DE102012003863B3 - Device, useful for determining effect of nanoparticle materials on living cells by measuring thermal power production of cells using chip-calorimeter, comprises flow measurement chamber, permanent magnet, pivot device, and thermopile - Google Patents

Abstract

The device comprises: a flow measurement chamber having a measuring channel, a feed channel for supplying a thermal power, a magnetic bead sample prepared with a nanoparticle material brought in contact with living cells, a discharge channel for the bead sample and a transport liquid provided for transporting the bead sample; a permanent magnet (18) that is positioned on a surface of the flow measurement chamber and engages, with its magnetic field strength, into the flow measurement chamber; and a pivot device (20) holding the permanent magnet. The device comprises: a flow measurement chamber having a measuring channel, a feed channel for supplying a thermal power, a magnetic bead sample prepared with a nanoparticle material brought in contact with living cells, a discharge channel for the bead sample and a transport liquid provided for transporting the bead sample; a permanent magnet (18) that is positioned on a surface of the flow measurement chamber and engages, with its magnetic field strength, into the flow measurement chamber; a pivot device (20) holding the permanent magnet; and a thermopile (24, 25, 26, 27) that is present opposite to the permanent magnet and the flow measurement chamber. The apparatus is assigned to: a holding device (14) at which the flow measurement chamber is vertically suspended from the feed channel and/or the discharge channel; a pump unit, which is arranged downstream of the discharge channel and serves to a selective removal of the transport liquid; and a bead sample trap, which is connected to the pump unit and is stored into the bead sample. A heat output measurement in the chip-calorimeter is conduced to the bead sample magnetically fixed into the flow measurement chamber after decay of thermal disturbances triggered by a bead sample transport. The thermal power produced by the cells is converted to electrical signals. The thermopile is connected to an evaluation device via signal lines, and the electrical signals are transmitted to the evaluation device. The electrical signals in the evaluation device containing a computer are processed by a program-technical unit and technical and biological algorithms that reflect a reaction of the living cells to contact with a material of the nanoparticles on the basis of the measured thermal power produced by the cells. The chip-calorimeter is integrated in a thermostat to which the flow measurement chamber is thermally coupled and causes a partial thermo-static control of the transport liquid required for the transport. The supply channel and the discharge channel represent cannulas and are realized as steel capillaries. The pivoting device contains a spring that is attached between the permanent magnet and the holding device, a chord attached to the spring, and a magnet movable with the permanent magnet. The chord serves to deflect the spring present away from the surface of the flow measurement chamber. The bead sample trap comprises a filter magnet that filters out the bead sample from the transport liquid. A T-piece is mounted between the pump unit and the bead sample trap. A syringe is mounted at the branch line and guided with a cleaning liquid into the bead sample trap for cleaning the bead sample that is occupied with the cells or residual cells. A temperature sensor is mounted in a region of the holding device in contact with the feed channel. A funnel for filling the transport liquid with the bead sample is assigned to the feed channel. The transport liquid is water or a liquid culture medium. The magnetic bead sample for making measurements before the introduction of the feed channel consists of an unprepared magnetic bead, and a magnetic bead prepared with living cells to compare measurements with the magnetic bead sample contacted with nanoparticles. The living cells have a time-defined contact treatment with nanoparticles or not contact treatment with nanoparticles before the introduction of the flow measurement chamber, where an influence of the material of the nanoparticle on the living cells changes their metabolic heat output. The magnetic beads are composed of individual particles of paramagnetic iron oxide, and have a bead-like shape or drop-like shape of an agglomeration, which is surrounded by a protective layer made of starch, where the aggregation of polyethyleneimine is coated as a ligand that is strong unspecific anion exchanger. Independent claims are included for: (1) a chip-calorimeter to measure the thermal output of magnetic samples; and (2) a method for determining an effect of nanoparticle materials on living cells by measuring a thermal power output of the cells using an apparatus.

Description

The invention relates to an apparatus and a method for determining the effect of nanoparticle materials on living cells by measuring the heat output production of the cells by means of a chip calorimeter.

The use of anthropogenically generated nanoparticle materials in engineering products, in pharmaceutical preparations as well as in many household goods, paints, car tires and diesel fuel is increasing. At the same time, however, this industrial production and spread are also subject to risks that are largely unpredictable. One of the reasons for this uncertainty is that, in particular, the toxic effect of these novel nanoparticle materials on living cells has largely not been investigated. In particular, there is still no in-depth understanding of the toxic effect at the molecular level. Therefore, targeted testing of the toxic effect is required by studying the effect on specific sections in the chain of action of nanoparticle materials.

Calorimetry, by means of which the thermal performance of biochemical activities can be determined, is one such method for determining the effect of a test substance on the physiological condition of a living cell.

It is estimated that a microbial cell produces about 3 pw to 4 pw of heat during the growth phase. The use of z. B. 10 ⁴ cells (eg., In 6 .mu.l volume) to 30 nW to 40 nW heat output, which is within the measurement resolution (<100 nW) of the chip calorimetry.

An advantage of chip calorimetry lies in the possibility of examining biofilms. Unicellular organisms, especially prokayotic cells, often live in their natural environment in the form of a biofilm - an aggregate of many cells (including various species) surrounded by a shell mainly consisting of polysaccharides. The investigation of the impairment of these biofilms is currently possible only under relatively high labor and expense. The fact that biofilms are of great importance not only in environmental microbiology but also in clinical For example, pathogenic species of microorganisms form biofilms with open wounds or deposits on artificial joints and catheters, underscoring the value of chip calorimetry as a measurement method.

A chip calorimeter with a measuring chamber with flow of liquids and with a sample aggregate therein is in the document DE 10 2008 011 839 B4 described in which a miniaturization of calorimetry has led to a rapid measurement method with increased sensitivity.

This in 1 illustrated conventional and horizontal chip calorimeter 1 has a chip carrier 2 , a chip applied to it 3 with a chip membrane with the measuring chamber placed thereon and loadable with thermal power-related, chemical or biological sample aggregates 4 , at the bottom of the chamber, a flow channel open to the bottom side 5 for heat transfer towards the underlying, with a thermopile assembly 6 provided chip membrane is introduced, which communicates with the chamber bottom in heat transfer connection. It is on the bottom side of the measuring chamber 4 a cover foil 7 for closing the ground-open flow channel 5 attached, with the cover 7 between the chamber bottom and the chip membrane for the spatial separation of the measuring chamber 4 and chip 3 and with the chip membrane in detachable connection by means of an operable mounting device 8th at the measuring chamber 4 attacks, stands. The measuring chamber prepared with the fixed sample aggregate 4 can on the chip 3 be held reversibly by the mounting device 8th the measuring chamber 4 on its top holds and presses on the chip membrane. The liquid 9 is passed through at least one inflow tube into the measuring chamber 4 transported and leaves after flow through the measuring chamber 4 through at least one spout the measuring chamber 4 , During the stay of the liquid 9 in the measuring chamber 4 the flowing liquid flows around it 9 the sample aggregate. In this case, a heat transfer takes place from the measuring chamber 4 towards the thermal power registering, several thermopile TP1, TP2, TP3 and TP4 having thermopile assembly 6 , By attaching the cover 7 can the chemical and / or biological sample aggregates by implementation in the provided measuring chambers 4 Prepared in stock.

