WO2017125475A1

WO2017125475A1 - Device, system, and method for manipulating objects, particularly micro- or nano-objects, and method for fabricating a device for manipulating objects, particularly micro- or nano-objects

Info

Publication number: WO2017125475A1
Application number: PCT/EP2017/051054
Authority: WO
Inventors: Patric Eberle; Mohammad Hadi Eghlidi; Dimos Poulikakos
Original assignee: Eth Zurich
Priority date: 2016-01-19
Filing date: 2017-01-19
Publication date: 2017-07-27

Abstract

The invention relates to a device (1) for manipulating objects (30), particularly micro- or nano-objects in a fluid, comprising a channel (10), having a maximal cross-sectional extension (emax) and comprising at least one constriction (14) with a minimal cross-sectional extension (emin) perpendicular to the longitudinal direction (L) of the channel (10), wherein the minimal cross-sectional extension (emin) is smaller than the maximal cross-sectional extension (emax), a first and a second electrode (20,21), which are adapted to generate an electric field in the channel (10) by means of a voltage applied between the first and the second electrode (20, 21), wherein the first and the second electrode (20,21) are positioned, in the channel (10) along the longitudinal direction (L) at opposite sides of the constriction (14). The invention further relates to a system and a method for manipulating objects (30) and a method for fabricating the device (1).

Description

Device, system, and method for manipulating objects, particularly micro- or nano- objects, and method for fabricating a device for manipulating objects, particularly micro- or nano-objects

The invention relates to the field of micro- and nanofluidics, particularly manipulating objects of small size (micro or nano objects) in a fluid, that is moving, accelerating and/ or restricting the movement of the objects.

Several methods for manipulating small objects are known from the prior art. For example, in a method termed dielectrophoresis, objects from a dielectric material can be moved by a force generated by a non-uniform electric field. However, this method is restricted to objects that can be polarized in the electric field, and is therefore not applicable to certain materials.

In addition, dielectrophoresis methods of the prior art are limited in their capability to accelerate, move, and restrict the movement of sub-micron particles. Furthermore, disadvantages of the prior art are exemplified by the need for intense fields, possibly harmful to biological species, the lack of versatility in handling different operating conditions and solutions of high ionic strength necessary for biological applications and the lack of compatibility with continuous lab-on-chip applications.

Therefore, the problem to be solved by the present invention is to provide a means of manipulating, particularly moving, accelerating and/ or restricting the movement of, small objects, which is improved with respect to the above-stated disadvantages of the prior art. The problem is solved by the subject matter of the independent claims 1 , 7, 8, and 9.

Embodiments of the invention are claimed by the dependent claims 2 to 6, and 10 to 17 and described hereafter.

According to a first aspect of the invention, a device for manipulating objects (also termed 'particles'), particularly micro- or nano-objects, in a fluid is provided, wherein the device comprises a channel, particularly a microfluidic or nanofluidic channel, wherein the channel has a maximal cross-sectional extension perpendicular to a longitudinal direction of the channel, and the channel comprises at least one constriction with a minimal cross-sectional extension perpendicular to the longitudinal direction of the channel, wherein the minimal cross-sectional extension is smaller than the maximal cross-sectional extension, particularly wherein the cross-sectional area at the constriction is smaller than the cross-sectional area at both sides adjacent to the constriction, a first electrode and a second electrode, wherein the first electrode and the second electrode are adapted to generate an electric field in the channel by means of a voltage applied between the first electrode and the second electrode, wherein the first electrode and the second electrode are positioned, particularly embedded, in the channel along the longitudinal direction of the channel at opposite sides of the constriction.

In certain embodiments, the section of the channel having the minimal cross-sectional extension (that is at the position of the constriction) forms a minimal cross-sectional area of the channel perpendicular to the longitudinal direction of the channel compared to the cross- sectional area of the channel neighbouring the constriction.

In particular, the device is adapted to accelerate, move, and/ or restrict the movement of an object, more particularly a micro or nano object (or particle), by means of the electric field.

The described device allows implementation of a switchable electrokinetic valve, particularly nanovalve, without moving parts, to confine and guide objects, particularly micro and nano objects suspended in a fluid, particularly a liquid, for example in a lab-on-chip environment. The operating principle of the device, particularly the electrokinetic valve, is based on spatiotemporal tailoring of the free energy landscape for an object, particularly micro or nano- object. This is achieved by an electric field modulated collaboratively by the topography (that is by the constriction between the electrodes) and addressable electrodes, particularly embedded electrodes, in the channel.

