Abstract:
A lab-on-chip device for the processing, in particular the separation, of a fluid mixture comprising two immiscible phases (liquid and/or solid), said device comprising a fluid line ( 1,2,5,6 ) which successively includes an inlet reservoir ( 1 ), a separation channel ( 2 ), a collection channel ( 5 ) and an outlet ( 6 ), said separation channel ( 2 ) being designed in a way as to allow a separation of the fluid mixture into said two phases.

Description:
FIELD OF INVENTION 
       [0001]    The present invention relates to a device and a method which may be advantageously used for separating whole blood cells and plasma/serum, and for collecting and analyzing the generated plasma/serum, e.g. on an absorbing material. The invention is particularly suitable for dried plasma spot analysis (DPS). The plasma/serum portion of the sample, progressively generated by depletion of blood cells, can be processed (e.g. concentrated or depleted from specific proteins) and/or analyzed after separation from the blood cells enriched portion. 
       STATE OF THE ART 
       [0002]    Current methods used to separate plasma from whole blood (i.e. centrifugation) generally involve several milliliters of blood. Sampling, storage, and processing of blood samples are many steps that can be improved in term of invasiveness, costs and time consumption. Today&#39;s trend in biological analysis is to reduce sampling volumes as well as reagent volumes, time, manipulation and handling of material to be analyzed. For these reasons a huge effort is conducted to develop effective point of care (POC) devices that allow on-site processing and/or analysis of biological samples. 
         [0003]    Although biological analysis is usually performed using plasma obtained by venipuncture, the use of dried blood spots (DBS) has grown in popularity in the clinical and pharmaceutical communities over the past decade as an alternative sampling procedure. The DBS sampling process is less invasive than conventional blood sampling because only a small volume (i.e. 20 uL) is collected and spotted onto a filter paper card after a small fingerprick. Because of the ease of collection, DBS can be obtained in a non-hospital environment by minimally trained technicians or even at home by the patients themselves. In addition, most of the pathogenic agents are deactivated on the filter paper during drying (reducing the risk of infection to a minimum) whereas the analytes of interest are stable over further months at room temperature. Since blood can be maintained on a credit-card format sample at room temperature, the cost of shipping and storage of filter paper cards is substantially reduced. 
         [0004]    The first biomedical application of DBS on filter paper dates back to 1963 when Professor Robert Guthrie introduced this alternative sampling method for detection of phenylketonuria in the newborn population. Detection of L-phenylalanine was based on a microbiological test that was sufficiently sensitive but with low analytical throughput. In the early 1990s, the development of PCR and immunoassays, including ELISA, RIA, or FIA, enabled the detection of DNA, viral RNA, antibodies, and hormones from DBS with an acceptable waiting time that was suitable for high-throughput analyses. More recently, DBS sampling form has been successfully applied to the monitoring of therapeutic agents, pharmacokinetic, and toxicokinetic studies based on liquid chromatography mass spectrometry (LC-MS). These combined advantages make the DBS procedure a patient-friendly tool for blood collection, especially in problematic and vulnerable patient populations. The ease of the process can also help in the recruitment of subjects (human and animal) for preclinical and clinical studies. 
         [0005]    Despite of its over-mentioned advantages, DBS sampling is rarely implemented in drug development, clinical analysis nor in a broader extent to people care. Filter paper is indeed a passive support that requires external manipulations to obtain accurate volume measurement and plasma analysis. Taken together, these external steps are tedious and make the DBS not competitive enough compared to conventional venipuncture. 
