WO2016029943A1 - Method for determining at least one physiological parameter of a biological sample - Google Patents
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- WO2016029943A1 WO2016029943A1 PCT/EP2014/068182 EP2014068182W WO2016029943A1 WO 2016029943 A1 WO2016029943 A1 WO 2016029943A1 EP 2014068182 W EP2014068182 W EP 2014068182W WO 2016029943 A1 WO2016029943 A1 WO 2016029943A1
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Classifications
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- 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
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- 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
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- G—PHYSICS
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- 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
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- G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
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- G—PHYSICS
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- 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
- G01N33/5002—Partitioning blood components
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
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- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
Definitions
- the invention relates to a method for determining at least one physiological parameter of a liquid biological sample by a flow device.
- physicians need different health parameters. For ex ⁇ ample, if they are severely bleeding or septic urgent pa ⁇ tients, this task can become a great challenge since the ex ⁇ pended time during diagnostics and treatment can have direct influence on the result. Severely bleeding patients or pa ⁇ tients with expectant sepsis thereby often do not get the sufficient therapy, which may result in increased mortality and invalidity of the patient cohort.
- haematocrit is measured in a haematology analyzer, a var- iant of the optical flow cytometer, and a haemoglobin value derived from it is calculated.
- the previous gold standard for the measurement of haemoglobin is the hemiglobin cyanide method.
- a spectroscopic measurement at 540 nm after staining with Drabkin' s reagent is performed on the sample. The method is applied in commer ⁇ cial applications such as the haematology analyzer, the blood gas analyzer and the co-oximeter.
- haematocrit and haemo- globin A quick test (“iSTAT”) for measuring haematocrit and haemo- globin is currently in commercial application.
- it is a conductivity measurement.
- the conductivity is de ⁇ pendent on the portion of cells, which are not conductive, thus the haematocrit, in a conductive medium, thus the blood plasma.
- the haemoglobin is determined via a conversion factor (0.34), wherein this factor is applied against the normal average corpuscular haemoglobin con ⁇ centration (MCHC) of a healthy patient.
- the scattering and absorption of the light beam is used as an assessment criterion of dif ⁇ ferent cell parameters.
- dilution of the blood is required since blood samples and many other cell mixtures are opaque liquids, in order that the employed light beam is able to penetrate the sample.
- Further methods for measuring the haemostasis do not perform count of the blood platelets, but measure the variation of a parameter, such as for example pressure, viscosity or imped ⁇ ance (cf. Lei et al .
- thrombocytes are also counted in a haematology analyzer in the central laboratory, but further differential diagnos- tics of the thrombocyte function has to be done afterwards.
- An object underlying the invention is the provision of a more efficient method for analyzing biological samples of a cell mixture.
- the invention is based on the idea of performing a dielectric measurement with the aid of a preset sensor geometry in a mi- crofluidic system utilizing the so-called Fahr3 ⁇ 4us effect (al- so: "Fahr3 ⁇ 4us-Lindqvist effect”)-
- the present invention allows simultaneous measurement of multiple physiological parameters of the biological sample, thus for example cellular and plas ⁇ matic parameters, such as for example blood parameters such as haematocrit, haemoglobin and thrombocyte number, without diluting and/or pre-analytic preparation of the biological sample being required.
- the method according to the invention is applicable to each type of liquid biological samples (in the following also re- ferred to as "sample"), which comprises a cell mixture of at least two different cell types.
- the cell types ide ⁇ ally differ in the cell size and/or the biological sample has different portions of the respective cell types as a charac ⁇ teristic feature.
- a first cell type the average cell diameter of which is preferably one fifth to two fifth of an average cell diameter of a further cell type, is present in a concentration, which is preferably one thou ⁇ sandth to five hundredth of a concentration of the cells of the further cell type.
- the biological sample preferably comprises at least two cell types, wherein the cells of a larger cell type are present in a distinct excess to a cell type of smaller cells.
- An exemplary biological sam- pie is composed of or comprises for example a whole blood sample, a plant cell mixture or a mixed bacterial population.
- the method according to the invention serves for determining at least one physiological parameter of a liquid biological sample by a flow device, for example for determining a haema- tocrit value and/or a haemostasis, or an activation state of a cell type of the cell sample.
- an interior space of a microfluidic channel of the flow device has a central area extending along a flow direc ⁇ tion and an detection area located between the central area and an inner wall of the microfluidic channel.
- a mi ⁇ crofluidic channel is a device suitable for a microfluidic flow measurement for guiding a liquid sample or a fluidic flow, which has for example a diameter or an extension in a range from 5 micrometers to 300 micrometers, preferably in a range from 5 micrometers to 50 micrometers or from 50 microm ⁇ eters to 300 micrometers.
- separation of the cell mixture of the sample is effected within the microfluidic channel by the liquid sample flowing through the microfluidic channel such that the cells of a first cell type of the sample are accumulated and flow in the detection area and cells of a further cell type of the sample are accumulat ⁇ ed and flow in the central area of the microfluidic channel.
- the biological sample is electrically isolated from the sensor device within the microfluidic channel.
- the passivation device is a protection of the sensor device, which can for example be configured as a protective layer, which is at least partially disposed on or at the in ⁇ ner wall.
- the passivation device is at least par- tially composed of a semi-metal oxide, for example of silicon oxide or aluminum oxide.
- the passivation device has at least partially a semi-metal oxide, for example silicon oxide or aluminum oxide.
- the passivation of at least one electrode of the at least one sensor device by the passivation device allows dielectrically acquiring the measured variable, which specifically increases the sensitivity to cellular membranes.
- conductivity of the biological sam- pie is not measured, but an electrical stray field.
- the meas ⁇ urement area can thus be adjusted to a predetermined area of the sample in the interior space of the microfluidic channel, for example to the central area or the detection area.
- the at least one sensor device ac ⁇ quires at least one measured variable, preferably at least two measured variables, of at least one of the cells of the further cell type and/or of a charge carrier, for example haemoglobin or chlorophyll, of the biological sample in the central area of the microfluidic channel.
- a charge carrier for example haemoglobin or chlorophyll
- the combination of acquisition of one or more physiological parameters and utilization of the Fahr3 ⁇ 4us effect in a dynamic microfluidic system allows simultaneous measurement of multi ⁇ ple parameters of an undiluted biological sample.
- the biological sample does not have to be expensively prepared or diluted before performing the method according to the invention such that specialized laboratories are not re ⁇ quired.
- measurement results are fast present.
- the integration of the measured parameters in the form of the cell function, cell concentration and an exemplary haemosta- sis test close to the patient opens improvement of the per- sonalized transfusion in the emergency care, in the intensive care unit and in elective surgeries. This method does not need preceding sample preparation for example for haemoglobin measurement, cell concentration and/or cell function test be ⁇ sides provision of the biological sample after sampling.
- labeling at least one of the cells of the first cell type of the biological sample with a cell-specific label can be effected, preferably with a magnetic label and/or within the detection area.
- a magnetic label has the advantage with respect to optical labels that a possible turbidity of the sample does not disadvantageously affect the measurement re ⁇ sult. If labeling is effected in the detection area, thus, an expensive labeling step in a separate sample vessel can be avoided and a possible contamination can be nearly completely prevented .
- At least one measured variable of at least one of the cells of the first cell type can be acquired in the detection area by a further sensor device during flow of the biological sample.
- the further sensor device comprises a
- At least one physiological parameter of the first cell type can be determined. This allows nearly simultaneous acquisition of various meas ⁇ ured variables and nearly simultaneous determination of mul ⁇ tiple physiological parameters. For example, this can include simultaneous counting at least one of the cells of the first cell type and/or quantifying a portion of activated cells of the first cell type and/or an aggregation characteristic of at least one of the cells of the first cell type with an aggregation molecule, for example with fibrin. According to a further embodiment of the method according to the invention, accordingly, the determination of the at least one physiological parameter of the first cell type can be effected by:
- the aggregation state describes a cell aggregate formation of the at least one acquired cell with an aggregation molecule.
- contact with at least one sensor device can be favored.
- fixing the at least one cell to a surface of the retaining device follows, wherein the retaining device has at least partially a
- the retaining de ⁇ vice can be at least partially disposed in the detection ar- ea.
- the passivation device is at least partially configured as the retaining device. This used modification layer serves for promoting the adhesion of cells.
- the acquisition of the measured variable of the at least one cell of the further cell type and/or of the charge carrier can preferably be effected with the aid of an impedance sen ⁇ sor device as the sensor device. This favors acquisition of the measured variable in the dielectric. In contrast to opti- cal measurement methods, expensive labeling or sample prepa ⁇ ration steps are not required.
- adaptation of at least one electrical parameter has proven particularly advantageous such that the sensor device becomes particularly sensitive to the adhesion of further cells.
- cells of the different accumulated cell types can also be cell type specific and independently acquired.
- the adaptation of the at least one electrical parameter can for example include adap ⁇ tation of a measurement voltage and/or a measurement frequen ⁇ cy, for example increasing the measurement voltage and/or the measurement frequency.
- the two end points basically are also considered to be encompassed by the respective range.
- the acquisition of the measured variable of the at least one cell of the further cell type can be effected at a measurement voltage in a range from 50 millivolts to 100 millivolts and/or at a measurement voltage in a range from 500 millivolts to 3000 millivolts.
- the acquisition of the measured variable of the at least one cell of the further cell type can be effected
- a first measurement frequency in a range from 100 kilo- hertz to 10 megahertz, preferably in a range from 100 kilo- hertz to 5 kilohertz or from 1.5 megahertz to 10 megahertz, and/or
- a measurement voltage respectively has a predeter ⁇ mined constant value.
- Such a measurement with the passivated sensor device for ex- ample an impedimetric measurement, has a particularly high specifity for the applied measurement frequency.
- the system is sensitive to capacitive resistances such as membranes.
- the measurement system becomes increasingly more sensitive to charge carriers both outside of and within the respective cell due to the decreasing capacitive re ⁇ sistance. Accordingly, at high frequencies, the portion of the soluble, charged carriers, such as for example haemoglo ⁇ bin, can in particular be measured.
- a pressure of a liquid column comprising the biological sample can be performed at a first point of time and/or a further point of time by the further sensor device. Based on the acquired pressure, a rhe- ological characteristic of the biological sample can be de ⁇ termined, for example a viscosity.
- an ac ⁇ tual end point of a measurement can be determined, in which for example a whole blood sample is completely coagulated.
- a flow device accord ⁇ ing to the invention with a microfluidic channel, wherein the interior space of the microfluidic channel of the flow device has a central area extending along a flow direction and an detection area located between the central area and an inner wall of the microfluidic channel.
- the flow device further comprises at least one sensor device disposed in the detec ⁇ tion area, and a passivation device, which is disposed on at least a partial area of the inner wall, on which the sensor device of the flow device is also disposed.
- the passivation device is adapted to electrically isolate the interior space from the sensor device.
- the flow device ac ⁇ cording to the invention can include a retaining device for fixing at least one cell of the biological sample, where- in the retaining device is at least partially disposed in the detection area, and wherein the retaining device at least partially has a coagulative substance.
- the flow device according to the invention can include a par- ticular sensor geometry and/or a particular geometry of the passivation device according to a further particularly preferred embodiment.
- the two end points basically are also considered to be encom ⁇ passed by the respective range.
- the flow de ⁇ vice is characterized in that the at least one sensor device comprises at least one interdigital electrode array, which preferably has an electrode distance of two electrodes of 3 micrometers to 30 micrometers or of 100 micrometers to 1000 micrometers, preferably of 300 micrometers.
- the flow device is characterized in that the passivation device has a height in a range from 15 nanometers to 400 nanometers.
- the flow device can include at least one further sensor de ⁇ vice, which preferably comprises a magnetoresistive sensor and/or a pressure sensor.
- the vari ⁇ ation of a distance of the measurement electrodes to each other and/or a thickness of the passivation layer allows adaptation of the flow device to a specific measurement problem, accord ⁇ ing to whether a user wants to measure a measurement of the entire volume (thus for example the haematocrit) or only a volume portion of the microfluidic channel close to surface (for example an adhesion of thrombocytes or thrombocytes dis ⁇ placed from the volume flow to the edge area of the volume flow by larger erythrocytes) .
- the assessment of the Fahr3 ⁇ 4us effect based on the combination of an exemplary impedimetric measurements for example of the primary haemostasis and/or the haematocrit in for example fully coagulating or stabilized undiluted whole blood is ef- fectively rendered more precisely by adapting the measurement frequency and/or the electrode geometry and/or the measure ⁇ ment voltage and/or a surface characteristic of an electrode.
