Abstract:
Microscale or nanoscale apparatus for stopped-flow, quenched flow or continuous flow reaction apparatus where fluids or gases are mixed in a device composed of parallel or serial assembly of the basic fluid-containing cell having a longitudinal axis, a cross-sectional area generally perpendicular to the longitudinal axis, and at least one connected crossing cell having a longitudinal axis, a cross-sectional area generally perpendicular to said longitudinal axis and a fluid motivating force interacting transversally with the fluids flow.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit under 35 U.S.C. Section 119(e) of the following co-pending and commonly-assigned U.S. patent application: 
         [0002]    U.S. Provisional Patent Application Ser. No. 60/910,154, filed on Apr. 4, 2007, by Caroline Cardonne, Frederic Bottausci, Carl Meinhart and Igor Mezic, entitled “STOPPED FLOW, QUENCHED FLOW AND CONTINUOUS FLOW REACTION METHOD AND APPARATUS,” attorneys docket number 30794.230-US-P1 (2005-040); 
         [0003]    which application is incorporated by reference herein. 
     
    
     BACKGROUND OF THE INVENTION 
       [0004]    1. Field of the Invention 
         [0005]    The present invention relates to stopped flow, continuous flow and quenched flow apparatus, and processes or methods for making the same. More particularly, the present invention relates to performing stopped flow, continuous flow or quenched flow experiments by mixing the solutions under low pressure and non-turbulent conditions. 
         [0006]    2. Description of the Related Art 
         [0007]    (Note: This application references a number of different publications as indicated throughout the specification by one or more reference numbers within brackets, e.g., [x]. A list of these different publications ordered according to these reference numbers can be found below in the section entitled “References.” Each of these publications is incorporated by reference herein.) 
         [0008]    Understanding the structure, the thermodynamic and kinetic properties of protein folding is crucial for comprehending the origin of a wide range of diseases. Many diseases appear to be the result of incomplete or misfolding of proteins. Folding and unfolding are the ultimate ways of generating and abolishing specific types of cellular activities [1]. Proteins that fail to fold correctly, or fail to remain correctly folded, might therefore give rise to malfunctioning living systems and hence to possible development of diseases [2-5, 6], like cystic fibrosis [5], or some types of cancer [7]. Misfolded proteins can also aggregate, which can result in several other diseases [10], like the Prion disease (Creutzfieldt-Jacob) [2, 8, 9], Alzheimer&#39;s [3] or Parkinson&#39;s disease [4]. 
         [0009]    Recently, several studies have been extended to structural investigations of larger biological assemblies, such as RNA (folding) [11], viruses (conformational changes) [11], or globular complexes (crystallization) [12]. 
         [0010]    The study of the kinetics of proteins is achieved by rapidly mixing the biological molecules with reactants. In contact with the chemicals, the unfolded proteins start to fold. Folding can be a very fast process. Protein folding, for example, can occur in a very short time, ranging from a few hundreds of nanoseconds to hundreds of milliseconds [13, 14, 15]. It is then essential to achieve a homogeneous mixing in a minimum amount of time in order to get reliable information from the start of the folding process. The purpose is to understand the reason for incomplete folding and misfolding of proteins. Mixing is thus a key component that has to be performed by one of the devices used in the process. Widely used mixing processes generally involve turbulence. The mixing is then achieved by combining different high velocity jets in order to achieve turbulent flows and rapid mixing. 
