GB2358361A - Method and apparatus for the separation of particles - Google Patents

Abstract

A method and apparatus for collecting and separating abiotic and/or biotic particles and/or chemicals in a liquid or gaseous sample. The apparatus includes a substrate (10) supporting at least one pair of electrodes (4,6) and defining a fluid channel (8) over said electrodes. The electrodes (4,6) are energised with at least two superimposed voltages having different predetermined frequencies which attract the particles, by dielectrophoresis, to the electrodes. By switching off one or more of the voltages, it is possible to selectively release one type of particle for subsequent collection and/or enumeration.

Description

2358361 8906:GH METHOD AND APPARATUS FOR THE SEPARATION OF PARTICLES The

present invention relates to a method and apparatus for collecting and separating abiotic and/or biotic particles, such as latex beads, mammalian cells or microbial cells, viruses, prions and chemicals or biochemicals using dielectrophoresis. It also relates to a method and apparatus for enumerating a particular particle present in a test sample.

It is well known that when an AC voltage is applied to a pair of electrodes which have a suspension of particles between them, the particles may polarise and have a force exerted upon them where the electric field is non-unifonn. This translational force (the dielectrophoretic force) may cause the particles to aggregate in areas of either high or low electric field gradient, dependant upon the polarisabilities of the particles and the suspending medium. The polarisability of the particle is a function of its conductivity and permittivity, and varies with the frequency of the electric field. Measuring the number of particles collected as the frequency of the voltage generating the electric field changes allows a collection spectrum to be plotted. This has been shown to be characteristic for individual species of biological cells and for abiotic particles, since the polarisability of a particle type is dependant upon its individual, unique structure.

This phenomenon, known as Dielectrophoresis (DEP), has been shown to be useful for particle and cell characterisation and also for the separation of a particle type from a mixed suspension. Previous DEP methods of cell separation have relied upon the application of a single, fixed-frequency, AC voltage to an electrode structure. The frequency of this sinusoid or other waveform is carefully chosen such that only the particle of interest experiences positive DEP (where the particles are forced to areas of high electric field gradient) and the other cell types undergo negative DEP (forcing the cells to areas of low, normally zero, electric field gradient. Since -ve DEP is a weaker force, a constant flow of the suspension can remove those particles undergoing -ve DEP, whereas those undergoing +ve DEP will remain in the areas of high field gradient and be separated from the suspension.

The disadvantage of this method of separation is that the choice of frequency, t@1 be applied to the electrodes requires prior knowledge of the electric properties of the p le ah and medium comprising the suspension. Furthermore, the method only allows the separation of one or a limited number of cell or particle types from the suspension.

WO 98/04355 describes an apparatus and method for characterising the prope, S j 1 i 1 of specified particle types or analysing the particle population in a fluid by placing a,:!, 1 suspension of the particles in a chamber in which an electrode array is arranged to g? ntrate a spectrum of different frequency dielectrophoretic fields. The electrode array comp i,, 011SOL series of spaced electrodes to which are applied electrical inputs of different frequeni(:.., to generate different dielectrophoretic fields in respective regions adjacent the electrod' The disadvantage of this arrangement is that it is complex and less convenient for separgin different particles and reducing the amount of background material.

It has now been found that by superimposing two or more voltages of differe t frequencies on at least one pair or a set of electrodes, passing or circulating a liquid 9, 11 containing different particles suspended therein past the electrodes, then switching o or more of the voltages it is possible to separate, identify and subsequently count the released particles.

It is an object of the invention to provide an improved dielectrophoretic meth.d "I apparatus for separating, collecting and counting abiotic and/or biotic particles.

According to one aspect of the invention there is provided a method of separ:i different particles present in a liquid or gaseous sample, the method comprising the sl e c f passing or circulating the sample through a region of non-unifonn electric field dens. produced by at least one pair of electrodes, energising said electrodes with a first vol having a first predetermined frequency selected to attract a first predetermined variet particle in the sample to said electrodes, superimposing on said electrodes a second c) age having a second predetermined frequency selected to attract a second predetermined n 1 iel y of particle in the sample, switching off either the first or the second voltage thereby releasing either the first or second variety of particle for subsequent collection and/or enumeration.

