WO2006072266A1

WO2006072266A1 - Method for attaching electrolyte reagents to a channel wall

Info

Publication number: WO2006072266A1
Application number: PCT/EP2005/000814
Authority: WO
Inventors: Jan Sudor; François Chatelain
Original assignee: Commissariat A L'energie Atomique
Priority date: 2005-01-05
Filing date: 2005-01-05
Publication date: 2006-07-13

Abstract

The present invention relates to a method for attaching a plurality of liquid electrolyte reagents (S1, S2, S3 … SN) onto a wall surface of a channel, such as a capillary of a microfluidic system. The invention also relates to a process for fabricating a microfluidic system, such as a DNA biochip, which is designed to detect biomolecules in order to perform localized biological and/or chemical reactions on a plurality of spatially separate probe zones attached onto said surface. This method comprises the following steps: (i) introducing said reagents into said channel, then (ii) transporting said introduced reagents along said channel to respective predetermined locations, and then (iii) stopping and simultaneously attaching said transported reagents to said wall surface at said locations, and this method is characterized in that step (i) comprises introducing sequentially said reagents, and in that step (ii) comprises transporting said reagents in a train of adjacent reagents which remain substantially unmixed from their introduction to their attachment, under a shear generated by a local electric field which is tangent to said surface.

Description

METHOD FOR ATTACHING ELECTROLYTE REAGENTS TO A CHANNEL WALL

The present invention relates to a method for attaching a plurality of liquid electrolyte reagents onto a wall surface of a channel, such as a capillary of a microfluidic system. The invention also relates to a process for fabricating a microfluidic system, such as a DNA biochip, which is designed to detect biomolecules in order to perform localized biological and/or chemical reactions on a plurality of spatially separate probe zones attached onto said surface.

The existence of DNA biochips revolutionized the experimental approach of molecular biology since the early nineties. DNA biochips, in the format of micro-arrays, are defined as monolithic, flat solid supports that bear multiple probe sites. Each of them contains a reagent whose molecular recognition of a complementary molecule can lead to a signal that is detected by an imaging technology, for instance by fluorescence. DNA arrays chips are very promising due to their ability to obtain information on nucleic acid levels and sequences in a faster, simpler, and less expensive way than traditional methods. DNA micro-arrays may play an important role in a better understanding of the role of DNA in the processes of life, because their numerous probe sites enable the simultaneous analysis of many genes. Other major applications that are well suited for DNA arrays are large-scale genotyping, re-sequencing and gene-expression profiling.

Two major parameters of a micro-array are the number of the different probe sites ("spots")^' per unit area, which reflects its information density, and the number of probe molecules per unit area within an individual probe site. More probes per micro-array means more information and a more powerful bio-analytical tool. In order to minimize array size, the probe sites and their spacing should be as small as possible. There are two possibilities for fabricating DNA array chips. First, oligonucleotides can be synthesized directly on the chips or, secondly, they can be p re-synthesized off the chips and, subsequently, end-grafted to a specific location on the chip. The size (diameter) of sample spots on such DNA array chips may vary between 20 μm and 200 μm, depending upon the employed technology for chip fabrication. The spot density lays in a range between 100 and 1 million spots per cm².

Although DNA array chips are well accepted by a wide community of biologist, their commercial and scientific potential has not been, however, totally achieved. One of their weak points, which relates to their planar geometry, is that their detection sensitivity is unconvincing. Consequently, such DNA array chips require relatively large quantities of sample materials (which often implies a sample amplification by "Polymerase Chain Reaction") and, therefore, these chips are less adapted for applications such as in diagnostics of infectious diseases or in multi-parametric measurements on a single cell. Along the same lines, the conventional DNA chip technology based on lithography is labor heavy for applications that require the synthesis of long DNA probes (20 - 60 bases) on a chip surface, as in the analysis of the expression pattern of mRNA.

The disadvantages of the conventional, i.e. planar, DNA chip technology can be overcome by introducing capillary-based biochips.

Micro-capillaries offer very high surface-to-volume ratios that can potentially prove beneficial for applications with very low amount of available material, as discussed above.

Patent document EP-B-969 083 presents a DNA capillary, comprising a light transmitting wall and a plurality of independent probe zones formed on the wall inner surface, thanks to an immobilization by a photochemical reaction of different DNA probe reagents onto these probe zones. One may note that this document fails to disclose any specific method for introducing and transporting these distinct probe reagents along the capillary.