In particular, on-demand and precise motion control of single adenoviruses, lipid vesicles, dielectric and metallic particles, of various sizes and inherent charges, suspended in electrolytes with ionic strengths up to biological buffer solution levels, can be achieved by means of the device according to the first aspect.

In order to operate the device, external voltages can be applied to the first and second electrode to regulate the free energy potential for the object to non-intrusively regulate its motion by the configuration of the electric field alone.

The device can be driven with either alternating current (AC) or direct current (DC) voltages. This allows the exploitation of different physical effects to exert sufficiently large forces on the objects, leading to a broad range of utility of the device (vis. particle type, size and charge, and liquid type). The two operational modes of the device can be understood by considering the free energy landscapes modulated by the combination of the first and second electrode and the topography (constriction) in the device. When exposed to a fluid such as water or an aqueous solution, the channel walls attain an equilibrium state with negative surface charge and, as a result, a double layer with a decaying electrostatic potential is formed at the surface with a characteristic Debye length κ^~ι oc l / . Here n₀ is the ionic strength of the electrolyte given by n₀ = 0-5 _^,. _,c_iz_i , where ς. and z. are the concentration and the valence of the /-th ionic species, respectively, and the summation is taken over all the ions. For low ionic strength electrolytes (n₀ < 0,5 mM) the double layers from a charged object and the channel wall can interact. At the channel constriction, because of increased interaction between the double layers of the channel walls and the object, an energy barrier AF_b = AF(max) - AF(min) for the object is formed. This barrier can be substantially higher than the inherent thermal energy k_BT (k_B, Boltzman constant) of the object and, therefore, can prevent its passage over the barrier (closed valve).

An electric field imposed along the channel by applying a voltage between the two electrodes in the DC mode bends the free energy potential profile and as a consequence the energy barrier is reduced. At sufficient field strengths the barrier is small enough so that the fluctuating thermal energy of the object exceeds the barrier and the particle is guided down the energy profile (open valve).

While the DC mode can handle charged particles in liquids with low ionic strengths with simplicity, it is not applicable for electrolyte ionic strengths >~0,5 mM. However, for a host of applications, particularly in biological experiments, in order to preserve the integrity of biological species, the electrolytic strength exceeds this threshold. In such a situation the effectiveness of the topographically induced energy barrier is lost due to weakly interacting double layers. Furthermore, insufficient charges on the particle and/or surface passivation of the channel walls can lead to the same effect. The AC mode overcomes these limitations and extends the operation to a wide range of ionic strengths and particle types. An imposed AC electric field along the channel induces a time-averaged free energy potential landscape of dielectrophoretic nature for the particle, stemming from the polarizability of the particle and the electrolyte, according to the following relation (Morgan, H. & Green, N. G. AC

Electrokinetics: Colloids and Nanoparticles. Research Studies Press, 2003; Pethig, R.

Dielectrophoresis: Status of the theory, technology, and applications. Biomicrofluidics 4, (2010)).

(A ) = -2^_mR³ Re(C )|E,_c|² (1 ), where z_m, R and E^are the permittivity of the electrolyte, the effective particle radius and σ — σ

the electric field from the applied AC voltage, respectively. CM =— is the Clausius- σ p + 2σ m

Mossotti factor, which is frequency dependent. The subscripts p and m denote particle and surrounding medium (electrolyte), respectively, and σ^* is the frequency-dependent complex electrical conductivity. For negative values of the real part of the complex Clausius-Mossotti factor, Re(CM), we obtain a potential barrier between the electrodes which is significantly amplified within the constriction due to the increased electric field intensity. Negative values of Re(CM) can be achieved by operating the device at frequencies where the conductivity of the electrolyte is larger than the conductivity of the particle. It is worth mentioning that this criterion cannot be easily fulfilled for metallic nanoparticles. However, charged metallic nanoparticles in sufficiently low ionic strength electrolytes can be handled using DC mode.

The cross-sectional area of the channel of the device may have various shapes, for example a rectangular shape or a circular shape. If the cross-sectional area has a circular shape, the term 'cross-sectional extension' corresponds to the diameter of the cross-sectional area. Alternatively, if the cross-sectional area has a rectangular shape, the term 'maximal cross- sectional extension' corresponds to the maximal width or height of the channel, depending on whether the width or the height is larger.

In certain embodiments, the channel has a width of about 500 nm, particularly 500 nm, and a depth of about 300 nm, particularly 300 nm.

In certain embodiments, at the constriction the channel becomes shallower with a depth of about 200 nm, particularly 200 nm.