More than two different voltages having different predetermined frequencies may be superimposed on and applied to the electrodes in order to attract all the particles in the liquid sample to them. The particles can then be subsequently released en masse by switching off all of the voltages, thus permitting a total particle count to be determined.

Alternatively, the particles may be released from the electrodes individually by type by switching off a selected voltage thus facilitating separation of the particles for subsequent collection, identification and/or enumeration.

The method may be used for separating different biotic particles such as micro organisms and/or different cell types and cell organelles including plasmids. The term micro-organism is intended to embrace bacteria, viruses, yeasts, algae, protozoa, fungi and prions. Abiotic: particles which may be separated include for example latex beads, metal particles or any inorganic or organic material, chemical or biochemical species can also be separated.

According to a second aspect of the invention there is provided an apparatus for separating particles present in a liquid sample the apparatus comprising a support defining a fluid flow channel through a region of non-uniform electric field density produced by at least one pair of spaced electrodes, circulating means for passing or circulating said sample containing said particles through said channel, a first AC source for applying a first voltage at a first frequency to said electrodes, said frequency being selected to cause a first predetermined type of particle to be attracted to said electrodes, a second AC source for applying a second voltage at a second frequency to said electrodes, said second frequency being selected to cause a second predetermined type of particle to be attracted to said electrodes and means for determining the quantity of either the first or second predetermined type of particle when either the first voltage or second voltage is not applied.

The use of multiple frequencies may be applied to any design of electrode. , t1le electrodes being shaped to generate maximum DEP effect. Thus, for example, the electrodes may be castellated or they may comprise an electrode array which may or y not define more than one fluid flow channel. Several pairs of electrodes may be use W 5 forming a set of electrodes which may be linked together.

According to yet another aspect there is provided the use of the method defin above or the use of the apparatus defined above for separating eukaryotic cells, bactori yeast, viruses, algae, protozoa, fungi, prions, inorganic or organic abiotic particles, plasmids, cell organelles, chemicals and biochemicals including nucleic acids, and chromosomes.

A method and apparatus for separating particles such as micro-organisms wil) i iow be described, by way of example, with reference to the accompanying diagrammatic drawings, in which:

te Figure I is an electrical and fluid circuit of an apparatus in accordance with tt, invention; and Figure 2 is a perspective fragmentary section taken through the collection blo'!of the apparatus of Figure 1; Figure 3 is a graph showing the number of released latex beads counted over 14 period of time when different voltages are applied; Figure 4 is a graph showing the number of released E.coli counted over a perip ot! time when different voltages are applied; Figure 5 is a graph showing the number of released latex beads and E.coli coq'In d' over a period of time when a 10 kHz voltage is applied; Figure 6 is a graph showing the number of released latex beads and E.coli coon d over a period of time when a 1 MHz voltage is applied; Figure 7 is a graph showing the number of released latex beads and E.coli coon 0 over a period of time when both 10 kHz and I MHz voltages are applied; and Figure 8 is a graph showing the number of released latex beads and E.coli cod I-, f - j over a period of time when both 10 kHz and I MHz voltages are applied and then disabled sequentially.

The apparatus shown in Figure 1 comprises a collection block 2 in which particles can be collected. The collection block 2 contains a pair of spaced electrodes 4 and 6 lying in a common plane and a fluid flow channel 8 positioned to cause a liquid to flow across the upper faces of the two electrodes.

The structure can be more clearly seen in Figure 2. As shown, the structure includes an electrically insulating substrate 10 on which two elongate electrodes 4 and 6 have been deposited in parallel but spaced relationship with each other. Two electrically insulating side walls 12 and 14 are attached to the substrate 10 and cover both the electrodes 4 and 6.

The side walls can be composed of a polyimide. The side walls 12 and 14 define the channel 8 which extends at right angles to the electrodes 4 and 6. A fin- ther electrically insulating layer (not shown) extends over the side walls 12 and 14 and the channel 8 to form the roof of the channel 8. The exposed face of each electrode may be covered with a thin electrically insulating layer as required.