One problem is that in a heat performance measurement of many sample aggregates, which also beads with z. B. may be cultivated bacteria as bead samples, but in each case the measuring chamber for each measurement 4 from the chip calorimeter 1 must be expanded to fix a bead sample in it. Despite the miniaturization of the measuring chamber 4 and the conventional chip calorimeter 1 associated assemblies take due to the frequent change of the measuring chamber 4 the measuring operations to perform each thermal power measurement for a long time.

The invention is therefore based on the object of specifying an apparatus and a method for determining the effect of nanoparticle materials on living cells by measuring the heat output of the cells by means of chip calorimeter, which are designed to be suitable from the sample aggregate consisting of magnetic beads and at least on living cells applied thereto, simplified, faster and more accurate to measure at least one of the thermopile transferable thermal power and to determine therefrom the effect of the material of cell-contacting nanoparticles on the living cells in a shorter time. In addition, time-consuming changes of the measuring chamber should be avoided.

• – eine Durchflussmesskammer mit einem Messkanal sowie mit einem Zuführungskanal zur Zuführung für mit Wärmeleistung produzierenden und mit mindestens einem Nanopartikelmaterial in Kontakt gebrachten lebenden Zellen präparierten, magnetischen Beadproben und mit einem Abführungskanal für die Beadproben und einer für einen Transport der Beadproben vorgesehenen Transportflüssigkeit,
• – einen Permanentmagnet, der an der Oberfläche der Durchflussmesskammer positioniert ist und der mit seiner magnetischen Feldstärke die Durchflussmesskammer durchgreift,
• – eine den Permanentmagnet halternde Schwenkeinrichtung, wobei die Schwenkung des Permanentmagneten von der Oberfläche weg bis hin zu einem vorgegebenen Abstand von der Oberfläche aus und zurück erfolgt, und
• – mindestens eine Thermosäule, die gegenüber dem angeordneten Permanentmagneten an der Oberfläche der Durchflussmesskammer anliegt, wobei des Weiteren der Vorrichtung zugeordnet sind
• – eine Halterungsvorrichtung, an der die Durchflussmesskammer zumindest am Zuführungskanal und/oder am Abführungskanal vertikal aufgehängt ist,
• – ein Pumpenaggregat, das dem Abführungskanal nachgeordnet ist und zu einer gezielten Abführung der Transportflüssigkeit dient, und
• – eine Beadprobenfalle, die an das Pumpenaggregat angeschlossen ist und in der die Beadproben abgelegt sind,

wobei die Wärmeleistungsmessung im Chip-Kalorimeter an in der Durchflussmesskammer magnetisch fixierten Beadproben nach Abklingen der durch den Beadprobentransport ausgelösten thermischen Störungen durchgeführt wird, wobei die von den Zellen produzierte Wärmeleistung in zumindest einer der Thermosäulen in elektrische Signale umgewandelt wird, wobei die Thermosäulen mit einer Auswerteeinrichtung über Signalleitungen verbunden sind und an die Auswerteeinrichtung die elektrischen Signale übermitteln, und
wobei die elektrischen Signale in der einen Rechner enthaltenden Auswerteeinrichtung mittels programmtechnischer Mittel und technischer sowie biologischer Algorithmen zu Darstellungen verarbeitet werden, die die Reaktion der lebenden Zellen auf den Kontakt mit dem Material der Nanopartikel auf der Basis der gemessenen, von den Zellen produzierten Wärmeleistung wiedergeben.The object is solved by the features of claims 1 and 16. The device for determining the effect of nanoparticle materials on living cells by measuring the heat output production of the cells by means of chip calorimeter contains at least
as a chip calorimeter

• A flow measuring chamber with a measuring channel and with a supply channel for supplying heat-producing and prepared with at least one nanoparticle material contacted living cell prepared magnetic bead samples and with a discharge channel for the Beadproben and provided for a transport of Beadproben transport liquid,
• A permanent magnet which is positioned on the surface of the flow measuring chamber and which penetrates the flow measuring chamber with its magnetic field strength,
• - A permanent magnet holding the pivoting device, wherein the pivoting of the permanent magnet away from the surface up to a predetermined distance from the surface and back, and
• - At least one thermopile, which rests against the arranged permanent magnet on the surface of the flow measuring chamber, wherein the further associated with the device
• A holding device, on which the flow-measuring chamber is suspended vertically at least on the feed channel and / or on the discharge channel,
• - A pump unit, which is arranged downstream of the discharge channel and serves for a targeted discharge of the transport liquid, and
• A bead sample trap which is connected to the pump set and in which the bead samples are stored,

wherein the thermal power measurement in the chip calorimeter is performed on bead samples magnetically fixed in the flow meter chamber after the thermal disturbances caused by the bead sample transport have been removed, the thermal power produced by the cells being converted into electrical signals in at least one of the thermopiles, the thermopiles being connected to an evaluation device are connected via signal lines and transmit to the evaluation device, the electrical signals, and
wherein the electrical signals are processed in the computer-containing evaluation means of programmatic means and technical and biological algorithms to representations that reflect the response of the living cells to the contact with the material of the nanoparticles on the basis of the measured, produced by the cells heat output.

The effect is indicated by the different response of the living cells primarily to the different thermal output in the form of different electrical signals as a result of the production of the material-related heat output. These signals evaluated by means of program technology and technical as well as biological algorithms can be used in representations for further interpretations, in particular the effect. The representations can be used as information or data z. B. output in tabular and / or curved form of the evaluation device.

• – eine Durchflussmesskammer mit einem Messkanal sowie mit einem Zuführungskanal zur Zuführung von Proben und mit einem Abführungskanal für die Proben, in denen ein Transport der Proben mittels einer Transportflüssigkeit über ein an den Abführungskanal angeschlossenes Pumpenaggregat erfolgt,
• – mindestens eine Thermosäule zur Erfassung der Wärmeleistung und Umwandlung in elektrische Signale,
• – eine über Signalleitungen mit den Thermosäulen in Verbindung stehende Auswerteeinheit, die die elektrischen Signale mittels vorgegebener programmtechnischer Mittel auswertet,

wobei erfindungsgemäß
der Messkanal der Durchflussmesskammer vertikal ausgerichtet ist und weiterhin angeordnet sind:
ein Permanentmagnet, der an der Oberfläche der Durchflussmesskammer positioniert ist und der mit seiner magnetischen Feldstärke in die Durchflussmesskammer eingreift und zur Fixierung der magnetischen Proben dient,
eine den Permanentmagnet halternde Schwenkeinrichtung, wobei die Schwenkung des Permanentmagneten von der Oberfläche weg bis hin zu einem vorgegebenen Abstand von der Oberfläche aus und zurück erfolgt zur Aufhebung der Fixierung der magnetischen Proben,
wobei mindestens eine Thermosäule zur Aufnahme der Wärmeleistung der magnetischen Probe gegenüber dem angeordneten Permanentmagneten an der Durchflussmesskammer anliegt.The chip calorimeter for measuring the heat output of magnetic samples can thus at least include

• A flow measuring chamber with a measuring channel and with a feed channel for supplying samples and with a discharge channel for the samples, in which the samples are transported by means of a transport liquid via a pump set connected to the discharge channel,
• At least one thermopile for detecting heat output and conversion to electrical signals,
• An evaluation unit connected to the thermopiles via signal lines and evaluating the electrical signals by means of predetermined program-technical means,

according to the invention
the measuring channel of the flow measuring chamber is vertically aligned and are further arranged:
a permanent magnet, which is positioned on the surface of the flow measuring chamber and which engages with its magnetic field strength in the flow measuring chamber and serves to fix the magnetic samples,
a pivoting device holding the permanent magnet, wherein the pivoting of the permanent magnet away from the surface up to a predetermined distance from the surface and back takes place to cancel the fixation of the magnetic samples,
wherein at least one thermopile for receiving the heat output of the magnetic sample relative to the arranged permanent magnet is applied to the flow measuring chamber.

