminescent detection are useful for a wide range of bioanalyses, offering sensitive detection over a wide range of analytes such as nucleic acids, polypeptides, hormones, drug substances, and the like. An exemplary class of bioanalyses are enabled by a technique know as the polymerase chain reaction (PCR). Some examples of bioanalyses that utilize the PCR technique include viral quantitation, quantitation of gene expression, drug therapy efficacy, DNA damage measurement, pathogen detection, and genotyping. 
     As previously mentioned, in various embodiments of apparatuses and methods for integrated bioanalysis systems, liquid processing, environmental control and detection are integrated functions that can be performed in individual conduits. Additionally, a liquid processing manifold including a plurality of conduits can be useful for high throughput liquid processing systems. The various embodiments of liquid processing manifold  100  depicted in  FIG. 1A  and  FIG. 1B  can be used with embodiments of integrated bioanalysis systems. Liquid processing manifold  100  of  FIG. 1A  and  FIG. 1B  can have a conduit assembly  120  having a plurality of conduits  110 . In various embodiments of liquid processing manifold  100  of  FIG. 1A  and  FIG. 1B , conduit  110  can be removable and replaceable. Conduit  110  has a body  112  which has a first end  114  and a second end  116 , and has a bore  118  extending through body  112 . In some embodiments of liquid processing manifold  100 , conduit assembly  120  can have conduits  110  that are arranged in a linear array. In some embodiments of liquid processing manifold  100 , conduit assembly  120  can have conduits  110  that can be arranged in numerous types of two-dimensional geometries. In some embodiments of liquid processing manifold  100 , conduit  110  can be fabricated from a polymeric material, for example, but not limited by, from classes of polymers such as polypropylene, polyethylene, polyhalohydrocarbon, polycarbonates, and polysilicones, and combinations thereof. In some embodiments of liquid processing manifold  100 , conduit  110  can be fabricated from an inorganic oxide material, for example, but not limited by such as quartz, fused silica, and sapphire, and combinations thereof. In some embodiments of liquid processing manifold  100 , conduit  110  can be fabricated from a metal, such as but not limited by stainless steel, titanium, and combinations thereof. In such embodiments, the metal may be lined with a polymer or inorganic oxide material. In general, attributes for conduits  100  of conduit assembly  120  include, but are not limited by, chemical, mechanical, and thermal stability for their intended use in bioanalysis. 
     In addition to conduit assembly  120 , various embodiments of liquid processing manifold  100  of  FIG. 1A  and  FIG. 1B  can have a plunger or piston assembly  150  to provide control for processing fluids. In some embodiments of liquid processing manifold  100  of  FIG. 1A , piston assembly  150  can function in a housing assembly  60 , that can have a plurality of piston housings  50 . Piston housing  50  has a body  52  having first end  54  and a second end  56 , with a bore  58  extending through body  52 . The second end  116  of conduit  110  can be fitted to first end  54  of the piston housing  50  so that piston housing bore  58  is in fluid communication with conduit bore  118 . Piston assembly  150  can have a plurality of pistons  140 . Piston  140  has a first end  142 , which sealably engages piston housing bore  58  and conduit bore  118 , and a second end  144 , which can be connected to mechanical means for moving the piston  140 , depicted by bar  146  in  FIG. 1A  and  FIG. 1B . As indicated in  FIG. 1B , which shows two variations for bar  146 , mechanical means for moving the piston  140  can be fashioned to move a plurality of pistons, or to move them individually. In  FIG. 1A and 1B , conduit  110  has first end  114  in which a liquid aliquot or slug  130  can be processed using various embodiments of liquid processing manifolds  100 . 
     In various embodiments of liquid processing manifold  100  of  FIG. 1A and 1B , the movement of piston  140  causes a displacement of fluid in conduit  110 , controlling the movement of fluids in conduit  110  thereby. Such control of fluids may be useful for many types of manipulations of fluids, such as, but not limited by aspiration, mixing, aliquoting, and dispensing, and the like. Moreover, various embodiments of liquid processing manifold  100  enable the processing of a few samples for low throughput processing or many samples for high throughput processing. 