- FIG 1 a schematic sketch of the principle of an embodi ⁇ ment of the method according to the invention
- FIG 2 a schematic sketch to the principle of the separa ⁇ tion step according to an embodiment of the method according to the invention, wherein a sensor device according to an embodiment of a device according to the invention for flow measurement with a microflu- idic channel is exemplary illustrated,
- FIG 3 a schematic sketch to a further embodiment of the method according to the invention with an optional labeling step
- FIG 4 a schematic sketch to a further embodiment of the method according to the invention with an impedimetric measurement method step, a schematic sketch to a further embodiment of the method according to the invention with an impedimetric measurement method step
- FIG 1 illustrates the principle of the method according to the invention based on a first embodiment.
- FIG 1 shows a section of a microfluidic channel 10 of an embodiment of a flow device 12 according to the invention, for example of
- the microfluidic channel 10 has a diameter of pref ⁇ erably 50 micrometers to 300 micrometers.
- an inner wall 14 of the microfluidic channel comprises a passivation device 16, which is at least partially or completely disposed on the inner wall 14 and for example is at least partially composed of silicon oxide or aluminum oxide.
- Corresponding coating methods for disposing such a passivation device 16 are known to the man skilled in the art of surface technique.
- the passivation device 16 allows electrically isolating the sample 5 from at least one sensor device 30 such that the sample 5 is present as a dielectric in the microfluidic chan ⁇ nel 10. Beving et al .
- the passivation device 16 can include a retaining device 42 or be configured as a retaining device 42.
- the retaining device 42 can at least partially or completely be composed of a coagulative substance, for exam- pie of fibronectin, collagen, fibrin and/or another adhesive protein known to the man skilled in the art from the prior art.
- the retaining device 42 allows fixing at least one cell 22 of a first cell type to the inner wall 14.
- the flow device 12 according to the invention comprises at least one sensor device 30 with at least one sensor, which can have at least one electrode 32. On and/or at the inner wall 14, at least one further sensor device 18 can be dis- posed.
- the at least one further sensor device 18 can include at least one sensor with for example two electrodes 20, as shown in FIG 1.
- the further sensor device 18 can preferably include at least one magnetic field based sensor, while the at least one sen ⁇ sor device 30 preferably can include at least one impedance sensor, or three impedance sensors, as exemplarily shown in FIG 1.
- Each two of the further sensor devices 30 can for ex- ample be used for an impedimetric measurement of haemostasis of the biological sample 5 (illustrated in flow direction F after the further sensor device 18 in FIG 1) .
- the measurement can be performed at a varying measurement voltage and a con ⁇ stant measurement frequency.
- separation of the cell mixture of the biological sample 5 is ef ⁇ fected in the microfluidic channel 10 (method step SI) such that the at least one cell 22 of the first cell type is urged to the inner wall 14 and the at least one cell 26 of the fur ⁇ ther cell type is accumulated in an area centrally extending in flow direction.
- This is achieved with the aid of the ar ⁇ chitecture of the microfluidic channel 10, as explained in more detail below to FIG 2.
- One of the exemplary sensor devices 30 can for example be used for the impedimetric measurement of a haematocrit value and/or a haemoglobin concentration of the exemplary blood sample (illustrated in flow direction F after the first two sensor devices 30 in FIG 1) .
- the measurement can be performed at a varying measurement frequency and a constant measurement voltage.
- the flow device 12 can include a further sensor device 18, which comprises a pressure sensor for de ⁇ termining a rheological characteristic of the biological sam- pie 5 (not illustrated in FIG 1) .
- the flow device 12 can include a magnet 34, which can be disposed on the inner wall 14 of the microfluidic channel 10.
- the biological sample 5 is liquid and preferably comprises an undiluted whole blood sample of a patient or an animal, plant or microbial cell sample.
- an exterior shape of the at least one cell 22 of the first cell type can have another shape in an activated state and a larger cell diameter than in a deactivated state.
- this is the case in a thrombocyte as the at least one cell 22 of the first cell type, which promotes a thrombocyte aggregation and initiates a secondary haemostasis in an activated state in blood coagulation.
- FIG 1 not all of the cells 22 are provided with reference signs, but are recog ⁇ nizable by an identical shape as correspondingly illustrated and identified cells 22.
- the cells 22 of the first cell type can be labeled with a label 24, for example a magnetic, chemiluminescent , fluorescent and/or plasmonic label 24, which can for example be bound to a cell- specific antibody.
- a label 24 for example a magnetic, chemiluminescent , fluorescent and/or plasmonic label 24, which can for example be bound to a cell- specific antibody.
- the man skilled in the art has avail ⁇ able usual methods from the prior art for providing cell- specific labels 24.
- the biological sample 5 is provided in a fluidic flow of the micro ⁇ fluidic channel 10, preferably in a laminar flow.
- the biolog ⁇ ical sample 5 further comprises at least one cell 26 of a further cell type, for example at least one erythrocyte or a photosynthetic plant cell.
- the at least one cell 26 of the further cell type can include a charge carrier 28 in the cell plasma, for example haemoglobin or chlorophyll.
- the charge carrier 28 can additionally or alternatively be present out ⁇ side of the at least one cell 26 of the further cell type in the sample liquid (not shown in FIG 1) .
- FIG. 1 For clarity, in FIG. 1
- FIG 1 illustrates the principle of separating a cell mixture (method step SI) and the principle of an exemplary integra ⁇ tion of for example multiple sensor devices 18, 30 for simul ⁇ taneous measurement (S3, S4, S5, S6, S7) of a physiological parameter of the biological sample 5, for example of a haema- tocrit value and/or a haemoglobin concentration.
- the integration of the two measurement methods can be performed in a flow device 12 according to the invention, for example a flow cell.
- Controlling an exemplary impedance measurement via con- trolling the different sensor geometries can be accomplished with the same component, for example with a controller of the flow device 12 as a control device 36 if the measurements are for example alternately performed.
- integration of at least one further sensor device 18 can be performed for acquiring at least one physiological parameter of the first cell type, for example a thrombocyte number and/or thrombo ⁇ cyte activation and/or a rheological characteristic of the sample 5.
- FIG 2 describes the effect occurring by the architecture of the microfluidic channel 10 and the flow of the sample 5, the so-called Fahr3 ⁇ 4us effect, based on the exemplary blood sample and for example in a microfluidic channel 10 with the sensor device 18 described to FIG 1 and an optional magnet 34.
- An interior space of the microfluidic channel 10 has a central area extending along the flow direction F and an detection area 11 located between the central area and an inner wall of the microfluidic channel 10, which preferably has a height as the extension into the interior space of the microfluidic channel 10 in a range from 0.5 micrometers to 5 micrometers, ideally from 3 micrometers to 5 micrometers.
- the cells 22 of the first cell type the exemplary thrombocytes, which have pref- erably a smaller volume than the cells 26 of the further cell type, impact the larger cells 26 of the further cell type, here red blood cells (erythrocytes) in the example, caused by the flow of the sample 5, and are urged into the detection area 11 of the microfluidic channel 10.
- the cells 26 of the further cell type have an about 1000-fold higher concentra ⁇ tion than the cells 22 of the first cell type in the exempla ⁇ ry blood sample. Thereby, a concentration gradient of the cells 22, 26 forms along a channel diameter.
- the concentration of the cells 22 of the first cell type is high ⁇ er at the edge of the microfluidic channel 10 than in the center, and the cells 26 of the further cell type have a high concentration in the central area.
- the detection area 11 can be considered as a depletion zone of the further cell type. Suitable conditions for a suitable flow rate and a suitable diameter of the microfluidic channel for promoting the Fahr3 ⁇ 4us effect are described in Alizadehrad et al .
- FIG 3 shows an optional labeling step S3 and an optional measurement of a physiological parameter of the at least one cell 22 according to a further embodiment of the method ac ⁇ cording to the invention.
- magnetic labels 24 preferably superparamagnetic nanoparticles , can be dis ⁇ claimed into the cell sample 5 and/or into the flow chamber.
- the exemplary at least one magnetic label 24 can be directed into the detection area 11 by the magnetic field to the inner wall 14 in the direction (M) such that efficient contacting of the label 24 with the at least one cell 22 of the first cell type and bonding or absorbing the marker 24 to or in the cell 22 can be effected due to the above described cell accumulation.
- the at least one labeled cell 22 moves further in the flow direction F and can thus be guided across the first sensor device 18.
- the first sensor device 18 for example comprises at least one magnetic field sensor with at least one elec ⁇ trode 20, for example at least one magnetooptical or
- the at least one further sensor device 18 can include an optical sensor.
- the further sensor device 18 can for example be controlled by the control device 36.
- the further sensor device 18 can be configured as a magnetoresistive sensor, for example as an AMR sensor, GMR sensor or TMR sensor.
- the at least one electrode 20 can pref ⁇ erably be configured as a strip electrode to allow very sen ⁇ sitive acquisition of a cell volume or a cell aggregate vol- ume .
- a portion of activated cells 22 of the first cell type can be determined. Additionally or alterna ⁇ tively, based on an acquired cell diameter, an activation state of the at least one exemplary labeled cell 22 and/or a portion of activated exemplary labeled cells 22 of an entire- ty of the acquired exemplary labeled cells 22 can be deter ⁇ mined .
- an aggregation characteristic of the at least one cell 22 of the first cell type with an aggregation molecule for example fibrin
- the aggregation state describes a cell aggregate for ⁇ mation of the at least one labeled cell 22 with the aggrega ⁇ tion molecule.
- FIG 4 shows a method step of an exemplary impedimetric meas ⁇ urement (S4, S5) according to an embodiment of the method ac ⁇ cording to the invention with the aid of the at least one sensor device 30, which preferably comprises at least one im ⁇ pedance sensor.
- determining a primary and/or secondary haemostasis as the physiological parameter of the exemplary blood sample can be effected.
- the aim is a differentiation of a layer of adhering cells 22 of the first cell type, for exam ⁇ ple a monolayer of the exemplary thrombocytes, and a clot, thus a cell aggregate of the exemplary thrombocytes as the cells 22 of the first cell type with an aggregation molecule 38, for example fibrin.
- the flow device 12 has a passivation device 16 with a retaining device 42 within the microfluidic channel 10, which has a coagulative surface and is at least partially composed of a coagulative substance, for example of silicon oxide and/or fibrin.
- the passivation device 16 preferably has a thickness in a range from 10 nanometers to 400 nanometers.
- the electrodes 32 for the impedimetric measure ⁇ ment of the coagulation by the exemplary thrombocytes can ei- ther be passivated by the electrical barrier layer and/or specifically be modified with coagulation factors consisting of organic and/or inorganic material, in their configura ⁇ tions. These used modification layers then serve for promot ⁇ ing and/or avoiding the adhesion of cells 22. Both a
- passivated and a non-passivated sensor device 30 can be used.
- the at least one sensor device 30 can for example include each one pair of electrodes 32 of an impedance sensor.
- ⁇ to, in particular an electrode 32 configured as a comb elec- trode is suitable as the electrode 32.
- the at least one sen ⁇ sor device 32 preferably comprises an interdigital electrode ( "interdigitated electrode", “IDES”) and/or an interdigital electrode array preferably having an electrode distance of 3 micrometers to 30 micrometers and adapted to be operated at least at two alternating measurement voltages.
- control of the electrode 32 can be effected via a control de ⁇ vice 36, for example a microcontroller of the flow device 12 or a controller external to device, which can be connected to the respective electrode 32 via a wireless or wired communi ⁇ cation link 37.
- a control de ⁇ vice 36 for example a microcontroller of the flow device 12 or a controller external to device, which can be connected to the respective electrode 32 via a wireless or wired communi ⁇ cation link 37.
- a layer of adhered labeled cells 22 can preferably be ac- quired at a low measurement voltage in the range from 50 mil ⁇ livolts to 100 millivolts (S4) since here the electric field lines of an electric field 39 do not penetrate very deeply, that is only between about 200 nanometers and 300 nanometers, into the analysis liquid, that is into the liquid sample 5.
- activated cells 22 of the exemplary blood sample After a layer of activated cells 22 of the exemplary blood sample has adhered to the surface of the sensor, they begin releasing the content of their granules from the interior of the cells 22. This process initiates the plasmatic coagula- tion among other things, in which soluble fibrinogen in the blood plasma is converted into insoluble fibrin. The fibrin surrounds the immobilized and activated cells and promotes the further adhesion of thrombocytes to already immobilized cells 22.
