DE102008062620A1 - Detection device i.e. quartz microbalance, for detecting e.g. protein in fluid sample, has flat metallic thick- or thin layer arranged as electrode at channel wall, where ligands above electrode are immobilized on measuring surface - Google Patents

Abstract

The device has a measuring channel (2) at which two openings (11, 12) are provided for supply and discharge of a fluid sample. A flat metallic thick- or thin layer is arranged as an electrode (1) at a channel wall between the openings, and electrically conductive strip-shaped thin layers are arranged as a set of electrodes opposite to the electrode. An electric voltage potential is maintained at the set of electrodes for forming an inhomogeneous electric field. Ligands above the electrode contact the sample, are in communication with the channel and are immobilized on a measuring surface. An independent claim is also included for a method for detecting analyte molecules in a fluid sample.

Description

The The invention relates to an apparatus and a method for detection of analyte molecules contained in liquid samples. These are in particular proteins or DNA.

there is usually done so that the liquid Sample flows through a measuring channel, in which for the respective analyte molecules specific ligands Measuring surfaces are immobilized, to which the analyte molecules can connect. After connection, a detection takes place in which it can be determined whether the respective analyte molecules are in the sample is included or not. It can also be a quantitative one Determination be carried out.

The Analyte molecules are more in the liquid sample or less evenly distributed and the measuring channel has a certain required volume. As a result flows due the liquid sample with a minimum layer thickness the measuring channel. But preferred is a complete filling of measuring channels. The analyte molecule transport too the immobilized ligand takes place essentially by Convection and diffusion. Near the surface of measuring surfaces where ligands are immobilized, a layer is formed in which diffusion essentially occurs. This is called the Nernst-type diffusion barrier layer. Of the Transport of analyte molecules to ligands is thereby hindered, this effect is as the thickness of the diffusion layer increases strengthened.

To counteract these disadvantages and to increase the binding rate of analyte molecules and accelerate the binding was in DE 10 2007 012 866 A1 proposed to guide through a flow channel a main stream formed with an inert liquid. In these main streams free of analyte molecules, a feed for liquid sample should then be arranged in front of the actual measuring surfaces. The main flow can be used to displace the liquid sample in the direction of the measuring surfaces with the ligands immobilized there. The liquid sample can thus flow as a thin film over the measuring surfaces.

It is obvious that by the required larger free cross section of the river channel an enlargement of the entire system. With the main stream can a dilution effect for the sample is not avoided become. Also, it can not be specific or selective the binding behavior of certain analyte molecules influence be taken.

Of Further, it is known that by means of dielectrophoresis a separation or sorting of nanoparticles or biomolecules is possible.

at Dielectrophoresis becomes an inhomogeneous electric field Manipulation of particles or molecules used in to induce a dipole moment in the particles / molecules which interacts with the electric field. So can a force effect be exerted on the particles / molecules that to a stop in a desired position as well can lead to a movement. Particles experience a translational force and move into areas of high field strength, so speaks one of positive dielectrophoresis. The particles move in Areas with low field strength are called negative Dielectrophoresis. The force acting on particles or molecules is proportional to the volume of particles or molecules. They can therefore be dependent on their volume captured in a "field cage" and be held in it.

For a polarizable part with a spherical shape, the following applies to a good approximation: F dep = 2π 3 ε 0 ε 1 R e {f cm } | E | 2 with f _cm - Clausius-Mossotti factor, ε - dielectric constant, E - electric field strength and a ³ - volume of the part.

The Clausius-Mossotti factor depends on the frequency, the electrical conductivities of the medium and the particles as well as the dielectric constant. Thus, in R _e {f _cm }> 0 positive deteetrophoresis occurs and in R _e {f _cm } <0 negative dieletrophoresis.

The achievable direction of acting on particles / molecules Forces depend on the frequency with which electric field is formed.

It The object of the invention is therefore the binding rate and the selectivity the binding of analyte molecules in liquid Samples are included to increase in immobilized ligands or to improve.

