METHOD FOR THE SELECTION AND/OR THE PROCESSING OF PARTICLES, IN PARTICULAR CELLS

TECHNICAL FIELD The present invention relates to a method for the selection and/or the processing of particles, in particular particles consisting of cells or including cells or cell material and finds its application mainly in the implementation of protocols with a resolution on a single cell. By cell "processing" is understood to mean, herein and below, any type of operations which can be carried out on a single particle or cell, or groups of the same. STATE OF THE ART PCT/WQ 00/69565 patent to G. Medoro describes an apparatus and. a method for the manipulation and the detection/recognition of particles through the use of closed dielectrophoretic potential cages and integrated sensors, if any. The method described teaches how to control the position of each particle independently of all the others in a two-dimensional space. The force used for trapping in suspension particles in a fluid medium is the negative dielectrophoresis. The single control on the manipulation operations takes place through the programming of memory elements and circuits associated with each element of an array of electrodes and sensors integrated within a same substrate. The device allows to

isolate cells, but it requires the displacement of these towards a second microchamber, fluidically isolated from the first one. Furthermore, a method for transforming the cells is not foreseen.. US 6,294,063 patent, Becker et al, describes a method and an apparatus for the manipulation of packages of solid, liquid or gaseous biological material, through a distribution of programmable forces. The patent also mentions the use of sensors. Also in this case the cell isolation can take place only by physically moving cells throughout the device.

A further force for the manipulation of particles is the viscous friction force generated by electro- hydrodynainic (EHD) flows, such as the electro-thermal flows (ETF) or the AC electroosmosis. In NG. Green, A. Ramos and H, Morgan, J. Phys. D: Appl. Phys. 33 (2000), EHDs are used for displacing particles. For example, PCT WO 2004/071668 Al discloses an apparatus for concentrating particles on some electrodes, by exploiting the so-called electro-hydrodynamic flows.

In the Italian patent application BG2005A000481, Medoro et al., some methods for manipulating particles with arrays of electrodes and some methods and apparatus for their detection are reported. Further, in the international patent application no. PCT/IT02/00524, a method in which first biological entities can be transformed by contacting the same with second biological

entities (for example DNA-containing liposomes, or microballs) is descirbed, wherein first biological entities are immobilized on a surface defined by a matrix of first electrodes at least partly selectively activable and addressable, arranged faced towards at least a second electrode, and are contacted with the second biological entities displaced by means of dielectrophoresis cages.

PCT IB 2006000636 patent application of the same Applicant relates to a method and an apparatus for the characterization and/or the counting of particles through non uniform, time varying force fields and integrated optical or impedance meter sensors _* Force fields can be of positive or negative dielectrophoresis, electrophoresis or electro-hydrodynamic motions, characterized by a set of stable points of equilibrium for the particles (solid, liquid or gaseous) ; the same method is suitable for the manipulation, of droplets

(liquid particles) by exploiting effects known as

Electrowetting on dielectric, with the aim of acting on the control of the position each particle existing in the sample, for the purpose of displacing such particles in a deterministic or statistical way, in order to detect their presence with the integrated optical or impedance meter sensors and/or characterize their type, for the purpose of counting or manipulating them in an efficient way.

In the Italian application in the same applicant's

name, no. TO2006A000226 dated 27.3-2006, methods and apparatus for the processing (for example washing, incubation, etc.) of particles are described, wherein the suspending particles in a first fluid are introduced in a laminar flow regime in at least a first microchamber or first region of the same, wherein a second fluid is introduced in a laminar flow regime in at least a second region of the micro-chamber or a second micro-chamber, in such a way not to mix itself with the first fluid, and wherein within the micro-chamber/s at least a force field (F) acting on the particles is activated, in order to cause a displacement of the particles only in a pre-fixed direction and transfer the same in suspension in the second fluid; there is preferably used an apparatus including at least three micro-chambers arranged in sequence to one another along a direction, each one connected with the micro-chamber immediately preceding and following through two orifices staggered to each other in a direction perpendicular to the sequence direction of the micro-chambers.