     In some bioanalyses, piston housing bore  58  and conduit bore  118  of  FIG. 1A and 18  may be other than an air/liquid interface for manipulating a liquid aliquot or slug  130  in order to provide an interface tension greater than that provided by an air/liquid interface. In some embodiments of liquid processing manifold  100 , first end  142  of piston  140  may come in direct contact with liquid aliquot or slug  130  to provide a solid/liquid interface. In some variations of liquid processing manifold  100 , the bore-space between first end  142  of piston  140  and liquid aliquot or slug  130  may be partially or totally filled with a fluid that is inert and immiscible and in contact with liquid aliquot or slug  130 , providing a liquid/liquid interface thereby. For example, since the vast majority of bioanalyses are aqueous-based, an example of such an inert, immiscible fluid can be an oil, such as a mineral oil. Additionally, it is desirable that the coefficient of expansion of the inert fluid be low, so as to minimize the impact of the change in volume of the inert fluid when thermostating system  200  is used in variations of integrated bioanalysis system  500 . 
     In various embodiments of liquid processing manifold  100  of  FIG. 1A and 1B , liquid aliquot or slug  130  positioned at first end  114  of conduit  110  can be finely manipulated and controlled. The phrase “positioned at first end  114 ” in reference to position of a liquid aliquot or slug  130  may include embodiments where liquid aliquot or slug  130  can be within the first end, and remains at a position proximal to first end  114 , as well as embodiments where the liquid aliquot or slug  130  can be at least partially extended from first end  114 . In some embodiments, liquid aliquot or slug  130  can be enveloped by an inert, immiscible fluid, such as an oil, for example a mineral oil, so that the protruding liquid can be an oil droplet or film. As will be discussed in more detail subsequently, liquid aliquot or slug  130  can be positioned at first end  114  so that it may be readily detected. 
     For various disclosed embodiments of integrated bioanalysis system  500 , a thermostating system  200  can be provided to conduit assembly  120  of the liquid processing manifolds  100  by providing one or a plurality of thermostating units, such as for example, thermostating units  252  and  254  of  FIG. 1A  or thermostating units  252 ,  254 ,  256  and  258  of  FIG. 1B . In addition to the thermostating units, such as  252  and  254  of  FIG. 1A , thermostating unit  200  may include additional components, such as thermisters and controllers. The plurality of thermostating units can provide discrete thermal zones for each conduit  110 , which discrete zones may be maintained at a desired temperature. In some embodiments of a thermostating system  200 , the thermostating units may be for example Peltier devices, providing the capability to heat or cool the discrete thermal zones to a desired temperature. In other embodiments of a thermostating system  200  the thermostating units may be, for example, heat blocks that can heat each discrete thermal zones to a desired temperature. An example of an integration of a liquid processing manifold with thermal control for heat cycling during PCR amplification can be found in U.S. Pat. No. 5,985,651 (Hunike-Smith; Nov. 16, 1999). 
     Various embodiments of liquid processing manifolds  100 , fitted with a thermostating system  200  may be incorporated into embodiments of integrated bioanalysis systems. Such systems are integrated to provide a complete range of liquid processing and detection adapted to conduit  110 , so that in addition to liquid processing, the conduit  110  serves as a reaction and detection vessel. Various embodiments of disclosed integrated bioanalysis systems provide flexibility to the end user by providing flexibility in throughput from a few samples to many, flexibility over the volume of liquid aliquot or slug  130  processed by selection of conduit inner diameter and slug length, and flexibility over assay format through selection of automated liquid processing providing control to individual or selected numbers of conduits. 