According to the invention to accomplish this task with a device having the features of the claim 1, solved. It can with a method according to claim 10 are worked. Advantageous embodiments and developments of the invention can be with in subordinate claims designated characteristics can be achieved.

By utilizing dielectrophoresis, analyte molecules distributed within a liquid sample are to be moved while flowing through a measurement channel by an achievable force action towards ligands immobilized within the measurement channel, so that they can bind to it. This can increase the binding rate, the time required is shortened and the selectivity improved.

at a device according to the invention are at the measuring channel, through which the liquid sample flows, at least two openings for the supply and removal of the sample available. In the area of the measuring channel is on a channel wall between the openings at least one metallic thick or thin film formed flat. This forms an electrode. Above This electrode has at least one measuring surface in the measuring channel formed on the ligands for analyte molecules im mobilized.

At the opposite of this electrode channel wall a plurality of electrically conductive strip-shaped thick or thin films, which are also electrodes form. These strip-shaped electrodes are connected to one electrical AC voltage source connected. Thereby the device becomes so operated and the connection of the strip-shaped electrodes takes place so that each juxtaposed strip-shaped Electrodes have a different electrical voltage potential. In this case, a negative electrical voltage potential is preferred on a strip-shaped electrode and on the next arranged strip-shaped electrode has a positive voltage potential available at the same time. The flat opposite arranged electrode has a constant electrical depending on the mode Potential on or is potential-free switched.

flows now the liquid sample through the measuring channel can at a given frequency specifically a force effect with the electric Field to be exerted on specific analyte molecules, so that these are immobilized in the direction of the measuring surface Ligands can be accelerated and connect to it.

To the binding can then be carried out the detection where different measuring principles can be applied.

So For example, below or above the at least one planar electrode be arranged an optical waveguide, be coupled into the monochromatic electromagnetic radiation can.

The Beam guidance and training of the optical waveguide can preferably be chosen so that at the interface of the optical waveguide in the direction of flat electrode preferably attenuated total reflection occurs.

This allows excitation of surface plasmon resonance, where the changing optical refractive index is a measuring signal can represent when the optical fiber below the flat Electrode is arranged. In this case, the thin film for the flat electrode preferably made of gold be formed.

It But there is also the possibility of fluorescence excitation This is then within the above the interface of the Fiber optic evanescent field with defined Penetration depth in the measuring channel is possible. analyte can do this with a suitable fluorophore dye be marked. For the detection then an optical Detector can be used. In this case, the optical waveguide is preferred be arranged above the planar electrode.

The Strip-shaped electrodes should be perpendicular to the flow direction the liquid sample flowing through the measuring channel aligned and be present in a number of at least 50. The strip-shaped electrodes are liquid Sample electrically isolated. The distance of the strip-shaped Electrodes to the planar electrode should be the same be great. This corresponds approximately to the height of the Measuring channel, which can be in the range 50 to 100 microns. she should also be arranged equidistantly to each other and each have the same dimensions. This is their length, Width and thickness. The distance from juxtaposed strip-shaped Electrodes can be selected in the range 5 to 50 μm become. The thickness can be in the range of 50 nm to 5 μm. The length should be at least approximately the width of the measuring channel correspond. The applied to the strip-shaped electrodes electrical voltage can be between 10 and 40 V. This can an electric field strength up to 800 kV / m can be achieved.

With Such training and choice of these parameters can Analyte molecules with a particle size be successfully manipulated in larger 2 nm.

The measuring surface with the immobilized ligands and / or the planar electrode should preferably not be arranged centrally over the length of the measuring channel. Under certain conditions, it is favorable to be able to choose an arrangement closer to an opening via the liquid sample from the measuring channel. With such an asymmetrical arrangement, a force effect can already be exerted with stripe-shaped electrodes arranged in front of and / or behind the flow direction of the sample, with which analyte molecules are moved in the direction of the corresponding channel wall of the measurement channel to which ligands are immobilized. In this case, the strip-shaped electrodes can be arranged on the channel wall opposite the measuring surface or on the same channel wall be. The particular arrangement can be taken into account by suitable choice of the frequency with which the electrical voltage is applied to the strip-shaped electrodes. Only in the area of the planar electrode should the strip-shaped electrodes be arranged on the opposite channel wall.