The chip calorimeter can be embedded in a thermostat, to which the flow measuring chamber is thermally coupled and causes a partial thermostating of the transport of the magnetic bead in the transport liquid used.

A magnetic bead sample can be made from at least unprepared magnetic beads, living cell-prepared magnetic beads, living magnetic beads already contacted with nanoparticles, or live nanoparticle-contacted cells to make measurements prior to introduction into the delivery channel Cell prepared magnetic beads. The magnetic bead samples from unprepared magnetic beads or living cell-prepared magnetic beads are used essentially for comparison measurements with the magnetic bead samples prepared from living magnetic beads already contacted with nanoparticles. The nanoparticles in the latter case are also on the magnetic bead.

A bead sample can thus consist of magnetic beads with adsorbed living cells applied to its surface, which had a time-defined contact treatment with nanoparticles or no contact treatment with nanoparticles before being introduced into the flow-measuring chamber, the influence of the material of the nanoparticles on the living cells can change metabolic heat output.

The magnetic beads used can be composed of a plurality of individual particles of superparamagnetic iron oxide (γ-Fe ₂ O ₃ , maghemite) and have a pearl-like shape or drop-like form of a cluster, which is surrounded by a protective layer. The aggregation may be enveloped by polyethylene imine as a ligand, which is a strong nonspecific anion exchanger. The magnetic beads used for the measurements can be 0.5 μm to 300 μm in size.

The feed channel and the discharge channel (bead sample transport channels) are cannulas and can be realized as steel capillaries.

The pivot means may include a spring mounted between the permanent magnet and the retainer, in particular a leaf spring and a tendon attached to the spring, the tendon for deflecting the spring and thus the permanent magnet away from the surface of the flow chamber.

However, in addition to the spring mounted between the permanent magnet and the holding device, the pivoting device can also have a movable displacement magnet corresponding to the permanent magnet, which can be moved by means of pivoting, ie. H. its mobility in the plane and in the direction of the permanent magnet lift the permanent magnet from the surface of the flow measuring chamber and lead out of its Magnetwirkungsbereich with respect to the flow measuring chamber and can lead depending on the polarity reversal of the displacement magnet back to the surface.

The Beadprobenfalle, which is connected to the pump unit, has at least one magnet, which filters out the magnetic bead samples from the flowing liquid and accumulates in itself.

Between the pump unit and the Beadprobenfalle a T-piece may be attached to the branch line, a syringe is mounted, with a cleaning liquid can be performed in the Beadprobenfalle for their cleaning.

In the region of the mounting device, a temperature sensor may be mounted in contact with the supply channel.

The feed channel may be associated with a funnel for filling transport liquid with the prepared bead sample therein into the feed channel.

In the method of determining the effect of nanoparticle materials on living cells by measuring the heat output production of the cells using a device including a chip calorimeter, comparative measurements between living cell-prepared magnetic bead samples are obtained in terms of the produced heat capacity of the living cells according to the characterizing portion of claim 16 the presence or absence of a time-defined contact between the respective cell structure and the respective material of the given nanoparticles to be examined, wherein the contact has either occurred time-defined before the introduction of prepared magnetic bead samples in the flow measuring chamber or permanently takes place when passing through the flow measuring chamber, wherein the Comparative measurements are evaluated by an evaluation device.

Contact treatment between the magnetic beads and the living cells and nanoparticles adsorbed thereon, both with different concentrations of the nanoparticles and considering the concentration ratio between living cells and nanoparticles, is performed according to the given evaluation modality.

• • die Kulturen in Form eines Biofilms vorliegen oder durch Kopplung über Antikörper-Antigen-Wechselwirkung oder elektrostatisch an magnetischen Beads immobilisiert sind oder
• • die Kulturen aus einer (klonal) oder mehreren (Mischkultur) Zell-Art(en) bestehen oder
• • die zu prüfenden Zellen prokarytischen oder eukarytischen Ursprungs sind oder
• • die zu prüfenden Zellen natürlich vorkommende Zellen oder gentechnisch veränderte Zeilen sind oder
• • die zu prüfenden Zellen künstlich aufrecht erhaltene Zellkulturen (z. B. He-La Zellen) sind.

By means of a computer associated with the evaluation device, the cytotoxic effect of the nanoparticle materials is represented by the calorimetric detection of the heat production of the biological objects in the form of a metabolic heat output and their change as a result of the action of the nanoparticle materials.
wherein for the cells to be examined / objects applies by the heat output of the cells is measured on cultures, wherein

• The cultures are in the form of a biofilm or are immobilized on magnetic beads by coupling via antibody-antigen interaction or electrostatically
• • the cultures consist of one (clonal) or more (mixed culture) cell type (s) or
• • the cells to be tested are of prokarytic or eukaryotic origin or
• • the cells to be tested are naturally occurring cells or genetically modified cells or
• • the cells to be tested are artificially maintained cell cultures (eg He-La cells).

In testing the effect of the material of nanoparticles on microorganisms, microorganisms are applied in the form of biofilms, which are cultivated on the magnetic beads as bead samples, wherein
the microorganisms cultured on the bead samples due to the high degree of heterogeneity represent realistic models for the assessment of the cytotoxic effect of nanoparticle materials in the environment and
culturing the biofilms on the magnetic bead samples allows for automated bead sample transfer through the chip calorimeter.

The operation of the device according to the invention will be explained in more detail below with reference to the following:
The flow measuring chamber is provided with at least one vertical channel for the supply and a vertically arranged channel for the discharge (bead sample transport channels) of at least one prepared magnetic bead sample. The flow metering chamber is suspended from cannulas implementing the bead sample transport channels in a fixture which is thermally coupled to the thermostat of the chip calorimeter and effects a partial thermostating of transport fluid required for the bead sample transport. The permanent magnet is pressed by a spring on the surface of the flow measuring chamber. To introduce a bead sample, it is flooded into the flow measuring chamber with the aid of the transport liquid stream (eg water or culture medium) and held in the height / plane of the permanent magnet. The measurement of the heat output produced by the cells of the bead sample in the measuring channel takes place at the bead sample located at the position level of the permanent magnet and after the thermal disturbances triggered by the bead sample transport at least in a thermopile also attached to the surface of the flow measuring chamber. The measured heat output is converted into electrical signals and fed to an evaluation device.

After completion of the measurement of the permanent magnet by means of a pivoting device, in the z. B. a spring is mechanically deflected over the thin tendon, lifted and the bead samples are discharged by means of the liquid flow from the measuring channel of the flow measuring chamber and collected outside the chip calorimeter in a Beadprobenfalle.

While measuring the heat output produced with a thermopile, the absolute voltage values are measured with additional subthreshold disturbance values, the advantage of measuring the produced heat output with at least two thermopile is that by subtracting the two thermopile voltage values, the disturbance values are eliminated.