Patent document WO-A-02/089972 in name of the Applicant presents a microfluidic device for nucleic acid and/or protein analysis, which comprises a capillary to the wall inner surface of which is attached an array of at least two reagents. It is mentioned in this document that the method for attaching these reagents onto this wall surface comprises either the step of sequentially immobilizing pre-synthesized reagents on said surface, or the step of synthesizing in situ said reagents.

One should note again that the method of attachment of reagents which is referred to in this document mainly deals with the sequential immobilization and not with a particular way for introducing and transporting these reagents along the capillary, before immobilizing them.

Patent document US-A-6, 107,038 discloses a method for attaching a plurality of liquid reagents based on oligonucleotides onto a wall inner surface of a channel, such as a capillary, comprising the following steps:

(i) introducing into this channel, between a leading electrolyte and a terminating electrolyte, a solution containing a mixture of these reagents, wherein the nucleotides have been modified (e.g. by electrical charges) to allow their subsequent electrophoretic separation,

(ii) separating by isotachophoresis into spatial zones within said channel said reagents from the mixture introduced in (i), according to their respective electrophoretic mobilities under a given electric field,

(iii) allowing the separated reagents to achieve by isotachophoresis a steady state speed of transport in the channel, and then

(iv) simultaneously attaching said transported reagents onto said wall surface on predetermined locations. One major drawback of the attachment method described in this document resides in the necessary introduction of a mixture of modified oligonucleotide reagents which need to be individually prepared and modified beforehand, so that the modified oligonucleotides may exhibit electrophoretic mobilities which differ from each other and that isotachophoresis separation may thus succeed, for all oligonucleotides are characterized by the same electrophoretic mobility.

As a result, such an attachment method, which requires both chemical modification and physical mixing steps prior to isotachophoretic separation and transport, appears to be time-consuming and cumbersome to carry out and, as a consequence, it involves a quite high implementation cost.

One purpose of the present invention is to overcome this disadvantages, and this is achieved in that the Applicant has surprisingly discovered that a method comprising:

(i) sequentially introducing a plurality of liquid electrolyte reagents into a channel, then

(ii) transporting said introduced reagents along said channel in a train of adjacent reagents, under a shear generated by a local electric field which is tangent to said wall surface, allows to subsequently stop and simultaneously attach the transported reagents onto said wall surface at separate locations while keeping the adjacent reagents substantially unmixed with each other in said train, from their introduction in the channel to their attachment thereto.

It will be noted that the method of the present invention involves a sequential (i.e. successive at time intervals) introduction of the individual reagents which are designed to respectively form probe zones onto the channel wall surface, such as a biochip capillary, contrary to that of document US-A-6, 107,038 which teaches the introduction of a mixture of reagents which are subsequently separated by isotachophoresis. It should also be noted that said reagents usable in the present invention are based on biomolecules which may have:

- either the same mobility under said predetermined electrical field, these biomolecules preferably consisting in this case of DNA fragments, nucleic acids or oligonucleotides, or

- different effective mobilities under said electrical field, such as DNA fragments or oligonucleotides which have been beforehand modified as in US-A-6, 107,038; peptides, glycopeptides, proteins, glycoproteins, oligosaccharides, polysaccharides, antibodies, antigens, etc. One should note that biomolecules having the same effective mobility represent an advantageous embodiment of instant invention, inasmuch as it allows to do without any chemical modification of such biomolecules, contrary to the teaching of document US-A-6, 107,038.

In the case of using biomolecules of different effective mobilities, said reagents should be introduced in an order ranging from the highest effective mobility to the lowest one.

According to another feature of the present invention, the molecules of each reagent are advantageously transported in step (ii) by electrosmosis in a unique predetermined direction.

According to a further aspect of the present invention, said reagents are transported in step (ii) with a flow velocity profile v which is substantially flat and independent upon a cross-dimension of said channel, such as its radial dimension in the preferential case of a capillary, and which is essentially defined by the relation:

v = °-^E (1), where ε represents the permittivity of each reagent, ε₀ represents the permittivity of the vacuum, ψ_z represents the zeta potential of said wall surface,

E represents the electric field intensity, and η is the viscosity of said reagent. It should be noted that the present invention is based on the advantageous selection of a very particular electrokinetic liquid transport by electrosmosis, i.e. an electrosmotic or plug-like flow, which is characterized by a flat velocity profile and a purely diffusive sample dispersion (see Rice, C. L., Whitehead, R., "Electrokinetic Flow in a Narrow Cylindrical Capillary", J. Phys. Chem., 69, 4017-4023, 1965).

