SMITH, Charles (21 Kimberley Road, Cambridge CB4 1HG, GB)
VIJENDRAN, Sanjay (27 Canterbury Street, Cambridge CB4 3QR, GB)
SMITH, Charles (21 Kimberley Road, Cambridge CB4 1HG, GB)
CLAIMS
1. An apparatus for driving small volumes of fluid, the apparatus comprising: a substrate; a first array of electrically conductive electrodes formed on the substrate; and a second array of electrically conductive electrodes formed on the substrate, the first and second arrays being interlaced and being arranged such that, in use, by applying a first alternating drive voltage thereto flow in a fluid adjacent to the arrays of electrodes can be generated; and means for generating negative dielectrophoresis in the fluid to repel microparticles therein from the surface of the electrodes.
2. The apparatus of claim 1 , further comprising means for providing the first variable alternating voltage to the first and second array of electrodes.
3. The apparatus of claim 1 or claim 2, wherein dielectric is provided over at least a portion of one or both of the electrode arrays to generate the negative dielectrophoresis.
4. The apparatus of claim 1 or claim 2 further comprising means for providing a second alternating voltage, at a frequency higher than that of the first alternating voltage, to the first and second array of electrodes in order to generate the negative dielectrophoresis.
5. An apparatus according to claim 1 or claim 3, wherein the relative dimensions of the first and second array of electrodes are selected with respect to the size of the micro-particles in the fluid such that negative dielectrophoresis is generated.
6. An apparatus according to any of the preceding claims, in which the electrodes and substrate are formed as part of a CMOS process.
7. An apparatus according to any of claims 1 to 6, arranged to be employed in a biochemical analysis process or drug manufacturing process.
8. A method for driving small volumes of fluid, the method comprising the steps of: providing a substrate; providing a first array of electrically conductive electrodes formed on the substrate and a second array of electrically conductive electrodes formed on the substrate, the first and second array being interlaced and being arranged such that application of a first alternating drive voltage thereto generates flow in a fluid adjacent to the arrays; and generating negative dielectrophoresis with the first and second electrodes to repel microparticles in the fluid from the surface thereof.
9. The method of claim 8, wherein the negative dielectrophoresis is generated by application of a second alternating voltage, at a frequency higher than the first alternating voltage, to the array of first and second electrodes.
10. A method according to claim 8, wherein the negative dielectrophoresis is generated by application of a dielectric on the surface of the first and second electrode arrays.
11. A method according to claim 8, wherein the negative dielectrophoresis is generated by selecting the relative dimensions of the electrodes of the first and second electrode or arrays with respect to the size of the micro particles in the fluid. |
Apparatus for driving small volumes of fluid
This invention relates generally to an apparatus such as an electronic device for the pumping of electrolyte solutions containing biomolecule-coated particles on substrates by electrical means. More specifically it relates to the selective use of applied frequencies and bead sizes to actuate the pumping while keeping the beads from binding to the electrodes. The device is capable of pumping solutions containing micrometre-sized particles without the need for special surface coatings on the pumping electrodes to eliminate the non-specific adhesion of beads to the electrode surface.
In chemical and biochemical analysis there is a need for the microparticles such as for pumping electrolytes containing beads or microspheres using AC electroosmotic flow on substrates for biochemical analyses while avoiding non-specific adhesion of the beads to the electrodes. Microparticles, also referred to as microspheres, or beads, are commonly used in biological assays as miniature solid supports for biomolecules. Microspheres have extremely high surface area to volume ratios by virtue of their small sizes and hence a solution containing a high concentration of microparticles offers a large surface area on which to bind biomolecules to enhance reaction kinetics in chemical or biological analyses. Furthermore, microspheres can be visualised easily using optical or fluorescence microscopy and hence read-out of the reaction products can be performed easily using the imaging methods described above. Such beads may be coated with biological molecules such as DNA strands, oligonucleotides, proteins or antibodies. The beads may alternatively encapsulate the biomolecules mentioned above.