- the geometry of the exemplary passivated impedance sensors, but also the electrical parameters, can be adapted such that they become sensitive to the adhesion of further cells 22, for example by increasing the measurement voltage and/or the measurement frequency.
- the further attachment of the exemplary thrombocytes to the monolayer and the formation of fibrin, illustrated as a cell aggregate 40 in FIG 4, can preferably be acquired at a high measurement voltage in the range from 500 millivolts to 3000 millivolts (S5) , since here the electric field lines pene- trate deeply, for example 1000 nanometers to 5000 nanometers, into the analysis liquid and therefore penetrate deeper into the cell plasma.
- the further sensor device 30 generates a signal upon contacting the at least one cell 22 of the first cell type. Increasing coverage of the exemplary impedance electrodes 32 is associated with variation of the impedance signal.
- a predetermined measurement frequency remains con ⁇ stant in both measurement steps S4, S5.
- a suitable measurement frequency for acquiring the cells 22 is known to the man skilled in the art from the prior art.
- FIG 5 and FIG 6 show an exemplary method step of an
- At least one physiological parameter of the biological sample 5 can be determined from it, for example a haematocrit value.
- the charge carrier 28, thus the exemplary haemoglobin can here be present in certain portions in the interior of the cells 26 or in the plasma surrounding the cells 26.
- the passivation of the electrodes 30 by the passivation de ⁇ vice 16 allows dielectric acquisition specifically increasing the sensitivity to cellular membranes.
- both the measurement voltage and the sensitive frequency range can be specifically predetermined.
- the impedimetric acquisition with passivated sensors 30 has a specifity for the applied meas- urement frequency.
- a measurement voltage depending on a breakdown voltage of the passivation device 16 remains constant and can preferably be in a range between 100 milli ⁇ volts and 10 volts.
- the man skilled in the art can determine a suitable measurement voltage depending on a layer thickness of the passivation device 16, for example a meas ⁇ urement voltage of 100 millivolts with a layer thickness of the passivation device 16 of 100 nanometers, with the aid of the general professional knowledge.
- the geometry of the sensor device 30 is adapted to measurement of the alternating current resistance.
- the acqui ⁇ sition of the alternating current resistance is effected in a specific frequency range.
- a first step S6 the acquisition of the alternating current resistance can be effected at a preset measurement voltage and a first measure ⁇ ment frequency in a range from 100 kilohertz to 10 megahertz, preferably in a range from 100 kilohertz to 5 kilohertz and/or from 1.5 megahertz to 10 megahertz.
- the membranes of the cells 26 of the further cell type are penetrated by the electric field 39 and the entire charge carrier portion of the sample 5 is acquired.
- the measure ⁇ ment system becomes increasingly unspecific to membranes and more sensitive to charge carriers both outside of and within the cellular analytes due to the decreasing capacitive re ⁇ sistance. Accordingly, the portion of the soluble, charged metabolites (such as haemoglobin) can particularly be measured at high frequencies.
- acquisition of the charge carrier 28 can be effected.
- the method steps S6 and S7 can be effected individu ⁇ ally or in any order or be alternately repeated several times one after the other.
- An advantageous electrode geometry describes a system with for example an IDES as the sensor device 32, which preferably has an electrode distance d in a range from 100 micrometers to 1000 micrometers.
- the at least one sensor device 30 is preferably adapted to be able to be operated at least at two alternating measurement frequencies.
- the control of the at least one sensor device 30 can for example be effected by the control device 36.
- FIG 7 shows an exemplary diagram of an exemplary impedance measurement according to an embodiment of the method accord ⁇ ing to the invention, as for example described to FIG 5 and FIG 6.
- an impedance in Ohm is plotted on an ordinate y of the coordinate system
- a haematocrit value in %HcT is plotted on an abscissa x of the coordinate system.
- a cell 26 of the further cell type thus an exemplary erythrocyte, is shown.
- the con ⁇ nected data points of the curve K for example represent meas ⁇ ured values, which are acquired at an exemplary measurement frequency of 500 kilohertz.
- the at least one sensor device 32 comprises for ex ⁇ ample two passivated impedance sensors, the electrode geome ⁇ try of which each has a geometry of 300 micrometers by 300 micrometers.
- an exemplary electrode geometry is shown in FIG 8, wherein each of the electrodes 20 preferably has a width b of 300 micrometers and/or they have a distance d from each other preferably of 300 micrometers.
- the arrows of FIG 8 mark the flow direction F of the liquid col ⁇ umn 44 of the biological sample 5.
- exemplary measurement parameters in which a meas ⁇ urement associated with the diagram of FIG 7 is performed, include an exemplary measurement voltage of 500 millivolts and/or a whole blood sample as the biological sample 5 and/or a passivation device 16, which is at least partially composed of aluminum oxide, with an exemplary layer thickness of 70 nanometers.
- the mapping of the measurement points in the curve K shows increase of the impedance with an increase of the haematocrit value.
- FIG 10 and FIG 12 each show an embodiment of a measurement of a pressure (S8) of a liquid column 44 of the biological sam- pie 5 by means of a further sensor device 18 at a center of pressure 50, which for example comprises a pressure sensor.
- An exemplary absolute pressure sensor or differential pres ⁇ sure sensor is known to the man skilled in the art from the prior art.
- the respective measurement can be per- formed with an undiluted biological sample 5.
- a rhe- ological characteristic of the biological sample 5 can be de ⁇ termined, for example a viscosity of the biological sample 5, whereby for example coagulation of the biological sample 5 can be inferred.
- Such an exemplary pressure measurement can serve as a control measurement.
- a non-stabilized sample 5 is used, thus for example a blood sample, in which coagulation occurs.
- the sample 5 coagulates, whereby hae- mostasis is effected, by which a rheological characteristic, for example the viscosity of the sample 5, varies.
- a rheological characteristic for example the viscosity of the sample 5
- FIG 10 shows a liquid column 44, which pushes an air column 46 in the direction F in front of it. If the viscosity of the sample 5 increases because the sample 5 coagulates, the pres ⁇ sure of the air column 46, which pushes the sample 5 in front of it, decreases because the flow velocity decreases.
- This pressure drop can be linear or exponential according to how the other parameters of the sample 5, for example a cell num ⁇ ber and/or concentration of plasmatic factors, are designed.
- the arising vacuum can be acquired by the further sensor device 18.
- a corresponding exemplary embodiment of a flow de- vice according to the invention is shown in FIG 9.
- the further sensor device 18 can be disposed at a piston 48, which is pulled in the direction F by a variation of the rhe- ological characteristic such that the further sensor device 18 can determine a pressure drop.
- a first absolute pressure "P(tl)” and at a further point of time a further absolute pressure "P(t2)" are acquired.
- FIG 12 shows a liquid column 44, which pushes an air column 46 in front of it.
- the coagulating sample 5 causes an in ⁇ crease of the viscosity in the system and thereby an increase of the resistance counteracting the extending piston 48, for example a piston 48 of a syringe.
- the applied force on the piston 48 on the part of a pump gets a measurable counter- force by the viscosity increase of the sample 5.
- a corre ⁇ sponding exemplary embodiment of a flow device according to the invention is shown in FIG 11.
- the further sensor device 18 can be disposed such that the further sensor device 18 can determine a pressure rise.
- a first absolute pressure "P(tl)" and at a further point of time a further absolute pressure "P(t2)" are acquired.
- Fig 13 and FIG 14 each show an exemplary pressure profile de- pending on a rheology of the biological sample 5. Therein, a pressure is respectively plotted on the ordinate y against a point of time on the abscissa y.
- the curve K shows a linear pressure drop in FIG 13.
- the measurement points of time are the exemplary points of time "tl" and "t2".
- a pressure drop of an exemplary whole blood sample with a high haematocrit value is for example shown.
- the curve K of FIG 14 shows an exemplary exponential pressure drop, for example of an exemplary whole blood sample with a low haematocrit value.
- FIG 15 shows severely schematized once again an embodiment of a flow device 12 according to the invention, for example a flow cell or a flow cassette.
- the flow device 12 comprises a microfluidic channel 10, a passivation device 16 and at least one sensor device 32.
- the flow device 12 can include a further sensor device 18 and/or a controller 38 with a communication link 37 for connecting to a respective sensor device 18, 32.
- the individual components are prefer ⁇ ably configured corresponding to the above description to the preceding figures.
- the flow device 12 can include a sample receiving container 52 and/or a sample collecting container 54.
- the above mentioned embodiments illustrate the principle of the method according to the invention, utilizing the Fahr3 ⁇ 4us effect and an adapted sensor geometry, to perform a simulta ⁇ neous measurement of multiple physiological parameters, for example cellular and plasmatic blood parameters such as haem ⁇ atocrit, haemoglobin and thrombocyte number, in for example undiluted whole blood as the biological sample 5, which pres- ently is only achievable by dilution or other pre-analytical efforts .
- physiological parameters for example cellular and plasmatic blood parameters such as haem ⁇ atocrit, haemoglobin and thrombocyte number
- an occurring Fahr3 ⁇ 4us effect is for example utilized in whole blood.
- the portion of cells in a preferably undiluted sample 5 (for ex ⁇ ample in whole blood, wherein for example red blood cells have the greatest cellular portion of 99%) is collectively measured with a physiological parameter, for example the con ⁇ centration of haemoglobin, the red dye within the red blood cells, on the one hand.
- the determination and/or the measure ⁇ ment of this at least one parameter can be realized by means of at least one sensor device 30, which for example comprises an impedance sensor.
- the number of cells 22 of a first cell type for example the thrombocytes, and/or the de ⁇ gree of activation thereof is quantified on the other hand.
- This measurement can for example be effected with the aid of a specific label of the cells 22 of the first cell type, for example with superparamagnetic nanoparticles , and subsequent read-out by means of for example a magnetoresistive sensor (for example a GMR, TMR, AMR and/or Hall sensor) based on travel time measurement.
- a magnetoresistive sensor for example a GMR, TMR, AMR and/or Hall sensor
- a pressure measurement can for example be inte ⁇ grated, which allows indirect measurement of the rheological characteristics, namely for example of the viscosity varia- tion of the biological sample.
- the integration of such a measurement serves for determining the actual end point of a measurement, at which the exemplary whole blood is completely coagulated and thereby the pressure in the system rapidly in ⁇ creases .
- the impedimetric measurement, and the exploitation of the Fahr3 ⁇ 4us effect in a dynamic microfluidic system allows the simulta- neous determination of various physiological parameters such as for example the thrombocyte number, the haematocrit, and the haemoglobin in the exemplary undiluted whole blood sample .
- the measurement can be per ⁇ formed in undiluted whole blood.
- a higher concentration of cells 22 of the first cell type, thus of the exemplary thrombocytes is located at the edge of for example a microfluidic channel 10 or a blood ves ⁇ sel than in the center.
- the sensor devices 18, 30 employed in this invention can be disposed at any side of the microfluid ⁇ ic channel 10.
- the integration of the above mentioned measurement parameters in the form of the exemplary cell function, cell concentra ⁇ tion and haemostasis test close to the patient opens an im- provement of the personalized transfusion in the emergency care, in the intensive care unit and in elective surgeries.
- the assessment of the Fahr3 ⁇ 4us effect based on the combination of for example an impedimetric measurement for example of the primary haemostasis, thus the adhesion of activated thrombo ⁇ cytes to a sensor surface, and the haematocrit, thus a per ⁇ centage portion of the red blood cells in the blood, prefera ⁇ bly in fully coagulating or stabilized undiluted whole blood, is allowed by adapting the measurement frequency, the elec- trode geometry, the measurement voltage and/or the surface characteristics of the electrodes 20, 32 on the one hand.
- the exemplary additional counting of the cells 22 of the first cell type can for example be allowed via a magnetoresistive sensor, for example by a time of flight measurement of un- rolling and/or immunomagnetically labeled cells 22.
- the vari ⁇ ation of the distance of the exemplary impedimetric measure ⁇ ment electrodes 20 to each other and/or the thickness of the passivation device 16 allows adaptation to a specific meas ⁇ urement problem according to whether the applicant wants to measure a measurement of the entire volume (thus for example the haematocrit) or only a volume portion of the microfluidic channel 10 close to surface (for example the adhesion of cells 22 of the first cell type or cells 22 of the first cell type, which are urged from the volume flow to the edge area of the volume flow by the larger cells 26 of the further cell type) .