When each electrolyte liquid reagent is adjacent to the channel wall surface, the latter can be charged through ionization of covalently bond surface groups (e.g. a glass surface produces SiO- surface groups and releases protons in the presence of water), or through adsorption of ions. In both cases, the wall surface gains a charge while the concentration of ions close to this surface becomes perturbed, i.e. the counter-ions are attracted to said surface and the co-ions are repelled therefrom. Such in the case of charged molecules in salty solutions, the surface charge leads to the formation of an electrical double layer with its thickness, also called Debye length, κ^'1 , described as follows: κ--⁷ = (ε_bεok_BT /2cz²e²)^1/2

(2) where:

S_b is the dielectric constant of the liquid reagent, ε₀ represents the permittivity of the vacuum,

/cs is the Boltzman constant, T is the absolute temperature, c is the electrolyte concentration, z is the charge of the ions in the electrolyte, and e is the charge of one electron.

For dilute solutions (ε_b = 78.49) at 25° C, this equation can be expressed as: K¹ = 0.304 z σ^1/2 (3) where C is the bulk z:z electrolyte concentration in mol/L and κ^'1 is given in nm. When an electric field E is applied along a channel, an electric force acts on the volume charge in the Debye layer that is made of excess of solvated counter-ions. Apart from this region, the liquid is electro-neutral and the net electric force which applied to the ions cancels out. However, since there is a net charge in the Debye (electrical double) layer, the local electric field that is tangent to the channel surface generates a body force on the liquid, induces a shear and sets the liquid inside the channel into motion. This liquid motion is called electroosmosis.

The velocity v (y) of electrosmotic flow is zero at the channel surface, increases over the size of Debye layer and becomes constant beyond this length scale, as resulting from the following relations:

v(y = 0) = 0 (4a) channel Surface

. -1 - εε . v(0 < y < K ) = — ^s-(ψ (y) - ψ ,)E (4b) electrical double layer V

— SS XI/ v(y —> oo ) = v_eo = — — E (4c) channel

V

The electrosmotic velocity is proportional to the electric field strength and surface (or zeta) potential and inversely proportional to the viscosity of liquid in the Debye layer, and it does not depend on the channel diameter d as long as d »κ^~1. Indeed, this is the case for most microfluidic applications as channel diameter is usually in μm-size range while the Debye thickness is in nm-s^'ize range. Thus, the electrosmotic flow is a plug-like flow with a flat velocity profile, contrary to the parabolic velocity profile of pressure- driven or laminar flows. The sample dispersion in electrosmotic flows is purely diffusive.

In addition, said electrosmotic velocity v does not depend on the channel radius r, as long as r » κ^~1 , while the velocity of laminar flows VL is proportional to the square of channel radius r (v_L ~ i²). This facilitates the use of small bore capillaries when this electrosmotic transport according to the invention is used. According to a further embodiment of the present invention, said reagents may be substantially contiguous in said train.

Alternatively, each pair of said reagents may be separated from each other in said train by a liquid spacer, such as a buffer of a variable length.

According to a still further embodiment of the present invention, step (i) may be carried out by sequentially introducing said reagents either by a pressure-driven flow or in an electrokinetic way.

According to a still further embodiment of the present invention, the attachment of said plurality of reagents to said wall surface is advantageously accomplished by a photochemical reaction.

According to another aspect of the present invention, there is provided a process for fabricating a microfluidic system, such as a DNA biochip, which is designed to detect biomolecules in order to perform localized biological and/or chemical reactions (e.g. PCR reactions) on a plurality of spatially separate probe zones located on a wall inner surface of at least one channel of said system, such as a capillary, said process comprising attaching a plurality of liquid electrolyte reagents onto said wall surface in order to respectively form said plurality of probe zones, by:

(i) introducing sequentially said reagents into said at least one channel,

(ii) transporting said introduced reagents along said at least one channel to respective predetermined locations, in a train of adjacent reagents which remain substantially unmixed from their introduction to their attachment, under a shear generated by a local electric field which is tangent to said surface, and then

