WO2000037920A1 - Method and device for electro-optical spectroscopy of individual particles - Google Patents

Abstract

In order to effect dielectric spectroscopy on at least one suspended particle (11) in a microsystem, the particle (11) is exposed to high frequency electric rotating fields in an electrode array (16) and maintained in the focus (14) of an optical trap.

Description

 Method and device for electro-optical single particle spectroscopy

The invention relates to methods for dielectric single particle spectroscopy in microsystems and devices for their implementation.

For the measurement of the passive electrical properties of particles or micro-objects suspended in liquids (such as latex particles, living cells etc.), rotary movements induced by electrical fields of rotation have long been used [overview m ZIMMERMANN, U. et al. , Electromampulation of Cells, CRC Press Inc., 1996]. This can be a rotational movement m or opposite to the direction of rotation of the field. Passive electrical properties can be inferred from the speed of rotation of the object as a function of the angular speed of the field (so-called rotation spectra). As a rule, the rotational speeds of the objects range from 100 revolutions per second up to 1 revolutions per minute, typically slower than 1 revolutions per second.

This method has proven to be comparable and high-resolution to impedance measurement, especially for biological-medical questions. The rotation of the object is proportional to the imaginary part of the Clausius-Mosotti factor, [cf. also JONES, T.B., Electromechanics of Particles, Cambridge University Press, Cambridge, 1995].

It is disadvantageous, however, that in addition to the induced torque, a force, the so-called dielectrophoresis, always occurs towards or away from the electrodes. Thereby - WO βσ / 37920 PCT / EP99 / 10278

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the particle misaligned or the measuring time is shortened uncontrollably. Both stand in the way of an automatic measurement.

Attempts to automatically record the rotation measurement have been described several times [DE 33 25 843; DD WP 281223 (1986)]. This is the alternating application of two excitation fields with different directions of rotation, in which the connection tents can be changed electronically. These are varied until the object comes to a standstill. The standstill of the object has so far also been determined exclusively by visual observation.

It is also known to detect the movement of complex structured objects using image processing systems. For this purpose, the microscopically generated image is electronically recorded and stored at various points in time and then an attempt is made to reconstruct the movement that has taken place via a corresponding spatial transformation of a large number of image points. These methods have the disadvantage of a high density of information processing and thus lengthy and complex computer processing. Particularly great difficulties arise when the object structure changes during the measurement, e.g. when the focus plane is shifted, and when objects are poorly contrasted.

Cell movement can also be determined automatically using dynamic light scattering methods [GIMSA, J., PRUGER, B., EPPMANN, P. and DONATH, E., Electrorotation of particles measured by dynamic light scattering - a new dielectπc spectroscopy technique, m " Colloids and Surfaces A ", Vol. 98, 243-249, 1995]. However, this method cannot be used on individual objects, but provides average values for all particles that are in the laser beam. As a rule, this is a few hundred or more. The exact positioning at one point in the electrical rotation field was achieved by using 3-dimensional electrode arrangements, so-called field cages, and the alternating application of a centering field and a rotation field. It turns out, however, that the rotation of the objects is slowed down to 1/10 to 1/40, which complicates evaluation and extends the measuring times (DE 196 53 659 Cl, as well as Schnelle, Th., Glasser, H., Fuhr, G., An opto-electronic technique for auto atic detection of electrorotational spectra of single cells, m "Cellular Engineering" Vol. 2, 33-41, 1997).

There are also known optical field traps, also called "optical tweezers", "laser tweezers" or "optical traps", which have been used for about two decades in the fields of biotechnology, medicine and molecular biology and in other technical fields for positioning and manipulating micrometer-sized and submicron-sized particles are used [G. Weber et al. in "Int. Rev. Cytol." Vol. 131, 1992, p. 1; S.M. Block m "Nonmvasive Techniques m Cell Biology", Wiley-Liss., New York 1990, p. 375]. The development of the laser tweezers goes back mainly to A. Ashkm [A. Ashkm m "Phys. Rev. Lett.", Vol. 24, 1970, p. 156]. The principle of particle capture by optically induced forces is based on the fact that, in addition to the light pressure, which always pushes a particle away from the light source, gradient forces occur which lead to a particle getting a focus or being held stable with it or with it is moved. The prerequisite is that the absorption and reflection of the particle is low, while the difference in the refractive index from the surrounding solution should be as large as possible.