- the depletion zone of the cells 26 of the further cell type in the edge area of the inner wall 14 can be dependent on for example a haematocrit, a channel cross-section and a flow velocity, however is in the range of few micrometers (3 micrometers to 5 micrometers) , which are for example occupied by smaller cells 22 such as for example TZ displaced from the volume flow.
- the method is characterized by the following characteristics:
- the biological sample 5 can for example be provided by means of canula after intravenous or arterial blood collection.
- the described method utilizes the Fahr3 ⁇ 4us effect, in which at least one cell type to be meas ⁇ ured (for example red blood cells and/or thrombocytes) in for example an undiluted whole blood sample as the biological sample 5 cluster to the edge in case of the cells 22 of the first cell type and in the center of a microfluidic channel 10 in case of the cells 26 of the further cell type (see FIG 2) .
- An exemplary magnetoresistive measurement for example of the primary or cellular haemostasis can for example be effected as follows: an optional specific labeling of the cells 22 of the first cell type with for example superparamagnetic nano- particles as the label 24 and/or the subsequent measurement of labeled cells 22 of the first cell type and
- microaggregates of cells 22 of the first cell type by means of for example a magnetoresistive sensor allows the simulta ⁇ neous counting of the cells 22 of the first cell type, the quantification of the portion of activated cells 22 of the first cell type and/or the aggregation characteristics of the cells 22 of the first cell type with an aggregation molecule, for example fibrin.
- the latter for example initiates the sec ⁇ ondary haemostasis.
- An exemplary impedimetric measurement for example of the pri ⁇ mary haemostasis thus the adhesion of exemplary activated cells 22 of the first cell type to a coagulative surface, can be effected as follows: in for example an undiluted sample 5, the cells 22 of the first cell type are urged to the edge of the volume flow of a microfluidic channel 10 by the 1000-fold higher concentration of the cells 26 of the further cell type (see FIG 4) .
- Exemplary impedance sensors located there can be used for measuring for example the coagulation, accordingly the adhesion of the cells 22 of the first cell type to one of the inner walls 14 (see FIG 4) .
- the electrodes 20 for the ex- emplary impedimetric measurement of the coagulation by the cells 22 of the first cell type can either be passivated in their configurations by dielectric barrier layers (with a thickness in an advantageous range from 10 nanometers to 400 nanometers) and/or specifically modified with coagulation factors (at least partially composed of organic and/or inor ⁇ ganic material) . These used modification layers then serve either for promoting and/or avoiding the adhesion of cells. Both passivated and non-passivated sensor elements can be used .
- An exemplary impedimetric measurement for example of the sec ⁇ ondary haemostasis can be effected as follows: after a layer of activated cells 22 of the first cell type has adhered to the surface of a sensor device 32 (see FIG 4), they begin to release the content of their granules from the interior of the cells. This process initiates the plasmatic coagulation among other things, in which for example soluble fibrinogen in the blood plasma is converted to insoluble fibrin. The fi ⁇ brin surrounds the immobilized and activated cells and pro- motes the further adhesion of cells 22 of the first cell type to already immobilized cells 22.
- the geometry of the exempla ⁇ ry passivated impedance sensor device 32, but also the elec ⁇ trical parameters, can be adapted such that they become sen ⁇ sitive to the adhesion of further cells 22. (For example by increasing the measurement voltage and/or the measurement frequency) .
- An exemplary measurement of a haematocrit value and/or a hae ⁇ moglobin concentration can be performed as follows: the pas ⁇ sivation of electrodes 32 allows dielectric measurement, which specifically increases the sensitivity to cellular mem- branes.
- both the measurement voltage and the sensitive frequency range can be specifically determined.
- the impedimetric measurement with a passivated sensor device 30 has a specifity for the applied measurement frequency (see FIG 5 and FIG 6) .
- an exemplary measurement of the haematocrit can be effected: the exemplary haematocrit value can for example be quantified by means of an impedimetric measurement, an adapted geometry of the sensor device 30 and/or a specific frequency range. At lower frequencies of the applied measurement voltage, prefer ⁇ ably in a range from 100 hertz to 100 kilohertz, the system is sensitive to capacitive resistances such as membranes. The conductivity of the cell plasma is not co-measured.
- haemoglobin can be effected: at high frequencies, preferably in a range from 100 kilohertz to 5 megahertz, the measurement system becomes increasingly un- specific to membranes and more sensitive to charge carriers 28 both outside of and within the cells 26 due to the de ⁇ creasing capacitive resistance. Accordingly, at high frequencies, in particular the portion of the soluble, charged me ⁇ tabolites (such as for example haemoglobin) can be measured.
- two measure ⁇ ment methods for example a magnetoresistive and an
- the Fahraeus effect can be used as a basis for assessing an analytic question, namely the fast and immediate counting of cells 22 of the first cell type and/or the assessment of the degree of activation thereof and/or the measurement of for example haematocrit and/or hae ⁇ moglobin as the physiological parameter in for example se ⁇ verely injured and traumatized patients.
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Abstract
The invention relates to a method for determining at least one physiological parameter of a liquid biological sample (5) by a flow device (12). A microfluidic channel (10) of the flow device (12) has a central area extending along a flow direction (F) and an detection area (11). Separation of the cell mixture is effected by the sample (5) flowing through the microfluidic channel (10) such that cells (22) of a first cell type are accumulated in the detection area (11) and cells (26) of a further cell type are accumulated in the central area. A passivation device (16) allows electrically isolating the sample (5) from at least one sensor device (30). During the flow, acquisition of a measured variable of a cell (26) of the further cell type and/or of a charge carrier (28) is effected in the central area and a physiological parameter (S4 - S8) is determined.
Description
Description
Method for determining at least one physiological parameter of a biological sample
The invention relates to a method for determining at least one physiological parameter of a liquid biological sample by a flow device. In order to stratify and purposefully treat patients in the clinic, physicians need different health parameters. For ex¬ ample, if they are severely bleeding or septic urgent pa¬ tients, this task can become a great challenge since the ex¬ pended time during diagnostics and treatment can have direct influence on the result. Severely bleeding patients or pa¬ tients with expectant sepsis thereby often do not get the sufficient therapy, which may result in increased mortality and invalidity of the patient cohort. Nowadays, the diagnostics of such patients is performed in the central laboratory in a hospital and is very often asso¬ ciated with organizational effort since the diagnostics has to be completed as fast as possible and these patients there¬ fore have be prioritized with respect to in-patients. A fur- ther problem therein is that blood samples of such patients have to be present in a stabilized form in order that coagu¬ lation does not occur during the transport time between the different stations in the central laboratory. Coagulated blood samples cannot be further processed in the central la- boratory.
In order to obtain a complete image of a blood sample, vari¬ ous parameters are determined with different approaches, e.g. the haematocrit is measured in a haematology analyzer, a var- iant of the optical flow cytometer, and a haemoglobin value derived from it is calculated.
The previous gold standard for the measurement of haemoglobin is the hemiglobin cyanide method. After removing the cell membranes of the red blood cells by lysis, a spectroscopic measurement at 540 nm after staining with Drabkin' s reagent is performed on the sample. The method is applied in commer¬ cial applications such as the haematology analyzer, the blood gas analyzer and the co-oximeter.
A quick test ("iSTAT") for measuring haematocrit and haemo- globin is currently in commercial application. Herein, it is a conductivity measurement. Herein, the conductivity is de¬ pendent on the portion of cells, which are not conductive, thus the haematocrit, in a conductive medium, thus the blood plasma. The more cells are in this medium, the lower the con- ductivity of the analyte is. The haemoglobin is determined via a conversion factor (0.34), wherein this factor is applied against the normal average corpuscular haemoglobin con¬ centration (MCHC) of a healthy patient. A haemoglobin concentration is determined with the formula: haemoglobin (g/dL) = haematocrit (% HcT) x 0.34.
In van Berkel et al . (2011) (van Berkel C, Gwyer J.D., Deane S., Green N.G., Holloway J., Hollis V., Morgan H.: "Integrat- ed systems for rapid point of care (PoC) blood cell analy¬ sis", Lab Chip 2011 Apr 7 ; 11 (7 ) : 1249-55. ) a conductivity measurement is performed in a microfluidic system after prep¬ aration of the cells. The most common method for counting blood platelets is the optical flow cytometry. Therein, cells within a liquid pass a light beam one after the other. The scattering and absorption of the light beam is used as an assessment criterion of dif¬ ferent cell parameters. Here, in any event, dilution of the blood is required since blood samples and many other cell mixtures are opaque liquids, in order that the employed light beam is able to penetrate the sample.
Further methods for measuring the haemostasis do not perform count of the blood platelets, but measure the variation of a parameter, such as for example pressure, viscosity or imped¬ ance (cf. Lei et al . , 2013: Lei K.F., Chen K.H., Tsui P.H., Tsang N.M.: "Real-time electrical impedimetric monitoring of blood coagulation process under temperature and hematocrit variations conducted in a microfluidic chip", PLoS One. 2013 Oct 7;8(10)), due to the coagulation of the stabilized whole blood comprising plasma and cells, by an activator. Exemplary commercially available products are here the products
PFA100®, Multiplate® and ROTEM®.
Existing methods, such as for example the optical flow cytometry, or methods operating according to the Coulter principle, require dilution or division of the blood into its cellular components. The platelet function measurement on single-cell level by means of the optical flow cytometry re¬ quires high pre-analytical effort in the form of lysis and centrifugation steps. Platelet function measurement close to the patient by aggregometry (impedance measurement) or PFA100 (pressure measurement) can be measured slightly faster. All of the systems close to the patient demonstrate that the test results have high dependency on the haematocrit and the platelet number.
However, these measurements are not possible in non- stabilized and undiluted whole blood samples. In addition, the thrombocytes are also counted in a haematology analyzer in the central laboratory, but further differential diagnos- tics of the thrombocyte function has to be done afterwards.
This is required up to now to get a more differentiated image of the coagulation status of a patient and accordingly to de¬ rive a therapy decision from it, for example a transfusion protocol or a first life-saving measure, which demands direct intervention. To this, it can be resorted to the aggregometry and further technologies and apparatuses, for example to the products PFA100®, Multiplate® or ROTEM®.
The above mentioned methods have in common that different blood parameters cannot be measured in parallel or in non- stabilized whole blood and require pre-analytics . Exactly this pre-analytics can entail that results are not present sufficiently fast or direct therapy for an urgent patient cannot be derived from it.
An object underlying the invention is the provision of a more efficient method for analyzing biological samples of a cell mixture.
The object is solved by the method according to the invention and the flow device according to the invention according to the independent claims. Advantageous developments of the in- vention are given by the dependent claims.
The invention is based on the idea of performing a dielectric measurement with the aid of a preset sensor geometry in a mi- crofluidic system utilizing the so-called Fahr¾us effect (al- so: "Fahr¾us-Lindqvist effect")- The present invention allows simultaneous measurement of multiple physiological parameters of the biological sample, thus for example cellular and plas¬ matic parameters, such as for example blood parameters such as haematocrit, haemoglobin and thrombocyte number, without diluting and/or pre-analytic preparation of the biological sample being required.
The method according to the invention is applicable to each type of liquid biological samples (in the following also re- ferred to as "sample"), which comprises a cell mixture of at least two different cell types. Therein, the cell types ide¬ ally differ in the cell size and/or the biological sample has different portions of the respective cell types as a charac¬ teristic feature. Ideally, therein, a first cell type, the average cell diameter of which is preferably one fifth to two fifth of an average cell diameter of a further cell type, is present in a concentration, which is preferably one thou¬ sandth to five hundredth of a concentration of the cells of
the further cell type. In other words, the biological sample preferably comprises at least two cell types, wherein the cells of a larger cell type are present in a distinct excess to a cell type of smaller cells. An exemplary biological sam- pie is composed of or comprises for example a whole blood sample, a plant cell mixture or a mixed bacterial population.
The method according to the invention serves for determining at least one physiological parameter of a liquid biological sample by a flow device, for example for determining a haema- tocrit value and/or a haemostasis, or an activation state of a cell type of the cell sample.