Recently, in the article A single cell electroporation chip. Lab on a Chip, 2005 _f 5 (1) _r 38 - 43, Michelle Khine, Adrian Lau, Cristiaπ Ionescu-Zanetti, Jeonggi Seo and Luke P. Lee it was described how to increment the permeability of cell membranes through electroporation carried out on single cells; in this way, polar substances which could not otherwise permeate the

plasmatic membrane (such as dyes, drugs, DNA, proteins, peptides and amino acids) can be introduced within the cell.

The article Flow-through micro-electrϋporation chip for high efficiency single-cell genetic manipulation, Sensors and Actuators A: Physical. Volume 104, Issue 3/ 15 May 2003, Pages 205-212, Yong Huang, Boris Rubinsky describes in particular the genetic manipulation of single cells, which is of a great interest in fields such , as biology and biotechnologies, obtained through an electroporation chip which uses micro-fluidic channels for accurately manipulating single cells; as it is known, the electroporation is a technique which uses strong electric fields for inducing structural rearrangements in the cell membra ne; pores are thus formed through the membrane when the trans-membrane potential overcomes the dielectric perforation voltage of the membrane (0.2-1.5V) allowing to external substances to penetrate the membrane and reach the cytoplasm contained therein. Eleσtroporation of single cells is a technique of interest also because it allows to study the variations occurring in a cell population cell by cell, as well as to study the intracellular chemistry, for example by providing specific phenotypes to single cells activating or blocking the expression of specific and single protein. Using a technology based on the use of matrixes implemented on chips it is therefore possible to carry

out HTS (high throughput screening) test equipments correlated both to the DNA and protein expression, and to chemical compounds (for example drugs) which are directed towards specific cellular targets (for example receptors) .

The electroporation of single cells is moreover an advantageous technology with respect to protocols of "in bulk" electroporation. normally used, which require very high voltages {>103 V) and which do not allow an effective control of the permeability of single cells, so that, for example, the re-closure of previously opened pores results difficult.

Attempts carried out so far in order to attain the electroporation of single cells include the use of miσroelectrodes made of carbon fiber (Lundqvist et al.,

1998) and other techniques, such as capillaries filled of electrolytes, micropipettes and micro-manufactured chips.

Micro-manufactured chips are ideal both for isolating single cells and for focusing the electric field.

Finally, the article "Controlling cell destruction using dielectrophoretic forces", A. Menachery and R. Pethig, IEE Proc.Nanobiotechnol. , Vol. 152, No, 4, August 2005, reports a study on lysis of cells for different kinds of cells in castellated or polynomial electrodes, and proposes the lysis or the differential electroporation of cells of different kinds existing in a

mixture (selecting such frequencies and amplitudes that lysis or electroporation of a type is performed, but another type is saved) .

However, as the electrodes are much greater than cells, the use of this approach is not proposed,, and probably it is not possible to use it for selectively destroying/electroporating single cells independently of their type. De facto, as the position with respect to relatively large electrodes (and accordingly the field intensity to which they are subjected) is remarkably varying, such method can not operate in a homogeneous way on different cells.

The lysis is preferably induced using fields in a frequency range included between the cross-over frequency (beyond which cells change from negative (nDEP) to positive (pDEP) electrophoresis, and lower than the frequency beyond which the membrane potential results weakened due to the overcoming of the relaxation constant of the membrane. Other documents of a recent publication, such as WO2005/075656 and US2005/0070018A1, relate to apparatus for electroporation of single cells based on the use of an array or a matrix of microelectrodes, on which cells are cultivated adherent (according to the international patent application) or equipped with conductive microwires for connecting cells to be electroporated to the electrodes and microfluidic channels for the cell

displacement. Such apparatus do not give the possibility to organize the cells in a deterministic way, and the position of the cells with respect to the electrodes results aleatory. Therefore, the electric field to which they are subjected at the moment of electroporation results quite varying, so that the applied stimulus is sometimes statistically excessive (causing the death of the cell) , or insufficient, lacking in the electroporation of the cell. The success percentage in the electroporation process then results sub-optimal and less effective. SUMMARY OF THE INVENTION

An object of the present invention is to provide a method and an apparatus for operating on fluid samples containing particles, typically cells, for carrying out the transformation of one or more cells, which is free of limitations and/or drawbacks described for the known art.