One of the difficulties associated with the movement of microparticles in an AC EOF-pumped microfluidic system is that the pumping mechanism necessitates the exposure of the electrodes to the fluid that is pumped through the system. The AC voltages that are applied to these electrodes to facilitate the pumping also strongly attract beads that are close to the electrodes, causing unwanted irreversible adhesion of the beads to the electrodes. The beads are drawn towards the
electrodes by positive dielectrophoretic forces, which are induced by the asymmetry in the lateral electric fields gradients over the electrodes.
According to the present invention there is provided an apparatus for driving small volumes of fluid, the apparatus comprising: a substrate; a first array of electrically conductive electrodes formed on the substrate; and a second array of electrically conductive electrodes formed on the substrate, the first and second arrays being interlaced and being arranged such that , in use, by applying a first alternating drive voltage thereto, flow in a fluid adjacent to the arrays of electrodes can be generated; and means for generating negative dielectrophoresis in the fluid to repel microparticles therein from the surface of the electrodes.
The present invention also provides a corresponding method. The fluid pumping is induced by AC electroosmotic flow (EOF), while the repulsion of beads from the electrodes is induced by negative dielectrophoresis (DEP). The two effects are induced simultaneously by a number of different methods.
One example of the present invention will now be described with reference to the accompanying drawings, in which:
Figure 1 is a schematic representation of an example device according to the present invention; and
Figure 2 is a schematic view of a second example device according to the present invention; and
Figures 3a and 3b are images of a prior art example device before and after use; and Figures 4a and 4b are images of a device before and after operation.
One example of this invention is the application of novel methods to resist adhesion of latex microspheres or other types of microparticles to the surfaces of electrodes used for AC electroosmotic flow (EOF) pumping in microchannels. AC EOF is a technique for moving small volumes of fluid in microchannels by the application of a
low frequency AC voltage to one or more sets of interdigitated asymmetric planar electrodes, which are patterned on a substrate such as glass, plastic or silicon. This AC flow has been successfully applied to low-voltage pumping in microchannels.
The present invention employs dielectrophoresis (DEP) which is a phenomenon where a particle in a solution is polarised by a non-uniform electric field and a net force is exerted on the particle. The force is dependent on the induced dipole and does not depend on the direction of the field, only on the field gradient. As such, AC fields can be used to manipulate particles, and this has the advantage that any electrophoretic force (due to any net charge on the particle) is reduced to zero. Thus both charged or uncharged particles will have the same DEP response. The DEP force is given by,
where r is the radius of the particle, ω is the frequency of the applied field, e m is the medium permittivity, 9t{K(ω)} is the real part of the Clausius-Mossotti factor and E^ s is the strength of the applied electric field. The Clausius-Mossotti factor is given by
— e.
/C (α>)=-
+2 e n
where e p and e m are the complex permittivities of the particle and medium, respectively. The complex permittivity is defined as
e - e~ J\ ~ ) where 7 = v— 1 , e is the permittivity and σ is the conductivity of the
dielectric.
The Clausius-Mossotti factor depends not only on the dielectric properties of the particle and medium but is also frequency dependent. For a particle, the real part of K(ω) can vary from 1 to -0.5. For 9t{K(ω)} > 0, the particle is more polarisable than the medium and experiences positive DEP resulting in a force towards the region of
highest field strength. Conversely, it undergoes negative DEP when 9t{K(ω} < O, and the particle is then repelled from the region of high field intensity to the region of minimum field strength. Thus the force on the particle can be controlled by the variation of the applied frequency.
In one example of this invention, shown in figure 1 , a device which initially consists of a glass substrate patterned with interdigitated metal electrodes 1 and 2, using microfabrication techniques, is incorporated into a microfluidic system, where the electrodes are used for pumping fluid through the microchannels in the system. The electrodes can be made out of Au or TiN or any conductive material that is inert to biological molecules and nonreactive to low concentration aqueous salt solutions. The optimum frequency of the applied voltage to the electrodes to actuate high flow rate pumping depends, amongst other things, on the dimensions 3 and 4 of the electrodes, but it is typically found to be between 5 kHz and 20 kHz for electrode widths between 5 μm and 2 μm in salt solutions of low conductivities (< 1 mS/m). If the device is used to pump an aqueous solution containing a concentration (> 1x10 5 particles/mL) of microparticles within the size range of 0.5 μm to 10 μm (shown as 6 in Figure 1), the particles within a few microns of the electrode surfaces will be attracted to the electrodes, especially the electrode edges where the electric field is the strongest, due to dielectrophoretic effects.