Therein, an interior space of a microfluidic channel of the flow device has a central area extending along a flow direc¬ tion and an detection area located between the central area and an inner wall of the microfluidic channel. Therein, a mi¬ crofluidic channel is a device suitable for a microfluidic flow measurement for guiding a liquid sample or a fluidic flow, which has for example a diameter or an extension in a range from 5 micrometers to 300 micrometers, preferably in a range from 5 micrometers to 50 micrometers or from 50 microm¬ eters to 300 micrometers. According to the method according to the invention, separation of the cell mixture of the sample is effected within the microfluidic channel by the liquid sample flowing through the microfluidic channel such that the cells of a first cell type of the sample are accumulated and flow in the detection area and cells of a further cell type of the sample are accumulat¬ ed and flow in the central area of the microfluidic channel.
By providing a passivation device on at least a partial area of the inner wall, on which at least one sensor device of the flow device is disposed, for example an impedance sensor, the biological sample is electrically isolated from the sensor device within the microfluidic channel.
Therein, the passivation device is a protection of the sensor device, which can for example be configured as a protective layer, which is at least partially disposed on or at the in¬ ner wall. Preferably, the passivation device is at least par- tially composed of a semi-metal oxide, for example of silicon oxide or aluminum oxide. In other words, the passivation device has at least partially a semi-metal oxide, for example silicon oxide or aluminum oxide. The passivation of at least one electrode of the at least one sensor device by the passivation device allows dielectrically acquiring the measured variable, which specifically increases the sensitivity to cellular membranes. Hereby, by the at least one sensor device, conductivity of the biological sam- pie is not measured, but an electrical stray field. The meas¬ urement area can thus be adjusted to a predetermined area of the sample in the interior space of the microfluidic channel, for example to the central area or the detection area. During flow of the sample, the at least one sensor device ac¬ quires at least one measured variable, preferably at least two measured variables, of at least one of the cells of the further cell type and/or of a charge carrier, for example haemoglobin or chlorophyll, of the biological sample in the central area of the microfluidic channel. Based on the ac¬ quired measured variable, for example an alternating current resistance of the charge carrier, at least one physiological parameter of the biological sample is determined, for example a haematocrit value.
The combination of acquisition of one or more physiological parameters and utilization of the Fahr¾us effect in a dynamic microfluidic system allows simultaneous measurement of multi¬ ple parameters of an undiluted biological sample. In other words, the biological sample does not have to be expensively prepared or diluted before performing the method according to the invention such that specialized laboratories are not re¬ quired. In addition, measurement results are fast present.
The integration of the measured parameters in the form of the cell function, cell concentration and an exemplary haemosta- sis test close to the patient opens improvement of the per- sonalized transfusion in the emergency care, in the intensive care unit and in elective surgeries. This method does not need preceding sample preparation for example for haemoglobin measurement, cell concentration and/or cell function test be¬ sides provision of the biological sample after sampling.
According to a preferred embodiment of the method according to the invention, labeling at least one of the cells of the first cell type of the biological sample with a cell-specific label can be effected, preferably with a magnetic label and/or within the detection area. This allows cell-specific detection, wherein a magnetic label has the advantage with respect to optical labels that a possible turbidity of the sample does not disadvantageously affect the measurement re¬ sult. If labeling is effected in the detection area, thus, an expensive labeling step in a separate sample vessel can be avoided and a possible contamination can be nearly completely prevented .
In order to allow simultaneous determination of physiological parameters of different cell types, according to a preferred embodiment of the method according to the invention, at least one measured variable of at least one of the cells of the first cell type can be acquired in the detection area by a further sensor device during flow of the biological sample. Preferably, the further sensor device comprises a
magnetoresistive sensor device. Based on the acquired meas¬ ured variable, then, at least one physiological parameter of the first cell type can be determined. This allows nearly simultaneous acquisition of various meas¬ ured variables and nearly simultaneous determination of mul¬ tiple physiological parameters.
For example, this can include simultaneous counting at least one of the cells of the first cell type and/or quantifying a portion of activated cells of the first cell type and/or an aggregation characteristic of at least one of the cells of the first cell type with an aggregation molecule, for example with fibrin. According to a further embodiment of the method according to the invention, accordingly, the determination of the at least one physiological parameter of the first cell type can be effected by:
- determining an activation state of at least one of the cells of the first cell type, and/or
- determining a portion of those cells of an entirety of the acquired cells of the first cell type, which have a predeter- mined cell diameter, and/or
- determining an aggregation state of the acquired cells of the first cell type, wherein the aggregation state describes a cell aggregate formation of the at least one acquired cell with an aggregation molecule.
By contacting at least one of the cells of the first cell type and/or the further cell type with a retaining device of the flow device according to a further preferred embodiment of the method according to the invention, contact with at least one sensor device can be favored. Thus, fixing the at least one cell to a surface of the retaining device follows, wherein the retaining device has at least partially a
coagulative substance, for example fibrin. The retaining de¬ vice can be at least partially disposed in the detection ar- ea. Preferably, the passivation device is at least partially configured as the retaining device. This used modification layer serves for promoting the adhesion of cells.
The acquisition of the measured variable of the at least one cell of the further cell type and/or of the charge carrier can preferably be effected with the aid of an impedance sen¬ sor device as the sensor device. This favors acquisition of the measured variable in the dielectric. In contrast to opti-
cal measurement methods, expensive labeling or sample prepa¬ ration steps are not required.
In combination with the passivated sensor device, adaptation of at least one electrical parameter has proven particularly advantageous such that the sensor device becomes particularly sensitive to the adhesion of further cells. Hereby, cells of the different accumulated cell types can also be cell type specific and independently acquired. The adaptation of the at least one electrical parameter can for example include adap¬ tation of a measurement voltage and/or a measurement frequen¬ cy, for example increasing the measurement voltage and/or the measurement frequency. In indication of ranges, in the fol¬ lowing, the two end points basically are also considered to be encompassed by the respective range.
According to a particularly preferred embodiment of the meth¬ od according to the invention, according to which for example a primary and/or secondary haemostasis of the cells can be determined in the detection area, the acquisition of the measured variable of the at least one cell of the further cell type can be effected at a measurement voltage in a range from 50 millivolts to 100 millivolts and/or at a measurement voltage in a range from 500 millivolts to 3000 millivolts.
According to a further particularly preferred embodiment of the method according to the invention, according to which for example a haemoglobin concentration and/or a haematocrit value can be determined in the central area of the microfluidic channel, the acquisition of the measured variable of the at least one cell of the further cell type can be effected
- at a first measurement frequency in a range from 100 kilo- hertz to 10 megahertz, preferably in a range from 100 kilo- hertz to 5 kilohertz or from 1.5 megahertz to 10 megahertz, and/or
- at a further measurement frequency in a range from 100 hertz to 800 kilohertz, preferably in a range from 0.5 kilo- hertz to 800 kilohertz or from 100 hertz to 100 kilohertz,
wherein a measurement voltage respectively has a predeter¬ mined constant value.
Such a measurement with the passivated sensor device, for ex- ample an impedimetric measurement, has a particularly high specifity for the applied measurement frequency. At lower frequencies of the applied measurement voltage, the system is sensitive to capacitive resistances such as membranes. At high frequencies, the measurement system becomes increasingly more sensitive to charge carriers both outside of and within the respective cell due to the decreasing capacitive re¬ sistance. Accordingly, at high frequencies, the portion of the soluble, charged carriers, such as for example haemoglo¬ bin, can in particular be measured. Additionally or alterna- tively, according to a further advantageous embodiment of the method according to the invention, a pressure of a liquid column comprising the biological sample can be performed at a first point of time and/or a further point of time by the further sensor device. Based on the acquired pressure, a rhe- ological characteristic of the biological sample can be de¬ termined, for example a viscosity.
With the aid of an integration of such a measurement, an ac¬ tual end point of a measurement can be determined, in which for example a whole blood sample is completely coagulated.
The above set object is also solved by a flow device accord¬ ing to the invention with a microfluidic channel, wherein the interior space of the microfluidic channel of the flow device has a central area extending along a flow direction and an detection area located between the central area and an inner wall of the microfluidic channel. The flow device further comprises at least one sensor device disposed in the detec¬ tion area, and a passivation device, which is disposed on at least a partial area of the inner wall, on which the sensor device of the flow device is also disposed. The passivation device is adapted to electrically isolate the interior space from the sensor device.
According to a preferred embodiment of the flow device ac¬ cording to the invention, it can include a retaining device for fixing at least one cell of the biological sample, where- in the retaining device is at least partially disposed in the detection area, and wherein the retaining device at least partially has a coagulative substance.
The flow device according to the invention can include a par- ticular sensor geometry and/or a particular geometry of the passivation device according to a further particularly preferred embodiment. In indication of ranges, in the following, the two end points basically are also considered to be encom¬ passed by the respective range.
According to one of these preferred embodiments, the flow de¬ vice is characterized in that the at least one sensor device comprises at least one interdigital electrode array, which preferably has an electrode distance of two electrodes of 3 micrometers to 30 micrometers or of 100 micrometers to 1000 micrometers, preferably of 300 micrometers.
According to a further of these preferred embodiments, the flow device is characterized in that the passivation device has a height in a range from 15 nanometers to 400 nanometers.
The flow device can include at least one further sensor de¬ vice, which preferably comprises a magnetoresistive sensor and/or a pressure sensor.
By the embodiments of the flow device according to the inven¬ tion, the advantages already mentioned above arise. The vari¬ ation of a distance of the measurement electrodes to each other and/or a thickness of the passivation layer (thus the mentioned height of the passivation device) allows adaptation of the flow device to a specific measurement problem, accord¬ ing to whether a user wants to measure a measurement of the entire volume (thus for example the haematocrit) or only a
volume portion of the microfluidic channel close to surface (for example an adhesion of thrombocytes or thrombocytes dis¬ placed from the volume flow to the edge area of the volume flow by larger erythrocytes) .
The assessment of the Fahr¾us effect based on the combination of an exemplary impedimetric measurements for example of the primary haemostasis and/or the haematocrit in for example fully coagulating or stabilized undiluted whole blood is ef- fectively rendered more precisely by adapting the measurement frequency and/or the electrode geometry and/or the measure¬ ment voltage and/or a surface characteristic of an electrode.
The invention is once again explained in more detail based on the attached drawings by specific embodiments. The shown ex¬ amples represent preferred embodiments of the invention.
Functionally identical elements have the same reference signs in the figures. In indication of ranges, in the following, the two end points basically are also considered to be encom- passed by the respective range. There shows:
FIG 1 a schematic sketch of the principle of an embodi¬ ment of the method according to the invention,
FIG 2 a schematic sketch to the principle of the separa¬ tion step according to an embodiment of the method according to the invention, wherein a sensor device according to an embodiment of a device according to the invention for flow measurement with a microflu- idic channel is exemplary illustrated,
FIG 3 a schematic sketch to a further embodiment of the method according to the invention with an optional labeling step,
FIG 4 a schematic sketch to a further embodiment of the method according to the invention with an impedimetric measurement method step,
a schematic sketch to a further embodiment of the method according to the invention with an
impedimetric measurement method step, a schematic sketch to a further embodiment of the method according to the invention with an
impedimetric measurement method step, an exemplary diagram of an exemplary impedance measurement according to a further embodiment of the method according to the invention, a schematic sketch to an exemplary sensor geometry a schematic sketch to a further embodiment of the device according to the invention, a schematic sketch to a further embodiment of the method according to the invention, a schematic sketch to a further embodiment of the device according to the invention, a schematic sketch to a further embodiment of the method according to the invention, an exemplary diagram of an optional pressure meas¬ urement according to a further embodiment of the method according to the invention, an exemplary diagram of an optional pressure meas¬ urement according to a further embodiment of the method according to the invention, and a schematic sketch to an embodiment of the device according to the invention for flow measurement with a microfluidic channel.
FIG 1 illustrates the principle of the method according to the invention based on a first embodiment. Therein, FIG 1 shows a section of a microfluidic channel 10 of an embodiment of a flow device 12 according to the invention, for example of a flow cytometer.
Therein, the microfluidic channel 10 has a diameter of pref¬ erably 50 micrometers to 300 micrometers. Therein, an inner wall 14 of the microfluidic channel comprises a passivation device 16, which is at least partially or completely disposed on the inner wall 14 and for example is at least partially composed of silicon oxide or aluminum oxide. Corresponding coating methods for disposing such a passivation device 16 are known to the man skilled in the art of surface technique. The passivation device 16 allows electrically isolating the sample 5 from at least one sensor device 30 such that the sample 5 is present as a dielectric in the microfluidic chan¬ nel 10. Beving et al . (1994) (Beving H., Eriksson L.E., Davey C.L., Kell D.B.: "Dielectric properties of human blood and erythrocytes at radio frequencies (0.2-10 MHz); dependence on cell volume fraction and medium composition.", Eur Biophys J. 1994 ; 23 (3) : 207-15. ) provide an overview to the dielectric characteristics of a blood sample.
