Abstract:
An integrated cartridge for automated sample manipulation and, particularly strand displacement amplification, is provided. The cartridge comprises a sealed, two-part device with internal fluid channels and chambers, as well as reagents. The cartridge performs the sequence of fluid transfers, reagent additions and heat transitions, such as those of the strand displacement amplification process, in a single, sealed device.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of the Invention  
         [0002]     The present invention relates to a device and method for the chemical processing of a biological sample and, more particularly, to an integrated cartridge for sample manipulation. The integrated cartridge comprises a sealed, two-part device with various internal fluid channels and chambers, some of which contain dried reagents. The integrated cartridge is capable of manipulating small liquid volumes through movement, reagents, filtering and heat. The integrated cartridge is well suited for process samples for high throughput screening and is particularly useful for performing assays such as nucleic acid sequence amplification.  
         [0003]     2. Discussion of the Background  
         [0004]     Determining the nucleic acid sequence of genes is important in many situations. For example, numerous diseases are caused by or associated with a mutation in a gene sequence relative to the normal gene. Such mutation may involve the substitution of only one base for another, called a “point mutation.” In some instances, point mutations can cause severe clinical manifestations of disease by encoding a change in the amino acid sequence of the protein for which the gene codes. For example, sickle cell anemia results from such a point mutation.  
         [0005]     Other diseases are associated with increases or decreases in copy numbers of genes. Although the determination of the presence or absence of changes in a sequence is important, determination of the quantity of such sequences in a sample can be used in the diagnosis of disease or the determination of the risk of developing disease. Moreover, variations in gene sequences of both prokaryotic and eukaryotic organisms has proven invaluable to identifying sources of genetic material (e.g., identifying one human from another or the source of DNA by restriction fragment length polymorphism).  
         [0006]     Certain infections caused by microorganisms or viruses may also be diagnosed by the detection of nucleic acid sequences peculiar to the infectious organism. Detection of nucleic acid sequences derived from viruses, parasites and other microorganisms is also important where the safety of various products is of concern, e.g., donated blood, blood products and organs in the medical field and the safety of food and water supplies.  
         [0007]     Identification of specific nucleic acid sequences by the isolation of nucleic acids from a sample and detection of the sought for sequences provides a mechanism whereby one can determine the presence of a disease, organism or individual. Generally, such detection is accomplished by using a synthesized nucleic acid “probe” sequence that is complimentary in part to the target nucleic acid sequence of interest.  
         [0008]     Although it is desirable to detect the presence of nucleic acids as described above, it is often the case that the sought for nucleic acid sequences are present in sample sources in extremely small numbers. The condition of small target molecule numbers causes a requirement that laboratory techniques be performed in order to amplify the numbers of the target sequences so that they may be detected. There are many known methods of amplifying targeted sequences. One such method is strand displacement amplification (SDA).  
         [0009]     The current format for performing SDA procedures is problematic for several reasons. First, SDA is currently performed in microtiter well plates. Such procedure typically uses two 96-well plate to process 94 liquid patient samples. The first plate, which is where the prime reaction takes place, typically contains short DNA sequences known as “primers” needed to initiate the subsequent amplification reaction, which occurs in the wells of the second(i plate. The two plates must be placed on separate heat blocks because the prime and amplification (“amp”) reactions require different temperatures. Furthermore, the user must work quickly after transferring the sample to the amplification plate because there is a time limit for placing the plate in the reader once amplification starts.  
         [0010]     Another problem with existing methods for performing SDA procedures is the requirement of manual pipetting. Currently, the user must transfer the liquid samples from the sample tube to the prime plate and later to the amplification plate using a multi-channel, manual pipette. Manual pipetting is tedious and time consuming because each transfer must be held steady for a few seconds while the pipette mixes the liquid in the wells. Manual pipetting also requires the user to keep track of which row of the plate he or she is on.  
         [0011]     A need, therefore, exists for an automated sample manipulation cartridge and, more particularly, for an automated SDA DNA amplification process in a miniaturized and sealed, disposable sample manipulation cartridge so as to overcome the deficiencies of current, manual SDA methods.  
       SUMMARY OF THE INVENTION  
       [0012]     The present invention relates to a device for automated sample manipulation. In particular, the present invention relates to a miniaturized and sealed disposable device for the automation of sample manipulation, especially the SDA DNA amplification process. The device of the present invention performs a sequence of fluid transfers, reagent additions and heat transitions in a unitized package. The device comprises a disposable cartridge and a means for driving the fluidics and heating functions of the cartridge. The cartridge comprises a sealed, two-part device with various internal fluid channels and chambers and dried reagents. The fluid channels connect a series of chambers that are used to measure an aliquot, provide heat and reagents for reactions, and control the position of the fluid bolus for each reaction step.  
