NL1040139C2 - High throughput capture device for biological objects. - Google Patents

High throughput capture device for biological objects. Download PDF

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Publication number
NL1040139C2
NL1040139C2 NL1040139A NL1040139A NL1040139C2 NL 1040139 C2 NL1040139 C2 NL 1040139C2 NL 1040139 A NL1040139 A NL 1040139A NL 1040139 A NL1040139 A NL 1040139A NL 1040139 C2 NL1040139 C2 NL 1040139C2
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porous support
biological objects
microsieve
hydrogel layer
antibodies
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NL1040139A
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Dutch (nl)
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Cornelis Johannes Maria Rijn
Jacob Baggerman
Ai Nguyen
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Aquamarijn Res B V
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Description

HIGH THROUGHPUT CAPTURE DEVICE FOR BIOLOGICAL OBJECTS
The present invention relates to a capture device for high throughput capture of biological objects from a sample fluid, comprising a porous support with fluidic pathways.
Direct capture of biological objects, such as cells, bacteria, viruses, organelles and biomolecules is facing limitations with respect to a high throughput of the sample volume in combination with a high sensitivity and/or selectivity. It is known to use techniques such as immunomagnetic bead separation for direct capture of biological objects from a sample fluid to discriminate selectively between different objects of interest. For larger sample volumes an upconcentration step of the biological objects can be applied, such as particle-density driven centrifugation or a size-based filtration technique. Centrifugation is well possible with sample tubes up to a volume of e.g. 100 ml. Biological objects with a higher density than the liquid will accumulate at the bottom of the tube and can be separated for further processing. Filtration offers the possibility of upconcentrating much larger sample volumes. A dead-end filtration method has the ability to remove all liquid from the sample, and capturing the biological objects of interest onto the membrane filter. The next step is then to deliver the captured biological objects into a clean buffer or transfer solution as small as 500-1000 microliters. The effectiveness of upconcentration however will be dependent on the presence of all particulate materials in the sample, and this can lead to a considerable buildup of a cake layer on the membrane and premature blockage during the upconcentration procedure.
An approach to loosen the cake layer of the membranes during the upconcentration step and to process more sample material is the use of mechanical forces, which are applied either continuously or periodically onto the membrane surfaces during the upconcentration process, and include techniques such as cross-flow, vibratory filtration, back-pulsing, frequent flow reversal, sonic irradiation or a combination of these techniques.
It is an object of the present invention to develop a flow-through capture device, comprising a porous support with fluidic pathways enabling the capture of biological objects from a sample fluid with a high throughput. The captured biological objects can be used directly or indirectly for analysis and/or diagnostic purposes.
The capture device according to the invention comprises a module with at least one inlet, at least one outlet and at least one porous support with pores, characterized in that at least a portion of the porous support is functionalized with capture reagents such as antibodies, RNA, DNA, aptamers, peptides, synthetic polymers and other molecules which can specifically bind to biological objects to selectively capture biological objects of interest from a sample fluid.
In order to minimize cake layer formation the porous supports should comprise sufficiently large pores to allow for easy passage of other particulate matter in the sample, such as protein granules and other molecular aggregates. The use of a non-fiinctionalized porous support with large pores (pore sizes are larger than biological objects) will also lead to a dramatic reduction of the retention of the biological objects of interest. For this can only be compensated by functionalization with capture reagents of the porous support material, capable in appropriate binding of the biological objects.
Surprisingly it has been found that micro-engineered membranes which are functionalized with capture reagents, in particular microsieves with a low flow resistance and a very short pore length, smaller than three times the mean pore size, have excellent capture properties with a high yield. Most of the biological objects are captured on the surface of the membrane and only a minor fraction in the pores themselves if the pore size is between a half to ten times, and preferably between one and five times, the size of the biological objects. To facilitate the microscopic manual counting procedure the membrane fields of the microsieve can be round shaped, having a diameter that fits with the optical field of view of the objective for the required magnification. Likewise to facilitate microscopic automated digital counting using a CCD camera the membrane fields of the microsieve can be rectangular shaped, having a length and a width that fits with the optical field of view of the CCD camera. In appropriate cases the individual membrane fields on the microsieve can be provided with individual alpha numeric characters for an easy retrieval of data and/or biological objects on the microsieve.
The use of the capture device according to the invention not only overcomes the diffusion limitation of the biological objects towards dense antibody functionalized surfaces, for example typically encountered in antibody-based planar microarrays, but also circumvents the complex washing and collection steps required in bead-based microarrays. Moreover, after staining the captured biological objects can be easily detected and counted with microscopic techniques, because all the biological objects on the planar surface of a microsieve type porous support can be easily brought in the focal plane of the microscope.