Advantageous it is, on a device according to the invention to provide an identification element, the data of the respective Sample can be taken. This should be contact and read without contact, which, for example, with a barcode or with RFID technology is possible.

As already mentioned may be the frequency and the strip-shaped Electrodes applied electrical voltage can be chosen so that the respective volume and the Clausius-Mossotti factor of the analyte molecules can be considered to be an improved selective Attaching certain analyte molecules to ligands to achieve.

It can work with negative or positive dielectrophoresis become.

at However, it is also particularly advantageous for the invention that a Replacement of unspecifically bound foreign molecules becomes possible. It can be used instead of the sample after the connection of molecules, as previously described, a rinsing liquid, in which no corresponding molecules are contained, the that is to say inert for the detection, flow through the measuring channel. At the same time, a force effect is achieved by means of dielectrophoresis Molecules exerted, the already attached foreign molecules from ligands and then these with the rinsing liquid can be removed from the measuring channel. Therefor is only with changed frequency of the electrical voltage to work or the previously potential-free area-trained Electrode to a constant electrical potential. To the Peeling off can be done in two stages and thereby in each case a frequency are selected, with the negative and then positive dielectrophoresis or vice versa occurs. For a positive dielectrophoresis should have a frequency range of 100 kHz to 1 MHz and for negative dielectrophoresis a frequency range from 1 MHz to 10 MHz.

The However, the device according to the invention can also be used in Form of a quartz microbalance be formed. In this case is on the lower side of the channel a flat system, formed with three layers arranged one above the other is available. These layers are an electrode, a quartz and another electrode. The upper electrode is flat Electrode formed, as explained in advance traveled has been.

at the implementation of the invention Procedure, the liquid sample also through several times be guided the measuring channel. The sample can be in circulation but also be pumped back and forth, so with flow alternating flow direction through the measuring channel. For this suction and / or pressure pumps can be used. To a device according to the invention can Container for liquid sample connected or also be trained. It should, however, be guaranteed be sure that liquid sample is safely completed and can not enter the environment in an uncontrolled manner.

following the invention is explained in more detail by way of example become.

there demonstrate:

1 an exploded view of an example of a device according to the invention;

2 in three sectional views, the procedure for binding of analyte molecules and subsequent detachment of non-specifically bound foreign molecules in an example and

3 in three sectional views, the procedure for binding of analyte molecules and subsequent detachment of non-specifically bound foreign molecules in another example.

With the exploded view of 1 an example of a device according to the invention will be clarified.

Here is a base part 6 present, on the upper side in the right part here a thin gold layer as a flat electrode 1 is trained. It is an optical fiber 5 integrated, the at least partially the flat electrode 1 overlaps.

There is also a recording 7 for an RFID chip 8th , as an identification element available.

On the surface of the base part 6 can a slide 9 are glued, in which an opening is formed, which the measuring channel 2 forms.

On the slide 9 can be a completion plate 10 glued on the underside, ie in the direction of the measuring channel, the strip-shaped electrodes 3 (not visible here) are formed. In the end plate 10 are two openings 11 and 12 formed, through the liquid sample into the measuring channel 2 and can be removed from it again. For the sealing are two sealing rings 13 and 14 intended. The whole thing can go up with a cover 15 be completed, which may be provided with a device for temperature control. All of these elements can be optically transparent.

For a temperature but also suitable lines on the top of the end plate 10 be arranged or trained on it.

For a tempering can be present through lines which flows a tempered liquid. It can but also be provided an electrical resistance heating, at the conductor tracks are formed as thin films.

In 2 three sectional views are shown. The liquid sample flows through the measuring channel 2 left to right. At the lower channel wall is the planar electrode 1 and then a measuring surface 4 , are immobilized on the ligands arranged.

At the upper channel wall of the measuring channel 2 are strip-shaped electrodes 3 trained and connected to an electrical AC voltage source that can be operated with adjustable frequency.