         [0011]    The time resolution of a fast mixing device is determined by the instrumental dead time, which depends critically on the time required to achieve complete mixing of the two reagents (mixing time), the flow velocity, and the volume between the mixing region and the point of observation (dead volume). 
         [0012]    To capture the thermodynamic and kinetic behavior of organic or inorganic compounds, several methods are used. 
         [0013]    Stopped-flow mixing techniques, originally developed in 1940 by Chance [16], have been widely used to induce rapid changes in concentration and trigger chemical reactions, thus getting real time information. These techniques use a reactor to rapidly mix together compounds which then react to form a new product. The main purpose of that technology is to study the formation of the product from the early stage on. The product then flows into an observation cell. A stop syringe is used to limit the volume of solution within the cell by abruptly stopping the flow. The product is then analyzed using optical properties and techniques (absorbance, fluorescence, light scattering, spectroscopic technique, etc.). The measurement of these optical properties is performed by system detectors which can be mounted either perpendicular or parallel to the path of incoming light. The technique has applications in several domains, including protein and macromolecule folding and unfolding [17, 18] or polymerization [19]. For instance, protein unfolding and refolding pathways have been extensively studied using fluorescence intensity [15, 20], circular dichroïsm [21], relaxation dispersion nuclear magnetic resonance [22], microcalorimetry [23], X-ray absorption spectroscopy [24], and scattering techniques [25]. The time resolution is often of the order of milliseconds or lower [6]. 
         [0014]    Chemical quench-flow is another method for studying the thermodynamic and kinetic behavior of organic or inorganic compounds [26, 27]. It allows direct measurement of the conversion of compounds into product. A basic chemical quench-flow allows the mixing of two reactants, followed (after a specified time interval) by quenching with a chemical agent (usually acid or base) [28]. The quenched sample is then collected and analyzed to quantify the conversion to product (usually the compounds are labeled and the product formation is determined by either gel electrophoresis or standard chromatography methods). The duration of the reaction is determined by the time given to the product to evolve before mixing it with the quenching chemical. 
         [0015]    Shastry et al. [29] have published a study on protein folding using a continuous flow mixing device. In that case, two (or more) compounds are rapidly mixed in a reactor. The product then flows in a transparent cell where it can be analyzed using the detection techniques mentioned previously. The evolution of the reaction occurs continuously while the product flows downstream (away from the mixer and along the flow direction). This can be translated into time knowing the flow rate and the dimensions of the flow channel. 
         [0016]    Rapid mixing in the techniques mentioned above is generally achieved through turbulent fluctuations. High pressure syringes are used to inject the compounds into the mixing cell with high velocities in order to create a turbulent flow. As the compounds are mixing, they are experiencing strong shear coming from the turbulent fluctuations and the high velocity confined flow. More recently, in order to reduce the volume of solution used for the study, the channels became smaller. Under these circumstances, the viscous effects become dominant and turbulence cannot be easily generated anymore. 