         [0013]     The integrated cartridge of the present invention has several advantages. While the integrated cartridge of the present invention contains the same fluidic metering, fluid transfer, reagent addition and heating functionality as current manual SDA methods, the present invention, unlike current SDA methods, is automated. A major advantage of the present invention, therefore, is the ease of use. For example, sample introduction can be done with a dropper instead of a pipette because the cartridge itself meters the required sample volume. Additionally, after sample input, the user simply seals the inlet well with a sealing means and places the cartridge in a cartridge processing device; no additional liquid handling needs to be done by the user. Moreover, the sealed, integrated cartridge of the present invention is better for evaporation, aerosol containment and sealed disposal of the completed test.  
         [0014]     The above and other features and advantages of the present invention will become more apparent from the following detailed description of the presently preferred embodiments, particularly when considered in conjunction with the drawings, and to the appended claims. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0015]      FIG. 1  is an unassembled view of the integrated cartridge of the present invention from the bottom thereof.  
         [0016]      FIG. 2  is an unassembled view of the integrated cartridge of the present invention from the top thereof.  
         [0017]      FIG. 3  is a perspective view of the integrated cartridge from the top thereof.  
         [0018]      FIG. 4  is fragmentary sectional view of the integrated cartridge of the present invention along line A-A of  FIG. 3 .  
         [0019]      FIG. 5  is an enlarged view along circle B of  FIG. 4 .  
         [0020]      FIG. 6  is a fragmentary sectional view along line CC of  FIG. 3 .  
         [0021]      FIG. 7  is a fragmentary sectional view of the integrated cartridge of the present invention along line D-D of  FIG. 3 .  
         [0022]      FIG. 8  is an enlarged view along circle E of  FIG. 7 .  
         [0023]      FIG. 9  shows the integrated cartridge of the present invention in a closed cartridge processing device.  
         [0024]      FIG. 10  shows the integrated cartridge of the present invention in an open cartridge processing device.  
         [0025]      FIG. 11  is an enlargement of the cartridge processing device showing the active components of thereof.  
         [0026]      FIG. 12  is a schematic drawing of the cartridge of the present invention and its drivers. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0027]     While this invention is satisfied by embodiments in many different forms, there will herein be described in detail preferred embodiments of the invention, with the understanding that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the embodiments illustrated and described. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents.  
         [0028]     In describing the present invention, various terms and phrases will be used herein. Generally, the meaning of these terms are known to those having skill in the art and are further described below. The definitions should not be understood to limit the scope of the invention. Rather, they should be used to interpret language of the description and, where appropriate, the language of the claims. These terms may also be understood more fully in the context of the description of the invention. If a term is included in the description or the claims that is not defined below, or that cannot be interpreted based on its context, then it should be construed to have the same meaning as it is understood by those of skill in the art.  
         [0029]     The term “amplification” refers to the increase in the number of copies of a particular nucleic acid target of interest.  
         [0030]     The term “amplicon” refers to the product of an amplification reaction, i.e., the copy of a particular nucleic acid target of interest.  
         [0031]     The term “amplification components” refers to the reaction materials such as enzymes, buffers and nucleic acids necessary to perform an amplification reaction to form amplicons of a target nucleic acid of interest.  
         [0032]     The term “sample” refers to a substance that is being assayed for the presence of one or more nucleic acids of interest. The nucleic acid, or acids, of interest may be present in a mixture of other nucleic acids. A sample containing the-nucleic acids of interest may be obtained in numerous ways. It is envisioned that the following could represent samples: cell lysates, purified genomic DNA, body fluids such as from a human or animal, clinical samples, food samples, etc.  
         [0033]     The present invention comprises a sealed, integrated cartridge with fluidic metering, fluid transfer, reagent addition and heating functionalities for sample manipulation. The cartridge of the present invention will first be described by reference to the figures. As seen in  FIG. 1 , the cartridge  10  of the present invention is a two-part assembly having internal fluidic cavities. Cartridge assembly  10  generally comprises a cartridge bottom  12  and a cartridge top  14 . Cartridge bottom  12  generally comprises a flat plate, while cartridge top  14  generally comprises a molded plate. The plates are preferably transparent. The plates may be made of any suitable material, but preferably are acrylic and, more preferably, are made of polymethylmethacrylate (PMMA) resin.  
         [0034]     Cartridge top  14  has fluidic features, which will be described in more detail later, including capillary channels, reaction chambers and the like, molded therein. Each reaction chamber is preferably shaped like a half cylinder with full-round ends and has a radius of about 0.060 inches. Each capillary channel, which connect the various fluid chambers, is preferably shaped like a half cylinder and has a radius of about 0.015 inches.  