         [0006]    International patent application WO2013/144743 discloses a lab-on-chip-based system for the direct and multiple sampling, control of the volume, fluid filtration, biochemical reactions, sample transfer, and dried spot generation on the conventional and commercial cards for dried fluid spot. Within an all-in-one integrated holder, this system allows the complete process required to ensure a quantitative analysis of blood, plasma or any other fluids, modification and enrichment of molecule subsets, and formation of a dried fluid spot on the specific spot location of a passive cellulose, non-cellulose, absorbent, or non-absorbent membrane material sampling. As described for this device, plasma is obtained from a filtration process. Consequently, a filter membrane is placed in the inlet of the microfluidic channel to filtrate the fluid and separate cells from whole blood. Although of interest, this process may be not efficient enough to generate the required volume of plasma. The use of membrane may lead to the lysis of blood cells, which is not intended. In addition, the passive collection of the generated plasma into the capillary may be compromised without assistance (e.g. manual or mechanical pumping). Moreover accurate generation of a predetermined volume is delicate since the capillary is in direct contact with the membrane while it is being filled with plasma. 
       GENERAL DESCRIPTION OF THE INVENTION 
       [0007]    The invention concerns a lab-on-chip device for the processing of a fluid mixture which contains at least two immiscible phases (liquid and/or solid). The device comprises a fluid line which successively includes an inlet reservoir ( 1 ), a separation channel ( 2 ), a collection channel ( 5 ) and an outlet ( 6 ), said separation channel ( 2 ) being designed in a way as to allow a separation of the fluid mixture into said two phases. 
         [0008]    In the present text the expression “channel being designed in a way as to allow a separation of the fluid mixture into said two phases” covers a large variety of solutions which, taken as such, are already known in the prior art. The separation may be induced with specific channel dimensions, the material that constitutes the channel wall, elements located within the channel, etc . . . More generally, any technical solution that makes it possible to separate the two phases within the separation channel can be used. 
         [0009]    The invention provides several improvements regarding the state of the art. It allows a passive separation of multiphasic fluids and suspensions (e.g. blood cells in whole blood), sample preparation, metering and isolation of processed fluid, generation of multiple dried plasma spots onto cellulose, non-cellulose, absorbent, or non-absorbent membrane material sampling without the use of a membrane or other filtration material. In addition, the volume of purified fluid (e.g. plasma/serum) which is transferred to the sampling material is controlled after its separation from raw material (e.g. blood cells and plasma/serum). It also allows the quantitative analysis of molecules on conventional dried-spots sampling cards. The device according to the invention may be used with conventional dried spot sampling supports such as #903® brand paper (Whatman, Inc., New Jersey USA), bond elut dried matrix spotting (Agilent, Germany) or treated filter papers, such as FTA and FTA Elute brand paper or DMPK A, B or C card (Whatman, Inc., New Jersey USA). 
         [0010]    According to a preferred embodiment of the invention the microfluidic channel is sized in a way to induce sedimentation and separation of the fluidic mixture (e.g. blood cells) and generation of purified fluid (e.g. blood-cell-free plasma or serum) by capillary action. 
         [0011]    In another embodiment of the invention the device comprises an air bubble actuator, for instance a soft push button, which, when actuated, generates an air bubble into the microfluidic separation channel in order to isolate a defined volume of processed fluid (e.g. plasma/serum). This active metering can afterwards assist the transfer of the said volume of fluid (e.g. plasma/serum) to the storage media. 
         [0012]    In another embodiment of the invention the holding element comprises several fluid channels. 
         [0013]    Several other embodiments of the invention are defined in the claims. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0014]    The invention will be better understood below, in particular with non-limiting examples illustrated by the following figures: 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0015]      FIG. 1  shows a first example (device top view A, 3D view B, channels top view C, channels cross section D) of a device according to the invention. 
           [0016]      FIG. 2  demonstrates the successive formation of the air bubble to delimitate a certain volume of plasma and to assist the transfer of the said volume of plasma towards the outlet. 
           [0017]      FIG. 3  shows another example (3D view) of a device according to the invention. 
           [0018]      FIG. 4  shows a dry plasma spot on a storage media. 
       