Alternatively or additionally, the passivation device 16 can include a retaining device 42 or be configured as a retaining device 42. The retaining device 42 can at least partially or completely be composed of a coagulative substance, for exam- pie of fibronectin, collagen, fibrin and/or another adhesive protein known to the man skilled in the art from the prior art. The retaining device 42 allows fixing at least one cell 22 of a first cell type to the inner wall 14. The flow device 12 according to the invention comprises at least one sensor device 30 with at least one sensor, which can have at least one electrode 32. On and/or at the inner wall 14, at least one further sensor device 18 can be dis-
posed. The at least one further sensor device 18 can include at least one sensor with for example two electrodes 20, as shown in FIG 1. The further sensor device 18 can preferably include at least one magnetic field based sensor, while the at least one sen¬ sor device 30 preferably can include at least one impedance sensor, or three impedance sensors, as exemplarily shown in FIG 1. Each two of the further sensor devices 30 can for ex- ample be used for an impedimetric measurement of haemostasis of the biological sample 5 (illustrated in flow direction F after the further sensor device 18 in FIG 1) . The measurement can be performed at a varying measurement voltage and a con¬ stant measurement frequency.
According to the method according to the invention, separation of the cell mixture of the biological sample 5 is ef¬ fected in the microfluidic channel 10 (method step SI) such that the at least one cell 22 of the first cell type is urged to the inner wall 14 and the at least one cell 26 of the fur¬ ther cell type is accumulated in an area centrally extending in flow direction. This is achieved with the aid of the ar¬ chitecture of the microfluidic channel 10, as explained in more detail below to FIG 2.
One of the exemplary sensor devices 30 can for example be used for the impedimetric measurement of a haematocrit value and/or a haemoglobin concentration of the exemplary blood sample (illustrated in flow direction F after the first two sensor devices 30 in FIG 1) . The measurement can be performed at a varying measurement frequency and a constant measurement voltage. Further, the flow device 12 can include a further sensor device 18, which comprises a pressure sensor for de¬ termining a rheological characteristic of the biological sam- pie 5 (not illustrated in FIG 1) .
According to an embodiment, the flow device 12 can include a magnet 34, which can be disposed on the inner wall 14 of the microfluidic channel 10. The biological sample 5 is liquid and preferably comprises an undiluted whole blood sample of a patient or an animal, plant or microbial cell sample. In the present example, an exterior shape of the at least one cell 22 of the first cell type can have another shape in an activated state and a larger cell diameter than in a deactivated state. For example, this is the case in a thrombocyte as the at least one cell 22 of the first cell type, which promotes a thrombocyte aggregation and initiates a secondary haemostasis in an activated state in blood coagulation. For clarity, in FIG 1, not all of the cells 22 are provided with reference signs, but are recog¬ nizable by an identical shape as correspondingly illustrated and identified cells 22. In the example of FIG 1, the cells 22 of the first cell type can be labeled with a label 24, for example a magnetic, chemiluminescent , fluorescent and/or plasmonic label 24, which can for example be bound to a cell- specific antibody. Now, the man skilled in the art has avail¬ able usual methods from the prior art for providing cell- specific labels 24. According to the method according to the invention, the biological sample 5 is provided in a fluidic flow of the micro¬ fluidic channel 10, preferably in a laminar flow. The biolog¬ ical sample 5 further comprises at least one cell 26 of a further cell type, for example at least one erythrocyte or a photosynthetic plant cell. The at least one cell 26 of the further cell type can include a charge carrier 28 in the cell plasma, for example haemoglobin or chlorophyll. The charge carrier 28 can additionally or alternatively be present out¬ side of the at least one cell 26 of the further cell type in the sample liquid (not shown in FIG 1) . For clarity, in FIG
1, not all of the charge carriers 28 are provided with refer¬ ence signs.
FIG 1 illustrates the principle of separating a cell mixture (method step SI) and the principle of an exemplary integra¬ tion of for example multiple sensor devices 18, 30 for simul¬ taneous measurement (S3, S4, S5, S6, S7) of a physiological parameter of the biological sample 5, for example of a haema- tocrit value and/or a haemoglobin concentration. The integration of the two measurement methods can be performed in a flow device 12 according to the invention, for example a flow cell. Controlling an exemplary impedance measurement via con- trolling the different sensor geometries can be accomplished with the same component, for example with a controller of the flow device 12 as a control device 36 if the measurements are for example alternately performed. In addition, integration of at least one further sensor device 18 can be performed for acquiring at least one physiological parameter of the first cell type, for example a thrombocyte number and/or thrombo¬ cyte activation and/or a rheological characteristic of the sample 5. FIG 2 describes the effect occurring by the architecture of the microfluidic channel 10 and the flow of the sample 5, the so-called Fahr¾us effect, based on the exemplary blood sample and for example in a microfluidic channel 10 with the sensor device 18 described to FIG 1 and an optional magnet 34. An interior space of the microfluidic channel 10 has a central area extending along the flow direction F and an detection area 11 located between the central area and an inner wall of the microfluidic channel 10, which preferably has a height as the extension into the interior space of the microfluidic channel 10 in a range from 0.5 micrometers to 5 micrometers, ideally from 3 micrometers to 5 micrometers.
In separating the cell mixture (SI), the cells 22 of the first cell type, the exemplary thrombocytes, which have pref- erably a smaller volume than the cells 26 of the further cell type, impact the larger cells 26 of the further cell type, here red blood cells (erythrocytes) in the example, caused by the flow of the sample 5, and are urged into the detection
area 11 of the microfluidic channel 10. The cells 26 of the further cell type have an about 1000-fold higher concentra¬ tion than the cells 22 of the first cell type in the exempla¬ ry blood sample. Thereby, a concentration gradient of the cells 22, 26 forms along a channel diameter. Accordingly, the concentration of the cells 22 of the first cell type is high¬ er at the edge of the microfluidic channel 10 than in the center, and the cells 26 of the further cell type have a high concentration in the central area. Thus, the detection area 11 can be considered as a depletion zone of the further cell type. Suitable conditions for a suitable flow rate and a suitable diameter of the microfluidic channel for promoting the Fahr¾us effect are described in Alizadehrad et al .
(2012) (Alizadehrad D., Imai Y., Nakaaki K., Ishikawa T., Ya- maguchi T.: "Quantification of red blood cell deformation at high-hematocrit blood flow in microvessels", J Biomech. 2012 Oct 11 ; 45 ( 15) : 2684-9) . In particular, the conditions de¬ scribed in figure 11 of this publication present preferred conditions of the method according to the invention.
FIG 3 shows an optional labeling step S3 and an optional measurement of a physiological parameter of the at least one cell 22 according to a further embodiment of the method ac¬ cording to the invention. For labeling the at least one cell 22 of the first cell type (S3) , for example, magnetic labels 24, preferably superparamagnetic nanoparticles , can be dis¬ pensed into the cell sample 5 and/or into the flow chamber. If the flow device 12 has a magnet 34 on and/or in the inner wall 14, the exemplary at least one magnetic label 24 can be directed into the detection area 11 by the magnetic field to the inner wall 14 in the direction (M) such that efficient contacting of the label 24 with the at least one cell 22 of the first cell type and bonding or absorbing the marker 24 to or in the cell 22 can be effected due to the above described cell accumulation.
The at least one labeled cell 22 moves further in the flow direction F and can thus be guided across the first sensor
device 18. The first sensor device 18 for example comprises at least one magnetic field sensor with at least one elec¬ trode 20, for example at least one magnetooptical or
magnetoresistive sensor or at least one Hall sensor. If the label 24 for example comprises a fluorescent label, thus, the at least one further sensor device 18 can include an optical sensor. The further sensor device 18 can for example be controlled by the control device 36. Preferably, the further sensor device 18 can be configured as a magnetoresistive sensor, for example as an AMR sensor, GMR sensor or TMR sensor. The at least one electrode 20 can pref¬ erably be configured as a strip electrode to allow very sen¬ sitive acquisition of a cell volume or a cell aggregate vol- ume . Preferably, counting individual labeled cells 22 or cell aggregates of the first cell type and/or acquisition of a size of a cell aggregate, for example of a blood clot, and/or a diameter of the at least one labeled cell 22 for example with the aid of a magnetic gradient field of the exemplary magnetoresistive sensor device 18 and by means of travel time measurement, for example a "time of flight" measurement, is effected .
Based on an acquired number of cells 22 of the first cell type, for example, a portion of activated cells 22 of the first cell type can be determined. Additionally or alterna¬ tively, based on an acquired cell diameter, an activation state of the at least one exemplary labeled cell 22 and/or a portion of activated exemplary labeled cells 22 of an entire- ty of the acquired exemplary labeled cells 22 can be deter¬ mined .
Additionally or alternatively, an aggregation characteristic of the at least one cell 22 of the first cell type with an aggregation molecule, for example fibrin, can be determined, wherein the aggregation state describes a cell aggregate for¬ mation of the at least one labeled cell 22 with the aggrega¬ tion molecule.
FIG 4 shows a method step of an exemplary impedimetric meas¬ urement (S4, S5) according to an embodiment of the method ac¬ cording to the invention with the aid of the at least one sensor device 30, which preferably comprises at least one im¬ pedance sensor.
Therein, determining a primary and/or secondary haemostasis as the physiological parameter of the exemplary blood sample can be effected. Therein, the aim is a differentiation of a layer of adhering cells 22 of the first cell type, for exam¬ ple a monolayer of the exemplary thrombocytes, and a clot, thus a cell aggregate of the exemplary thrombocytes as the cells 22 of the first cell type with an aggregation molecule 38, for example fibrin.
Preferably, the flow device 12 has a passivation device 16 with a retaining device 42 within the microfluidic channel 10, which has a coagulative surface and is at least partially composed of a coagulative substance, for example of silicon oxide and/or fibrin. Therein, the passivation device 16 preferably has a thickness in a range from 10 nanometers to 400 nanometers. The electrodes 32 for the impedimetric measure¬ ment of the coagulation by the exemplary thrombocytes can ei- ther be passivated by the electrical barrier layer and/or specifically be modified with coagulation factors consisting of organic and/or inorganic material, in their configura¬ tions. These used modification layers then serve for promot¬ ing and/or avoiding the adhesion of cells 22. Both a
passivated and a non-passivated sensor device 30 can be used.
The at least one sensor device 30 can for example include each one pair of electrodes 32 of an impedance sensor. Here¬ to, in particular an electrode 32 configured as a comb elec- trode is suitable as the electrode 32. The at least one sen¬ sor device 32 preferably comprises an interdigital electrode ( "interdigitated electrode", "IDES") and/or an interdigital electrode array preferably having an electrode distance of 3
micrometers to 30 micrometers and adapted to be operated at least at two alternating measurement voltages. Therein, the control of the electrode 32 can be effected via a control de¬ vice 36, for example a microcontroller of the flow device 12 or a controller external to device, which can be connected to the respective electrode 32 via a wireless or wired communi¬ cation link 37.
A layer of adhered labeled cells 22 can preferably be ac- quired at a low measurement voltage in the range from 50 mil¬ livolts to 100 millivolts (S4) since here the electric field lines of an electric field 39 do not penetrate very deeply, that is only between about 200 nanometers and 300 nanometers, into the analysis liquid, that is into the liquid sample 5.
After a layer of activated cells 22 of the exemplary blood sample has adhered to the surface of the sensor, they begin releasing the content of their granules from the interior of the cells 22. This process initiates the plasmatic coagula- tion among other things, in which soluble fibrinogen in the blood plasma is converted into insoluble fibrin. The fibrin surrounds the immobilized and activated cells and promotes the further adhesion of thrombocytes to already immobilized cells 22. The geometry of the exemplary passivated impedance sensors, but also the electrical parameters, can be adapted such that they become sensitive to the adhesion of further cells 22, for example by increasing the measurement voltage and/or the measurement frequency. The further attachment of the exemplary thrombocytes to the monolayer and the formation of fibrin, illustrated as a cell aggregate 40 in FIG 4, can preferably be acquired at a high measurement voltage in the range from 500 millivolts to 3000 millivolts (S5) , since here the electric field lines pene- trate deeply, for example 1000 nanometers to 5000 nanometers, into the analysis liquid and therefore penetrate deeper into the cell plasma.