In particular, an object of the present invention is to act on the control of the position of each particle existing in the sample, for the purpose of displacing such particles in a deterministic way, for operating in a selective way on each cell and/or performing in a more effective way operations such as electroporation.

Here and in the following, by terms "particles" or ^' "particle", natural or artificial, micrometric or nanometeric entities are intended, such as cells, sub- cell components, viruses, liposomes, niosomes,

microballs. Sometimes, the term cell will be used, but where not otherwise specified it should be intended as a non limiting example of the use for the detection and characterization of particles in the widest sense above described.

The present invention therefore relates to a method as specified in the claim 1.

In particular, non uniform, time varying force fields and integrated optical sensors are used. The force fields can be of positive or negative dielectrophoresis, electrophoresis or _ electro-hydrodynamic motions, characterized by a set of stable points of equilibrium for the particles (solid, liquid or gaseous) -

In this way, restrictions of the known art are overcome by the present invention.

The implementation of the method according to the invention allows to transform cells in an effective and selective way, for example with the introduction of exogenous genetic material. Furthermore, it allows to accurately purify a sample of cells, possibly transformed, also from contaminants existing in a low percentage. Finally, it allows to quickly isolate a few cells of interest from a heterogeneous sample. The whole through the adoption of a single technology based on a same array of microelectrodes which are used both for moving cells within a micro-chamber, in which the array of electrodes is incorporated, and for producing the

electroporation thereof.

Further features and advantages of the invention will result evident from the following description of some non limiting embodiments of the same, which is carried out with reference to the figures of the enclosed drawings . BRIEF DESCRIPTION OF THE FIGURES

Figure 1 diagrammaticalIy shows the steps of the method according to the invention carried out in a manipulation apparatus shown in a sectional and elevation view;

Figure 2 shows with photographic sequences taken in a top plan view with respect to the apparatus of figure 1, the practical execution of the method of figure 1; Figure 3 shows with photographic sequences some cells before and after the electroporation;

Figures 4, 5a, 5b diagrammatically show possible execution protocols of the method according to the invention. DETAILED DESCRIPTION

The object of the present invention is to carry out a method for the manipulation and/or the transformation and/or the study of the optimal electroporation conditions and/or the detection of particles. The method of the invention is based on the use of a non uniform force field (F) through which it attracts single particles or groups of particles (BEADS) towards

positions o£ stable equilibrium (CAGE) . Such field can be for eκample a negative (NDEP) or a positive (PDEP) dielectrophoresis field (DEP) , an electrophoretiσ field (EF) or an eleσtro-hydrodynamiσ (EHD) motions field. The processing operated on cells is based on the application of localized electric fields capable of causing the temporary permeabilization of the cell membrane.

The method can also use integrated sensors, preferably of an optical and/or impedance meter type, for example in all those steps in which it is necessary to check the type of particles in proximity of certain electrodes. Alternatively, a similar information can be obtained through non-integrated optical sensors, coupled with a microscope which allows to examine the content of the micro-chamber. Generation of forces

There are different methods for the generation of forces for displacing particles, according to the known art, through arrays of electrodes (EL) , carried on a substrate. Typically, according to preceding patent rights of the same Applicant (figure 1) , a cover (LID) is used, which can be in turn an electrode, which delimits a micro-chamber within which particles (BEADS) are typically found in a liquid suspension. In case of dielectrophoresis (DEP) , voltages applied are in phase periodic voltages (Vphip) shown by the addition symbol