In certain embodiments, at the constriction the channel becomes shallower and narrower with a depth of about 40 nm, particularly 40 nm, and width of about 70 nm, particularly 70 nm. This results in an improved manipulation of very small objects (e.g. quantum dots with diameters of 10-20 nm). In certain embodiments, the constriction has a depth of about 200 nm, particularly 200 nm.

In certain embodiments, at least a part of an inner wall, particularly the surface of at least a part of the inner wall, of the channel is adapted to exhibit a surface charge when brought into contact with a fluid, particularly water and/ or an aqueous solution.

In certain embodiments, the inner wall, particularly the surface of the inner wall, of the channel comprises Si0₂. In particular, this material exhibits a surface charge when brought into contact with water or an aqueous solution.

In certain embodiments, the maximal cross-sectional extension (e_max) of the channel is between 10 nm and 200 μηη.

In certain embodiments, the maximal cross-sectional extension is between 10 X 10 nm² and 200 X 200 m².

In certain embodiments, the device comprises a first constriction, a further constriction and a third electrode, wherein the first electrode is positioned between the first constriction and the second constriction, and wherein the second electrode is positioned at the opposite side of the first constriction in relation to the first electrode, and wherein the third electrode is positioned at the opposite side of the further constriction in relation to the first electrode. That is, according to this embodiment, three electrodes are positioned along the channel and two constrictions are positioned between the electrodes.

In particular, the first and the second electrode are adapted to generate an electrical field at the first constriction if a voltage between the first and the second electrode is provided, and/ or the first and the third electrode are adapted to generate an electrical field at the further constriction if a voltage between the first and the third electrode is provided. In particular, such a device can have the effect of two valves connected in series. Therefore, in particular, an object can be trapped at a position in the channel adjacent to the first electrode if both valves are in the closed state. Therefore, such a device is also designated as 'trap-in-channel' device

If operated in DC mode, the respective valve is open when a voltage is applied and closed when no voltage is applied between the respective pair of electrodes. Alternatively, if operated in AC mode, the respective valve is open when no voltage is applied and closed when a voltage is applied between the respective pair of electrodes.

In certain embodiments, the device comprises a first channel comprising a first constriction, wherein the first electrode is positioned adjacent to the first constriction, a second channel comprising a second constriction, wherein the second electrode is positioned adjacent to the second constriction, a third channel comprising a third constriction, a third electrode, which is positioned adjacent to the third constriction, a junction, wherein the first channel, the second channel, and the third channel are connected by the junction, such that a flow connection between the first channel, the second channel, and the third channel is established, and a fourth electrode, which is positioned at the junction.

In certain embodiments, the channel of the device comprises a first segment comprising a first constriction, wherein the first electrode is positioned adjacent to the first constriction, a second segment comprising a second constriction, wherein the second electrode is positioned adjacent to the second constriction, a third segment comprising a third

constriction, a third electrode, which is positioned adjacent to the third constriction, a junction, wherein the first segment, the second segment, and the third segment are connected by the junction, such that a flow connection between the first segment, the second segment, and the third segment is established, and a fourth electrode, which is positioned at the junction. That is, according to this embodiment, the device comprises a Y-shaped structure

comprising three channels (or segments) connected at a central junction, and four electrodes, wherein a central electrode (that is, the fourth electrode) is positioned at the central junction of the Y-shape, and three electrodes (the first, second, and third electrodes) are positioned in the three channels (or segments) of the Y-shaped device, and wherein the device comprises three constrictions in the three channels (or segments) between the central electrode and the respective electrode positioned in the respective channel (or segment).

In particular, this device can be used as a three-way valve, wherein the valve comprises three connected arms corresponding to the channels (or segments), and wherein each arm comprises a valve which can selectively be closed and opened by applying a voltage between respective pairs of electrodes.

In particular, the first and the fourth electrode are adapted to generate an electrical field at the first constriction if a voltage between the first electrode and the fourth electrode is provided, the second electrode and the fourth electrode are adapted to generate an electrical field at the second constriction if a voltage between the second electrode and the fourth electrode is provided, and third electrode and the fourth electrode are adapted to generate an electric field at the third constriction if a voltage between the third electrode and the fourth electrode is provided.

For example, such a device can be used to trap an object at the junction adjacent to the central fourth electrode. Therefore, such a device is also designated as 'trap-in-junction' device. In addition, an object trapped at the position of the fourth electrode can be released into a specified channel of the device by opening a valve in one of the arms (channels).

Furthermore, it is possible to provide the object in one of the channels, and direct the movement of the object into one of the other channels. In particular, this allows sorting objects into selected fluid paths. In certain embodiments, at least a part of an inner wall, particularly at least a part of the surface of an inner wall of the channel comprises a passivation agent, particularly bovine serum albumin or polyethylene glycol.