It is an important insight according of the invention that biological objects deviate from their flow path when they approach the pore of a microsieve. Instead of going with the flow and passing the pore, the biological objects will experience a fluidic (Stokes) force that deviate the biological objects towards the edge and further surroundings of the pore, in particular towards the surface of the microsieve membrane at the inlet side of the sample. This effect is more pronounced if the size of the biological objects approaches the pore size.
It is a further insight that there is an optimum void area of the pores in the membrane in relation to the dense (non-open) area of the membrane surface. The ratio of the void area and the dense area of the membrane is between 0.1 and 35% and with preference between 1 and 20%. A high ratio will reduce the necessary area for biological objects to bind to the functionalized membrane surface, whereas a low ratio will create large areas between the pores without substantial binding.
The proper functionalization of the porous supports according to the invention is crucial. For example in case the capture reagent is an antibody, the capture efficiency is determined by the binding density and the orientation of the antibodies. Also the required interaction time of the antigens to bind with the antibodies is important and will be dependent on the flow rate through the porous support. All these factors codetermine the capture efficiency of the biological objects.
Surprisingly it has been found that microsieves functionalized with antibodies attached to a relatively thick hydrogel layer between 10 nm and 10 pm showed excellent capture properties with a high yield. Most of the biological objects have been captured on the surface of the membrane when the antibodies are bound to a hydrogel layer composed of polymer chains, with a length typically between 100 nm and 5 pm, capable in binding a large number of antibodies per polymer chain. The coating preferably provides a three-dimensional surface structure in which the chains of the hydrophilic polymer are aligned at least partly vertical to the membrane surface, i.e. brushlike. Due to their increased surface compared to planar structures, such brush-like hydrogel surfaces show a particularly enhanced immobilization capacity for capture reagents, such as antibodies and other affinity molecules which are capable of binding the biological objects. It has been found that brush-like structured hydrogel coatings, in particular those which comprise or consist of certain polycarboxylate polymers (i.e. polymers with carboxylate groups, such as polyacrylic, polymethacrylic or polymaleic acid), provide an excellent surface for attaching antibodies. With preference the porous support is provided with a hydrogel layer having a thickness that is about 5 -50 % of the mean diameter of the pores. The optimum thickness of the hydrogel coating according to the invention is also related to the (mean) size of the micro-objects, typical the ratio of the size of the biological objects and the thickness of the hydrogel coating is between 20 and 0.5, and with preference between 1 and 10 times.
Very good results have been obtained with a slightly cross linked and very open polycarboxylate network layer provided on the microsieve. It is an insight according to the invention that part of the sample flow will pass through the open network, herewith increasing the probability of close contact between the antibodies immobilized in the open network and the biological objects in the sample fluid. Typically the open porosity (is fraction of the volume of voids over the total volume) of the open network should be between 30 and 99.9%, and with preference between 80 and 99%.
To reduce the number óf non-specific binding events to the antibody functionalized polymeric network layer two additional measures have been found advantageously. The first measure is to form polycarboxylate polymers with side groups of polyethyleneglycol. The second measure is to form polycarboxylate polymers with side groups of zwitterionic moieties, such as phosphocholine, sulfobetaine and carboxybetaine groups.
The high-throughput capture of biological objects from a sample fluid on a microsieve functionalized with antibodies is suitable in combination with polymerase chain reaction (PCR), Reverse transcription polymerase chain reaction (RT-PCR), Fluorescent in-situ Hybridisation (FISH) or comparable DNA and RNA including ribosomalRNA, messengerRNA and microRNA analysis methods. FISH techniques in which the shape of the biological objects is maintained and only the outer cell membrane is made permeable for the FISH probes gives very good results. Especially the detection of ribosomal RNA via FISH represents an efficient way of utilizing sensitivity and specificity of DNA-probes without having to use a nucleic acid amplification test (NAAT) step.
The capture device according to the invention can also be realized as a microarray wherein individual membrane fields of the porous support or microsieve comprises are spotted with a number of different antibodies.
Fig. 1 illustrates a capture device according to the invention.
Fig. 2 illustrates a detail of an antibody functionalized microsieve.
Fig. 3 shows microscopic images of a microsieve with bacteria..
Fig. 4 shows bacterial counting results
Fig. 5 shows CTC counting results obtained according to certain embodiments of the present disclosure.
In Fig. 1 a capture device is shown according to the invention comprising a module (1) with one inlet (2), one outlet (3) and at least one porous support (4) with pores. A portion of the porous support (4) is functionalized with an antibody rich hydrogel layer (5) to selectively capture antigen-presenting micro-objects (6) of interest from a sample liquid. Non specific antigen-presenting species (7,8,9) are not captured on the membrane.
Fig. 2 shows a detail of Fig. 1 of the porous support (4) with a hydrogel layer (5) composed of brushlike polycarboxylate polymers (10) with specific antibodies (11) directed to specific antigens (12) of the target micro-object (6).
