Classifications

• • 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
  • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C1/00—Magnetic separation
  • B03C1/005—Pretreatment specially adapted for magnetic separation
  • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C1/00—Magnetic separation
  • B03C1/02—Magnetic separation acting directly on the substance being separated
  • B03C1/28—Magnetic plugs and dipsticks
  • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles
• • 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
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0652—Sorting or classification of particles or molecules
• • 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
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0668—Trapping microscopic beads
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0848—Specific forms of parts of containers
  • B01L2300/0851—Bottom walls
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C2201/00—Details of magnetic or electrostatic separation
  • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C2201/00—Details of magnetic or electrostatic separation
  • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications

Definitions

• the invention relates to an arrangement for quantifying cells of a cell suspension with a fluid channel for conducting the cell suspension and a magnetic sensor on the fluid channel for counting magnetically labeled cells in the cell suspension.
• optical flow cytometry can be used for single-cell detection.
• FACS fluorescence activated cell sorting
• the separated cells are electrically charged to a different degree and deflected from charged plates into different collecting containers.
• magnet-based cell detection can also be used.
• the cells are labeled with superparamagnetic markers and transported over a magnetoresistive component, such as GMR. The aim is to be able to arrange the analytes at the highest possible cell concentration in the smallest possible distance from each other and to detect them individually.
• the arrangement according to the invention for quantifying cells of a cell suspension has a fluid channel for conducting the cell suspension. Furthermore, the arrangement comprises a magnetic sensor on the fluid channel. This is based insbesonde ⁇ re on GMR, AMR, etc. and is designed to count magnetically labeled cells in the cell suspension. Suitably, the magnetic sensor is in close proximity to or within the fluid channel.
• the fluid channel has an enrichment region with an enlarged cross-section.
• a magnet is arranged on at least one side of the enrichment area.
• the magnet can be an electromagnet.
• a permanent magnet comes to a ⁇ set.
• a fluid passage having a first cross-section and an enhancement-type region with respect to the first cross-sectional enlarged second sectionalmayge ⁇ is, the cells are in the fluid channel for enrichment Area and there led to a magnetic sensor for counting magnetically labeled cells in the cell suspension and finally the cells are drawn in the enrichment area by a magnet to one side of the fluid channel.
• the magnet ensures an enrichment of the magnetically marked cells in the enrichment area. Since the non-magnetically labeled cells do not react to the magnetic field, they are also not enriched and flow freely in the fluid channel away from the magnet. It is expedient if the magnetic sensor is arranged in the enrichment area. So can be beneficial in addition to the measurement of cells also a subsequent separation of the cells and a removal of the magnetically labeled cells take place. The magnetically labeled cells are enriched and a subsequent step for sorting magnetically labeled cells from unlabelled is unnecessary.
• the magnet is preferably arranged such that the cells are extracted from the present outside of the enhancement region ⁇ cross-section of the fluid channel in the enriching section. In other words, the cells are pulled out of the flow present in the fluid channel. Cells that are in the flow-calmed parts of the fluid channel in the enrichment area are much easier influenced by the force of the field generated by the magnet and are not so easily carried away by the flow otherwise present in the fluid channel.
• the fluid channel has in flow direction of the cell suspension ⁇ a concave shape designed to capture the magnetically-labeled cells that are drawn from the magnet in the enhancement region.
• Fluid channel in the extended cross-section towards the end of a pocket or similar shape which is so gestal ⁇ tet that once there cells are largely cut off from the flow in the fluid channel and could only get into the fluid channel by against the otherwise move prevailing flow.
• FIG. 1 shows an arrangement of a Si-based GMR sensor and a fluid channel for cell quantification and enrichment
• An arrangement 10 for single-cell detection and subsequent separation of the cells 17, 18 according to FIG. 1 consists of a mixing chamber (not shown in FIG. 1) and a fluid channel 11 for enrichment of the cells and guidance via a magnetoresistive GMR sensor 12.
• the GMR sensor 12 is applied to a silicon wafer, which in turn is arranged on a permanent magnet 13.
• the fluid channel 11 has a first cross section 14 away from the permanent magnet 13.
• ⁇ 13 of the fluid passage 11 is widened and has a second cross-section 15 which is larger than the first cross-sectional ⁇ fourteenth
• the increase of the cross-section 14 widens the fluid channel 11 so that it extends to the silicon wafer with the GMR sensor 12.
• the fluid channel 11 is at a distance from the permanent magnet 13.
• the enrichment region 21, which results from the second cross-section 15, is formed in the view according to FIGS. 1 to 4 in the manner of a parallelogram. As a result, a kind of pocket forms in the flow direction of the cells 17, 18 toward the end of the enrichment region 16.
• the enrichment region 21 is formed exclusively to the side, which is the permanent magnet 13 is ⁇ supplied. In the other directions, the fluid channel 11 in the region with the second cross section 15 is unchanged from the region with the first cross section 14.
• Fig. 1 shows a set of cells 17, 18. A part of cell h ⁇ len 17 is not magnetically labeled. The rest of the cells 18 are magnetically marked, for example with superparamagnetic beads. The marked and unmarked cells 17, 18 are mixed together and flow in the fluid channel 11 to the region of increased cross section 15. For this purpose, a pump, which is not shown in FIG. 1, generates a suitable flow in the fluid channel 11.