The device according to the invention can by means of the evaluation, which processes the electrical signals with their programmatic means and technical and biological algorithms, for the analysis of the effect, also referred to as toxic effect of the material of nanoparticles on both microbial test systems (planktonic cultures, biofilm) as also be used on eukaryotic cell cultures, provided that they are immobilized on magnetic beads. In the latter case, the method can be used both to study the toxic effect on living healthy cells and to evacuate the use of nanoparticle materials in the treatment of living diseased cells, e.g. As for the treatment of tumor cells used.

Due to the development of the device according to the invention for performing the chip calorimetry, the use of the device described here for the above purposes, in particular the (fast and inexpensive) screening of new nanoparticle materials, particularly suitable.

Further developments and more advantageous embodiments of the invention are specified in further subclaims.

The invention will be explained in more detail using an exemplary embodiment by means of several drawings.

Show it:

1 1 is a schematic representation of a chip calorimeter according to the prior art,

2 an enlarged schematic representation of the measuring chamber according to 1 ,

3 a schematic representation of a device according to the invention with a chip calorimeter according to the invention,

4a an enlarged view section of the measuring range with measuring channel when filling a cell-prepared, magnetic bead sample in the supply channel according to 3 .

4b an enlarged view section of the measuring range with measuring channel at the moment of the thermal power measurement of the magnetically locked, cell-prepared magnetic bead sample and

4c an enlarged view section of the measuring range with measuring channel during the discharge of the magnetically arrested, cell-prepared magnetic bead sample from the flow measuring chamber of the chip calorimeter and pivoting of the permanent magnet.

5a typical course of the thermoelectric voltage, ie course of the thermal voltage with cyclic dosing of water,

5b typical course of the temperature, measured by the temperature sensor, at the mounting device, ie temperature increase at the mounting device made of copper,

5c typical course of the control performance of the inner thermostat, ie the course of the decrease in heating power during dosing,

6a a measuring curve for the thermoelectric voltage when measuring 5 · 10 ⁶ cfu in the measuring channel,

6b a measuring curve for the thermoelectric voltage when measuring 5 · 10 ⁵ cfu in the measuring channel,

6c a measurement curve for the thermoelectric voltage of only cellless bead samples in the measuring channel zero measurement.

• – eine Durchflussmesskammer 11 mit einem Messkanal 47 sowie mit einem Zuführungskanal 12 zur Zuführung für mit Wärmeleistung produzierenden und mit mindestens einem Nanopartikelmaterial in Kontakt gebrachten lebenden Zellen präparierten, magnetischen Beadproben 16 und mit einem Abführungskanal 13 für die Beadproben 16 und der für einen Transport der Beadproben eingesetzten Transportflüssigkeit 17,
• – einen Thermostaten 15, an den die Durchflussmesskammer 11 thermisch gekoppelt ist und eine teilweise Thermostatisierung der für den Transport eingesetzten Transportflüssigkeit 17 bewirkt,
• – einen Permanentmagnet 18, der an der Oberfläche 19 der Durchflussmesskammer 11 positioniert ist und der mit seiner magnetischen Feldstärke in die Durchflusskammer 11 eingreift,
• – eine den Permanentmagnet 18 halternde Schwenkeinrichtung 20, wobei die Schwenkung des Permanentmagneten 18 von der Oberfläche 19 weg bis hin zu einem vorgegebenen Abstand von der Oberfläche 19 erfolgen kann, und
• – vier Thermosäulen 24, 25, 26, 27, die gegenüber dem angeordneten Permanentmagnet 18 an der Durchflussmesskammer 11 anliegen,

wobei des Weiteren der Vorrichtung 10 zugeordnet sind

• – eine Halterungsvorrichtung 14, an der die Durchflussmesskammer 11 zumindest am Zuführungskanal 12 und/oder am Abführungskanal 13 vertikal aufgehängt ist,
• – ein Pumpenaggregat 28, das dem Abführungskanal 13 nachgeordnet ist und zu einer gezielten Abführung der Flüssigkeit 17 dient, und
• – eine Beadprobenfalle 29, die an das Pumpenaggregat 28 angeschlossen ist und in der die Beadproben 16 abgelegt sind,

wobei die Wärmeleistungsmessung im Chip-Kalorimeter 40 bei in der Durchmessflusskammer 11 magnetisch fixierter Beadprobe 16 nach Abklingen der durch den Beadprobentransport ausgelöstes thermischen Störungen durchgeführt wird, wobei die von den Zellen produzierte Wärmeleistung in zumindest einer der Thermosäulen 24, 25, 26, 27 in elektrische Signale umgewandelt wird, wobei die Thermosäulen 24, 25, 26, 27 mit der Auswerteeinrichtung 39 über Signalleitungen 42, 43, 44, 45 verbunden sind und an die Auswerteeinrichtung 39 die elektrischen Signale übermitteln, und
wobei die elektrischen Signale in der einen Rechner 41 enthaltenden Auswerteeinrichtung 39 mittels programmtechnischer Mittel und technischer sowie biologischer Algorithmen zu Darstellungen verarbeitet werden, die die Reaktion der lebenden Zellen auf den Kontakt mit dem Material der Nanopartikel auf der Basis der gemessenen, von den Zellen produzierten Wärmeleistung wiedergeben.In the 3 illustrated device 10 for determining the effect of nanoparticle materials on living cells by measuring the heat output production of the cells by means of a chip calorimeter 40 contains at least as a chip calorimeter 40

• - a flow measuring chamber 11 with a measuring channel 47 as well as with a supply channel 12 magnetic bead sample prepared for delivery to thermal energy producing living cells contacted with at least one nanoparticle material 16 and with a discharge channel 13 for the bead samples 16 and the transport fluid used for transporting the bead samples 17 .
• - a thermostat 15 to which the flow measuring chamber 11 thermally coupled and a partial thermostating of the transport liquid used for transport 17 causes
• - a permanent magnet 18 that at the surface 19 the flow measuring chamber 11 is positioned and with its magnetic field strength in the flow chamber 11 intervenes
• - One the permanent magnet 18 holding swivel device 20 , wherein the pivoting of the permanent magnet 18 from the surface 19 away up to a given distance from the surface 19 can be done, and
• - four thermopiles 24 . 25 . 26 . 27 facing the arranged permanent magnet 18 at the flow measuring chamber 11 issue,

further comprising the device 10 assigned

• - A mounting device 14 at the flowmeter 11 at least at the feed channel 12 and / or on the discharge channel 13 is hung vertically
• - a pump unit 28 that the discharge channel 13 is downstream and to a targeted discharge of the liquid 17 serves, and
• - a bead sample trap 29 connected to the pump unit 28 is connected and in which the bead samples 16 are stored,

where the thermal power measurement in the chip calorimeter 40 in the flow-through chamber 11 magnetically fixed bead sample 16 is performed after the decay of the Beadprobentransport triggered thermal disturbances, wherein the heat output produced by the cells in at least one of the thermopiles 24 . 25 . 26 . 27 is converted into electrical signals, the thermopiles 24 . 25 . 26 . 27 with the evaluation device 39 via signal lines 42 . 43 . 44 . 45 are connected and to the evaluation 39 transmit the electrical signals, and
where the electrical signals in the one computer 41 containing evaluation 39 By means of programmable means and technical as well as biological algorithms are processed into representations, which reproduce the reaction of the living cells on the contact with the material of the nanoparticles on the basis of the measured, produced by the cells heat output.