       FIG. 2A  is a perspective view of integrated bioanalysis system  500  according to various embodiments of the present teachings. The integrated bioanalysis system  500  can have instrument support unit  300  which includes instrument support housing  310 , which can be a housing for instrument control system  320 . Additionally, instrument support unit  300  can act as a mount for liquid processing manifold  100  using liquid processing manifold chassis  312 , stage  330 , and detection system  400 . Instrument control system  320  can control the operation of liquid processing manifold  100 , control thermostating system  200 , as well as the control the movement of stage  330 , and the operation of detection system  400 . Additionally, instrument control system  320  may provide data processing and report preparation functions. All such instrument control functions may be dedicated locally to the integrated bioanalysis system  500 , or instrument control system  320  may provide remote control of part or all of the control, analysis, and reporting functions. 
     The detection system  400  of  FIG. 2A  has excitation source  410 , detector  430 , and an optical train including filter  450 , first mirror  452 , second mirror  454 , and motor  456  that can be used to control the position of first mirror  452  and second mirror  454 . According to various embodiments, a detection system can comprise one or more excitation sources, detectors, operational amplifiers, and current control circuits. Such components may have temperature dependent properties, meaning that their properties (e.g., LED intensity) can change with temperature variations, which will be discussed in more detail subsequently. In  FIG. 2A , excitation source  410  is depicted as an array of light emitting diodes (LEDs), though different embodiments of detection system  400  may use various excitation sources. An excitation source  410  is used to excite chemical or biochemical species in liquid aliquot or slug  130  positioned at first end  114  of conduit  110 , which first end serves as a reaction and detection vessel. The terms “excitation source,” “irradiation source,” and “light source” are used in the art interchangeably. 
     The term “LED” or “light emitting diode” is used herein to refer to conventional light-emitting diodes, i.e., inorganic semiconductor diodes that convert applied electrical energy to light, as well as organic light emitting diode (OLEDs). Conventional LEDs include, for example, aluminum gallium arsenide (AlGaAs), which generally produce red and infrared light, gallium aluminum phosphide, which generally produce green light, gallium arsenide/phosphide (GaAsP), which generally produce red, orange-red, orange, and yellow light, gallium nitride, which generally produce green, pure green (or emerald green), and blue light, gallium phosphide (GaP), which generally produce red, yellow and green light, zinc selenide (ZnSe), which generally produce blue light, indium gallium nitride (InGaN), which generally produce bluish-green and blue light, indium gallium aluminum phosphide, which generally produce orange-red, orange, yellow, and green light, silicon carbide (SIC), which generally produce blue light, diamond, which generally produce ultraviolet light, and silicon (Si), which are under development. LEDs are not limited to narrowband or monochromatic light LEDs; LEDs may also include broad band, multiple band, and generally white light LEDs. Organic LEDs can be polymer-based or small-molecule-based (organic or inorganic), edge emitting diodes (ELED), Thin Film Electroluminescent Device s(TFELD), Quantum dot based inorganic “organic LEDs,” and phosphorescent OLED (PHOLED). In addition to LEDs and OLEDs, some embodiments of integrated bioanalysis system  500  may utilized excitation sources such as lasers, for example solid state lasers, such as YAG lasers, gas lasers, such as helium neon (HeNe) lasers, and diode lasers as well as lamps, such as for example, deuterium or mercury lamps. 
     According to some embodiments of detection system  400  of  FIG. 2A  of integrated bioanalysis system  500 , excitation source  410  can illuminate an entire conduit assembly  120 . In other embodiments, detection system  400 , excitation source  410  can be directed to illuminate portions of first ends  114  of conduit assembly  120  (see  FIGS. 1A and 1B ). An excitation source  410  can include, for example, a combination of two, three, or more LEDs, OLEDs, laser diodes, and the like that are positioned to illuminate all or a portion of conduit assembly  120 . In some embodiments, the LEDs may be white light LEDs that illuminate all or a portion of conduit assembly  120 . In some embodiments, all or a portion of conduit assembly  120  may be illuminated by LEDs having a first relatively short wavelength in the visible range of the electromagnetic spectrum (e.g., UV-blue within the range of 380 nm to 495 nm), a second longer wavelength LED (e.g., green within the range of 450 nm to 495 nm), or a third longer wavelength LED (e.g., red within the range of 620 nm to 750 nm). In various embodiments, excitation source  410  of  FIG. 2A  that illuminates all or a portion of conduit assembly  120  may include combinations of LEDs having different wavelengths in the UV-visible range of the electromagnetic spectrum of between about 380 nm to about 750 nm. 