Laser tweezers have become more widespread in recent years because, with the same, always strong focusing of the light beam, both particles larger than that Wavelength (so-called Mie particles), as well as particles that are smaller than the wavelength (so-called Rayleigh particles) can be caught. These are primarily biological Whether _j such as cells, organelles and other cell components and large molecules ects (such as DNA) and artificial kropartikel M [SM Block et al. in "Nature", 1990, p. 348; JM Colon et al. in "Fertility and Steπlity", vol. 57, 1992, pp. 695 ff.].

By G. Fuhr et al. "Topics m Current Chemistry", vol. 194, Springer-Verlag Berlin, 1998, p. 83 ff. describes a microelectrode system in which electrical, optical or hydrodynamic forces act on suspended particles. The implementation of hybrid processes is described in which particles are manipulated alternately with electrical or optical forces. This use of electrical or optical forces is aimed at fulfilling certain goals in particle handling. The electrical forces are used in particular to carry out electrical measurements on the particles, as mentioned above. The optical forces, on the other hand, serve to manipulate the particles before or after the electrical measurement. The optical forces are exerted, for example, as a function of the result of the electrical measurement (sorting). The simultaneous effect of electrical and optical forces is not intended.

The interaction of electrostatic and optical forces in the detection of gradual adjustments of biological cells in microsystems is described by M. Nishioka et al. m "IEEE Transactions on Industry Application", vol. 33, 1997, p. 127 ff. Under the effect of the radiation pressure of a laser, the cells to be examined are printed against a cover glass over an electrode arrangement, with which switchable electrostatic fields are generated. The cells are examined in relation to their orientation in the electrical fields. The manipulation of the cells remains with this so-called optoelectrostatic technique limited to low adjustment rates. It was found that at adjustment speeds with typical times of 0.5 s the cell movement no longer follows the desired manipulation steps. Another disadvantage of the method described by M. Nishioka et al. The technique described consists of restricting it to non-spherical particles or particles with an inhomogeneous structure. The above-mentioned measurement of passive electrical properties by recording rotation spectra is not possible with optoelectrostatic technology.

The invention has for its object to provide new methods for dielectric single particle spectroscopy in microsystems and devices for their implementation, with which the above. Problems can be solved and, in particular, the particles in a rotating field, regardless of whether attractive or repulsive dielectrophoretic forces occur, are kept floating in a solution at any point with an accuracy below the particle radius in the rotating field, without reducing the speed of rotation.

This object is achieved by the combination of an optical catching beam (“optical tweezers”) and one or more rotating electrical fields of variable angular velocity with the features of claims 1 and 6, respectively. Advantageous embodiments and applications of the invention result from the dependent claims.

Important aspects of the invention consist in particular in that a suspended particle (particle), which can be artificial or also biological in nature, is caught in a strongly focused laser beam, as is known from optical tweezers. The capture point of the laser with the particles in it is now guided between microelectrodes, which are usually planarly applied to a smooth substrate, until the Particles are located in the area of the electric field spreading out in the solution, provided the electrodes are exposed to high-frequency, phase-shifted alternating voltage signals in a suitable manner. The particle is in a free suspension state. It is held by the interaction of electrical and optical forces.

The laser focus is expediently positioned on a line which is perpendicular to the point which denotes the field minimum between the electrodes. Even if forces are developed by the rotating electric field that want to pull the particle to the electrodes, they act at this location in all electrode directions pretty evenly, so that, according to the invention, only very small forces are required to keep the particle stable in spite of the field-induced attractive forces Hold laser focus. On the other hand, the intensity of the laser must be chosen so high that the particle is raised. If the particle is repelled by the electrodes via the electrically induced polarization forces, the forces of the optical field can be selected even less, since the particle itself centers on the designated line of symmetry. Here, however, it is raised and pushed out of the electrode area. This force must be compensated for by the choice of the intensity of the laser beam. This particle, which is caught very optically by m free solution, experiences a torque through the rotating electrical field and can be shifted depending on the frequency m in the manner known per se m slow rotation.

It is therefore a method and an electro-optical device for automatic rotation measurement on individual microparticles, in particular for measuring the speed of rotation of living cells as a function of the rotation frequency of an electrical field, the object being held in the rotation field using a laser focus he follows. A particular advantage of this method is that both forces (optical and electrical) complement each other almost without interaction and can be alternately reduced. The optical capture field is also independent of the conductivity of the suspension solution, so that m conductive to poorly conductive solutions can be used in an unlimited manner, which was previously not possible. A further advantage is then that, according to the invention, electrodes which are very close together can be used and can therefore be measured with lower amplitudes depending on the application. Furthermore, new electrode shapes can be used to generate field gradients, which was previously impossible. This considerably extends the field of application of dielectric spectroscopy.