The representations can be used as information or data z. B. issued in tabular and / or curved form.

• – eine Durchflussmesskammer 11 mit einem Messkanal 47 sowie mit einem Zuführungskanal 12 zur Zuführung von Proben 16 und mit einem Abführungskanal 13 für die Proben 16, in denen ein Transport der Proben 16 mittels einer Transportflüssigkeit 17 über ein an den Abführungskanal 13 angeschlossenes Pumpenaggregat 28 erfolgt,
• – mindestens eine Thermosäule 24, 25, 26, 27 zur Erfassung der Wärmeleistung und Umwandlung in elektrische Signale,
• – eine über Signalleitungen 42, 43, 44, 45 mit den Thermosäulen 24, 25, 26, 27 in Verbindung stehende Auswerteeinheit 38, die die elektrischen Signale mittels vorgegebener programmtechnischer Mittel auswertet,

wobei erfindungsgemäß
der Messkanal 47 der Durchflussmesskammer 11 vertikal ausgerichtet ist und weiterhin angeordnet sind:
ein Permanentmagnet 18, der an der Oberfläche 19 der Durchflussmesskammer 11 positioniert ist und der mit seiner magnetischen Feldstärke in die Durchflussmesskammer 11 eingreift und zur Fixierung der magnetischen Proben 16 dient, eine den Permanentmagnet 18 halternde Schwenkeinrichtung 20, wobei die Schwenkung des Permanentmagneten 18 von der Oberfläche 19 weg bis hin zu einem vorgegebenen Abstand von der Oberfläche 19 aus und zurück erfolgt zur Aufhebung der Fixierung der magnetischen Proben 16,
wobei mindestens eine Thermosäule 24, 25, 26, 27 zur Aufnahme der Wärmeleistung der magnetischen Probe 16 gegenüber dem angeordneten Permanentmagneten 18 an der Durchflussmesskammer 11 anliegt.This includes the chip calorimeter 40 in 3 for measuring the heat output of magnetic samples 16 at least

• - a flow measuring chamber 11 with a measuring channel 47 as well as with a supply channel 12 for feeding samples 16 and with a discharge channel 13 for the samples 16 in which a transport of the samples 16 by means of a transport liquid 17 via a to the discharge channel 13 connected pump set 28 he follows,
• - At least one thermopile 24 . 25 . 26 . 27 for recording heat output and conversion into electrical signals,
• - one via signal lines 42 . 43 . 44 . 45 with the thermopiles 24 . 25 . 26 . 27 related evaluation unit 38 which evaluates the electrical signals by means of predetermined program-technical means,

according to the invention
the measuring channel 47 the flow measuring chamber 11 is vertically aligned and still arranged:
a permanent magnet 18 that at the surface 19 the flow measuring chamber 11 is positioned and with its magnetic field strength in the flow measuring chamber 11 engages and fix the magnetic samples 16 serves, one the permanent magnet 18 holding swivel device 20 , wherein the pivoting of the permanent magnet 18 from the surface 19 away up to a given distance from the surface 19 out and back takes place to cancel the fixation of the magnetic samples 16 .
where at least one thermopile 24 . 25 . 26 . 27 for absorbing the heat output of the magnetic sample 16 opposite the arranged permanent magnet 18 at the flow measuring chamber 11 is applied.

The magnetic samples 16 can be magnetic bead samples, as stated above.

A magnetic bead sample 16 can be used to carry out the measurements before insertion into the feed channel 12 consist of at least unprepared magnetic beads, magnetic beads prepared with living cells, magnetic beads prepared with living cells already contacted with nanoparticles, or magnetic beads prepared with living cells still to be contacted with nanoparticles.

A bead sample 16 Thus, it may consist of magnetic beads with adsorbed living cells deposited on its surface, which prior to introduction into the flow measuring chamber 11 optionally had a time-defined contact treatment with nanoparticles or no contact treatment with nanoparticles, whereby the influence of the material of the nanoparticles on the living cells can change their metabolic heat output.

The magnetic beads used can be composed of a plurality of individual particles of paramagnetic iron oxide (γ-Fe ₂ O ₃ , maghemite) and have a pearl-like shape or drop-like form of a cluster, which is surrounded by a protective layer. The aggregation may be enveloped by polyethylene imine as a ligand, which is a strong nonspecific anion exchanger. The magnetic beads used for the measurements may be 0.5 μm to 200 μm in size.

The feed channel 12 and the discharge channel 13 in the chip calorimeter 40 the device 10 represent cannulas and can be realized as steel capillaries.

The pivoting device 20 can one between the permanent magnet 18 and the holding device 21 attached leaf spring 22 and one on the leaf spring 22 attached tendon 23 contain, with the tendon 23 for the deflection of the leaf spring 22 away from the surface 19 the flow measuring chamber 11 serves.

The pivoting device 20 can also be a between the permanent magnet 18 and the holding device 21 attached spring 22 and one with the permanent magnet 18 corresponding movable displacement magnets (not shown), by means of its mobility in the direction of the permanent magnet 18 the permanent magnet 18 from the surface 19 lifts off and from its Magnetwirkungsbereich with respect to the flow measuring chamber 11 leads out and depending on the polarity reversal of the displacement magnet back to the surface 19 approach leads.

The bead sample trap 29 connected to the pump unit 28 is connected, has at least one filter magnet 30 . 31 . 32 on, who the Beadproben 16 from the flowing liquid 17 filters out, absorbs and attaches to itself.

Between the pump unit 28 and the bead sample trap 29 can be a tee 33 be attached to the branch line 34 a syringe 35 is attached, with a cleaning fluid 36 into the bead sample trap 29 for cleaning the bead samples still occupied with cells or residual cells 16 or nanoparticles can be performed.

In the area of the mounting device 14 can come into contact with the feed channel 12 a temperature sensor 37 be attached, the electrical signals of the evaluation are supplied.

The feed channel 12 can be a funnel 38 for filling transport liquid 17 with the prepared bead sample contained therein 16 in the feed channel 12 be assigned.

The determination of the effect of the material of the nanoparticles consists essentially in at least one comparison measurement of living cell-prepared magnetic bead samples 16 with respect to the produced heat capacity of the living cells based on the presence or absence of a time-defined contact between the respective cell structure and the respective material of the nanoparticles to be investigated. The contact can thus either be time-defined before being introduced into the flow measuring chamber 11 have taken place or permanently when passing through the flow measuring chamber 11 occur.

The contact treatment between the magnetic beads and the living cells and the nanoparticles adsorbed thereon can, for. B. be carried out with different concentrations of the nanoparticles as well as taking into account the concentration ratio between living cells and nanoparticles depending on the given evaluation modality.