     The term “detector” refers to devices that convert electromagnetic energy into an electrical signal, and may include both single element, multi-element and array optical detectors. As previously mentioned, excitation source  410  is used to excite chemical or biochemical species in liquid aliquot or slug  130  positioned at first end  114  of conduit  110 . For the phenomenon of luminescent detection, such excited chemical or biochemical species emit electromagnetic radiation of a longer wavelength than the excitation source. As such, detector  430  is a device capable of monitoring the electromagnetic (e.g., optical) signal from the chemical or biochemical species in liquid aliquot or slug  130  positioned at first end  114  of conduit  110 , providing an electrical output signal or data related to the monitored electromagnetic (e.g., optical) signal. Such devices include, for example, but not limited by photodiodes, including avalanche photodiodes, phototransistors, photoconductive detectors, linear sensor arrays, CCD detectors, CMOS optical detectors (including CMOS array detectors), photomultipliers, and photomultiplier arrays. According to certain embodiments, an optical detector, such as a photodiode or photomultiplier, may contain additional signal conditioning or processing electronics. For example, an optical detector may include at least one pre-amplifier, electronic filter, or integrating circuit. Suitable preamplifiers include integrating, transimpedance, and current gain (current mirror) pre-amplifiers. 
     As shown in  FIG. 2A , detector  430  may be mounted from liquid processing manifold chassis  312 , though detector  430  can be mounted from numerous locations, such as, for example, stage  330  or a free-standing mount, so as to be positioned over second mirror  454 . Detector  430  is shown as a CCD camera, though various embodiments of integrated bioanalysis system  500  of  FIG. 2A  may use a variety of detectors as previously described. Light emitted from conduits  110  of liquid processing manifold  100  is reflected from first mirror  452  to second mirror  454  to be detected by detector  430 . If specificity of the wavelength of electromagnetic energy reaching detector  430  is indicated for some embodiments of integrated bioanalysis system  500 , a filter  450  can be utilized in various embodiments the detection system  400 . Additionally, control system  320  can control motor  456  for adjusting first mirror  452  and second mirror  454 , as well as a motor or motors (not shown) for controlling the positioning of stage  330 . Such control may be important not only for focusing the emitted light from liquid aliquot or slug  130  positioned at first end  114  of conduit  110 , but for other functions, as will be discussed in more detail subsequently. 
       FIG. 2B  is a cross-section of a side view depicting a liquid aliquot or slug  130  positioned at first end  114  of conduit  110  using the control of piston  140  and illuminated by excitation source  410 , depicted as LEDs, though as previously described, capable of being a variety of devices. The light emitted by excited chemical or biochemical moieties in liquid aliquot or slug  130  is reflected from first mirror  452  and second mirror  454  to detector  430 , as indicated by the hatched line. As previously discussed, the phrase “positioned at first end  114 ” in reference to position of liquid aliquot or slug  130  for the purpose of detection may include embodiments where liquid aliquot or slug  130  can be within the first end, and remains at a position proximal to first end  114 , as well as embodiments where liquid aliquot or slug  130  can be at least partially extended from first end  114 , as depicted in  FIG. 2B . In some embodiments, liquid aliquot or slug  130  can be enveloped by an inert, immiscible fluid, such as an oil, for example a mineral oil, so that the protruding liquid is an oil droplet or film. Most importantly, liquid aliquot or slug  130  can be positioned at first end  114  so that it may be readily detected by detector  430 . 