         [0035]     Cartridge  10  also comprises a filter membrane  16  located in a cavity (not shown) inside of cartridge bottom  12  and a waste trap absorbent pad  18  located in a recess  20  in the bottom of cartridge bottom  12 . Absorbent pad  18  is covered by a vacuum chamber cover  22 . PIG.  8  is an enlarged view showing filter membrane  16 , absorbent pad  18  and vacuum chamber cover  22 .  
         [0036]      FIG. 2  is a unassembled view of cartridge  10  from the top of the cartridge.  FIG. 3  is a perspective view of assembled cartridge  10  as seen from the top thereof. Cartridge top  14  includes sample inlet well  24  into which the user inputs a liquid sample. While the shape of liquid inlet well  24  is preferably cylindrical, the shape is not critical and other shapes are possible. Cartridge top  14  also includes three tapered luer ports that are used to connect fittings (not shown) for tubing from various pumping means of a cartridge processing device, which will be described in more detail later, to cartridge  10 . Air drive entry port  26  is the cartridge interface point for a means of pumping air, such as an air pump  100  ( FIG. 12 ) into the cartridge to move the liquid sample throughout the various chambers and channels. A vacuum pump  102  ( FIG. 12 ) is connected to vacuum port  28  and is used to pull the liquid sample through filter membrane  16  into absorbent pad  18 . Input port  30  is used to introduce wash buffers and other amplification components into the fluidic features of cartridge  10 . Cartridge top  14  further includes a prime and amplification heating surface  32 , a denature heating surface  34  and a take-out well  36 .  
         [0037]      FIG. 4  is a cross-sectional, side view of the integrated cartridge of the present invention showing a portion of the fluid path. The fluid path of cartridge  10  starts with liquid inlet well  24  and flows sequentially through the various capillary channels and reaction chambers, into a desalt filter  38  ( FIGS. 6 and 7 ) and into take-out well  36  ( FIG. 3 ). Liquid inlet well  24  connects to a chamber entry  40 . Chamber entry  40  connects to a liquid input chamber  42 . On either side of liquid input chamber  42  are capillary channels  44 ,  46 . Capillary channel  46  connects liquid input chamber  42  to a sensing chamber  48 . In sensing chamber  48 , the liquid sample is observed by optical through-beam sensors (not shown) to identify the meniscus and locate the leading edge of the liquid sample, or the “fluid bolus.” The curvature of the meniscus momentarily deflects the beam and causes a detectable drop in the transmission, allowing detection. Sensing chamber  48 , which is of the same dimensions as the reaction chambers, contains no reagents that would disrupt the optical quality of the liquid or hinder the flow of the liquid sample.  
         [0038]     The next chambers connected in sequence by capillary channels are a prime chamber  50  and an amplification chamber  52 .  FIG. 5  is a detailed view of prime chamber  50  and its associated capillary channels  54 ,  56 . Prime chamber  50  and amp chamber  52  contain reagents required for the reactions that occur therein. Preferably, the reagents are dried down in their respective chambers. The fact that the reagents are dried down in the cartridge itself can drastically change the surface wetting properties of the liquid sample, which can in turn change the flow characteristics. Typically, reagents can reduce the surface tension to the point of defeating the capillary locks. Drying down the reagents tends to eliminate this problem; however, dried coatings too close to the edges of the capillaries can still wick the fluid into the next chamber. This undesirable effect is overcome by interspersing small, uncoated, chambers  59  ( FIG. 4 ) between the coated chambers.  
         [0039]      FIG. 7  is a cross-sectional, side view of the cartridge assembly of the present invention showing the remaining portion of the fluid path. The remainder of the fluid path comprises desalt filter  38 , which includes filter membrane  16 . Located below filter membrane  16  is absorbent pad  18  to trap the liquid that comes through. The final chamber of cartridge  10  is a denature chamber  58 , which preferably is used to strip apart the DNA strands into single strands with heat. Denature chamber  58  also functions as a second meniscus sensing chamber because the movement of the fluid off of filter membrane  16  is not consistent, and the leading edge of the fluid bolus must be located again. To do so, capillary channel  60  preceding denature chamber  58  is placed between the ends of a second optical sensor of the cartridge processing device to relocate the meniscus. Like sensing chamber  48  ( FIG. 4 ), therefore, denature chamber  58  contains no reagents. Capillary channels  60 ,  62  are located on either side of denature chamber  58 . Again, the small cross section of the capillary channels reduces evaporation, and the capillary lock feature keeps the fluid centered. After denaturing is complete and the meniscus has been relocated, the completed sample is pushed to take-out well  36  where it is manually transferred to another instrument, such as a NanoChip™ cartridge by Nanogen, Inc. of San Diego, Calif., for analysis. Instruments such as the NanoChip™ cartridge are for hybridizing and reading the amplification products but do not themselves perform the amplification process. In an alternative embodiment of the present invention, the product design would integrate the amplification cartridge with the reader so that the transfer step could be eliminated.  