The different electrical voltage potentials of the strip-shaped electrodes 3 are indicated by the characters "plus" and "minus".

With the top representation, the procedure for binding molecules to ligands should be recognizable. In this case, a frequency of the electric voltage for the strip-shaped electrodes 3 chosen, occurs at the negative dielectrophoresis. It becomes clear that the molecules contained in the liquid sample are able to move in the direction of the lower channel wall and consequently the measuring surface due to the force effect achievable with the electric field and the dipole moment 4 to be moved.

After binding of molecules to ligands, an inert rinse liquid, for example a buffer solution, is passed through the measurement channel 2 guided and the frequency of the electrical voltage for the strip-shaped electrodes 3 chosen so that positive dielectrophoresis occurs. As a result, certain non-specifically bound foreign molecules can be replaced by ligands again. The acting force on these foreign molecules is opposite to the direction of force when tying. It can only be replaced foreign molecules. The binding of analyte molecules to ligands is retained (see middle section of 2 ).

Alternatively, the frequency of the electric voltage for the strip-shaped electrodes 3 chosen so that negative dielectrophoresis occurs again. (see the lower illustration of 2 , In this case, the flat electrode is formed 1 to a constant electrical potential, which sets a gradient field, which leads to the detachment of bound foreign molecules.

With 3 Another possibility for attachment and detachment of molecules to ligands will be shown in three sectional views.

The liquid sample flows through the measuring channel again 2 left to right. At the lower channel wall is the planar electrode 1 and then a measuring surface 4 , are immobilized on the ligands arranged.

At the upper channel wall of the measuring channel 2 are strip-shaped electrodes 3 above the planar electrode 1 and the measuring surface 4 trained and connected to an electrical AC voltage source that can be operated with adjustable frequency.

In this example, however, strip-shaped electrodes are in the flow direction of the liquid sample in front of the planar electrode 1 and the measuring surface 4 arranged on the lower channel wall.

The different electrical voltage potentials of the strip-shaped electrodes 3 are indicated by the characters "plus" and "minus".

In the uppermost diagram, the procedure for attaching molecules to ligands should be recognizable. In this case, a frequency of the electric voltage for the strip-shaped electrodes 3 located on the lower channel wall of the measuring channel 2 are arranged, in which positive dielectrophoresis occurs. It can be seen that the molecules contained in the liquid sample are able to move in the direction of the lower channel wall and consequently of the measuring surface due to the force effect achievable with the electric field and the dipole moment 4 to be moved. In this case, arranged on the upper channel wall strip-shaped electrodes 3 floating.

After binding of molecules to ligands, an inert rinse liquid, for example a buffer solution, is passed through the measurement channel 2 guided and the frequency of the electrical voltage for the strip-shaped electrodes 3 which are arranged on the upper channel wall are chosen so that positive dielectrophoresis occurs. As a result, certain non-specifically bound foreign molecules can be replaced by ligands again. The acting force on these foreign molecules is opposite to the direction of force when tying. It can only be replaced foreign molecules. The binding of analyte molecules to ligands is retained. This is from the middle representation of 3 out.

Alternatively, the frequency of the electric Voltage for the strip-shaped electrodes 3 chosen so that negative dielectrophoresis occurs (see lower section of 3 ). In this case, the flat electrode is formed 1 placed on a constant / fixed electrical potential, which sets a gradient field, which leads to the detachment of bound foreign molecules.

QUOTES INCLUDE IN THE DESCRIPTION

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Cited patent literature

• DE 102007012866 A1 [0004]

Claims (16)