Advantageously, a passivation agent avoids sticking of the object, particularly biological object, to the inner wall, particularly to the surface of the inner wall, of the channel. This facilitates more reliable manipulation of the object by means of the device. In this embodiment, it is especially advantageous to use the AC mode of the device, because the passivation agent reduces the surface charge of the channel wall, which is required for operation in the DC mode. According to a second aspect of the invention, a system for manipulating objects, particularly micro- and/or nano-objects, comprising at least two devices for manipulating objects, particularly micro- and/or nano-objects, according to the first aspect of the invention is provided, wherein the respective channels of the at least two devices are in flow connection with each other, and wherein an electric field in the respective channel of the respective device can be provided by means of a voltage applied between the respective first electrode and the respective second electrode.

In certain embodiments, each device comprises a channel having a respective maximal cross-sectional extension, and wherein each channel comprises a constriction with a respective minimal cross-sectional extension, wherein the respective minimal cross-sectional extension is smaller than the respective maximal cross-sectional extension, and wherein each device comprises at least a first electrode and a second electrode, wherein an electric field in the respective channel of the respective device can be provided by means of a voltage applied between the respective first electrode and the respective second electrode.

In the system according to the second aspect, multiple independently addressable valves can be combined to achieve various on-demand functionalities, such as single entity control in trapping and sorting/combining of nanoscopic objects. In particular, the on-chip design of the proposed concept allows parallelization of nanofluidic processes and large scale, seamless integration of nanofluidic-based devices into existing microfluidic systems.

In certain embodiments, in each channel the respective first electrode and the respective second electrode are positioned at opposite sides of the respective constriction along a longitudinal direction of the respective channel.

In particular, possible arrangements of the devices in the system include devices (or valves) connected in series, devices (or valves) connected in parallel, more particularly in two parallel channels, and devices arranged as a three-way valve (that is three valves connected to each other at a central junction).

According to a third aspect of the invention, a method for fabricating a device for

manipulating objects, particularly micro- and/or nano-objects, according to the first aspect of the invention is provided, wherein the method comprises the steps of providing a surface, particularly a silicon wafer, providing a first layer, particularly comprising Si0₂, on the surface, particularly by depositing the first layer on the surface, more particularly by plasma- enhanced chemical vapour deposition, low pressure chemical vapour deposition, or thermal oxidation of silicon, generating at least one recess in the first layer, particularly by generating a pattern on the first layer by means of a first electron beam lithography process or photolithography process, and etching the at least one recess from the first layer or into the first layer at locations patterned by the first electron beam lithography process or photolithography process, providing at least one electrode by depositing a metal layer onto the first layer at the location of the at least one recess, particularly by vapour deposition, more particularly by electron beam evaporation, depositing a second layer, particularly comprising Si0₂, onto the first layer and/ or onto the metal layer, providing at least one constriction by generating at least one recess in the second layer, particularly by generating a pattern on the second layer by means of a second electron beam lithography process or photolithography process, and etching the at least one recess from the second layer at locations patterned by the second electron beam lithography or photolithography process. In particular, the described layer structure forms a first wall of the channel of the device. In particular the metal layers deposited in the recesses of the first layer form the electrodes of the device. In particular, the constriction of the channel is formed by generating the recesses in the second layer, wherein more particularly at least one section of the second layer, into which no recess is introduced, forms at least a part of the constriction of the channel. Advantageously, by means of the described method a device comprising a channel with embedded electrodes and a constriction between the electrodes can be generated at the small size (in the nanometer to micrometer range) required to manipulate micro and nano objects.

In certain embodiments, a passivation layer, particularly a Si0₂ layer, more particularly a 2 nm thin Si0₂ layer, is deposited onto the second layer. This advantageously provides electrochemical passivation of the electrodes, particularly nanoelectrodes, such that less electrochemical reactions occur in the fluid contacting the electrodes.

In certain embodiments, a top wall structure is positioned on the second layer. Thereby, a closed channel structure can be generated. For example, the top wall structure may be a glass slide, wherein particularly the channel is sealed by anodic bonding of the glass slide to the top facet of the structure.

In certain embodiments, a third layer, particularly comprising Si0₂ and/ or of a thickness < 10 nm, is deposited onto the second layer.

In certain embodiments, a third layer comprising Si0₂ is deposited onto the second layer. In certain embodiments, a third layer of a thickness < 10 nm, is deposited onto the second layer.