         [0017]    Over the past decades, the study of microfluidic micromixers enabled more efficient mixing through passive mixers [35, 38] or active mixers [36, 37, 39]. Laminar microfluidic mixers might be a solution to this problem [1, 7]. The use of such devices allows small volume consumption and enables rapid mixing with low shear rates and low costs. 
         [0018]    What is needed are improved methods and apparatuses for efficient, rapid, accurate and low-cost mixing of small amounts of compounds (e.g., proteins, DNA, RNA, molecules) using fluid flow manipulations. The present invention satisfies that need, especially when applied to stopped-flow, quenched flow and continuous flow analysis. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0019]    Referring now to the drawings in which like reference numbers represent corresponding parts throughout: 
           [0020]      FIG. 1  is a block diagram of the basic cell microfluidic system, wherein  FIG. 1(   a ) is a top view of the micromixer, and  FIG. 1(   b ) is a schematic diagram of the microfluidic system (pumps, syringes and manifold) and connections. 
           [0021]      FIG. 2  is a picture of the mixing channel crossing by two pairs of side channels (the basic cell), wherein the second pair is activated, fluids are flowing from left to right, amplitude and frequency are optimized, the Reynolds number is Re=2.6 and the flow rate is fixed at 277 pl/s. 
           [0022]      FIG. 3(   a ) (left) shows the Mixing Variance Coefficient (MVC) for the particles solution with diffusivity D ps =2.2 μm 2 /s, wherein good mixing is achieved for low value of the MVC, complete mixing is achieved in 10 ms over 200 microns in the region of high frequency and amplitude, and  FIG. 3(   b ) (right) is a block of three pictures showing the flow in the main channel after mixing corresponding to three sets of parameters (Amplitude, Frequency) of oscillation ( 1 , 2  and  3 ). 
           [0023]      FIG. 4  ( a ) is an experimental velocity field at the intersection of the mixing channel and the side channel, as a function of position in the intersection, after a quarter of a period and  FIG. 4  ( b ) is a computed velocity field under the same conditions, where amplitude and frequency are respectively Â=2.15 and {circumflex over (f)}=2.2. 
           [0024]      FIG. 5  ( a ) is a picture of the flow in one of the transverse channels, after a few oscillations, where fluids are mixed by Taylor-Aris dispersion, and  FIG. 5  ( b ) is a picture of the flow in the main channel where mixed fluids are injected from the side channel. 
           [0025]      FIG. 6  is a schematic of a micromixer device, comprising one pair of transverse channels perpendicular to the mixing chamber and two injection channels from which the fluid(s) to be mixed are injected, wherein in each pair the flow oscillates to create structures in the main channel, and a valve at the end of the main channel enables the flow to be stopped. 
           [0026]      FIG. 7  is a schematic of a micromixer configuration comprising four injection channels, three micromixers, and a valve, wherein a product is formed in parallel using the micromixers A and B and then these products (from A and B) can be mixed together using the micromixer C, and the valve at the end of the main channel enables the flow to be stopped. 
           [0027]      FIG. 8  is a schematic of a micromixer configuration comprising three injection channels, two micromixers, and a valve, wherein a product is formed using the micromixer A, then this product can be mixed together with another reactant using the micromixer B, and the valve at the end of the main channel enables the flow to be stopped. 
           [0028]      FIG. 9  is a schematic of a micromixer configuration comprising five injection channels, one micromixer, and a valve, wherein a product is formed by mixing simultaneously the five reactants, and the valve at the end of the main channel enables the flow to be stopped. 
       