         [0040]      FIG. 9  shows integrated cartridge  10  of the present invention closed within a cartridge processing device  80 , while  FIG. 10  shows cartridge  10  in open cartridge processing device  80 .  FIG. 11  shows the various active components of cartridge processing device  80 . Referring first to  FIGS. 10 and 11 , cartridge processing device  80  includes denature heat blocks  82 ,  84  and prime and amp heat blocks  86 ,  88 . Cartridge  10  is placed in a cartridge nest  90  of cartridge processing device  80  such that assembly alignment pin  35  ( FIG. 6 ) lines up with cartridge alignment pin  91  of cartridge processing device  80 . Cartridge processing device  80  also includes through-beam optical sensors (“meniscus sensors”)  92 ,  94  that examine the non-reagent chambers to find the meniscus. These are preferably fiber optic tips. The curvature of the meniscus causes a shadow that is detectable by the sensor. Cartridge processing device  80  further includes a sonicator  96  and its associated sonicator probe  98 .  
         [0041]     Referring now to  FIG. 9 , when cartridge processing device  80  is closed and locked, it places the heat blocks against cartridge  10  under light spring pressure such that heat blocks  86 ,  88  sandwich the prime and amp chambers, while heat chambers  82 ,  84  sandwich the denature chamber. The heat blocks preferably are copper blocks with resistance heaters and RTD sensors that allow precise temperature control. The heat blocks are spring loaded over cartridge  10  directly over and under the reaction chambers and extending out approximately 0.250 inches on all sides of the chambers.  
         [0042]      FIG. 12 , which is a schematic drawing of the cartridge and drivers, shows the logical sequence of all the active chambers and the external driving and sensing devices.  
         [0043]     In operation, a user places the patient sample into liquid inlet well  24 . Chamber entry  40  connecting liquid inlet well  24  to liquid input chamber  42  allows the liquid sample to flow down into the chamber and fill it. When the sample reaches capillary channels  44 ,  46  at each end of chamber  42 , it is pulled through to the opposite ends of the channels and stops. Surface tension prevents the liquid from flowing past the sharp transition from the capillary channel to the next cavity. This feature is referred to as a “capillary lock” and is described in more detail in co-pending U.S. patent application Ser. No. (Attorney Docket No. 20187-112). In general, the capillary locks allow the fluid bolus to be roughly positioned in the cavities and then self-center and lock in place.  
         [0044]     After input chamber  42  has measured and locked the required volume for processing, the remainder of the input volume accumulates and remains in inlet well  24  above it. The user places a sealing means (not shown), preferably tape or a self-adhesive label, over the entrance  25  of inlet well  24  to form a vacuum and retain the excess liquid there when the sample in chamber  42  is moved forward. Application of a positive pressure through air drive entry port  26  moves the sample out of input chamber  42  and leaves the excess liquid trapped in inlet well  24 . This self-metering input allows crude filling on the user&#39;s part, while accurate metering is performed by the self-metering volume input device. Input metering through the capillary locks and the sealing of the inlet well, therefore, eliminates the requirement for accurate pipetting. The volume of the sample processed can be varied by changing the size of the input metering chamber. This self-metering volume input device is described in more detail in copending U.S. patent application Ser. No. ______ (Attorney Docket No. 20187-113).  
         [0045]     The reaction sequence moves the single bolus of liquid through the sequence of chambers along the capillary channels. The fluid bolus is moved into the chambers one by one where the reagents are dissolved, and the external heat blocks of the cartridge processing device maintain reaction temperatures in the reaction chambers of the cartridge.  