    
    
     NUMERICAL REFERENCES USED IN THE FIGURES 
       [0000]    
       
           1 . Inlet reservoir 
           2 . Separation (sedimentation) channel 
           3 . Restriction element 
           4 . Soft button 
           5 . Collection channel 
           6 . Outlet 
           7 . Blood cells 
           8 . Air bubble 
           9 . Plasma 
           10 . Card 
           11 . Dried Fluid Spot 
           12 . Metering channel 
           13 . Outlet vertical channel 
       
     
         [0032]    In the following examples the devices are used for the separation of blood. The invention is of course not limited to such a use. Any suitable multiphasic fluid or particle suspension may be contemplated. 
         [0033]    The device illustrated in  FIG. 1  comprises a blood supply reservoir  1 , a separation channel  2  (also named “sedimentation channel” in the present examples), a restriction element  3 , a plasma collection channel  5 , a soft push button  4  communicating with the separation channel  2  through a metering channel  12 , and an outlet  6  located on the device lateral side. 
         [0034]    A blood droplet (typically 10-50 μL) is deposited into the inlet reservoir  1 . Blood cells  7  and plasma  9  are passively separated into a sedimentation channel  2  (See  FIG. 1 -C). The generated plasma  9  is collected in a collection channel  5 . A restriction element  3 , for instance a baffle, is located between the sedimentation channel  2  and the collection channel  5 . The restriction element  3  ensures a proper orientation of the fluid in the direction of the collection channel  5 . The geometry of the sedimentation channel  2  (length, cross section, shape, etc . . . ) has an effect on the blood dynamic within the channel  2  and thus the sedimentation properties. 
         [0035]    Preferably, the channel geometry is chosen in a way as to induce a capillary effect, i.e. a driving force, to the fluid sample and consequently the sedimentation of blood cells before the restriction element  3  location. After the restriction element  3 , the generated plasma  9  is collected into the collection channel  5  that has a determined volume. The geometry of the collection channel  5  may be adapted to modify either the plasma speed into the sedimentation channel  2  or the capillary forces that drive the fluid. As for the sedimentation channel  2 , the collection channel  5  length, shape, and material are chosen in a way as to induce a capillary effect, i.e. a driving force, to the plasma  9  which is entering the channel  5 . 
         [0036]    The collection channel  5  is advantageously calibrated to receive a predetermined volume of plasma  9  (typically 1-10 μL). This volume can be modified with respect to the analysis requirements. 
         [0037]    After the filling of the collection channel  5 , a soft button  4  is manually or automatically activated by pressure or any other means. Activation of the soft button  4  induces the formation of an air bubble  8  through a metering channel  12  that ends at the entrance of the collection channel  5 . The air bubble  8  sequentially allows the separation between the sedimentation channel  2  and the collection channel  5 . It also allows the collection channel  5  to be mechanically emptied from the outlet  6  (see  FIG. 2 ). Advantageously the outlet  6  may be located on the card upper side (see  FIG. 3 ). In this case a vertical channel  13  is formed between the collection channel  5  and the outlet  6  (see  FIG. 3 ). 
         [0038]    A card  10  ( FIG. 4 ), which may incorporate cellulose and/or non-cellulose storage media, is then applied on the outlet  6  to collect the determined volume of plasma, either by directly pressing the card  10  with the fingers or by any other mean, including for instance a lid which may be clipped to the device. The contact between the card  10  and the fluid at the outlet  6  generates a dried fluid spot  11  on the card. 
         [0039]    In the example of  FIG. 3  a vertical channel  13  is defined between the collection channel  5  and the outlet  6 . Advantageously, the device may contain several vertical and parallel channels (not illustrated) which are set in-line to allow multiple and independent samplings according to the number of spot locations  11  of the collection media  10 . Each channel is preferably designed to produce a dried plasma spot within those spot locations  11 . 
         [0040]    The outlet  6  may have different geometries, which may be circular or non-circular. 
         [0041]    The invention is of course not limited to the devices shown in the above examples.