Example 1 Detection of Salmonella
The capture efficiency of antibody-coated microsieves with a pore size of 3.5 pm was examined. Anti-Salmonella antibodies were attached to a polycarboxylate-coated microsieve. The antibodies were anti-Salmonella CSA-1 antibodies. The polycarboxylate chains had a linear chain length of ca. 530 nm. Uncoated microsieves were used of 0.45 pm and 3.5 pm pore size. Next a diluted Salmonella-containing solution of 1 ml was filtered through all the devices, and the captured Salmonella were stained by an FITC-labeled anti-Salmonella antibody, and observed and analyzed on the microsieve surface by an automated fluorescence microscope. Fig. 3 shows a bright field image of a coated (Fig.3 left) and an uncoated microsieve (Fig.3 right) with a pore size of 3.5 pm after the filtration run. In Fig. 4 counting results are depicted. Microsieve 1-4 shows the salmonella counts for an uncoated 3.5 pm microsieve, microsieve 5-7 shows the counts for an anti -Salmonella antibody coated 3.5 pm microsieve, and microsieve 8-10 shows the counts for an uncoated 0.45 μιη microsieve with a 100% retention.
Example 2 Detection of CTCs
The capture efficiency of antibody-coated microsieves with a pore size of 15 pm was examined to capture SKBR-3 cells with a mean cell size of 12 pm. Anti-EpCAM (VU1D9) antibodies were attached to a polycarboxylate-coated microsieve. The polycarboxylate chains had a linear chain length of ca. 1500 nm. The polycarboxylate chains were partly provided with polyethyeleneglycol side groups to reduce unspecific binding. Uncoated microsieves were also used of 5 pm and 15 pm pore size. Next 1 ml blood samples were spiked 125 SKBR-3 cells and were filtered and DNA stained by DAPI, and subsequently observed and analyzed on the microsieve surface by an automated fluorescence microscope. In Fig. 5 counting results are depicted. Microsieve 1-2 shows the EPCAM expressing CTC cell counts for an uncoated 15 pm microsieve, microsieve 3-5 shows the counts for the VU1D9 coated 15 pm microsieve, microsieve 6-7 shows the counts for a non-EPCAM expressing cell line filtered through a coated 15 pm microsieve, and microsieve 8-10 shows the EPCAM expressing CTC cell counts for an uncoated 5 pm microsieve.

Claims (16)

1. Werkwijze voor high-throughput vangst van antigenpresenterende biologische objecten uit een monster vloeistof gebruikmakend van een apparaat met een module met minstens één ingang, minstens één uitgang en minstens één vlakke poreuze drager met poriën, met het kenmerk dat het oppervlak van de poreuze drager aan de ingangszijde is gefunctionaliseerd met bindingsreagentia zoals antilichamen, RNA, DNA, aptameren, peptiden, synthetische polymeren en andere moleculen voor selectieve binding van objecten zoals cellen, bacteriën en virussen aan het oppervlak van het poreuze substraat aan de ingangszijde.A method for high-throughput capture of antigen-presenting biological objects from a sample liquid using a device with a module with at least one entrance, at least one exit and at least one flat porous carrier with pores, characterized in that the surface of the porous carrier is functionalized on the input side with binding reagents such as antibodies, RNA, DNA, aptamers, peptides, synthetic polymers and other molecules for selective binding of objects such as cells, bacteria and viruses on the surface of the porous substrate on the input side. 2. Apparaat voor de werkwijze volgens conclusie 1, met het kenmerk, dat de gemiddelde poriegrootte van de poreuze drager 1 tot 10 keer, en bij voorkeur een tot vijf maal de grootte van de biologische objecten heeft.Device for the method according to claim 1, characterized in that the average pore size of the porous support has 1 to 10 times, and preferably one to five times the size of the biological objects. 3. Apparaat voor de werkwijze volgens conclusie 1 of 2, met het kenmerk, dat de verhouding tussen het open en dicht oppervlak van de poreuze drager tussen 0,1% en 35% is en bij voorkeur tussen 1% en 20%.Apparatus for the method according to claim 1 or 2, characterized in that the ratio between the open and closed surface of the porous support is between 0.1% and 35% and preferably between 1% and 20%. 4. Apparaat voor de werkwijze volgens conclusie 1,2 of 3, met het kenmerk, dat de poreuze drager een micro-gefabriceerd membraan is, in het bijzonder een microzeef met een gemiddelde porie lengte kleiner dan 3 maal de gemiddelde diameter van de porie.Apparatus for the method according to claim 1, 2 or 3, characterized in that the porous support is a micro-fabricated membrane, in particular a microsieve with an average pore length of less than 3 times the average diameter of the pore. 5. Apparaat voor de werkwijze volgens conclusie 4, met het kenmerk, dat de poreuze drager of microzeef omvat afzonderlijke membraanvelden met een ronde of rechthoekige vorm passend bij de grootte van het gezichtsveld van een optische microscoop, en dat de individuele membraanvelden zijn voorzien van individuele alfanumerieke tekens voor het eenvoudig verkrijgen van gegevens en / of biologische objecten op de microzeef.Apparatus for the method according to claim 4, characterized in that the porous support or microsieve comprises individual membrane fields with a round or rectangular shape corresponding to the size of the field of view of an optical microscope, and in that the individual membrane fields are provided with individual alphanumeric characters for easily obtaining data and / or biological objects on the microsieve. 