    
    
     SUMMARY OF THE INVENTION 
       [0029]    The present invention discloses a device where stopped flow, continuous flow and quenched flow experiments are performed under low pressure, non-turbulent conditions. The device includes an active mixer capable of mixing under laminar flow conditions. Stopped flow experiments may be performed in parallel under the low pressure, non-turbulent conditions. 
         [0030]    The present invention also discloses a process where two or more reactants are brought together in a device to perform stopped flow, continuous flow and quenched flow experiments under low pressure, non-turbulent conditions. 
         [0031]    The present invention also discloses a process where ELISA assay is performed using a microchannel mixing setup. 
         [0032]    The present invention discloses a process where cDNA experiments are performed using a microchannel mixing setup. 
         [0033]    An apparatus for mixing two or more fluids in accordance with the present invention comprises a fluid containing cell for containing two or more main fluid flows, and at least one side cell for introducing a secondary fluid flow into the fluid containing cell at an intersection between the side cell and the fluid containing cell, wherein the secondary fluid flow perturbs the main fluid flows in the fluid containing cell and introduces recirculating motion in the fluid containing cell. 
         [0034]    Such an apparatus further optionally comprises the two or more fluids introduced into the fluid containing cell upstream of the intersection are not mixed, the secondary fluid flow creates a mixing of the two or more fluids, and two or more fluids are fully mixed downstream of the intersection, the secondary fluid flow being an oscillatory flow resulting from fluid motion oscillations at specific amplitudes and frequencies driven in the side cell which perturbs the two or more main fluid flows in the fluid containing cell to enhance the mixing of the two or more fluids in the fluid containing cell, the mixing being under laminar flow conditions, the apparatus being a laminar flow mixer for one or more of the following applications: stopped flow mixing, quenched flow mixing, continuous flow mixing, ELISA testing, c-DNA experimentation, and Kinase-based assays, there being a pair of side cells, the side cell being perpendicular to the fluid containing cell, a secondary fluid in the secondary fluid flow being identical to one of the fluids, and a secondary fluid in the secondary fluid flow being a mixture of the fluids. 
         [0035]    A method in accordance with the present invention comprises perturbing two or more fluid flows with one or more oscillating side flows, wherein the oscillating side flow causes the two or more fluid flows to homogeneously mix under laminar conditions and within 100 milliseconds. 
         [0036]    Such a method further optionally comprises the oscillating side flow creating recurrent circulating flows within the two or more fluid flows, the oscillating side flow being a jet flow having a frequency and amplitude of oscillation which enhances mixing by creating vortices, which in turn create multiple fluid layers, and Taylor-Aris dispersion in the two or more fluids flows, the mixing being used to perform one or more of the following: stopped flow analysis, quenched flow analysis or continuous flow analysis, the method being used to perform ELISA testing or c-DNA experimentation, the mixing being fast enough to observe protein folding and unfolding, and a device for implementing the method. 
         [0037]    Another apparatus for mixing two or more fluids in accordance with the present invention comprises a main channel for containing a first fluid stream of a first fluid and a second fluid stream of a second fluid, in the main channel, and a secondary channel for introducing a transverse oscillating jet flow into the main channel at an intersection between the secondary channel and the main channel, wherein the intersection provides an intersection for the first fluid stream, second fluid stream, and the transverse oscillating jet flow, a first inlet to the main channel, upstream of the intersection, for introducing the first fluid into the main channel, a second inlet to the main channel, upstream of the intersection, for introducing the second fluid into the main channel, and an outlet for the main channel, downstream of the intersection. 