• FIG. 2 shows the situation at a time when the cells 17, 18 have almost reached the region of enlarged cross section 15.
• the magnetically marked cells 18 are first pulled under the influence of the permanent magnet 13 in the direction of the permanent magnet and concentrated on the corresponding side of the fluid channel 11.
• FIG. 3 shows the situation at a time when the cells 17, 18 reach the area of enlarged cross section 15. They go through there - from the fault through the
• Permanent magnet 13 apart - the imaginary continuation 20 of the fluid channel 11 in the region with the second cross section 15.
• the magnetically marked cells 18 are further pulled out under the influence of the permanent magnet 13 from the imaginary continuation 20 of the fluid channel 11 in the enrichment area 21. It is also possible that some unmarked cells 17 are dragged by mutual friction. However, the plurality of unlabelled cells 17 remain in the imaginary continuation 20 and are carried on by the flow in the fluid channel 11 on.
• the magnetically marked cells 18 are carried by the flow via the GMR sensor 12 and thereby trigger signals, on the basis of which a count of the labeled cells is possible.
• FIG. 3 shows the situation at a later point in time when the cells 17, 18 increase the end of the area b
• the labeled cells 18 collect in the bag in Anreich ceremoniessbe ⁇ rich 21 and could return from there only in the fluid channel 11, by moving against the flow. They therefore largely remain in the pocket in the enrichment area 21. However, the unmarked cells 17 are carried away with the flow in the fluid channel 11.
• a marked accumulation of the labeled cells 18 in the enrichment region 21 thus takes place through the described arrangement 10.
• Unmarked cells 17 are washed away.
• the labeled cells 18 may subsequently be taken, for example, and used to conduct follow-up examinations.
• this can be achieved in certain test sequences, a significant reduction of the steps.
• a septum 22 punctcture membrane
• An example of such a test sequence is an examination of the lymphocytopenia, that is to say the insufficient number of lymphocytes.
• the arrangement 10 is implemented within a point-of-care device.
• the lymphopenia can example ⁇ as occur in the use of corticosteroids in the course of HIV infection (CD4 + T-helper cells), a lot of stress, rheumatoid arthritis or idiopathic CD4 + lymphopenia (less than 300 CD4 + T cells / ⁇ blood).
• FIG. 5 shows states of the cells 17, 18 that are achieved in this example for the quantification by specific measuring steps.
• a first state 501 is reached after the cells 17, 18, both labeled and unmarked, have been passed over the GMR sensor 12 in a first flow direction 52. They have already been separated as described for FIGS. 1 to 4 and drawn in the enrichment region 21 to the permanent magnet 13.
• the cells 17, 18 are concentrated at one end of the enrichment area 21. Is then carried out a reversal of the flow direction, and thus 18 flow through the cells 17 in a second direction of flow 52 to the other end of the accumulation area 21. In this case, about ⁇ they traverse again the GMR sensor 12, and thereby results in the second state 502. possibility to be counted again.
• the magnetic guide structures 51 thereby ensure that the cells 18 are aligned with the center of the enrichment region 21 in the case of the magnetically marked cells 18, so that these cells are increasingly guided individually and one after the other via the GMR sensor.
• the unmarked cells 17 do not react to the magnetic guide structures 51 and can thus leave the enrichment area 21 again-separated from the marked cells 18.
• the third state 503 results when reversed
• the cells to be examined 17, 18 can be removed after quantification in the assembly 10 for further follow-up examination of the septum 22.
• An example of such a follow-up is in connection with HIV infections, for example, the following: In the early stages of HIV infection, the number of free in the blood viruses is very low and usually not recognizable. However, CD4 + cells that are infected may already contain a precursor of HI virus after infection (proviruses). At this stage of the infection no conspicuous number of CD4 + cells is measurable (conspicuous: below 500 / ⁇ 1 - normal: 600-1600 / ⁇ 1) and the symptoms of the infection are indistinguishable from conventional flu.
• CD4 + Cells are counted in the presented invention, these can then be removed and further tested for a possible HIV in ⁇ fection in the early stage towards.
• the arrangement 10 is also used as part of a point-of-care device for measuring Thrombo cytes ⁇ to investigate the process of hemostasis.
• the number of platelets is very important, especially in the context of thrombocytopenia, ie too small a number of platelets.
• Known methods record either only the relative change in the platelet count (cellular branch of the hemostasis) or only the plasmatic branch of the blood coagulation, ie without detecting the number of platelets.
• the assembly 10 described herein is able to measure both ⁇ s ⁇ te hemostasis (cellular and plasmatic) by the number of platelets is determined at the beginning of the measurement.
• the steps of the survey and the conditions that occur are shown in FIG.
• the cells 17, 18 of the cell suspension are initially passed several times over the sensor area as already described for Figure 5.
• cell aggregates 630 and finally a clot with fibrin 640 are formed.
• the formation of a clot may e.g. can be achieved by additional surface-sensitive impedance sensors. The sample is passed over the sensor several times, with more and more cells depositing on the surface in the course of time, which is characterized by an increase in the impedance.

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• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
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• Biotechnology (AREA)
• Fluid Mechanics (AREA)
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• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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• 2012
  • 2012-07-04 DE DE102012211626.5A patent/DE102012211626A1/de not_active Withdrawn
• 2013
  • 2013-06-24 CN CN201380034979.8A patent/CN104380080A/zh active Pending
  • 2013-06-24 WO PCT/EP2013/063128 patent/WO2014005869A1/de active Application Filing
  • 2013-06-24 US US14/412,543 patent/US20150209784A1/en not_active Abandoned
  • 2013-06-24 EP EP13733996.6A patent/EP2839262A1/de not_active Withdrawn

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