(+) and counter-phase periodic voltages ⁽ Vphin) shown by the subtraction symbol {-) . By the term ^counter-phase voltages", voltages out of phase of 180 degrees are meant. The field generates a force which acts on the particles in a space region (CAGE) , attracting them towards a point of equilibrium (PEQ) - In case of negative DEP (NDEP) , it is possible to generate closed force cages, according to the known art, if the cover (LID) is a conductive electrode; in this case, the point of equilibrium (PEQ) is established in correspondance with each electrode connected to Vphin (-) if the adjacent electrodes are connected to the opposite phase Vphip {+) and if the cover (LID) is connected with the phase Vphin (-) . Such point of equilibrium (PBQ) is normally spaced apart in the liquid compared to the electrodes, whereby particles (BEADS) are, in a steady state, in levitation. In case of positive DEP (PDEP) , the point of equilibrium (PEQ) is normally found in correspondance with the surface on which the electrodes are provided, and the particles (BEADS) are, in a steady state, contacting with the same. For the PDEP, the presence of further electrodes in the cover, (lid) is not necessary, because the points of equilibrium of the PDξP correspond to the maxima of the electric field. For the eleσtro- hydrodynamic (EHD) motions, configurations of the electrodes generate sortie flows which drive the particles towards points of minimum of the flow.

In the following, for easiness, the use of closed negative dielectrophoresis cages as an execution force for the displacement of particles in the description of t _h e methods of the invention is merely considered by way of non limiting example for the purposes of the present invention (for this reason, it is necessary to use a lid acting as an electrode) . To those skilled in the art with ordinary skill, it is apparent how to generalize methods and apparatus described below for the use of different execution forces and different types of particles.

Electroporation of particles assisted by_ dielectrophoretic manipulation

Particles are positioned in proximity of the gap between two electrodes by means of one of the execution forces above mentioned, for example by energizing the electrodes themselves with sinusoidal voltages of a first amplitude and frequency. The gap is preferably lower than 10 μin, and typically of the order of 1-2 μm, such that also a low voltage stimulus, compatible with the feed voltage of an integrated circuit (examples 2.5, 3.3 or 5 V) is sufficient to determine a trans-membrane potential capable of causing the reversible permeabilization of the particle.

This stimulus preferably consists of a sinusoidal impulse train of a second amplitude and a second frequency.

Figure 1 shows in a sectional view the temporal

evolution of force fields and voltage "patterns' ⁷ (or the complex of configurations of (+) or (-) state of the electrodes) applied to the electrodes according to a preferred embodiment of the invention. In Figure l(a) cells (CELL) are in nPEP, in suspension in the liquid in a first point of equilibrium (MPEQ). In Figure l(b), the pattern of voltages applied to the electrodes (EL) changes, as well as the frequency and optionally the amplitude of the applied voltages, such that also the force to which the cells (CELL) are subjected changes in pDEP (FZAP) . However, thanks to the change of voltage pattern only the cell to be electrophorated (CELLZ) is subjected to a significant force, therefore it is attracted towards a. new stable point of equilibrium (EPEQ) . In proximity of such point, the electric field is the highest and the frequency is $uch that a transmembrane potential sufficient to eleσtroporate the cell is determined. In this way, at least a compound (PL) existing in suspension is able to penetrate within the cell.

Such compound can be a chemical compound (dye, drug, peptide) in solution in the fluid in which particles are dipped, or it could be, for example, a plasmide, etc. according to the known art, or second PL particles of desired type and quantity could be present in suspension within the fluid.

Figure 2 shows the electroporation. of a single cell.