         [0038]    Such an apparatus further optionally comprises the main channel having a length which is at least ten times h where h=100 μm, the dimensions of the secondary channel being chosen so that flow in the secondary channel has a Reynolds number greater than 11, and the transverse oscillating jet flow has an amplitude and frequency of oscillation causing a mixing variance coefficient less than 0.02. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0039]    In the following description of the preferred embodiment, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. 
         [0040]    Overview 
         [0041]    The mechanism of the rapid laminar mixing of fluids induced by transversal jet flows is described numerically and experimentally herein. By means of a simplified model, namely, a microfluidic device comprised of a main channel (mixing cell) and transversal channels, it is shown that jet flows induce the formation of a recirculation region providing a dynamical mechanism to produce rapid and efficient mixing. The numerically predicted dynamical phenomenon is demonstrated experimentally. 
         [0042]    An exemplary application of this invention would be to mix a protein (e.g. cytochrome C) and a basic or acid buffer, to analyze its folding or unfolding structure and kinetic behavior. Current methods require turbulent mixing and are generally high compound consuming. Another use might be to analyze the polymerization of molecules like ethene or propene. Another use might be to study the RNA folding. 
         [0043]    The present invention comprises a device that can produce mixing under laminar conditions for applications in stopped flow, continuous flow and quenched flow analysis. The present invention also comprises a method that can produce mixing under laminar conditions where the manipulation of the fluids to achieve mixing is done by the action of the jet flow coming from one or more transverse cells. The present invention also comprises a process that can produce mixing under laminar conditions where a least one solution resulting from the mixing of reagents has been created. 
         [0044]    Technical Description 
         [0045]    This disclosure first introduces an apparatus and a procedure where two fluids are introduced, then mixed, in a micromixer device. The micromixing produced by a microfluidic device comprises of a main fluid containing cell (main channel) and a pair of transverse channels. Jet flows resulting from the oscillations in the transverse channels perturb the main stream to create vortices and generate an efficient mixing. 
         [0046]    The design of the mixer is composed of a main channel where the fluids are injected, and one or more transversal channels to manipulate the fluids and increase the mixing [31, 32, 36, 37]. 
         [0047]    An example of the basic cell mixing device  100  design is shown  FIG. 1(   a ) and  FIG. 1(   b ).  FIG. 1(   a ) is a top view of the 16 mm by 16 mm mixing device  100  microfabricated using conventional techniques. The device  100  was deep reactive ion etched in silicon, and anodically bonded to a cover glass. The mixing device  100  consists of a main channel  102  where the two fluids  104 ,  106  to be mixed are injected at Inlet A  108  and Inlet B  110 . The two fluid streams  112 ,  114  move from left to right in the main channel  102 . Three pairs of secondary channels ( 116 A,  116 B), ( 118 A,  118 B) and ( 120 A,  120 B) are positioned perpendicular to the main channel  102 . The fluid motion in these secondary channels ( 116 A,  116 B), ( 118 A,  118 B) and ( 120 A,  120 B) is driven at specified amplitudes and frequencies, and perturbs the flow field in the main channel  102  to enhance mixing. In the present study only the central pair of secondary channels ( 118 A,  118 B) is activated. The main channel  102  is 2h wide and L=13.5h long. The secondary side channels  116 (A,B),  118 (A,B) and  120 (A,B) are h/2 wide, 5h long and separated from each other by a distance of 3h. The depth of the channels  102 , ( 116 A,  116 B), ( 118 A,  118 B) and ( 120 A,  120 B) is h where h=100 μm. 
         [0048]    The flow in the secondary channels  118 (A,B) is driven by high-frequency oscillating syringe pumps  122  ( FIG. 1(   b )) and a syringe pump  124  injects the two fluids  104 ,  106  to be mixed into the device  100  ( FIG. 1(   b )). 
         [0049]    Two fluids  104 ,  106  are injected in the device  100  through inlet A  108  and B  110  ( FIG. 1 ) at steady and constant flow rate. The flow  112 ,  114  is fully developed when reaching the intersection  124  of the side channels  118 (A,B) [31]. The fluids  104 ,  106  are then manipulated by the transverse oscillating jet flow. The velocity condition V SC =V 0  sin(ωt) is imposed at the entrance  126  of the side channels  118 (A,B). The Reynolds number associated with the secondary channel flow is defined as 
         [0000]    
       