In prior art arrangements the strong attraction results in an eventual permanent adhesion of a large number of these particles to the electrode surfaces, which reduces the efficiency of the AC pumping as well as the availability of the particles for their intended utilisation in the microfluidic system (for example, for biological assay purposes). The particles generally remain attached to the electrodes even after removal of the electric field, thus rendering the device unsuitable for further use. There are some applications where bead adhesion in this manner is actually intended and required for the proper functioning of the device. However in other applications, as in a microfluidic system for conducting assays in the solution phase, bead adhesion to the electrodes is not a requirement and is detrimental to the proper operation of the device. The level of adhesion of the particles to the electrode surfaces depends amongst other things, on the material that the electrodes and the particles are composed of, as well as any chemical or biological surface coatings on the particles such as DNA or proteins. In general, hydrophobic
interactions between biomolecule-coated particles, especially protein-coated particles, and metal electrodes are sufficiently strong to result in irreversible coating of the electrodes by the particles. Furthermore, strong non-covalent interactions such as Van der Waals forces also contribute to electrode-particle adhesion.
Current methods to reduce the fouling of electrodes in biodevices commonly involve coating the electrodes with a biocompatible material that resists adhesion of proteins and other biomolecules. An example of a widely used material is poly (ethylene glycol) or PEG. While the coating may well serve to eliminate bead or biomolecule adhesion, it would affect the operation of an AC EOF-pumped device, due to the sensitivity of the AC pumping mechanism to the surface chemistry at the pumping interface as well the as any change in the total capacitance of the device due to the surface coating. In light of such difficulties, this invention seeks to simplify the protection of the electrodes by eliminating any need for special coatings which may involve complex chemistries and difficult fabrication methods. As described earlier, particles which are close to the electrodes are attracted by strong positive DEP forces when frequencies in the kHz range are used in conjunction with particle sizes in the micrometre range. Therefore, the present invention provides for an apparatus and method to keep the particles away from the electrodes by ensuring that they experience a negative DEP force repelling them from the electrodes during the fluid pumping, rather than an attractive positive DEP force. This is achieved in one example by adding a high frequency AC signal to the low frequency signal that is used for the pumping. As explained earlier, microparticles experience either positive of negative DEP depending on their size, the permittivity of the surrounding medium and the Clausius-Mossotti factor, K(ω). For example, in the case of uniform latex microspheres, there is a cross-over frequency at which the force goes from attractive to repulsive which typically occurs at frequencies much higher than 5 kHz for particles in the micrometer size range. Figure 2 shows an example device with high and low (7,8) frequency drivers to generate pumping in conjunction with negative DEP.
By applying a signal which is made up of an addition or superposition of a 5 kHz signal and a 5 MHz signal to the pumping electrodes, the device will induce AC EOF while simultaneously inducing negative DEP above the electrodes to repel the beads.
Figure 3(a) shows a dark-field optical micrograph of a device with 5 nm/100 mm thick Ti/Au interdigitated electrodes with lateral dimensions of 5 μ m for the narrow electrode 9 and 25 μ m for the wide electrode 10, patterned on a glass substrate. The images in Figure 3 have been inverted to show up the microparticles more clearly (as dark spots). After 5 minutes of pumping a solution of IxIO "4 M NaNO 3 containing 1 μm latex microspheres with a concentration of 1x10 6 particles/mL at an applied voltage of 1.8 V 1711 ,. and frequency of 5 kHz, it is clear from Figure 3(b) that the electrodes are significantly coated with the microspheres especially at the edges where the electric field is the strongest. The microspheres remain attached to the electrodes even after the applied voltage is turned off and cannot be removed even with vigorous washing in deionised water. Some particles are removable after ultrasonic cleaning, however the majority remain, demonstrating the strong interactions that occur when the particles contact the electrode surface under positive DEP.