(iii) stopping and simultaneously attaching said transported reagents onto said wall surface at said locations. Said fabrication process is further characterized in that:

- the molecules of each reagent are transported in a unique predetermined direction in step (ii), such as by electrosmosis;

- said reagents are transported in step (ii) with a flow velocity profile v which is substantially flat and independent upon a cross-dimension of said channel or capillary, such as its radial dimension, and which is essentially defined by the hereinabove recited relation (1);

- said reagents may be substantially contiguous in said train or each pair of said reagents may be separated from each other in said train by a liquid spacer, such as a buffer of a variable length;

- step (i) is carried out by sequentially introducing said reagents either by a pressure-driven flow or in an electrokinetic way; and

- said introduced and transported reagents advantageously include non-modified biochemical molecules having all the same mobility under said electrical field, such as DNA fragments, nucleic acids and oligonucleotides.

According to another embodiment of the fabrication process according to the present invention, such a channel or capillary provided with a plurality of probe sites may be fabricated after one or more than one cycle(s) of introduction, transport and attachment of said plurality of reagents.

The above-mentioned characteristics of the present invention, along with others, will be understood more clearly on reading the following description of several examples of the invention, which are given for illustrative purposes and are not intended to limit the invention, said description referring to the attached drawings, wherein: figure 1 is a schematic diagram of one embodiment of the present invention illustrating a train of adjacent liquid electrolyte reagents Sj being transported inside a capillary of a microfluidic system, such as a biochip; figure 2 is a schematic diagram of another embodiment of the present invention illustrating a train of these reagents Sj being separately transported by some neutral spacers N, in such a capillary; figure 3 is a schematic diagram of a flat flow velocity profile v characterizing the electrosmotic transport of reagents in a capillary according to the present invention; figures 4 and 5 are two graphs respectively showing the difference between a pressure-driven transport of said reagents, which is not according to the invention, and an electrosmotic transport thereof, in terms of UV absorbance (AU) in function of migration time (minutes); figures 6, 7, 8 are three graphs respectively showing the different peak shapes, in terms of UV absorbance (AU) in function of migration time (minutes), for reagents being:

- introduced and transported along the capillary by a pressure- driven flow (figure 6),

- introduced in the capillary in an electrokinetic way and transported therein by a pressure-driven flow (figure 7), and

- introduced in the capillary in an electrokinetic way and transported therein according to the invention by electrosmosis (figure 8); and figure 9 is a graph showing a sequence of 21 oligonucleotide reagents, which are separated by 20 buffer spacers and which are introduced and transported inside a capillary according to the invention by electrosmosis, in terms of UV absorbance (AU) in function of migration time (minutes).

The schematic diagram of figure 1 shows a train of contiguous liquid electrolytes reagents Sj flowing inside a biochip capillary of 10 cm long. Si is the first reagent, S₂ is the second reagent, S₃ is the third reagent ... and S_N is the N^th reagent. The schematic diagram of figure 2 shows a train of separated liquid electrolytes reagents which flow inside a biochip capillary of 10 cm long and which are separated by neutral spacers Nj, wherein Ni is the first neutral spacer, Na is the second neutral spacer, N₃ is the third neutral spacer ... and NN-I is the N-1^th neutral spacer.

The schematic diagram of figure 3 shows an electrosmotic, flat flow profile according to instant invention inside the capillary, wherein d is the channel diameter and κ^~1 is the thickness of the electrical double layer. It will be noted that electrosmosis advantageously involves a minimized dispersion during the transport process, in comparison to laminar flows.

COMPARATIVE AND INVENTION EXAMPLES:

There was fabricated a biochip capillary presenting the following features according to a transport method of pressure-driven flow type (i.e. not according to the invention) and according to an electrosmotic method (i.e. according to the invention).

1) Experimental conditions:

a) The fused cylindrical silica capillaries (Polymicro Technologies, Phoenix, USA) were UV-light transparent with a 100 μm inner diameter and a 365 μm outer diameter. These capillaries were covalently modified with a linear polyacrylamide, according to Hjerten, S., J. Chromatogr., 347, 191 , 1985., in order to decrease the electrosmotic flow. The length of the capillary biochip was typically of a few centimeters.

b) Oligonucleotides labeled with Cy3 fluorophore were covalently and locally attached to the inner surface of the capillary biochip. The concentration of the Cy3-labeled oligonucleotide was 1 nM.