     Additional designs of detection systems for integrated bioanalysis system  500  are illustrated by various embodiments of detection system  400  of  FIG. 3A  and  FIG. 3B , as well as by various embodiments of detection system  400  of  FIG. 4A  and  FIG. 4B . Various embodiments of detection system  400  of  FIG. 3A  utilize direct detection of light emitted from excited chemical or biochemical species in liquid aliquots or slugs  130  positioned at first ends  114  of conduit assembly  120  (see  FIG. 1A and 1B ) by positioning detector  430  directly in view of first ends  114 . Various embodiments of detection system  400  indicated by  FIG. 3B  utilize a dichroic filter  458 . Such filters can be selected to reflect light of specific wavelength range to excite chemical or biochemical moieties in liquid aliquot or slug  130  positioned at first end  114  of conduit  110 , and then pass the emitted light from first end  114  to detector  430 . In  FIG. 4A , detection system  400  can be positioned on stage  330 . In some embodiments of integrated bioanalysis system  500  of  FIG. 4A , detection system  400  can be attached to stage  330 , and stage  330  can move detection system  400  into position to detect all or a subset of first ends  114  of conduit assembly  120 . In other embodiments of integrated bioanalysis system  500  of  FIG. 4A , detection system  400  can be moved along stage  330  to position detection system  400  to detect all or a subset of the first ends  114  of conduit assembly  120 . 
     Various embodiments of detection system  400  of  FIG. 4B  utilize of two, three, or more LEDs, OLEDs, laser diodes, and the like that are positioned to illuminate all or a subset of the first ends  114  of conduit assembly  120  and have additionally two, three, or more detecting devices such as photodiodes, phototransistors, photoconductive detectors, linear sensor arrays, such as CMOS array detectors positioned to detect the light emitted by chemical or biochemical moieties in liquid aliquots or slugs  130  for all or a subset of first ends  114  of conduit assembly  120  (see  FIG. 1A and 1B ). Embodiments of integrated bioanalysis system  500  that can utilize various embodiments of detection system  400  of  FIG. 5  are exemplary of a detection system that can be positioned and moved either along stage  330  or using stage  330 . For some embodiments of a movable detection system  400  of  FIG. 5  at least one excitation source, such as  430 ,  432 , and  434 , as well as at least one detector  410 , and at least one dichroic filter, such as  450 ,  452 ,  454 , and  456  can be used. Additionally, other optical elements, such as a focusing lens  460  may be incorporated in some embodiments of a movable detection system  400  of  FIG. 5 . An example of a detection system adaptable to embodiments of detection system  400  of  FIG. 5  can be found in US 2006/0121602 (Hoshizaki, et al.; Jun. 8, 2006). 
     According to the various embodiments of a detection system  400  given in the above, such detection systems can comprise one or more excitation sources  410 , such as LEDs, OLEDs, laser diodes, lasers, lamps, and the like, as well as one or more detectors  430 , such as photodiodes, CCD detectors, and CMOS optical detectors, and the like. Additionally, optical systems may include operational amplifiers, and LED-current control circuits. Such components may have temperature dependent properties, meaning that their properties (e.g., LED intensity) can change with temperature variations. In that regard, variations of detection systems  400  for use with embodiments of integrated bioanalysis systems  500  may utilize a temperature compensation system that can, for example, maintain some or all of these components at a constant temperature to eliminate or reduce changes in the temperature dependent property or properties. The temperature dependent property may also include properties that are a derived or indirect function of a temperature dependent property. Thus, for example, if electrical resistance is a temperature dependent property, current or voltage, which would be functions of the resistance, could also be temperature dependent properties. Other temperature dependent properties may include, for example, temperature dependent properties of an optical detector, such as a photodiode. For example, the “dark current” or noise of a detector may be temperature dependent. Temperature sensors may thus include electronic circuits and signal measurement devices or elements configured to monitor, for example, dark current or noise. 