Therein, the further sensor device 30 generates a signal upon contacting the at least one cell 22 of the first cell type. Increasing coverage of the exemplary impedance electrodes 32 is associated with variation of the impedance signal.
Therein, a predetermined measurement frequency remains con¬ stant in both measurement steps S4, S5. Therein, a suitable measurement frequency for acquiring the cells 22 is known to the man skilled in the art from the prior art.
FIG 5 and FIG 6 show an exemplary method step of an
impedimetric measurement with the aid of the at least one sensor device 30 for acquiring at least one measured varia¬ ble, for example quantifying cells 26 of a further cell type and/or a concentration of a charge carrier 28, for example a haemoglobin concentration, of the biological sample 5. At least one physiological parameter of the biological sample 5 can be determined from it, for example a haematocrit value. The charge carrier 28, thus the exemplary haemoglobin, can here be present in certain portions in the interior of the cells 26 or in the plasma surrounding the cells 26.
The passivation of the electrodes 30 by the passivation de¬ vice 16 allows dielectric acquisition specifically increasing the sensitivity to cellular membranes. Depending on a materi¬ al composition of the passivation and its thickness, both the measurement voltage and the sensitive frequency range can be specifically predetermined. The impedimetric acquisition with passivated sensors 30 has a specifity for the applied meas- urement frequency. Therein, a measurement voltage depending on a breakdown voltage of the passivation device 16 remains constant and can preferably be in a range between 100 milli¬ volts and 10 volts. Therein, the man skilled in the art can determine a suitable measurement voltage depending on a layer thickness of the passivation device 16, for example a meas¬ urement voltage of 100 millivolts with a layer thickness of the passivation device 16 of 100 nanometers, with the aid of the general professional knowledge.
Therein, the geometry of the sensor device 30 is adapted to measurement of the alternating current resistance. The acqui¬ sition of the alternating current resistance is effected in a specific frequency range.
According to a particularly preferred embodiment of the meth¬ od according to the invention, in a first step S6 (FIG 5) , the acquisition of the alternating current resistance can be effected at a preset measurement voltage and a first measure¬ ment frequency in a range from 100 kilohertz to 10 megahertz, preferably in a range from 100 kilohertz to 5 kilohertz and/or from 1.5 megahertz to 10 megahertz. At a measurement frequency in one of the mentioned ranges, the membranes of the cells 26 of the further cell type are penetrated by the electric field 39 and the entire charge carrier portion of the sample 5 is acquired. At high frequencies, the measure¬ ment system becomes increasingly unspecific to membranes and more sensitive to charge carriers both outside of and within the cellular analytes due to the decreasing capacitive re¬ sistance. Accordingly, the portion of the soluble, charged metabolites (such as haemoglobin) can particularly be measured at high frequencies. In the method step S7, as shown in FIG 6, at a preset meas¬ urement voltage and a first measurement frequency in a range from 100 hertz to 800 kilohertz, preferably in a range from 0.5 kilohertz to 800 kilohertz or from 100 hertz to 100 kilohertz, acquisition of the charge carrier 28 can be effected. With such a low measurement frequency, in particular in a particularly preferred range from 500 hertz to 800 kilohertz, membranes are not pierced and only charge carriers 28 outside of the cells 26 of the further cell type affect the signal acquisition. Accordingly, the system is sensitive to capaci- tive resistances as membranes. A conductivity of the cell plasma is not co-acquired.
Therein, the method steps S6 and S7 can be effected individu¬ ally or in any order or be alternately repeated several times one after the other. An advantageous electrode geometry describes a system with for example an IDES as the sensor device 32, which preferably has an electrode distance d in a range from 100 micrometers to 1000 micrometers. The at least one sensor device 30 is preferably adapted to be able to be operated at least at two alternating measurement frequencies. The control of the at least one sensor device 30 can for example be effected by the control device 36.
FIG 7 shows an exemplary diagram of an exemplary impedance measurement according to an embodiment of the method accord¬ ing to the invention, as for example described to FIG 5 and FIG 6. Therein, an impedance in Ohm is plotted on an ordinate y of the coordinate system, and a haematocrit value in %HcT is plotted on an abscissa x of the coordinate system. For il- lustrating the haematocrit value, a cell 26 of the further cell type, thus an exemplary erythrocyte, is shown. The con¬ nected data points of the curve K for example represent meas¬ ured values, which are acquired at an exemplary measurement frequency of 500 kilohertz.
Therein, the at least one sensor device 32 comprises for ex¬ ample two passivated impedance sensors, the electrode geome¬ try of which each has a geometry of 300 micrometers by 300 micrometers. Therein, an exemplary electrode geometry is shown in FIG 8, wherein each of the electrodes 20 preferably has a width b of 300 micrometers and/or they have a distance d from each other preferably of 300 micrometers. Therein, the arrows of FIG 8 mark the flow direction F of the liquid col¬ umn 44 of the biological sample 5.
Therein, exemplary measurement parameters, in which a meas¬ urement associated with the diagram of FIG 7 is performed, include an exemplary measurement voltage of 500 millivolts
and/or a whole blood sample as the biological sample 5 and/or a passivation device 16, which is at least partially composed of aluminum oxide, with an exemplary layer thickness of 70 nanometers. Therein, the mapping of the measurement points in the curve K shows increase of the impedance with an increase of the haematocrit value.
FIG 10 and FIG 12 each show an embodiment of a measurement of a pressure (S8) of a liquid column 44 of the biological sam- pie 5 by means of a further sensor device 18 at a center of pressure 50, which for example comprises a pressure sensor. An exemplary absolute pressure sensor or differential pres¬ sure sensor is known to the man skilled in the art from the prior art. Ideally, the respective measurement can be per- formed with an undiluted biological sample 5. Thereby, a rhe- ological characteristic of the biological sample 5 can be de¬ termined, for example a viscosity of the biological sample 5, whereby for example coagulation of the biological sample 5 can be inferred. Such an exemplary pressure measurement can serve as a control measurement.
Ideally, therein, a non-stabilized sample 5 is used, thus for example a blood sample, in which coagulation occurs. Over a certain period of time, the sample 5 coagulates, whereby hae- mostasis is effected, by which a rheological characteristic, for example the viscosity of the sample 5, varies. The fur¬ ther the coagulation of the sample 5 proceeds, the higher the viscosity is and the slower the liquid column 44 flows within the microfluidic channel 10. This effect affects in the pre- sented application such that the flow velocity of the sample 5 in the microfluidic channel 10 decreases.
FIG 10 shows a liquid column 44, which pushes an air column 46 in the direction F in front of it. If the viscosity of the sample 5 increases because the sample 5 coagulates, the pres¬ sure of the air column 46, which pushes the sample 5 in front of it, decreases because the flow velocity decreases. This pressure drop can be linear or exponential according to how
the other parameters of the sample 5, for example a cell num¬ ber and/or concentration of plasmatic factors, are designed. The arising vacuum can be acquired by the further sensor device 18. A corresponding exemplary embodiment of a flow de- vice according to the invention is shown in FIG 9. Therein, the further sensor device 18 can be disposed at a piston 48, which is pulled in the direction F by a variation of the rhe- ological characteristic such that the further sensor device 18 can determine a pressure drop. Hereto, for example, at a first point of time, a first absolute pressure "P(tl)" and at a further point of time a further absolute pressure "P(t2)" are acquired.
FIG 12 shows a liquid column 44, which pushes an air column 46 in front of it. The coagulating sample 5 causes an in¬ crease of the viscosity in the system and thereby an increase of the resistance counteracting the extending piston 48, for example a piston 48 of a syringe. The applied force on the piston 48 on the part of a pump gets a measurable counter- force by the viscosity increase of the sample 5. A corre¬ sponding exemplary embodiment of a flow device according to the invention is shown in FIG 11. Therein, the further sensor device 18 can be disposed such that the further sensor device 18 can determine a pressure rise. Hereto, for example, at a first point of time a first absolute pressure "P(tl)" and at a further point of time a further absolute pressure "P(t2)" are acquired.
Fig 13 and FIG 14 each show an exemplary pressure profile de- pending on a rheology of the biological sample 5. Therein, a pressure is respectively plotted on the ordinate y against a point of time on the abscissa y.
The curve K shows a linear pressure drop in FIG 13. Therein, the measurement points of time are the exemplary points of time "tl" and "t2". In the example of FIG 13, therein, a pressure drop of an exemplary whole blood sample with a high haematocrit value is for example shown. The curve K of FIG 14
shows an exemplary exponential pressure drop, for example of an exemplary whole blood sample with a low haematocrit value.
FIG 15 shows severely schematized once again an embodiment of a flow device 12 according to the invention, for example a flow cell or a flow cassette. The flow device 12 comprises a microfluidic channel 10, a passivation device 16 and at least one sensor device 32. In addition, the flow device 12 can include a further sensor device 18 and/or a controller 38 with a communication link 37 for connecting to a respective sensor device 18, 32. Therein, the individual components are prefer¬ ably configured corresponding to the above description to the preceding figures. In addition, the flow device 12 can include a sample receiving container 52 and/or a sample collecting container 54.
The above mentioned embodiments illustrate the principle of the method according to the invention, utilizing the Fahr¾us effect and an adapted sensor geometry, to perform a simulta¬ neous measurement of multiple physiological parameters, for example cellular and plasmatic blood parameters such as haem¬ atocrit, haemoglobin and thrombocyte number, in for example undiluted whole blood as the biological sample 5, which pres- ently is only achievable by dilution or other pre-analytical efforts .
According to the present invention, an occurring Fahr¾us effect is for example utilized in whole blood. Therein, the portion of cells in a preferably undiluted sample 5 (for ex¬ ample in whole blood, wherein for example red blood cells have the greatest cellular portion of 99%) is collectively measured with a physiological parameter, for example the con¬ centration of haemoglobin, the red dye within the red blood cells, on the one hand. The determination and/or the measure¬ ment of this at least one parameter can be realized by means of at least one sensor device 30, which for example comprises an impedance sensor. For example, the number of cells 22 of a
first cell type, for example the thrombocytes, and/or the de¬ gree of activation thereof is quantified on the other hand. This measurement can for example be effected with the aid of a specific label of the cells 22 of the first cell type, for example with superparamagnetic nanoparticles , and subsequent read-out by means of for example a magnetoresistive sensor (for example a GMR, TMR, AMR and/or Hall sensor) based on travel time measurement. The underlying measurement principle for the last mentioned measurement is known to the man skilled in the art from the prior art.
Furthermore, a pressure measurement can for example be inte¬ grated, which allows indirect measurement of the rheological characteristics, namely for example of the viscosity varia- tion of the biological sample. The integration of such a measurement serves for determining the actual end point of a measurement, at which the exemplary whole blood is completely coagulated and thereby the pressure in the system rapidly in¬ creases .
The combination described here for example of two measurement methods, namely for example a magnetoresistive and an
impedimetric measurement, and the exploitation of the Fahr¾us effect in a dynamic microfluidic system, allows the simulta- neous determination of various physiological parameters such as for example the thrombocyte number, the haematocrit, and the haemoglobin in the exemplary undiluted whole blood sample . In order to be able to correctly measure the above mentioned parameters in the present system, the measurement can be per¬ formed in undiluted whole blood. With the occurring Fahr¾us effect, a higher concentration of cells 22 of the first cell type, thus of the exemplary thrombocytes, is located at the edge of for example a microfluidic channel 10 or a blood ves¬ sel than in the center. The sensor devices 18, 30 employed in this invention can be disposed at any side of the microfluid¬ ic channel 10.
The integration of the above mentioned measurement parameters in the form of the exemplary cell function, cell concentra¬ tion and haemostasis test close to the patient opens an im- provement of the personalized transfusion in the emergency care, in the intensive care unit and in elective surgeries.