• – Eine Durchflussmesskammer 11 wird mit einem Messkanal 47 sowie mit mindestens einem vertikal angeordneten Kanal 12 für die Zuführung und einem Kanal 13 für die Abführung mindestens einer zellenpräparierten, magnetischen Beadprobe 16 versehen.
• – Die Durchflussmesskammer 11 wird an die Beadprobentransportkanäle t2, t3 realisierenden Kanülen in der Halterungseinrichtung 14 aufgehängt, die an den Thermostaten 15 des Chip-Kalorimeters 40 thermisch gekoppelt ist und eine teilweise Thermostatisierung für den Beadprobentransport erforderlichen Transportflüssigkeit 17 bewirkt.
• – Der Permanentmagnet 18 wird über eine Feder 22 auf die Oberfläche 19 der Durchflussmesskammer 11 gedrückt. Um den Permanentmagneten 18 abzuheben, wird die Feder 22 mechanisch über eine dünne Sehne 23 ausgelenkt.
• – Zur Einführung einer Beadprobe 16 wird die Beadprobe 16 mit Hilfe des Flüssigkeitsstromes (z. B. Wasser oder Kulturmedium) 17 über den Zuführungskanal 12 in die Durchflussmesskammer 11 eingeschwemmt und in Höhe des an die Oberfläche 19 angelegten Permanentmagneten 18, dessen magnetische Feldstärke in die Durchflusskammer 11 eingreift, gehalten.
• – Die Beadprobe 16 befindet sich dann innerhalb der Durchflussmesskammer 11 im Bereich zumindest einer an der Oberfläche 19 anlegenden Thermosäule 24, 25, 26, 27.
• – Die Wärmeleistungsmessung erfolgt bei der auf dem Positionsniveau des Permanentmagneten 18 fixierten Beadprobe 16 nach Abklingen der durch den Beadprobentransport ausgelösten thermischen Störungen in zumindest einer auch an der Oberfläche der Durchflussmesskammer 11 angebrachten Thermosäulen 24, 25, 26, 27.
• – Nach Beendigung der Messung der Wärmeleistung wird der Permanentmagnet 18 von der Oberfläche 19 abgehoben und die Beadprobe 16 wird mit Hilfe des Flüssigkeitsstromes 17 ausgeschleust und außerhalb des Chip-Kalorimeters 40 in einer Beadprobenfalle 29 aufgefangen.

The operation of the device according to the invention 10 is using the 3 and 4a . 4b and 4c explained in more detail. In 4a is an enlarged view of the measuring range 46 with a measuring channel 47 upon delivery of the cell-prepared magnetic bead sample 16 via the supply channel according to 3 shown. In 4b is an enlarged view of the measuring range 46 with the measuring channel 47 at the moment of thermal power measurement of the magnetically arrested, cell-prepared magnetic bead sample 16 and in 4c is an enlarged view of the measuring range 46 with the measuring channel 47 in removing / discharging the magnetically arrested, cell-prepared magnetic bead sample 16 from the flow metering chamber 11 of the chip calorimeter 40 belonging measuring channel 47 and swinging away the permanent magnet 18 shown. The following is done:

• - A flow measuring chamber 11 is with a measuring channel 47 as well as with at least one vertically arranged channel 12 for the feeder and a canal 13 for the removal of at least one cell-prepared magnetic bead sample 16 Mistake.
• - The flow measuring chamber 11 is to the Beadprobentransportkanäle t2, t3 realizing cannulas in the mounting device 14 hung on the thermostat 15 of the chip calorimeter 40 thermally coupled and a partial thermostating necessary for the Beadprobentransport transport liquid 17 causes.
• - The permanent magnet 18 is about a spring 22 on the surface 19 the flow measuring chamber 11 pressed. To the permanent magnet 18 take off, the spring 22 mechanically over a thin tendon 23 deflected.
• - To introduce a bead sample 16 will be the bead sample 16 with the aid of the liquid flow (eg water or culture medium) 17 over the feed channel 12 into the flow measuring chamber 11 flooded and at the level of the surface 19 applied permanent magnet 18 , its magnetic field strength in the flow chamber 11 engages, held.
• - The bead sample 16 is then located inside the flow measuring chamber 11 in the area of at least one on the surface 19 applying thermopile 24 . 25 . 26 . 27 ,
• - The heat output is measured at the on the position level of the permanent magnet 18 fixed bead sample 16 after the thermal disturbances triggered by the bead sample transport have decayed in at least one of the surfaces of the flow measuring chamber 11 attached thermopiles 24 . 25 . 26 . 27 ,
• - After completion of the measurement of the heat output becomes the permanent magnet 18 from the surface 19 lifted off and the bead sample 16 is using the liquid flow 17 discharged and outside the chip calorimeter 40 in a bead sample trap 29 collected.

The following is the signal generation at dosages of lines for bead samples 16 will be presented in a brief explanation. The signal behavior or the thermal behavior of the chip calorimeter 40 is at a dosage via the bead sample transport channel 12 - 47 - 13 examined. By determining the thermal voltage difference .DELTA.U of two adjacent thermopiles 25 . 26 In the signal evaluation, a corrected signal is generated. Thus, disturbing effects such. B. a baseline drift or noise can be eliminated. For this purpose, the measuring signal of the thermopile 25 from the measuring signal of the thermopile 26 subtracted. The Beadprobe acting as a heat source 16 is on the thermopile 26 and next to the thermopile 25 positioned. So are both thermopiles 26 . 25 exposed to the same disturbances. In this way, a high resolution of 20 nW (0.1 μV) can be achieved. In the 5a is a typical course of the thermoelectric voltage and in 5b is a typical curve of the temperature measured by the temperature sensor 37 , on the mounting device 14 as in 5c is a typical curve of the control power of the internal thermostat 15 shown. The increase in temperature indicates that the transport fluid 17 one to the set temperature of the thermostat 15 has higher temperature. Accordingly, the temperature control reacts by lowering the control power. The strongly exothermic changes of the thermopile signals show that the heat introduced by the increased liquid temperature is not completely before entering the measuring channel 47 the flow measuring chamber 11 is compensated. However, it should be noted that the relaxation of the temperature disturbance is already completed about 7 minutes after the start of dosing and thus the heat output of the bead sample 16 with reference to the bead-free measuring channel 47 can be measured. If a culture medium (LB medium) is metered instead of water, no other effects than previously mentioned occur.

In 6a is a measurement curve for 5 × 10 ⁶ cfu on bead samples 16 shown. In comparison, a measurement curve is shown, in contrast, only 5 × 10 ⁵ cfu in the flow measuring chamber 11 are included ( 6b ). Are bacteria in the measuring channel 47 , after the decay of the dosing effect, a time-limited constant signal (plateau) sets in. The amount of the thermovoltage difference ΔU with respect to the baseline increases with the number of bacteria in the measurement channel 47 to ( 6a ). Likewise, a renewed decay of the measurement signal can then be detected. The fewer bacteria in the measuring channel 47 the longer the plateau phase lasts ( 6b ).

The differences in the course of the traces are explained as follows:
Regardless of how many bacteria are in the measurement channel 47 they can only produce a limited heat of 450 kJ per O ₂ under aerobic conditions. As long as oxygen and carbon substrate are in excess, substrate saturation is present. Thus, the growth rate under given conditions is maximal and thus constant if the amount of biomass is considered constant for a short measuring time. The amount of bacteria only determines the heat output, ie how fast the oxygen is metabolised and thus how high the exothermic measurement signal is. The more bacteria in the measuring channel 47 The faster the oxygen is consumed and the shorter the plateau. In 6a Only a time of only about 3 minutes passed until the metabolic activity and thus the heat production of the bacteria subsided. In addition, in the 6a to see that the measurement signal is not directly due to the baseline, which suggests that the bacteria have adjusted to aerobic living conditions. The 6c shows the trace of a zero measurement. Only bead samples without bacteria are dosed. The measuring signal returns to the baseline immediately after the end of dosing.