Device for detecting analyte molecules contained in liquid samples, which bind to ligands immobilized on at least one measuring surface, thereby passing the sample through a measuring channel ( 2 ) flows, at the opposite at least two openings ( 11 . 12 ) are present for an intake and a removal of liquid sample, in the region of the measuring channel ( 2 ) on a channel wall between the openings ( 11 . 12 ) a flat metallic thick or thin layer, as an electrode ( 1 ) and the electrode ( 1 ) opposite to a further channel wall a plurality of electrically conductive strip-shaped thin films as further electrodes ( 3 ) are arranged, which are connected to an electrical AC voltage source, wherein at juxtaposed electrodes ( 3 ) a different electrical voltage potential, for the formation of inhomogeneous electric fields, is respected; while the ligands above the one electrode ( 1 ), with contact to the liquid sample and in communication with the measuring channel ( 2 ), on a measuring surface ( 4 ) are immobilized. Apparatus according to claim 1, characterized in that below the at least one planar electrode ( 1 ) an optical waveguide ( 5 ) in which monochromatic electromagnetic radiation can be coupled in such a way that at the interface to the at least one electrode ( 1 ) attenuated total reflection occurs and on the surface of the at least one electrode ( 1 ) Surface plasmon resonance occurs. Apparatus according to claim 1, characterized in that above the at least one planar electrode ( 1 ) an optical waveguide ( 5 ) is arranged in the monochromatic electromagnetic radiation can be coupled so that at the interface to at least one electrode ( 1 ) attenuated total reflection occurs, so that a fluorescence excitation can be achieved for a coupled to analyte molecules Fluorophorfarbstoff. Device according to one of the preceding claims, characterized in that the device as a quartz microbalance is trained. Apparatus according to claim 1 or 2, characterized in that the strip-shaped electrodes ( 3 ) aligned with their longitudinal axes perpendicular to the flow direction of the liquid sample and each with the same distance to at least one electrode ( 1 ) are arranged. Device according to one of the preceding claims, characterized in that the strip-shaped electrodes ( 3 ) are arranged equidistant to each other and each formed the same with their dimensions. Device according to one of the preceding claims, characterized in that at least 50 strip-shaped electrodes ( 3 ) each having a width and a distance from each other in Be rich 5 to 50 microns and a thickness in the range 50 nm to 5 microns are present. Device according to one of the preceding claims, characterized in that in the flow direction of the liquid sample, before and / or behind the one electrode ( 1 ), strip-shaped electrodes ( 3 ) on the same channel wall and in the region of one electrode ( 1 ) are arranged on the opposite channel wall. Device according to one of the preceding claims, characterized in that an identification element ( 8th ) is available. Method for detecting analyte molecules contained in liquid samples, which are attached to at least one measuring surface ( 4 ) immobilized ligand, while the sample flows through a measuring channel ( 2 ), with one in the area of the measuring channel ( 2 ) arranged on a channel wall flat metallic thick or thin film, as an electrode ( 1 ) and arranged on a further channel wall a plurality of electrically conductive strip-shaped thin films as further electrodes ( 3 ), which are connected to an electrical AC voltage source, inhomogeneous electric fields are formed and exerted by dielectrophoresis a force on the analyte molecules to increase the binding rate towards immobilized ligands and after binding of analyte molecules detection is performed. A method according to claim 10, characterized in that the frequency and electrical voltage with which the strip-shaped electrodes ( 3 ), taking into account the volume and the Clausius-Mossotti factor of the respective analyte molecules. A method according to claim 10 or 11, characterized in that the frequency is first selected so that the respective analyte molecules move in the direction of immobilized ligands and subsequently at a changed frequency or changed electrical potential of the flat electrode ( 1 ) by dielectrophoresis a replacement unspecifically bound foreign molecules of ligands is achieved. Method according to claim 12, characterized in that that for detachment of nonspecifically bound foreign molecules working with positive and negative dielectrophoresis. A method according to claim 12 or 13, characterized in that when detaching non-specifically bound foreign molecules, a rinsing liquid through the measuring channel ( 2 ) to be led. Method according to one of claims 10 to 14, characterized in that with positive dielectrophoresis in the frequency domain 100 kHz to 1 MHz and with negative dielectrophoresis in the frequency domain 1 MHz to 10 MHz is worked. Method according to one of claims 10 to 15, characterized in that the strip-shaped ( 3 ) Electrodes applied an electrical voltage in the range 10 to 40 V and the flat electrode ( 1 ) is set floating or at a constant electrical potential.