     Liquid processing manifolds, such as various embodiments of disclosed liquid processing manifold  100 , process liquids taken from samples and reagents held in containing means, for example, but not limited by microtiter plates, as well as various containers such as, but not limited by, vials, tubes, ampoules, and cuvettes, and the like, that are held in holders, such as racks. As one of ordinary skill in the art is apprised, many high-throughput bioanalyses are adapted to a microtiter plate format, for example based on a 8 by 12 array of wells, yielding 96 wells per plate, or higher orders of wells per plate based on a multiple of the 96 well pattern. In a typical operation, liquid processing manifold  100  is used primarily for the dispensing of fluids, while the bioanalysis steps of reacting and detecting are done in containing means. Mixing a reagent or reagents with a sample is necessary to the step of reacting. In that regard, various embodiments of methods for on-conduit mixing of a plurality of liquids using embodiments of liquid processing manifold  100 , enabling on-conduit reactions thereby are depicted in  FIGS. 6A ,  6 B, and  6 C and  FIGS. 7A ,  7 B, and  7 C. 
     In various embodiments of a method depicted by  FIGS. 6A ,  6 B, and  6 C, first liquid slug  132  and second slug  134  can be drawn into conduit  110  from a containing means, such as  160 , in which the sample or reagent, such as  162 , has been dispensed ( FIG. 6A ). As depicted, first slug  132  and second slug  134  are separated by a segment of another fluid with which they are both immiscible, e.g., air. First slug  132  and second slug  134  can be drawn through conduit  110  and as depicted in  FIG. 6B , into a second, wider bore, e.g., piston housing bore  58 , using piston  140 . In various embodiments of a method depicted by  FIGS. 6A ,  6 B, and  6 C, piston housing bore  58  has a diameter that is different than that of conduit bore  118 . In  FIGS. 6B , and  6 C, as slugs  132  and  134  are drawn first into piston housing bore  58  and then moved back into conduit bore  118 , they are mixed to form mixed slug  136 . In various embodiments, the mixing of the first fluid and the second fluid can be increased by drawing mixed slug  136  into the second, wider bore and moving it back again into conduit bore  118 . Other embodiments for a method of mixing a plurality of slugs based on the difference in bore diameter of a conduit, housing, or combination thereof, can utilize, for example, a tapered conduit, housing or combination thereof. Various embodiments of a method for mixing a plurality of liquid slugs depicted in  FIGS. 7A ,  7 B, and  7 C utilize the movement of liquid slugs between conduit bore  118  and first end  114  for on-conduit mixing of a plurality of liquid slugs. In  FIG. 7A , a first liquid slug  132  can be drawn into conduit  110  from a containing means, such as  160 , in which the sample or reagent, such as  162 , has been dispensed. A second slug  134  can be drawn into first end  114  of conduit  110  as depicted in  FIG. 7B . Using piston  140 , first slug  132  and second slug  134  can be drawn up into conduit bore  118  as depicted in  FIG. 7C , and then a portion of the combined first slug  132  and second slug  134  can be controllably exuded at first end  114  as depicted in  FIG. 7B , effecting the mixing of first slug  132  and second slug  134  thereby to form mixed slug  136 . Though various embodiments of the methods depicted by  FIGS. 6A ,  6 B, and  6 C and  FIGS. 7A ,  7 B, and  7 C have been demonstrated with a first and second slug, the variations of embodiments of the methods can be extended to mixing higher orders of liquid slugs for numerous samples and reagents. In addition to mixing, other benefits may be realized in the use of various embodiments of on-conduit manipulations of liquid aliquots or slugs. For example, sample preparation steps, such as, but not limited by, nucleic acid shearing may be done on-conduit. 