With the device according to the invention 10 including one computer 41 containing evaluation 39 With regard to the measured heat output, a method for testing the cytotoxic effect of nanoparticle materials on living cells (microorganisms and bacteria) as well as on eukaryotic cells / living beings is carried out by means of program technology and technical and biological algorithms on the basis of the measured, each produced by the cells heat output.

• • Die Kulturen können aus einer (klonal) oder mehreren (Mischkultur) Zell-Art(en) bestehen.
• • Die zu prüfenden Zellen können prokarytischen oder eukarytischen Ursprungs sein.
• • Die zu prüfenden Zellen können natürlich vorkommende Zellen oder gentechnisch veränderte Zellen sein.
• • Die zu prüfenden Zellen können künstlich aufrecht erhaltene Zellkulturen (z. B. HeLa Zellen) sein.

This is done by means of the computer 41 a proof of the cytotoxic effect of the nanoparticle materials by the calorimetric detection of the heat output production of the biological objects (metabolic heat output) and their change as a result of the action of the nanoparticle materials. The following applies to the cells / objects to be examined:
The heat output of the cells can be measured on cultures which are in the form of a biofilm or otherwise immobilized on magnetic beads (eg by coupling via antibody-antigen interaction or electrostatically).

• • The cultures may consist of one (clonal) or more (mixed culture) cell type (s).
• • The cells to be tested may be of prokaryotic or eukaryotic origin.
• • The cells to be tested can be naturally occurring cells or genetically modified cells.
• • The cells to be tested may be artificially maintained cell cultures (eg HeLa cells).

• – eine empfindliche Detektion der Wärmeleistung (< 100 nW Messauflösung),
• – geringe Geräte-Kosten,
• – schnelle durchführbare Messungen,
• – Verwendung geringer Mengen an Prüf-Organismen, was im Falle von eukaryotischen Zellkulturen eine weitere wesentliche Kosteneinsparung mit sich bringt,
• – Anwendung auf eine Bestimmung der Wirkung auf Biofilme ist möglich,
• – es besteht die Möglichkeit der online-Bestimmung und damit einer schnellen Bestimmung der Wirkung der Prüf-Organismen.

The measurement of the metabolic heat output is carried out as already described by means of the chip calorimeter according to the invention 40 , The advantages of using the chip calorimeter according to the invention 40 regarding the test organisms are:

• - a sensitive detection of the heat output (<100 nW measurement resolution),
• Low equipment costs,
• - fast feasible measurements,
• Use of small amounts of test organisms, which in the case of eukaryotic cell cultures brings about a further substantial cost saving,
• - Application to a determination of the effect on biofilms is possible
• - there is the possibility of online determination and thus a rapid determination of the effect of the test organisms.

LIST OF REFERENCE NUMBERS

1

Chip calorimeter according to the prior art

2

chip carrier

3

chip

4

measuring chamber

5

river channel

6

Thermopile arrangement

7

cover

8th

support means

9

liquid

10

Device according to the invention

11

Flow measurement chamber

12

feed channel

13

discharge channel

14

mounting device

15

thermostat

16

Magnetic bead sample

17

transport liquid

18

permanent magnet

19

surface

20

Pivot means

21

holder

22

feather

23

tendon

24

First thermopile

25

Second thermopile

26

Third thermopile

27

Fourth thermopile

28

pump unit

29

Beadprobenfalle

30

First filter magnet

31

Second filter magnet

32

Third filter magnet

33

Tee

34

branch line

35

syringe

36

cleaning fluid

37

temperature sensor

38

funnel

39

evaluation

40

Inventive chip calorimeter

41

computer

42

First electrical signal line

43

Second electrical signal line

44

Third electrical signal line

45

Fourth electrical signal line

46

measuring range

47

measuring channel

Claims (20)