In contrast to the prior art of figure 3, when a 5 MHz signal with a magnitude of 1.4 V nJ18 is superimposed on a 5 kHz, 1.0 V rms signal, almost no beads are attached to the electrodes (apart from those already attached prior to the experiment) after pumping the same salt solution for 5 minutes, as is evident in Figure 4, where (a) shows a micrograph of the electrode prior to pumping the bead solution, and (b) shows the same location after 5 minutes of pumping with the dual frequency signal of a device of figure 2. Although there are a few additional beads located on some of the electrodes, they are insignificant when compared to the bead adhesion that occurs after pumping with only a single frequency. This thus demonstrates a very simple but effective method of avoiding particle adhesion while pumping fluids in microchannels using low-voltage AC EOF.
In another example of the present invention, microparticles can be pumped without adhesion to the pumping electrodes by a judicious choice of the size of the microparticles and the dimensions of the electrodes. The cross-over frequency from positive to negative DEP is dependent on the diameter of the particles in question. This allows a single frequency to be used for both pumping as well as to induce negative DEP above the electrodes if the dimensions of the electrodes as well as the beads are selected such that the optimal pumping frequency is higher than the cross-over frequency from positive to negative DEP for the size of beads that are to
be pumped. This therefore further simplifies the protection of the electrodes from adhesion of the microspheres, as it does not require a source of multiple AC frequencies. For example, using electrodes with the same dimensions as those shown in Figure 1, one can choose to pump latex microspheres with diameters of 10 μm, which will experience negative DEP forces near the electrodes at the pumping frequency of 5 kHz. A useful consequence of using the same applied voltage and frequency for the pumping as well as particle repulsion is that as the applied voltage is increased to speed up the flow, the magnitude of the repulsive DEP force is also increased hence improving the resistance of the electrodes to particle adhesion. One limitation of this technique however is that in the limit of small particle sizes (~3 μm or less), the frequency that is required to induce negative DEP is so high (> 200 kHz) that the mechanism of AC pumping, which is highly frequency dependent, is unable to be sustained.
In a further example of this invention, smaller microparticles (< 3 μm in diameter) can be pumped without adhesion to the pumping electrodes by a judicious choice of the size of the microparticles, the dimensions of the pumping electrodes and the thickness or dielectric properties of a dielectric layer (not shown) that is coated over the electrodes. Optimum AC pumping frequency for electrodes that are coated with a dielectric layer depends on the properties of the dielectric layer, including the thickness and the dielectric constant of the layer. This is due to the fact that the dielectric layer serves to lower the capacitance of the double layer of ions that form above the electrodes and as such, the frequency at which the maximum pumping velocity occurs increases significantly. Therefore, although AC EOF normally occurs between frequencies of 100 Hz to 100 kHz the dielectric layer, which may be 50 - 500 μm thick, but more preferably 100-200 μm thick and composed of a material such as silicon dioxide or silicon nitride, shifts the operating frequency range up into the 100's of kHz or even into the MHz range, depending on the material and the thickness of the layer. These frequencies are also the appropriate frequencies to induce negative DEP over the electrodes for particles in the 3 μm down to 0.5 μm size range, thus repelling the microparticles while simultaneously producing AC fluid flow over electrodes coated in such a manner.
The mechanism of pumping in this example at these high frequencies, is still AC EOF rather than electrothermal or other forms of electrohydrodynamic flows, due to
the existence of the dielectric layer which reduces the overall capacitance of the electrode-electrolyte interface. There is a limit however, to how high the frequency of operation of the device can be increased using this technique, as increasing thickness of the dielectric also causes a larger voltage drop to occur across the electrode-electrolyte interface, hence the limiting the density of ions in the double layer and the corresponding pumping speed.