The employed reagent sample was a 23-mer oligonucleotide (GAGGTGTCCGCA l l l l l l l l l l l ; Apibio, France). The original solution (500 μM) was diluted with MiIiQ water (Millipore, France) 25-times to a final concentration of 20 μM. Sodium Phosphate buffer (30 mM, pH 8.5) was used for the experiments. The sample dispersion was measured on P/ACE MDQ capillary electrophoresis instrument (Beckman, USA). The oligonucleotides were detected by absorption at 254 nm. The measured electrosmotic mobility in such capillaries was inferior to 1 x 10^~5 cm²/V.s. The reagent sample was injected three times consecutively (field strength, 50 V/cm; injection time 10 s). The buffer was injected, as a spacer, between each injection (field strength, 50 V/cm; injection time 120 s).

c) The injected samples were transported over 20 cm of capillary by a pressure-driven flow (0.2 psi, corresponding to figure 4) and by electrosmosis (field strength, 300 V/cm, corresponding to figure 5).

2) Results:

a) Figures 4 and 5 compare the dispersion of an injected reagent sample zone during pressure-driven and electrosmotic transports. It can be clearly seen from Figures 4 and 5 that the sample dispersion is much smaller in the case of electrosmotic transport (Figure 5), as compared to the pressure-driven flow one (Figure 4). In fact, the reagent sample disperses during electrosmosis by diffusion, while it disperses faster in laminar flows due to the flow parabolic profile.

b) Figures 6, 7, 8 show the different peak shapes for:

- figure 6: a reagent sample being injected and transported along the capillary by a pressure-driven flow (injection: 0.2 psi, 10 s; transport: 0.2 psi),

- figure 7: a reagent sample being introduced electrokinetically and transported along the capillary by a pressure-driven flow (injection: 50

V/cm, 10 s; transport: 0.2 psi), and for - figure 8: a sample being introduced electrokinetically and transported along the capillary by electrosmosis (injection: 50 V/cm, 10 s; transport: 300 V/cm). The other conditions were the same as for Figure 5.

Figures 6 and 7 compare the peak width of samples introduced by pressure (Figure 6) and by the electric field (Figure 7). The peak width of the sample injected by pressure is 21.9±1.9 mm, while it is 21.3±1.3 mm for the electrokinetic introduction.

It is to be noted that in both cases the introduced reagent samples were transported inside the capillary from the capillary extremity to the detector (20 cm) by a pressure-driven flow.

This result suggests that either the sample pre-concentration is negligible during the electrokinetic injection or that the sample dispersion during the transport is the pre-dominant process.

On the other hand, the sample dispersion is much smaller when the electrosmotic transport is employed, see Figure 8. The peak width at half-height is 4.2±0.3 mm, which is approximately 5-times less than when the sample is transported in a pressure-driven flow (Figure 7).

c) Finally, 21 sample reagents were injected and transported according to instant invention by electrosmosis, during 20 cm and detected on-line by UV absorption at 254 nm (see Figure 9). The oligonucleotide reagent zones were separated from each other by buffer zone.

Figure 9 is a graph showing a sequence of these 21 sample reagent zones, separated by buffer zones, introduced electrokinetically and transported by electrosmosis in a capillary. The sample was injected 21 -times consecutively under the following conditions:

- Electrokinetic sample injection: 33.33 V/cm, 6 s; and

- Electrokinetic buffer (spacer) injection: 33.33 V/cm, 90 s. The sample transport was effected by electrosmosis under

300 V/cm. The other conditions were the same as for Figure 5.

Claims

1. Method for attaching a plurality of liquid electrolyte reagents (S-i, S₂, S3 ... S_N) onto a wall inner surface of a channel, comprising the following steps:

(i) introducing said reagents into said channel, then (ii) transporting said introduced reagents along said channel to respective predetermined locations, and then

(iii) stopping and simultaneously attaching said transported reagents to said wall surface at said locations, characterized in that step (i) comprises introducing sequentially said reagents, and in that step (ii) comprises transporting said reagents in a train of adjacent reagents which remain substantially unmixed from their introduction to their attachment, under a shear generated by a local electric field which is tangent to said surface.

2. Method according to claim 1, characterized in that the molecules of each reagent (S-i, S₂, S₃ ... SN) are transported in a unique predetermined direction in step (ii), such as by electrosmosis.