     As previously mentioned, an exemplary class of bioanalyses are enabled by a technique know as the polymerase chain reaction (PCR). One type of PCR reaction is known to those skilled in the art as real-time PCR, which has become a widely used in bioanalyses. An example of a system and method for real time PCR amplification can be found in U.S. Pat. No. 5,928,907 (Woudenberg, et al.; Jul. 27, 1999). A range of embodiments of real-time PCR methods can be performed using various embodiments of an integrated bioanalysis systems  500 , as indicated by  FIG. 4A ,  FIG. 4B  and  FIG. 5 . In  FIG. 5  conduit bore  118  can be at least partially filled with an oil, such as a mineral oil. Sample and reagents for conducting a quantitative PCR method have been mixed according to variations of methods for on-conduit mixing previously described, and can be formed as slug  130 , which can be thermocycled, i.e., taken through a plurality of thermal cycles, using thermal system  200  for the purpose of amplification of targeted nucleic acid species. 
     Some embodiments of thermal system  200  of  FIG. 5  can have between about 2 heating blocks to about 4 heating blocks, each of which are controlled to a targeted temperature to create a separate targeted heat zone in conduit  110 . In some embodiments of a quantitative PCR method, a thermal setting of about 95° C. can be maintained for heating block  252 , a thermal setting of about 109° C. can be maintained for heating block  254 , a thermal setting of about 47° C. can be maintained for heating block  256 , and a thermal setting of about 60° C. can be maintained for heating block  258 . In some embodiments of apparatuses and methods for an integrated bioanalysis system  500 , in order to decrease the cycle time, pairing heating blocks for the denaturation portion of the real-time PCR cycle and the extension/annealing portion of the real-time PCR cycle can be done. For example, for the denaturation portion of a PCR cycle, slug  130  can be moved into a thermal zone of about 109° C. of heating block  254  until the desired temperature for slug  130  of about 95° C. is reached, and then slug  130  can be moved into a thermal zone of about 95° C. of heating block  252  for the duration of the denaturation portion of the cycle. Similarly, during the extension/annealing portion of a PCR cycle, slug  130  can be moved into a thermal zone of about 47° C. of heating block  256  until the desired temperature for slug  130  of about 60° C. is reached, and then slug  130  can be moved into a thermal zone of about 60° C. of heating block  258  for the duration of the extension/annealing portion of the cycle. After each cycle, slug  130  is either in position at first end  114  for detection, or can be readily positioned at first end  114  for detection before the next cycle is initiated. As previously discussed, the phrase “positioned at first end  114 ” in reference to position of a liquid aliquot or slug  130  may include embodiments where liquid aliquot or slug  130  can be within the first end, and remains at a position proximal to first end  114 , as well as embodiments where liquid aliquot or slug  130  can be at least partially extended from first end  114 . In some embodiments, liquid aliquot or slug  130  can be enveloped by an inert, immiscible fluid, such as an oil, for example a mineral oil, so that the protruding liquid can be an oil droplet or film  131 , as depicted in  FIG. 5 . 
     Though various embodiments of detection system  400  have been illustrated in various embodiments of figures presented, it is recognized by one of ordinary skill in the art that detection of slug  130  can be done on conduit  110  at a location other than the first end  114 . For example, detection of slug  130  could be done in any location along conduit  110  using, for example, fiber optic cables both from an excitation source and to a detector. 
     The principles of luminescent detection in integrated bioanalysis systems have been described in connection with exemplary embodiments. Accordingly, it should be understood that these descriptions are made for the purpose of illustration, and are not intended to limit the scope of the disclosure. In that regard, what is disclosed herein is not intended to be exhaustive or to limit the illustrations and descriptions to the precise forms depicted. Many modifications and variations will be apparent to the practitioner skilled in the art. What is disclosed was chosen and described in order to best explain the principles and practical application of the disclosed embodiments of the art described, thereby enabling others skilled in the art to understand the various embodiments and various modifications that are suited to the particular use contemplated. It is intended that the scope of what is disclosed be defined by the following claims and their equivalence.