         
           
             
               
                 Re 
                 SC 
               
               = 
               
                 Afb 
                 / 
                 v 
               
             
             , 
             
               
 
             
              
             where 
           
         
       
       
         
           
             A 
             = 
             
               
                 ∫ 
                 0 
                 
                   
                     1 
                     / 
                     2 
                   
                    
                   f 
                 
               
                
               
                 
                   
                     V 
                     SC 
                   
                    
                   
                     ( 
                     t 
                     ) 
                   
                 
                  
                 
                    
                   t 
                 
               
             
           
         
       
     
         [0050]    and b=50 μm is the secondary channel width. The present invention makes the various flow parameters non-dimensional, thereby obtaining a non-dimensional amplitude Â=A/a, frequency 
         [0000]    
       
      
       {circumflex over (f)}=f 
       ab 
       /v 
      
     
         [0000]    and velocity 
         [0000]    
       
      
       {circumflex over (v)}=V 
       v 
       /Aab&#39; 
      
     
         [0051]    where a=2h is the chamber  102  width and b=h/2 the side channel  118 A, B width. The unsteadiness in the flow in the secondary channels is described by the Strouhal Number, 
         [0000]    
       
      
       St=fh/U&#39; 
      
     
         [0000]    where f is the oscillation frequency and U is the flow velocity. 
         [0052]    The main flow consists of two unmixed, miscible fluids  104 ,  106  entering the chamber  102 . The working fluids are degassed and deionized water. In order to distinguish the two fluid streams  112 ,  114 , one stream was seeded with 98 nm dia. fluorescent polystyrene particles, with a diffusion coefficient of D ps =2.2 μm 2 /s. As they enter the mixing chamber, the interface is clear and only a slight amount of mixing occurs due to molecular diffusion (diffusion length is calculated to be 0.7 μm). When the side channels  118 (A,B) are activated for Re SC  above the threshold of 11, the two fluid streams (or flows)  112 ,  114  (which are not mixed just before the intersection  124 ) are manipulated by the transverse flow from the side channels  118 (A,B) and become fully mixed downstream of the intersection  124 . Also shown is the outlet  128  of the device  100 . A similar analysis applies to activation of additional side channels  116 (A,B) and  120 (A,B). 
         [0053]      FIG. 2  is a picture of a basic cell comprising the mixing channel  200  crossing by two pairs of side (secondary) channels ( 202 A,  202 B) and ( 204 A,  204 B), wherein the second pair ( 204 A,  204 B) is activated, fluids are flowing from left to right, amplitude and frequency are optimized, the Reynolds number is Re=2.6 and the flow rate is fixed at 277 pl/s (picoliters per second). 
         [0054]      FIG. 2  is an instantaneous snapshot (top view) of the mixing performance for optimized frequency and amplitude. As the fluids approach the intersection  206  from the left, two well discernible bands  208 ,  210  of fluid are visible, and within about 100 microns downstream of the intersection  206  the fluids are thoroughly mixed. 
         [0055]    The degree of mixing can be quantified by the so-called Mixing Variance Coefficient function Φ (MVC) [31-33]. Complete mixing is achieved when Φ=0 and no mixing corresponds to Φ=0.25. The optimum amplitude and frequency of the secondary channel oscillations were determined systematically by measuring the MVC as a function of non-dimensional frequency 0&lt;{circumflex over (f)}&lt;2.5 and amplitude 1&lt;Â&lt;3.  FIG. 3(   a ) shows the MVC as a function of {circumflex over (f)} and Â. In Region C, where high frequencies and amplitudes are achieved, the mixing is excellent (MVC˜0.01).  FIG. 3(   b ) is a block of three pictures  1 ,  2  and  3 , showing the flow (MVC) at the same location in the main channel  300  after mixing corresponding to the three sets of parameters (Amplitude, Frequency) of oscillation  1 , 2  and  3  marked in  FIG. 3(   a ). 
         [0056]    In order to improve the present invention&#39;s understanding of the mixing mechanism, the present invention examines the fluid motion and scalar fields at the intersection of the secondary channel and the main channel. 