3. Method according to claim 1 or 2, characterized in that said reagents (S-i, S₂, S₃ ... S_N) are transported in step (ii) with a flow velocity profile which is substantially flat and independent upon a cross-dimension of said channel, such as its diameter.

4. Method according to claim 3, characterized in that said flow velocity profile v is essentially defined by the relation:

v= ⁹^-^Z-E , where ε represents the permittivity of each reagent, ε₀ represents the permittivity of the vacuum, ψ_z represents the zeta potential of said wall surface,

E represents the intensity of said electric field, and η represents the viscosity of said reagent (S-i, S₂, S₃ ... S_N).

5. Method according to any of claims 1 to 4, characterized in that said reagents (S-i, S₂, S₃ ... SN) are substantially contiguous in said train.

6. Method according to any of claims 1 to 4, characterized in that each pair of said reagents (S-i, S₂, S3 ... SN) are separated from each other in said train by a liquid spacer (Ni, N₂, N₃ ... NN-I), such as a buffer.

7. Method according to any of claims 1 to 6, characterized in that step (i) is carried out by sequentially introducing said reagents (S₁, S₂, S₃ ... SN) by a pressure-driven flow.

8. Method according to any of claims 1 to 6, characterized in that step (i) is carried out by sequentially introducing said reagents (S-i, S₂, S₃

... SN) in an electrokinetic way.

9. Method according to any of the preceding claims, characterized in that said introduced and transported reagents (Si, S₂, S₃ ... S_N) include biochemical molecules all having the same mobility under said predetermined electrical field, such as DNA fragments, nucleic acids and oligonucleotides.

10. Method according to any of the preceding claims, characterized in that said channel is a capillary which is usable for a microfluidic system, such as a DNA biochip.

11. Process for fabricating a microfluidic system, such as a

DNA biochip, designed to detect biomolecules in order to perform localized biological and/or chemical reactions on a plurality of spatially separate probe zones located along a wall inner surface of at least one channel of said system, such as a capillary, said process comprising attaching a plurality of liquid electrolyte reagents (S-i, S₂, S₃ ... S_N) to said wall surface in order to respectively form said plurality of probe zones, by:

(i) introducing said reagents into said at least one channel, (ii) transporting said introduced reagents along said at least one channel to respective predetermined locations, and then (iii) stopping and simultaneously attaching said transported reagents to said wall surface at said locations, characterized in that step (i) comprises introducing sequentially said reagents, and in that step (ii) comprises transporting said reagents in a train of adjacent reagents which remain substantially unmixed from their introduction to their attachment, under a shear generated by a local electric field which is tangent to said surface.

12. Process according to claim 11 , characterized in that the molecules of each reagent (S-i, S₂, S₃ ... SN) are transported in a unique predetermined direction in step (ii), such as by electrosmosis.

13. Process according to claim 11 or 12, characterized in that said reagents (S-i, S₂, S₃ ... S_N) are transported in step (ii) with a flow velocity profile which is substantially flat and independent upon a cross-dimension of said channel, such as its diameter.

14. Process according to claim 13, characterized in that said flow velocity profile v is essentially defined by the relation:

v= ^~εε°^Ψz E , where ε represents the permittivity of each reagent, ε₀ represents the permittivity of the vacuum, ψ_z represents the zeta potential of said wall surface, E represents the intensity of said electric field, and η represents the viscosity of said reagent (S-i, S₂, S₃ ... SN).

15. Process according to any of claims 11 to 14, characterized in that said reagents (S-i, S₂, S₃ ... S_N) are substantially contiguous in said train.

16. Process according to any of claims 11 to 14, characterized in that each pair of said reagents (S-i, S₂, S₃ ... S_N) are separated from each other in said train by a liquid spacer (N₁, N₂, N₃ ... NN-I), such as a buffer.

17. Process according to any of claims 11 to 16, characterized in that step (i) is carried out by sequentially introducing said reagents (S-i, S₂, S₃ ... S_N) by a pressure-driven flow.

18. Process according to any of claims 11 to 16, characterized in that step (i) is carried out by sequentially introducing said reagents (S-i, S₂, S3 ... SN) in an electrokinetic way.

19. Process according to any of claims 11 to 18, characterized in that said introduced and transported reagents (Si, S₂, S₃ ...

S_N) include biochemical molecules having all the same mobility under said predetermined electrical field, such as DNA fragments, nucleic acids and oligonucleotides.