         [0046]     External pumps, preferably syringe pumps, are used to move the fluid bolus through the cartridge and add reagents to it. Pump  100  connects to air drive entry port  26  adjacent liquid inlet well  24 . This pump pushes only air, which moves the fluid bolus from input chamber  42  through the sensing, prime and amp chambers and onto the desalt filter. The capillary locks allow the drive fluid to be air. The compliance of air prevents accuracy in other systems and causes other systems to use deionized water as a system fluid for stiffness. The fluid movement pump moves only the volume in the liquid input chamber forward into the cartridge for processing. Any excess fluid in inlet well  24  remains trapped there by the sealing means placed over its opening  25 . When the fluid movement sequence starts, pump  100  moves the fluid forward slowly into sensing chamber  48 , where an optical sensor detects the meniscus. The instrument then knows the exact location of the leading edge of the liquid and proceeds with the predetermined number of steps to move the liquid into prime chamber  50 . When the liquid bolus is roughly centered in prime chamber  50 , the controller stops and opens a solenoid valve (not shown) to vent the tubing from the pump. This allows the capillary locks to center the bolus in prime chamber  50 . The preferred method of moving the liquid is to not use the optical sensors at all. If the input chamber capillary locks function properly, then the starting position will be known, and the air volume needed to reach the prime chamber will be consistent. One or both of the meniscus sensing optics may, therefore, be eliminated by knowing the starting position of the fluid.  
         [0047]     Prime chamber  50  contains dried-down reagents. When the fluid bolus is moved into prime chamber  50 , it is held there for a specified time to allow for dissolution and reaction of the reagents. The bolus is then moved to amp chamber  52 , which also contains dried-down reagents, and held there for dissolution and reaction. Both prime chamber  50  and amp chamber  52  require an elevated temperature. Heat blocks  86 ,  88  span the area covering these two chambers and maintain them at a constant temperature. When the fluid bolus is in the heated chambers, the capillary channels on both sides are vented to prevent pressure buildup that would move the fluid bolus out of position. The small exposed surface inside of the capillary channels also essentially eliminates evaporation.  
         [0048]     After the amplification is complete, pump  100  moves the fluid bolus onto the face of desalt filter  38  where it passes through filter membrane  16  of desalt filter  38 . Filter membrane  16  is preferably polysulfone. The capillary channels are vented, and vacuum pump  102  is started. It takes less than about  10  minutes to pull the liquid through the filter and into waste trap  18 . The pore size of filter membrane  16  allows all liquids and salt ions to pass through, but traps the DNA amplicons on its surface. The amplicons tend to embed themselves into the pores because of the high fluid pressure. The amplicons are freed from the filter by agitation achieved with sonicator probe  98  pressed against the face of cartridge  10  above filter membrane  16 . Input port  30  is used to add various buffers, rinse fluids and the like to the cartridge. A pump  104  uses a selector valve to draw wash buffer, preferably about  100  microliters, followed by air. By closing the valve of pump  100 , the fluid introduced into input port  30  flows toward die filter and not backwards. Pump  104  pushes the wash buffer slowly onto the filter so that it can be pulled through by vacuum pump  102 .  
         [0049]     Pump  104  then uses a selector valve to push a small volume of buffer, plus the mechanical release of the sonication, to resuspend the DNA, followed by driving air onto the filter. By resuspending in a smaller volume of buffer, the DNA can also be concentrated during desalting, which increases sensitivity. If this volume is half the original sample volume, then the DNA concentration is nearly doubled (some of the DNA is lost to binding) when it is resuspended. The DNA recovery from the filter is enhanced by mechanical agitation in the form of sonication. Sonicator probe  98  is held in contact with the upper wall of the filter chamber. This is energized briefly before the resuspension buffer is moved onto the next step. The sonication improves the DNA recovery from about 50% to greater than about 80%. The wash and resuspension solutions can be varied in both composition and volume.  
         [0050]     In the manufacture of the cartridge assembly, the prime and amp reagents are dried down in their respective reaction chambers in a vacuum oven. The filter membrane is inserted into its cavity, and a retaining ring is heat-formed down over the edge of the membrane. The two plates of the cartridge are then bonded together. Preferably, the bonding is done by silk screening an adhesive pattern onto the cartridge bottom. Silk screening is preferred because it is less abusive to the reagent dry downs than other bonding techniques, particularly ultrasonic welding. The adhesive pattern is about 0.005 inches thick and matches the outline of the walls of the molded plate. The pattern is preferably set back from the inside edges of the channels by about 0.020 inches so that it does not squeeze into them during assembly. The two plates are then clamped together and exposed to ultraviolet light to cure the adhesive. After the bonding is complete, the waste trap absorbent pad is placed into its recess in the bottom of the assembled cartridge, and the vacuum chamber cover glued on over it, preferably with the same ultraviolet adhesive. Porous, sintered plastic plugs may then be pressed into the luer ports in the cartridge top to prevent liquid contamination of the driving instrument. The assembled cartridge is packaged, preferably with a desiccant sachet, in a foil laminate pouch as a light and moisture barrier.  
         [0051]     Having now fully described the invention with reference to certain representative embodiments and details, it will be apparent to one of ordinary skill in the art that changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.