Contraption ( 10 ) for determining the effect of nanoparticle materials on living cells by measuring the heat output production of the cells by means of a chip calorimeter ( 40 ) containing at least a chip calorimeter ( 40 ) - a flow measuring chamber ( 11 ) with a measuring channel ( 47 ) and with a supply channel ( 12 ) for delivery to heat-producing and living cell contacted with at least one nanoparticle material prepared magnetic bead samples ( 16 ) and with a discharge channel ( 13 ) for the bead samples ( 16 ) and one for a transport of bead samples ( 16 ) provided transport liquid ( 17 ), - a permanent magnet ( 18 ) on the surface ( 19 ) of the flow measuring chamber ( 11 ) and with its magnetic field strength into the flow measuring chamber ( 11 ), - a permanent magnet ( 18 ) holding swiveling device ( 20 ), wherein the pivoting of the permanent magnet ( 18 ) from the surface ( 19 ) away to a predetermined distance from the surface ( 19 ) and back, and - at least one thermopile ( 24 . 25 . 26 . 27 ), which are opposite the arranged permanent magnet ( 18 ) at the flow measuring chamber ( 11 ), further comprising the device ( 10 ) - a holding device ( 14 ), at which the flow measuring chamber ( 11 ) at least at the supply channel ( 12 ) and / or at the discharge channel ( 13 ) is suspended vertically, - a pump unit ( 28 ), which is the discharge channel ( 13 ) and to a targeted discharge of the transport liquid ( 17 ), and - a bead sample trap ( 29 ) connected to the pump unit ( 28 ) and in which the bead samples ( 29 ) are stored. wherein the thermal power measurement in the chip calorimeter ( 40 ) at the in the flow measuring chamber ( 11 ) magnetically fixed bead sample ( 16 ) is performed after the decay of the thermal sample induced by the bead sample transport, wherein the thermal power produced by the cells in at least one of the thermopiles ( 24 . 25 . 26 . 27 ) is converted into electrical signals, the thermopiles ( 24 . 25 . 26 . 27 ) with an evaluation device ( 39 ) via signal lines ( 42 . 43 . 44 . 45 ) and to the evaluation device ( 39 ) transmit the electrical signals, and wherein the electrical signals in one computer ( 41 ) evaluation device ( 39 ) By means of programmable means and technical as well as biological algorithms are processed into representations, which reproduce the reaction of the living cells on the contact with the material of the nanoparticles on the basis of the measured, produced by the cells heat output. Device according to claim 1, characterized in that the chip calorimeter ( 40 ) in a thermostat ( 15 ) to which the flow measuring chamber ( 11 ) is thermally coupled and a partial thermostatting of the transport liquid used for transport ( 17 ) causes. Device according to claim 1, characterized in that the supply channel ( 12 ) and the discharge channel ( 13 ) Represent cannulas and are realized as steel capillaries. Apparatus according to claim 1, characterized in that the pivoting device ( 20 ) one between the permanent magnet ( 18 ) and the holding device ( 21 ) mounted spring ( 22 ) and one on the spring ( 22 ) attached tendon ( 23 ), the tendon ( 23 ) for the deflection of the spring ( 22 ) away from the surface ( 19 ) of the flow measuring chamber ( 11 ) serves. Apparatus according to claim 1, characterized in that the pivoting device ( 20 ) one between the permanent magnet ( 18 ) and the holding device ( 21 ) mounted spring ( 22 ) as well as one with the permanent magnet ( 18 ) has corresponding movable magnet, which by means of its mobility in the direction of the permanent magnet ( 18 ) the permanent magnet ( 18 ) from the surface ( 19 ) and from its magnetic action area with respect to the flow measuring chamber ( 11 ) leads out and depending on the polarity reversal of the magnet back to the surface ( 19 ). Apparatus according to claim 1, characterized in that the Beadprobenfalle ( 29 ) connected to the pump unit ( 28 ) is connected, at least one filter magnet ( 30 . 31 . 32 ) containing the bead samples ( 16 ) from the flowing transport liquid ( 17 ) filter out and attach to itself. Apparatus according to claim 6, characterized in that between the pump unit ( 28 ) and the bead sample trap ( 29 ) a tee ( 33 ) is attached, at whose branch line ( 34 ) an injection ( 35 ) is attached, with the cleaning liquid ( 36 ) into the bead sample trap ( 29 ) for cleaning the bead samples still occupied by cells or residual cells ( 16 ) can be performed. Apparatus according to claim 1, characterized in that in the region of the holding device ( 14 ) in contact with the supply channel ( 12 ) a temperature sensor ( 37 ) is attached. Device according to claim 1, characterized in that the supply channel ( 12 ) a funnel ( 38 ) for filling transport liquid ( 17 ) with the prepared bead sample ( 16 ) in the supply channel ( 12 ) assigned. Apparatus according to claim 9, characterized in that for the flooding and removal of Beadproben ( 16 ) provided transport fluid ( 17 ) Is water or a liquid culture medium. Device according to claim 1, characterized in that a magnetic bead sample ( 16 ) for carrying out the measurements before the introduction into the feed channel ( 12 ) consists of at least one unprepared magnetic bead, one living magnetic cell prepared magnetic bead, one living magnetic bead already contacted with nanoparticles, or one prepared with living nanoparticle contacting cells. Device according to claim 11, characterized in that the magnetic bead samples ( 16 ) of at least one unprepared magnetic bead or of a living cell prepared magnetic bead substantially for comparison measurements with the magnetic bead samples of a living bead already contacted with nanoparticles prepared magnetic bead and / or living cells permanently contacted with nanoparticles Serve prepared magnetic bead. Device according to claim 11 or 12, characterized in that a magnetic bead sample ( 16 ) thus consists of a magnetic bead with adsorbed living cells applied to the surface thereof, which had either a time-defined contact treatment with nanoparticles or no contact treatment with nanoparticles before being introduced into the flow-measuring chamber, wherein the influence of the material of the nanoparticles on the living cells their metabolic Heat output changed. Device according to claims 11 to 13, characterized in that the magnetic beads used are composed of a plurality of individual particles of paramagnetic iron oxide (γ-Fe ₂ O ₃ , maghemite) and have a pearl-like shape or drop-like form of aggregation, that of a protective layer surrounded by strength, the aggregation of Polyethylene imine is coated as a ligand, which is a strong nonspecific anion exchanger. Chip calorimeter ( 40 ) for measuring the heat output of magnetic samples ( 16 ), comprising at least - a flow measuring chamber ( 11 ) with a measuring channel ( 47 ) and with a supply channel ( 12 ) for feeding samples ( 16 ) and with a discharge channel ( 13 ) for the samples ( 16 ), in which a transport of the samples ( 16 ) by means of a transport liquid ( 17 ) via a to the discharge channel ( 13 ) connected pump set ( 28 ), - at least one thermopile ( 24 . 25 . 26 . 27 ) for detecting the heat output and conversion into electrical signals, - one via signal lines with the thermopile ( 24 . 25 . 26 . 27 ) associated evaluation unit ( 38 ), which evaluates the electrical signals by means of predetermined program-technical means, characterized in that the measuring channel ( 47 ) of the flow measuring chamber ( 11 ) is vertically aligned and further arranged: - a permanent magnet ( 18 ) on the surface ( 19 ) of the flow measuring chamber ( 11 ) and with its magnetic field strength into the flow measuring chamber ( 11 ) and for fixing the magnetic samples ( 16 ) serves, - a permanent magnet ( 18 ) holding swiveling device ( 20 ), wherein the pivoting of the permanent magnet ( 18 ) from the surface ( 19 ) away to a predetermined distance from the surface ( 19 ) back and forth takes place to cancel the fixation of the magnetic samples ( 16 ), and wherein at least one thermopile ( 24 . 25 . 26 . 27 ) for absorbing the heat output of the magnetic sample ( 16 ) with respect to the arranged permanent magnet ( 18 ) at the flow measuring chamber ( 11 ) is present. Method for determining the effect of nanoparticle materials on living cells by measuring the heat output production of the cells using a device ( 10 ) according to claims 1 to 14 including a chip calorimeter ( 40 ) according to claim 15, characterized in that comparison measurements between living cell-prepared magnetic bead samples ( 16 ) with respect to the produced thermal power of the living cells based on the presence or absence of a time-defined contact between the respective cell structure and the respective material to be investigated of the given nanoparticles, wherein the contact either time-defined before the introduction of prepared magnetic bead samples ( 16 ) into the flow measuring chamber ( 11 ) or permanently during passage through the flow measuring chamber ( 11 ) takes place, wherein the comparative measurements of an evaluation device ( 39 ) be evaluated. A method according to claim 16, characterized in that a contact treatment between the magnetic beads and the living cells adsorbed thereon and the nanoparticles both with different concentrations of the adsorbed cells and the nanoparticles and taking into account the concentration ratio between living cells and nanoparticles depending on the given evaluation modality performed become. A method according to claim 16 or 17, characterized in that by means of one of the evaluation device ( 39 ) associated computer ( 41 ) a representation of the cytotoxic effect of the nanoparticle materials by the calorimetric detection of the heat production of biological objects in the form of a metabolic heat output and their change due to the action of the nanoparticle materials is carried out, which applies to the cells / objects to be examined by the heat output of the cells measured on cultures where: • the cultures are in the form of a biofilm or are immobilized by coupling through antibody-antigen interaction or electrostatically immobilized on magnetic beads; or • the cultures consist of one (clonal) or more (mixed culture) cell type (s), or • the cells to be tested are of prokarytic or eukaryotic origin or • the cells to be tested are naturally occurring cells or genetically modified cells or • the cells to be tested are artificially maintained cell cultures (eg He-La cells). Method according to claims 16 to 18, characterized in that, when testing the effect of the material of nanoparticles on microorganisms, the microorganisms are applied in the form of biofilms which are bound to magnetic beads as bead samples ( 16 ), which are • due to biofilm formation on the bead samples ( 16 cultured microorganisms, because of the high degree of heterogeneity, are realistic models for assessing the cytotoxic effect of nanoparticle materials in the environment, and • culturing the biofilms on the magnetic bead samples ( 16 ) an automated bead sample transfer through the chip calorimeter ( 40 ). Process according to claims 16 to 19, characterized in that the thermal power measurement in the chip calorimeter ( 40 ) of the device ( 10 ) at least one in the Flow measuring chamber ( 11 ) magnetically fixed bead sample ( 16 ) after the decay of the bead sample transport in the thermopiles ( 24 . 25 . 26 . 27 ) triggered thermal disturbances at least in one of the on the surface ( 19 ) of the flow measuring chamber ( 11 ), with one computer ( 41 ) evaluation device ( 38 ) via signal lines ( 42 . 43 . 44 . 45 ), and thermal energy produced by the living cells, are equivalent to thermopiles ( 24 . 25 . 26 . 27 ) is carried out.

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