6. Apparaat voor de werkwijze volgens conclusie 1,2,3,4 of 5, met het kenmerk, dat de poreuze drager gefunctionaliseerd is met antilichamen gebonden aan een hydrogellaag met een dikte tussen 10 nanometer en 10 micrometer.Apparatus for the method according to claim 1,2,3,4 or 5, characterized in that the porous support is functionalized with antibodies bound to a hydrogel layer with a thickness between 10 nanometers and 10 micrometers. 7. Apparaat volgens conclusie 6, met het kenmerk, dat de hydrogellaag bestaat uit polymeerketens, met een lengte typisch tussen 100 nanometer en 5 micrometers, die een groot aantal antilichamen per polymeerketen kunnen binden.Device according to claim 6, characterized in that the hydrogel layer consists of polymer chains, typically between 100 nanometers and 5 micrometers in length, that can bind a large number of antibodies per polymer chain. 8. Apparaat volgens conclusie 6 of 7, met het kenmerk, dat de hydrogellaag is samengesteld uit hydrofiele polymeren, die ten minste gedeeltelijk loodrecht op het membraanoppervlak zijn uitgelijnd en de vorm van een borstel aannemen.Device according to claim 6 or 7, characterized in that the hydrogel layer is composed of hydrophilic polymers, which are at least partially aligned perpendicular to the membrane surface and take the form of a brush. 9. Apparaat volgens conclusie 6, 7 of 8, met het kenmerk, dat de hydrogel laag bestaat uit polycarboxylaatpolymeren, zoals polyacrylzuur, polymethacrylzuur of polymaleïnezuur.Device according to claim 6, 7 or 8, characterized in that the hydrogel layer consists of polycarboxylate polymers, such as polyacrylic acid, polymethacrylic acid or polymaleic acid. 10. Apparaat volgens conclusie 6, 7, 8 of 9, met het kenmerk, dat de hydrogellaag bestaat uit polycarboxylaatpolymeren met zijgroepen die polyethyleenglycol en / of zwitterionen, zoals fosfocholine, sulfobetaïne en carboxybetaïne, bevatten.Device according to claim 6, 7, 8 or 9, characterized in that the hydrogel layer consists of polycarboxylate polymers with side groups containing polyethylene glycol and / or zwitterions, such as phosphocholine, sulfobetaine and carboxybetaine. 11. Apparaat volgens een van de voorgaande conclusies, met het kenmerk, dat de poreuze drager gefunctionaliseerd is met antilichamen gekoppeld aan een relatief dikke hydrogellaag met een dikte in de order van 5% tot 50% van de gemiddelde diameter van de poriën.Device according to any of the preceding claims, characterized in that the porous support is functionalized with antibodies coupled to a relatively thick hydrogel layer with a thickness in the order of 5% to 50% of the average diameter of the pores. 12 Apparaat volgens een van de voorgaande conclusies, waarin individuele membraanvelden van de poreuze drager of microzeef zijn gespot met verschillende bindingsreagentia.Apparatus according to any of the preceding claims, wherein individual membrane fields of the porous support or microsieve are spotted with different binding reagents. 13 Apparaat volgens een van de voorgaande conclusies waarbij de poreuze drager met verzamelde biologische objecten wordt geanalyseerd met microscopische technieken.Apparatus according to any of the preceding claims, wherein the porous support with collected biological objects is analyzed by microscopic techniques. 14 Apparaat volgens een van de voorgaande conclusies waarbij de verzamelde biologische objecten op poreuze drager worden geanalyseerd door toepassing van PCR, RT-PCR, FISH of vergelijkbare DNA en RNA zoals rRNA, mRNA en miRNA analysemethoden.Apparatus according to any of the preceding claims, wherein the collected biological objects on porous support are analyzed using PCR, RT-PCR, FISH or similar DNA and RNA such as rRNA, mRNA and miRNA analysis methods. 15. Gebruik van een apparaat volgens een van de voorgaande conclusies.Use of an apparatus according to one of the preceding claims. 16. Diagnostische kit omvattende een apparaat volgens een van de voorgaande conclusies.A diagnostic kit comprising an apparatus according to any one of the preceding claims.
NL1040139A 2013-04-01 2013-04-01 High throughput capture device for biological objects. NL1040139C2 (en)

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