Due to the extremely precise (to a micrometer and less) precise positioning of the particles, automatic measuring methods can now be m very easily, e.g. Adapt the image recognition and the scattered light measurement etc. for the automatic detection of the rotation spectra of the particles. This is done in a known manner, e.g. through microscopic observation.

In the following, the essential features of the invention are explained in the exemplary embodiments shown in the drawings. Show it:

FIG. 1: an overview representation for holding a particle according to the invention in a quadrupole arrangement for dielectric spectroscopy with laser tweezers;

Figure 2 is a graph showing the dependence of the torque or the pushing or pulling forces on the frequency f of

Field of rotation (ω = 2 π f); FIG. 3: an overview representation for the combination according to the invention of a microsystem for dielectric spectroscopy with a microscope arrangement, and

Figure 4: a schematic illustration of a microsystem for Screenmg tasks.

Figure 1 shows a perspective view of the arrangement. Details of the microsystem that are known per se are not shown. A particle 11, suspended in an ambient solution 12, is located in the radiation field of a strongly focused laser beam 13 and is caught in the focus 14. Four planar electrodes 16a to 16d, which are usually planar on a substrate 15, are phase-shifted signals by 90 degrees (phase angle 0 °, 90 °, 180 °, 270 °) of the same frequency (amplitudes, for example, about 1 to 20 V) controlled so that a rotating field m of the xy plane is created. Correspondingly, due to the strong frictional forces, the captured particle rotates much more slowly than the field rotates compared to the suspension liquid 12. The speed of rotation of the object as a function of frequency is determined by measuring or observing the particle and provides the desired rotation spectra. Alternatively, 3 or more electrodes can also be used in one plane, of greater thickness as well as in a multilevel arrangement.

The microsystem shown in FIG. 1 can advantageously be equipped with resonance devices for forming a resonant increase or damping of the field strength of the alternating electrical fields at predetermined frequencies, as described in PCT / EP96 / 05244. The content of patent application PCT / EP96 / 05244 is hereby incorporated in its entirety by express reference to the content of the present description. This applies in particular to all measures for generating resonance Nanzeschemungen m particle suspensions in microelectrode arrangements.

FIG. 2 shows a rotation spectrum (curve 21, describing the spectrum of a living cell) and the associated dielectrophoretic force (curve 22). It can be seen that the cell was pulled to the electrodes without the optical capture field in the frequency range (ω) between 20 Hz and 1 GHz. The curves shown are a measurement on a 20 μm cell in an aqueous solution with a conductivity of ImS / m, as is typical for algae. As a result, it has hitherto not been possible to measure in this frequency range or only with reduced accuracy, as was explained above. According to the invention, the force force represented by curve 22 is compensated for with the laser tweezers. Accordingly, the laser tweezers are used with such operating parameters that a sufficiently large trapping force is exerted on the particle.

FIG. 3 shows a device that is transparent at least on one side, with further details with which the rotation measurements can be carried out. Planar electrodes 32a to 32d are processed on a substrate 31 (for example glass) using the means of semiconductor technology and AC signals are applied via the feed lines 33a to 33d for generating the rotating field. A channel is formed through the side walls 34a, 34b and the cover plate 35, which is less than 250 μm thick, through which the particle suspension can be wound (36, arrow direction). The channel ceiling is made of glass, so that a lens 37 with a high numerical aperture, for example also as an olim ersionsobj ektiv (01 38) can generate a highly focused laser focus in the channel interior, with which the particle 39 is captured. Analogously, further electrodes can be inserted and the laser beam or the channel can be shifted relative to one another. The entire system can be implemented as an addition to a microscope. A preferred application of the invention is explained below with reference to FIG. 4. FIG. 4 shows a microsystem which is designed for the construction of a test system (assay system) for the high-throughput screenmg. The microsystem 40 has a channel structure 41a, 41b. A suspension with particles that are to be tested flows through the first channel 41a. The particles include, for example, biological cells or modified synthetic particles or combinations of biological cells and synthetic particles. The second channel 41b, through which a solution or suspension of a test substance flows, flows into the first channel 41a. The test substance preferably comprises ligands, for example antibodies.