Cells, figure 2 (a) are trapped in nDEP (frequency - 50 kHz, peak-to-peak voltage to electrodes 3.3 V, peak-to- peak voltage to the lid 6,6 V). Figure 2(b) shows the fluorescence imagine. The field is then mutated in pDEP (two 500 kHz impulses, amplitude of peak-to-peak voltage to electrodes 2 V, amplitude of peak-to-peak voltage to the lid 4 V) _r and the selected cell undergoes a pore opening which causes the release of calcein (previously loaded within cells, for the purpose of carrying out the experiment) and the fluorescence decrease (Figure 2(c)).

With the optical-type integrated sensors with fluorescence detection (see previous patents of the same Applicant) the effective transformation can be checked, for example through the expression of Green Fluorescent Protein (GFP) .

Method for the optimization of electroporation stimuli upon the array of electrodes, cells can be placed and stimuli with different amplitude and/or frequency parameters can be tried on each of them or groups of them, in order to rapidly determine the more effective stimulus.

For example, the electroporation can be checked with integrated optical sensors, as above described, or with the monitoring of a possible introduction of a dye (dye or colouring matter) within the cell (for example Trypan blue), normally unable to penetrate the membrane.

Figure 3 shows cells in Trypan blue before, Figure

3 ₍ a ₎ and after, Figure 3(b), the electroporation according to the protocol above described. In Figure 3 (b) , it can be noted that Trypan blue is penetrated within the cell. With integrated optical sensors the inlet of the dye can be detected as well as the occurrence of the electroporatxαn.

Alternatively, the dispersion of a dye present within the cell caused by the diffusion of the dye outside at the pore opening can be controlled. For example, fluorescent oalcein within the cell could be released outside and determine a decrease of the cell fluorescence, as above described.

The method foresees to iteratively apply stimuli of growing intensity on single cells, measure the number of electroporated cells for defining the dependence curve of the electroporation percentage as a function of the applied stimuli.

Method for the adjustment of electroporation stimuli for each cell On the array of electrodes, cells can be positioned and stimuli of growing intensity can be tried thereon and the possible inlet of a dye normally unable to penetrate the membrane can be controlled with integrated optical . sensors. Based on what has been described, it is apparent that the invention allows to carry out the selection or the processing of first particles sensitive to the

application of an external stimulus and provides the step of producing, through the application of such external stimulus, the permeabilization of at least one said first selected particle; in particular, it provides the steps of: a) bringing first particles (CELL) in proximity of electrodes (EL) of an array of selectable electrodes having dimensions comparable or lower than those of said particles, to which a first configuration (PMAW) of voltages is applicable for organizing said first particles (CELL) through a first force field (FMAN) by selectively energising said electrodes (EL) ; b) applying to said electrodes a second configuration (PZAP) of voltages, so as generate a second force field (FZAP) _r substantially positioned in proximity of at least one first selected particle to be permeabilised (CELL) and such to produce the application to said at least one first selected particle of a stimulus suitable for bringing said at least one first selected particle in a permeabilized state for at least a fraction of the length of said stimulus.

The method according to the invention further includes the step of: c) contacting said at least one first selected particle in a permeabilised state with at least a second particle; wherein steps b) and c) are carried out so as to produce the penetration of said at least one second

particle within said at least one first selected particle in a permeabilized state, as it is diagrairanatically shown in figure 1,

First particles are dipped in a fluid and the step c) is carried out by dipping second particles within the fluid and extending said step b) for a time sufficient to allow that at least one said second particle penetrates within said at least one first selected particle, which is in a permeabilized state. Alternatively, second particles are dipped in the fluid and then at least one selected said second particle is displaced towards said at least one first selected particle kept in a permeabilized state, preferably by applying to at least said selected second particle a force field (FMAN) obtained by selectively energizing the electrodes (EL) of the array of selectable electrodes applying to the electrodes a sequence of voltage configurations properly selected.

The stimulus applied by the field can be such to maintain the selected first particles in a permeabilized state for a pre-determined time also after the removal of the stimulus itself; step c) can therefore be carried out by dipping second particles within the fluid in which first particles are dipped and by moving first particles in a permeabilized state towards second particles applying to the same a force field (FMAN) obtained by selectively energizing the electrodes (EL) of the array

of selectable electrodes applying to the electrodes a sequence pr e-fixeded voltage configurations.