         [0057]    The secondary channels  204 A,  204 B are perpendicular to the main channel  200  and have sharp corners  212 . This creates a sudden expansion for flow being injected into the main channel  200  from the secondary channels  204 A and  204 B as shown in  FIG. 2 . For sufficiently large Reynolds numbers, Re SC &gt;11, nonlinear effects are prominent at the intersection  206  and two recirculation vortices  400  appear (see  FIGS. 2 and 4 ). 
         [0058]      FIG. 4(   a ) is an instantaneous snapshot of the velocity field at the intersection of the mixing channel  402  and the side channel  404 , after a quarter of a period of oscillation, obtained by micro Particle Image Velocimetry [34] (μPIV), and  FIG. 4(   b ) is a direct numerical simulation of the flow for the same configuration and conditions using Fluent™ (Lebanon, N H) (see Bottausci et al. [31] for details). 
         [0059]    The vortices develop and grow as the flow velocity reaches its maximum velocity in the side channel (data not shown). As the jet flow slows down, the recirculation does not stay symmetrical, because of the influence of the velocity in the main channel, U. The vortices vanish just after the velocity in the secondary channels reaches zero. 
         [0060]    The mixing process is a combination of two factors: 
         [0061]    1) The vortices creating multiple layers of fluids. 
         [0062]    2) The Taylor-Aris dispersion taking place mainly in the side channels  500 ,  FIG. 5(   a ), but also in the main channel  502  ( FIG. 5(   b )).  FIG. 5(   a ) is a picture of the flow in one of the transverse channels  500 , after few oscillations, where fluids are mixed by Taylor-Aris dispersion, and  FIG. 5(   b ) is a picture of the flow in the main channel  502  where mixed fluids are injected from the side channel  500 . 
         [0063]    The layers entering the side channel are stretched, due to the contraction of the secondary channel. Inside the secondary channels, there is significant Taylor-Aris dispersion due to high shear rates, which further enhances the mixing process. Every half cycle, mixed fluid coming from the side channel is injected in the main channel. 
         [0064]    Depending on the applications, a valve can be added at the end of the main channel to rapidly stop the flow. The valve can be, for example, a mechanical valve (piezohydraulic, pneumatic and thermopneumatic or pressure driven), or a multiphase valve (bubble valve or two phase flow where the flow is suddenly stopped by freezing a section of the main channel). 
         [0065]      FIG. 6  illustrates a mixing apparatus  600  (micromixer device) for mixing two or more fluids ( 602 ,  604 ), comprising a fluid containing cell  606  (or main channel), injection channels  608 ,  610  for injecting two or more main fluid flows  602 A,  604 A (of fluids  602  and  604 , respectively) into the fluid containing cell  606 , a pair of side cells  612 ,  614  for introducing a secondary fluid flow  616  into the fluid containing cell  606  at an intersection  618  between the side cells  612 ,  614  and the fluid containing cell  606 , and a valve  620  at the end of the main channel  606  enabling flow to be stopped. Upstream of the intersection  618 , fluids  602  and  604  are unmixed. The secondary fluid flow  616 , which oscillates, perturbs the main fluid flows  602 A,  604 A in the fluid containing cell  606  (to create structures in the flows  602 A,  604 A, leading to a mixing of the flows  602 A,  604 A) so that downstream of the intersection  618 , the fluids flows  602 A,  604 A are fully mixed. 
         [0066]    The mixing process and apparatus described here constitute an efficient and rapid micromixer that can be viewed as a module to be incorporated in already existing or to-be-developed apparatuses. This work opens the door to more sophisticated hydrodynamic behavior and apparatus design for micromixing applied to stopped flow, quenched flow or continuous flow apparatuses. Specifically, the basic unit described above can be operated in parallel to provide for multiple reactions where a specified cascade of reactions is to occur, such as in experimental studies of gene regulatory networks. Examples of such operation are provided in  FIGS. 7 and 8 . 
         [0067]      FIG. 7  is a schematic of a micromixer configuration  700  comprising three mixing apparatuses A, B and C, wherein micromixer A has two injection channels  702 ,  704  and a pair of transverse channels  706 ,  708  for oscillations, micromixer B has injection channels  710 ,  712  and a pair of transverse channels  714 ,  716 , micromixer C has transverse channels  718 ,  720 , a product is formed in parallel using the micromixers A and B mounted in parallel and then these products (from A and B) can be mixed together using the micromixer C mounted in series with micromixers A and B, and the valve  722  enables the flow to be stopped. 