A reservoir 20, for containing a sample of liquid to be analysed, is connected by a duct 22 to the upstream end of the channel 8. A duct 24 connected to the downstream end of the channel 8 feeds liquid from the channel 8 through a pump 26 back to the reservoir 20.

The pump 26 is advantageously a peristaltic pump to prevent any contamination to the sample liquid and particles therein. The liquid in the reservoir 20 may be agitated by bubbling air or other gas therethrough to keep the particles in suspension.

Two signal generators 28 and 30, which supply two voltages of different frequencies, are inductively coupled using an isolation transformer 32 and the voltages from the signal generators 28 and 30 are applied to the pair of electrodes 4 and 6.

To separate a suspension of particles, for example latex beads and E.coli bat( 'a, and pumped b3,p mj) from a liquid sample, the liquid sample is placed in the reservoir 20 26 via duct 22 through the channel 8 over electrodes 4 and 6.

Latex beads are known to collect well on the electrodes when an output freqi(iic3, of 10 kHz is appliedto the electrodes4 and 6. Signal generator 30 is therefore set tg Itave an output frequency of 10 kHz. Latex beads do not collect well at 1 MHz. Incontre:'E coli bacteria collect well at both frequencies. Signal generator 28 is set to have an ()iii U frequency of 1 MHz.

In order to assess any particle collection at the electrodes 4 and 6, they were i mounted beneath a microscope to which a charge coupled device (CCD) camera w4 tte d.

The microscope was focused slightly downstream of the electrodes. The camera fe'i to an image analysis card fitted in a computer, which labelled and counted the number of particles within the plane of focus five times per second. Other methods of countin, tl'e particles may be utilised here, eg spectrophotometric, impedance analysis. i To determine whether particle collection had occurred, one or both of the vot e inputs were applied for a period of time, while the image analysis program counted:! particles in order to determine the background level. The voltage was then turned o, If any particles had collected at the electrodes they would then be released and pass be cath the microscope, leading to an increase in the number of particles counted as the rele d packet of particles flowed by. The number of counted particles would then drop ba( tne background level. Changing the phase/contrast of the microscope allowed either tho:, 1 beads or the E.coli to be labelled and counted separately.

In an initial test which was performed using separate suspensions of latex be aid E.coli, the 10 kHz voltage was applied for 8 seconds then disabled while the image; n ysis system counted the particles flowing beneath the microscope. This was performed X v 1 times and the particle count was averaged. This was then repeated for the 1 MHz vOit ge and with both the 10 kHz and I MHz voltages applied simultaneously. The results for both particle types are shown in Figures 3 and 4.

The latex beads collected well at 10 kHz, as there was a significant increase in the number counted once the voltage was disabled and the particles were released, before the count dropped back to a background level. At 1 MHz there was not collection, with the count remaining at a background level throughout the experiments. The application of both voltages resulted in collection of the latex, but at a lower level than the 10 kHz alone.

The E.coli collected equally well at 10 kHz and I MHz, but there was a significant increase in collection with both voltages applied.

This demonstrates that by appl3ing two frequencies to a pair of electrodes it was possible to generate an increase in the number of E.coli cells collected at the electrodes, as compared with a decrease in latex collection when both frequencies were applied.

The inifial test was repeated using a mixed suspension of latex beads and E.coli.

Figures 5 to 7 show the results.

At 10 kHz both particles collected well, as was seen from the individual suspensions, and at 1 WIz, as expected, the E.coli collected at a similar level as for 10 kHz, whereas the latex beads did not. When both voltages were applied both particles collected, with the E.coli reaching a greater level.

In a further experiment, the effect of disabling the voltages subsequently was studied to find out how easily the collected particles could be separated.

Both voltages were applied simultaneously for 8 seconds. Then the 10 kHz voltage was disabled. Ten second later the 1 MHz voltage was disabled. The results are shown in.

Figure 8.

When the 10 kHz field was disabled and the I MHz field remained applied,', latex beads that collected on the electrodes were released. No E.coli were released, t Ihi, time. When the I MHz field was later disabled, the E.coli were released. It shouldi j noted that no latex was counted at this time, indicating that the beads were all releas,e, when the 10 kHz field was disabled.