With reference to figures 4, 5a, 5b first particles can be dipped in a first fluid, for example they can already be contained in suspension in the first fluid; and the step c) can be carried out by dipping second particles in a second fluid contiguous to the first fluid

(in which second fluid, for example, second particles can already be dipped) and by displacing particles in a permeabilized state (electroporated) present in the first fluid, from the first fluid to the second one by applying to the particles a suitable force field (FMαN) energising the electrodes by applying to the same electrodes a sequence of pre-fixed voltage configurations. Subsequently, first particles transformed for receiving within them second particles can be brought back in the first fluid, again applying a suitable configuration or sequence of voltage configurations to the electrodes.

When the first particles are dipped in a fluid, the method described can alternatively include the step of c) introducing within the fluid in which first particles are dipped at least a solution compound; and d) allowing said compound to penetrate within first particles which are in a permeabilized state (previously electroporated through the electrodes) ,

When first particles are dipped in a first fluid contained in a micro-chamber equipped with the array of

electrodes, the method according to the invention ⁽ figure 4) includes the steps of: σ) introducing within the micro-chamber a second fluid by operating in a laminar motion such that the second fluid is not mixed with the first one (figure 5a ⁾ ; d) introducing in the second fluid at least a dissolved compound (an operation that can obviously be carried out also before the introduction step of the second fluid in the micro-chamber, figure 5a) ; e) displacing first particles in a permeabilized state from the first fluid to the second one by applying to the particles a force field (PMAW) obtained by selectively energizing said electrodes (EL) of said array of selectable electrodes by applying to said electrodes a sequence of pre-fiκed voltage configurations (figure 5b) ; f) allowing said compound to penetrate within selected first particles in a permeabilized state, subject to electroporation of such selected first particles, an operation which can take place while the first particles are dipped in the second liquid/fluid or, if the imparted stimulus is suitable for maintaining the first particles to which it is applied in a permeabilized state for a predetermined time also after the removal of the stimulus, while the first particles are still in the first fluid, in order to then displace them in the second fluid while they are still in a permeabilized state.

Generally speaking, the first particles can be

dipped in a plurality of different fluids contained in a micro-chamber equipped with said array of electrodes and which have been introduced in said micro-chamber by operating in a laminar motion such that said different fluids do not mix together.

Preferably, as above described, the first particles are biological entities having a permeabilizable and lysable membrane, for example a cell, and the stimulus consists of bringing the trnas-membrane potential of the selected particles at such a value to produce the permeabilization of the membrane. The fluid in which, according to the invention, particles to be electroporated are dipped is always selected so as to present a relatively low electric conductivity, such that it is possible to pass to pDEP in order to attract cells in the maxima of the field, sufficient to cause such a trans-membrane potential to create pores.

Finally, according to a possible variant of the method of the invention, it provieds to apply stimuli of a growing intensity to a plurality of said selected first particles and to measure, for each applied stimulus, the number of particles existing in a permeabilized state through sensors integrated with said array of electrodes, in a single chip, For example, first particles are dipped in a fluid and the fluid contains a compound suitable for being detected by the sensors at least when it penetrates within those said first particles which are in a

permeabilized state.

Method for adjusting a priori electroporation stimuli for each cell

Should information about the optimal value of stimuli, dependently on the cell characteristics, be available (for example following to characterizations as per the above methods) , the following method can be adopted "a priori".

On the array of electrodes, it is possible to previously detect by the sensors (preferably optical and/or impedance meter sensors integrated within the chip) the characteristics of each cell to be electroporated, such as for example the dimensions, and to programme stimuli to be locally applied, so as to optimize, locally site by site, or electrode by electrode, the success percentage of the electroporation for each specific cell.