         [0068]      FIG. 8  is a schematic of a micromixer configuration  800  comprising two mixing apparatuses A and B mounted in series, wherein micromixer A has injection channels  802 ,  804  and transverse channels  806 ,  808 , wherein a product is formed using the micromixer A, then this product can be mixed together with another reactant (introduced in injection channel  810 ) using the micromixer B mounted in series with micromixer A, and the valve  812  at the end of micromixer B enables the flow to be stopped. Micromixer B has transverse channels  814  and  816  for oscillations. 
         [0069]    In addition, simultaneous reactions of multiple species can be pursued using the invention embodiment shown in  FIG. 9 .  FIG. 9  is a schematic of a mixing apparatus  900  comprising five injection channels  902 ,  904 ,  906 ,  908 ,  910 , a main channel  912  and a pair of transverse channels  914  and  916  for oscillations, wherein a product is formed by mixing simultaneously the five reactants inputted through the injection channels  902 - 910  and the valve  918  at the end of the main channel  912  enables the flow to be stopped. 
         [0070]    This invention thus introduces a method of mixing and a device, that can produce mixing under laminar conditions for applications in stopped flow, continuous flow and quenched flow analysis. Additionally, designs utilizing the basic cell to operate in parallel with other such cells can be used to perform c-DNA experimentation and ELISA testing, as well as Kinase-based assays. 
         [0071]    Possible Modifications and Variations 
         [0072]    The fluid containing cell and side cells may be channels having a longitudinal axis and a cross-sectional area, wherein the fluid flows generally along the longitudinal axis. The fluid-containing cell cross-sectional area may be symmetrical or non-symmetrical. The cross-sectional area of the main fluid-containing cell and the side cells may be identical or different. 
         [0073]    The angle between the main fluid-containing cell and the side cell may be 90 degrees or any other angle. One or more walls of the mixing apparatus may be smooth or have asperities. The fluid containing cell and side cell may have microscale dimensions or less. At least one pair of transverse side cells is necessary. 
         [0074]    The secondary fluid flow may perturb flow in the main fluid containing cell in a variety of ways. For example, the secondary fluid flow may create recurrent circulating fluid flow within the fluid containing cell or cause laminar flow conditions in the fluid containing cell. The secondary fluid flow may be oscillatory, for example, vary in time or intensity, which oscillation may be applied by an external mechanism or an internal mechanism. The secondary fluid flow may introduce a fluid motivating shear into the transverse cell and or into the main cell. 
         [0075]    Fluids may be reagents introduced individually, sequentially, or by pair in the main fluid containing cell. The introduction of reagents may be delayed in time. The introduction of at least one reagent may be downstream from the previous reagent introduction. 
         [0076]    There are no limitations on the nature of the fluids that may be contained in the fluid containing cell. For example, the fluid may be a liquid or a gas. The fluid in the transverse cell may be identical, different or a mixture of one of the fluids in the main cell. Two or more fluids may be injected in the main fluid containing cell before the intersection with the side/intersecting cell. 
         [0077]    The present invention may also comprise a system of one or more mixing apparatuses mounted in series or in parallel, so that one or more mixing processes may be performed in series or in parallel. 
         [0078]    Consequently, the present invention discloses a method of a method of manipulating two or more fluids, comprising perturbing two or more fluid flows with one or more oscillating side flows, wherein the oscillating side flow causes the two or more fluid flows to homogeneously mix under laminar conditions and within 100 milliseconds. 
       REFERENCES 
       [0079]    The following references are incorporated by reference herein:
   1. Dobson C. M.  Protein folding and misfolding  NATURE 426: 884 2003.   2. DeArmond, S. J., and Prusiner, S. B. 1997. Molecular neuropathology of prion diseases. In  The molecular and genetic basis of neurological disease . R. N. Rosenberg, S. B. Prusiner, S. DiMauro, and R. L. Barchi, editors. Butterworth-Heinemann. Boston, Mass., USA. 145-163.   3. Selkoe, D. J. 2002. Deciphering the genesis and fate of amyloid-β protein yields novel therapies for Alzheimer disease.  J. Clin. Invest.      4. Conway, K. A., Harper, J. D., and Lansbury, P. T. 2000. Fibrils formed in vitro from α-synuclein and two mutant forms linked to Parkinson&#39;s disease are typical amyloid.  Biochemistry.  39:2552-2563.   5. Thomas, P. J., Qu, B. H. &amp; Pedersen, P. L. Defective protein folding as a basis of human disease.  Trends Biochem. Sci.  20, 456-459 (1995).   6. Fadiel A, Eichenbaum K D, Hamza A, et al.  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       CONCLUSION 
       [0123]    This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.