This demonstrated the effectiveness of the technique as a method of particl&, separation, as all of the latex beads were released at a different time than the E.coli.

Any number of signal generators may be inductively coupled to apply more:, frequencies of voltage to the pair of electrodes or superimposing using other metho4,s By using an appropriate number of frequencies it should be possible to collect every tye f particle in a suspension.. Following this it would be possible to determine the numbr f particles collected with the image analysis technique or another method. This woulj, er j be a measure of the total number of particles in the test suspension.

Impedance matching may be used to make the isolation transformers and thq], electrode structure match the 500 impedance of any coaxial cables used to connect system. This will create a constant voltage across the electrode gap within the rang(r: c 20 frequencies to be used.

The apparatus according to the invention may be computer controlled to enaol rapid, easy control of the signal generators. This will be of particular benefit if all p I ch I types are to be collected from a sample and released individually by changing the ap p1l id 25 frequencies.

Apart from the image analysis technique described other methods including 1 spectrophotometric (including fluorescence), laser, impedance analysis and radiomet' may be used to count the number of particles collected at or released from the electr 4d s30 With appropriate choice of frequencies for separating particle types the method and apparatus defined above may be used to separate a single species from the test suspension by either collecting all cell types bar the desired one, or by only collecting the desired species. Another option may be to collect all of the cells in the suspension and then release them one at a time by either turning off one frequency or by changing one or more of the frequencies such that one cell type no longer collects. This method could be used to separate any number of species in the suspension, by collecting them all and selectively releasing them individually.

It may also be possible to collect any number of the species from a large suspension onto the electrodes by appropriate choice of applied frequencies. Following the collection it will be possible to elute the collected cells into a much smaller volume suspension, effectively concentrating the suspension. In addition, the collected cells could be eluted into a different suspending medium as a further form of sample preparation. 15 In addition, by applying more than one frequency of voltage it may be possible to change the DEP collection, generating a marked increase in the number of collected particles. Such a method could be used to enhance the collection of any number of particles in a suspension. It will also be possible to choose the frequencies such that a number of particle types experience -ve DEP and are repelled from the electrodes when other are collected producing an additional form of sample separation.

Claims (6)

1. A method of separating different particles present in a liquid or gaseous sarj , I tie method comprising the steps of passing or circulating the liquid or gaseous sample I h 'ugh a region of non-uniform electric field density produced by at least one pair of electp,)( s, i i I i i I energising said electrodes with a first voltage having a first predetermined frequenq, i selected to attract a first predetermined variety of particle in the sample to said elec( 4e,;, superimposing on said electrodes a second voltage having a second predetermined frequency selected to attract a second predetermined variety of particle in the samp 10 switching off either the first or second voltage, thereby releasing either the first or sq nd j. variety of particle for subsequent separation, collection, identification and/or enump iolr

2. The method according to Claim 1, wherein more than two different voltages! g predetermined frequencies are superimposed on and applied to said pair of electrodd,$.

3. Apparatus for separating particles present in a sample liquid the apparatus form comprising a support defining a fluid flow channel through a region of non-uni ct -ic field density produced by at least one pair of spaced electrodes, circulating means ft$ circulating said sample containing said particles through said channel, a first AC soi rc)r applying a first voltage at a first frequency to said pair of electrodes, said frequency bt 8 selected to cause a first predetermined type of particle to be attracted to said electroo I a second AC source for applying a second voltage at a second frequency to said elec S2 tr ' I said second frequency being selected to cause a second predetermined type of partic I e attracted to said electrodes and means for determining the quantity of either the first second predetermined type of particle when either the first voltage or second voltag([ i no t applied.

4. Use of the method according to Claim 1 or Claim 2, or of the apparatus accoo g 0 Claim 3, for separating eukaryotic cells, bacteria, viruses, yeasts, algae, protozoa, prions, plasmids, chromosomes, cell organelles, abiotic particles, chemicals and biochemicals including nucleic acids.

5. The method of separating particles present in a liquid or gaseous sample substantially as hereinbefore described. 5

6. Apparatus for separating particles present in a liquid sample substantially as hereinbefore described, with reference to the accompanying drawings.