Upstream of the junction of the second channel 41b, a first electrode system 42a is attached to the first channel 41a, which is constructed, for example, like the microelectrode system according to FIG. 1. A second electrode system 42b is arranged downstream of the mouth point. The electrode systems 42a, 42b are designed for rotational measurements on the particles 43 before or after the interaction with the test substance. Each electrode arrangement is equipped with a laser device and a microscope arrangement for generating the optical traps (see FIG. 3). A test procedure could be implemented as follows, for example.

First, the inflowing particles 43 in the first electrode system 42a are subjected to a first rotation measurement (with simultaneous holding with an optical trap). After mixing with the test substance there are interactions (eg particles 43a), the influence of which on the particle properties is detected by the second rotation measurement in the electrode system 42b. The changed properties can be absolutely by characteristic spectra or relatively by comparing the spectra of the first and second rotation measurements can be determined. After the second rotation measurement, the particles are released from the second electrode system, possibly subjected to further rotation measurements in further electrode arrangements (not shown) and, depending on the measurement result, subjected to a sorting process.

Typical applications for a test system according to FIG. 4 are binding studies and the determination of kinetic parameters (association constant, dissociation constant) in the context of the characterization of interactions between biomolecules with one another or with small organic or inorganic molecules. Biomolecules and associated application examples can be: antigen antibodies, in studies for the development of immunoreagents and immunoassays, epitope mapping and screen mg from phage libraries; Ligands and their cell membrane receptors; Cell adhesion molecules and their ligands, for example in studies of the affinity between cadherms and integers and their receptors located in the cell membrane; Membrane molecules, such as lipids or glycoprotems, when studying the interaction of these molecules with other soluble or cell membrane-containing biomolecules; extracellular matrix molecules and their soluble or cell membrane-bound ligands; intracellular messenger substances, in the investigation of signal transmission within the cell through the interaction of molecules in a signal transmission cascade; soluble proteins and peptides, in the monitoring of the production of proteins and peptides in a biotechnological process; Enzymes and their substrates and cofactors, for example in the study of blood coagulation factors; Proteins or peptides and DNA or RNA molecules, for example in mutation analysis by binding differently strong DNA to certain proteins or studies of transcription factors; and surface molecules of viruses, bacteria, fungi, pias Moiden or other pathogenic microorganisms and the cell membrane of a host cell, for example for the development of anti-infective strategies.

A test system according to the invention can also be constructed from a multiplicity of microsystems according to FIG. 4, which interact in series or in parallel.

Claims

PATENT CLAIMS 1. A method for dielectric spectroscopy on at least one particle in a microsystem, in which the particle in an electrode arrangement is exposed to high-frequency electrical rotating fields and at the same time is freely suspended in the focus of an optical trap, with the spectra rotating spectra by measuring the rotational speed as a function of Frequency of the rotating electrical fields can be determined. 2. The method according to claim 1, wherein the optical trap is generated with a laser device which is operated with such a radiation intensity that the optical forces in the optical trap are stronger than field-induced attraction or repulsion forces of the electrode arrangement. 3. The method of claim 1, wherein a plurality of optical traps are used to hold a plurality of particles and are subjected to a rotation spectra measurement simultaneously or serially. 4. The method according to claim 1 or 2, in which a first and a second rotation spectra measurement are carried out on the particle, the particle being brought into interaction with a test substance between the rotation spectra measurements and after the second rotation spectra measurement the particle undergoing a sorting process as a function of a modification of the dielectric properties of the particle is subjected to the interaction. 5. The method according to claim 4, wherein at least one further test substance is supplied after the second rotation spectrum measurement and at least one further rotation spectrum measurement is carried out. 6. Device for dielectric spectroscopy on at least one suspended particle in a microsystem, which comprises at least one electrode arrangement for forming electrical rotation fields and a device for forming at least one optical trap in the effective range of the electrode arrangement. 7. The device according to claim 6, which has a laser device and a microscope arrangement for forming the optical trap in the microsystem. 8. The device according to claim 6 or 7, in which a first channel (41a) with a first (42a) and at least a second (42b) electrode arrangement is provided in the microsystem, each with a first and a second laser device for forming optical traps, wherein between the electrode arrangements (42a, 42b) m the first channel (41a) at least one second channel (41b) for supplying test substances and a sorting device is provided downstream of the electrode arrangements. 9. Use of an optical trap (laser tweezers) to hold particles freely suspended in electrical rotating fields.