The assessment of the Fahr¾us effect based on the combination of for example an impedimetric measurement for example of the primary haemostasis, thus the adhesion of activated thrombo¬ cytes to a sensor surface, and the haematocrit, thus a per¬ centage portion of the red blood cells in the blood, prefera¬ bly in fully coagulating or stabilized undiluted whole blood, is allowed by adapting the measurement frequency, the elec- trode geometry, the measurement voltage and/or the surface characteristics of the electrodes 20, 32 on the one hand. The exemplary additional counting of the cells 22 of the first cell type can for example be allowed via a magnetoresistive sensor, for example by a time of flight measurement of un- rolling and/or immunomagnetically labeled cells 22. The vari¬ ation of the distance of the exemplary impedimetric measure¬ ment electrodes 20 to each other and/or the thickness of the passivation device 16 allows adaptation to a specific meas¬ urement problem according to whether the applicant wants to measure a measurement of the entire volume (thus for example the haematocrit) or only a volume portion of the microfluidic channel 10 close to surface (for example the adhesion of cells 22 of the first cell type or cells 22 of the first cell type, which are urged from the volume flow to the edge area of the volume flow by the larger cells 26 of the further cell type) . The depletion zone of the cells 26 of the further cell type in the edge area of the inner wall 14 can be dependent on for example a haematocrit, a channel cross-section and a flow velocity, however is in the range of few micrometers (3 micrometers to 5 micrometers) , which are for example occupied by smaller cells 22 such as for example TZ displaced from the volume flow.
The method is characterized by the following characteristics:
This method does not need a preceding sample preparation for the exemplary haemoglobin measurement, cell concentration and/or cell function test. The biological sample 5 can for example be provided by means of canula after intravenous or arterial blood collection. The described method utilizes the Fahr¾us effect, in which at least one cell type to be meas¬ ured (for example red blood cells and/or thrombocytes) in for example an undiluted whole blood sample as the biological sample 5 cluster to the edge in case of the cells 22 of the first cell type and in the center of a microfluidic channel 10 in case of the cells 26 of the further cell type (see FIG 2) . This separation of the concentration of the cellular com- ponents of the biological sample 5 along the cross-section of a microfluidic channel 10 is now utilized to be able to as¬ sess the status of the same cells via for example two meas¬ urement methods combined with each other. An exemplary magnetoresistive measurement for example of the primary or cellular haemostasis can for example be effected as follows: an optional specific labeling of the cells 22 of the first cell type with for example superparamagnetic nano- particles as the label 24 and/or the subsequent measurement of labeled cells 22 of the first cell type and
microaggregates of cells 22 of the first cell type by means of for example a magnetoresistive sensor allows the simulta¬ neous counting of the cells 22 of the first cell type, the quantification of the portion of activated cells 22 of the first cell type and/or the aggregation characteristics of the cells 22 of the first cell type with an aggregation molecule, for example fibrin. The latter for example initiates the sec¬ ondary haemostasis. An exemplary impedimetric measurement for example of the pri¬ mary haemostasis, thus the adhesion of exemplary activated cells 22 of the first cell type to a coagulative surface, can be effected as follows: in for example an undiluted sample 5,
the cells 22 of the first cell type are urged to the edge of the volume flow of a microfluidic channel 10 by the 1000-fold higher concentration of the cells 26 of the further cell type (see FIG 4) . Exemplary impedance sensors located there can be used for measuring for example the coagulation, accordingly the adhesion of the cells 22 of the first cell type to one of the inner walls 14 (see FIG 4) . An increasing coverage of the exemplary impedance electrodes 20 is associated with the var¬ iation of the impedance signal. The electrodes 20 for the ex- emplary impedimetric measurement of the coagulation by the cells 22 of the first cell type can either be passivated in their configurations by dielectric barrier layers (with a thickness in an advantageous range from 10 nanometers to 400 nanometers) and/or specifically modified with coagulation factors (at least partially composed of organic and/or inor¬ ganic material) . These used modification layers then serve either for promoting and/or avoiding the adhesion of cells. Both passivated and non-passivated sensor elements can be used .
An exemplary impedimetric measurement for example of the sec¬ ondary haemostasis can be effected as follows: after a layer of activated cells 22 of the first cell type has adhered to the surface of a sensor device 32 (see FIG 4), they begin to release the content of their granules from the interior of the cells. This process initiates the plasmatic coagulation among other things, in which for example soluble fibrinogen in the blood plasma is converted to insoluble fibrin. The fi¬ brin surrounds the immobilized and activated cells and pro- motes the further adhesion of cells 22 of the first cell type to already immobilized cells 22. The geometry of the exempla¬ ry passivated impedance sensor device 32, but also the elec¬ trical parameters, can be adapted such that they become sen¬ sitive to the adhesion of further cells 22. (For example by increasing the measurement voltage and/or the measurement frequency) .
An exemplary measurement of a haematocrit value and/or a hae¬ moglobin concentration can be performed as follows: the pas¬ sivation of electrodes 32 allows dielectric measurement, which specifically increases the sensitivity to cellular mem- branes. Depending on the material composition of the pas¬ sivation device 16 and/or its thickness, both the measurement voltage and the sensitive frequency range can be specifically determined. The impedimetric measurement with a passivated sensor device 30 has a specifity for the applied measurement frequency (see FIG 5 and FIG 6) .
An exemplary measurement of the haematocrit can be effected: the exemplary haematocrit value can for example be quantified by means of an impedimetric measurement, an adapted geometry of the sensor device 30 and/or a specific frequency range. At lower frequencies of the applied measurement voltage, prefer¬ ably in a range from 100 hertz to 100 kilohertz, the system is sensitive to capacitive resistances such as membranes. The conductivity of the cell plasma is not co-measured.
An exemplary measurement of haemoglobin can be effected: at high frequencies, preferably in a range from 100 kilohertz to 5 megahertz, the measurement system becomes increasingly un- specific to membranes and more sensitive to charge carriers 28 both outside of and within the cells 26 due to the de¬ creasing capacitive resistance. Accordingly, at high frequencies, in particular the portion of the soluble, charged me¬ tabolites (such as for example haemoglobin) can be measured. With the aid of the combination for example of two measure¬ ment methods, for example a magnetoresistive and an
impedimetric measurement, the Fahraeus effect can be used as a basis for assessing an analytic question, namely the fast and immediate counting of cells 22 of the first cell type and/or the assessment of the degree of activation thereof and/or the measurement of for example haematocrit and/or hae¬ moglobin as the physiological parameter in for example se¬ verely injured and traumatized patients.
Claims
1. Method for determining at least one physiological param¬ eter of a liquid biological sample (5) by a flow device (12), wherein an interior space of a microfluidic channel (10) of the flow device (12) has a central area extending along a flow direction (F) , and an detection area (11) located be¬ tween the central area and an inner wall (14) of the micro¬ fluidic channel (10), comprising the steps of:
- separating a cell mixture of the sample (5) by the liquid sample (5) flowing through the microfluidic channel (10) such that cells (22) of a first cell type are accumulated in the detection area (11) and cells (26) of a further cell type are accumulated in the central area of the microfluidic channel (10),
- electrically isolating the biological sample (5) from at least one sensor device (30) within the microfluidic channel (10) by providing a passivation device (16) on at least a partial area of the inner wall (14), on which the sensor de- vice (30) of the flow device (12) is disposed, and
- during the flow of the sample (5) acquiring at least one measured variable of at least one of the cells (26) of the further cell type and/or of a charge carrier (28) of the bio¬ logical sample (5) in the central area by the at least one sensor device (30) and determining at least one physiological parameter of the biological sample (5, S4, S5, S6, S7, S8) from the acquired measured variable.
2. Method according to claim 1,
characterized by
- labeling at least one of the cells (22) of the first cell type of the biological sample (5) with a cell-specific label (24, S2), preferably with a magnetic label (24) and/or within the detection area (11) .
3. Method according to any one of the preceding claims, characterized in that
during the flow of the biological sample (5) at least one measured variable of at least one of the cells (22) of the first cell type is acquired by a further sensor device (18) in the detection area (11, S3), preferably by a
magnetoresistive sensor device (18), and determining at least one parameter of the first cell type.
4. Method according to claim 3,
characterized in that
the determination of the at least one physiological parameter of the first cell type (S3) is effected by determining
- an activation state of at least one of the cells (22) of the first cell type, and/or
- a portion of those cells (22) of an entirety of the ac- quired cells (22) of the first cell type, which have a prede¬ termined cell diameter, and/or
- an aggregation state of the acquired cells (22) of the first cell type, wherein the aggregation state describes a cell aggregate formation of the at least one acquired cell (22) with an aggregation molecule (38) .
5. Method according to any one of the preceding claims, characterized by
contacting at least one of the cells (22, 26) of the first cell type and/or of the further cell type to a retaining de¬ vice (42), which is at least partially disposed in the detec¬ tion area (11), and fixing the at least one cell (22, 26) on a surface of the retaining device (42), wherein the retaining device (42) has at least partially a coagulative substance.
6. Method according to any one of the preceding claims, characterized in that the acquisition of the measured varia¬ ble of the at least one cell (26) of the further cell type and/or of the charge carrier (28) is effected with the aid of an impedance sensor device as the at least one sensor device (30) .
7. Method according to any one of the preceding claims, characterized in that the acquisition of the measured varia¬ ble of the at least one cell (26) of the further cell type is effected at a measurement voltage in a range from 50 milli- volts to 100 millivolts (S6) and/or at a measurement voltage in a range from 500 millivolts to 3000 millivolts (S5) .
8. Method according to any one of claims 6 or 7,
characterized in that the acquisition of the measured varia- ble of the at least one cell (26) of the further cell type is effected
- at a first measurement frequency in a range from 100 kilo- hertz to 10 megahertz, preferably in a range from 100 kilo- hertz to 5 kilohertz or from 1.5 megahertz to 10 megahertz, and/or
- at a further measurement frequency in a range from 100 hertz to 800 kilohertz, preferably in a range from 0.5 kilo- hertz to 800 kilohertz or from 100 hertz to 100 kilohertz, wherein a measurement voltage each has a predetermined con- stant value.
9. Method according to any one of the preceding claims, characterized by
acquiring a pressure of a liquid column comprising the bio- logical sample (5) at a first point of time and/or a further point of time by the further sensor device (18) and determin¬ ing a rheological characteristic of the biological sample (5) .
10. Flow device (12) with a microfluidic channel (10), where¬ in an interior space of the microfluidic channel (10) of the flow device (12) has a central area extending along a flow direction (F) and an detection area (11) located between the central area and an inner wall (14) of the microfluidic chan- nel (10), further comprising:
- at least one sensor device (32) disposed in the detection area (11), and
- a passivation device (16), which is disposed on at least one partial area of the inner wall (14), on which the sensor device (30) of the flow device (12) is disposed, for electri¬ cally isolating the interior space of at least one sensor de- vice (30) within the microfluidic channel (10) .
11. Flow device (12) according to claim 10,
characterized by:
- a retaining device (42) for fixing at least one cell (22, 26) of the biological sample (5) , wherein the retaining de¬ vice (42) is at least partially disposed in the detection ar¬ ea (11), and wherein the retaining device (42) has at least partially a coagulative substance.
12. Flow device (12) according to claim 10 or 11,
characterized in that
the at least one sensor device (30) comprises at least one interdigital electrode array, which preferably has an elec¬ trode distance of two electrodes (20) of 3 micrometers to 30 micrometers or of 100 micrometers to 1000 micrometers, preferably of 300 micrometers.
13. Flow device (12) according to any one of claims 10 to 12, characterized in that
the passivation device has a height in a range from 15 na¬ nometers to 400 nanometers.
14. Flow device (12) according to any one of claims 10 to 13, characterized by at least one further sensor device (18), which preferably comprises a magnetoresistive sensor and/or a pressure sensor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/EP2014/068182 WO2016029943A1 (en) | 2014-08-27 | 2014-08-27 | Method for determining at least one physiological parameter of a biological sample |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/EP2014/068182 WO2016029943A1 (en) | 2014-08-27 | 2014-08-27 | Method for determining at least one physiological parameter of a biological sample |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050087000A1 (en) * | 2001-12-21 | 2005-04-28 | Reinder Coehoorn | Sensor and method for measuring the areal density of magnetic nanoparticles on a micro-array |
| US20080314749A1 (en) * | 2006-01-04 | 2008-12-25 | Koninklijke Philips Electronics N.V. | Microelectronic Device with Magnetic Excitation Wires |
-
2014
- 2014-08-27 WO PCT/EP2014/068182 patent/WO2016029943A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050087000A1 (en) * | 2001-12-21 | 2005-04-28 | Reinder Coehoorn | Sensor and method for measuring the areal density of magnetic nanoparticles on a micro-array |
| US20080314749A1 (en) * | 2006-01-04 | 2008-12-25 | Koninklijke Philips Electronics N.V. | Microelectronic Device with Magnetic Excitation Wires |
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