In certain embodiments, a third layer of a thickness < 10 nm, is deposited onto the second layer, wherein the third layer comprises Si0₂. According to a fourth aspect of the invention, a method for manipulating objects, particularly micro- and/or nano-objects, by means of a device for manipulating objects according to the first aspect or a system for manipulating objects according to the second aspect is provided, wherein the method comprises the steps of providing at least one object, particularly a micro- and/or nano-object, in a fluid in at least one channel of a device according to the first aspect of the invention or a system according to the second aspect of the invention, providing a voltage between the electrodes, particularly the first electrode and the second electrode, the first electrode and the third electrode and/ or the first electrode and the fourth electrode, of the device or the system, and moving and/or accelerating the at least one object and/ or restricting the movement of the at least one object by means of an electric field generated by the electrodes.

In particular for micro- or nano-objects, the term 'restricting the movement' comprises restricting the space in which the object moves, for example due to Brownian motion.

Therefore, in this case, the object still moves, but the movement is spatially confined.

By means of the at least one constriction of the at least one channel, the electric field between the first and the second electrode on opposite sides of the respective constriction the free energy landscape of the object is shaped such that a mechanical force is exerted on the particle. As described above for the first aspect of the invention, this can be achieved either by providing a direct voltage (or direct current, DC) between the electrodes, wherein the charged object and the charged channel walls interact such that the object is moved past the respective constriction, or by providing an alternating voltage (or alternating current, AC) between the electrodes, wherein the polarized object is prevented from moving past the respective constriction by a dielectrophoretic force acting on the object.

In certain embodiments, the at least one object is moved by means of the electric field.

In certain embodiments, the at least one object is accelerated by means of the electric field. In certain embodiments, the movement of the at least one object is restricted by means of the electric field.

In certain embodiments, an alternating voltage is provided between the electrodes, which results in an alternating electric field in the at least one channel, such that movement of the at least one object through the at least one constriction is prevented by means of a dielectrophoretic force acting on the at least one object as a result of the alternating electric field. Advantageously, this mode of operation allows use of a fluid of high ionic strength, which may be required in particular if the object is a biological object. In certain embodiments, a surface charge on at least a part of an inner wall, particularly at least a part of the surface of an inner wall, of the at least one channel is provided, such that a repulsive force between the inner wall, particularly the surface of the inner wall, of the respective channel and the object is generated, such that a movement of the object through the respective constriction is restricted by means of the repulsive force, particularly at an ionic strength of the fluid of < 0,5 mM.

In certain embodiments, a direct current voltage between the electrodes is provided, wherein an electrostatic force on the object is generated, such that the object is moved through the respective constriction. In particular, this mode of operation has the advantage that the method can also be applied on metallic objects.

In certain embodiments, the object is a particle from a conductive material, particularly a gold particle, or a particle from a dielectric material, particularly a polysterene bead, or a biological particle, more particularly a prokaryotic or eukaryotic cell, a bacterium, a protein, a complex comprising proteins, most particularly an exosome, an organelle, most particularly an endosome, a DNA molecule, an RNA molecule, a virus, a bacteriophage, a lipid vesicle, a combination thereof, a part thereof, or a combination of parts thereof.

In certain embodiments, the object has a maximum extension between 1 nm and 100 μηη.

In certain embodiments, the object has a size between 1 nm and 100 μηη.

In certain embodiments, at least two selected objects, particularly nano-objects, are brought into contact with each other and/or contained at a location by means of a device for manipulating objects, particularly micro- and/or nano-objects, according to the first aspect of the invention, wherein particularly a chemical reaction, more particularly a biochemical reaction, is triggered between the at least two selected objects.

In certain embodiments, at least two selected objects, particularly nano-objects, are brought into contact and/or positioned at nearly the same location in the at least one channel or junction, contained at a location by means of a system for manipulating objects, particularly micro- and/or nano-objects, according to the second aspect of the invention, wherein particularly a chemical reaction, more particularly a biochemical reaction, is triggered between the at least two selected objects. In certain embodiments, the at least two selected objects are contained at a location by means of a system for manipulating objects.

In certain embodiments, the path of movement of at least one object is controlled by applying a voltage to the electrodes comprised in the system for manipulating nano-objects. In certain embodiments, the path of movement of at least one object is controlled by applying a voltage to the electrodes of the respective devices comprised in the system for

manipulating nano-objects.

In certain embodiments, at least a part of an inner wall, particularly at least a part of the surface of the inner wall, of the at least one channel is coated with a passivation agent, particularly bovine serum albumin or polyethylene glycol.

In certain embodiments, the fluid comprises a surfactant, particularly polyethylene glycol sorbitan monolaurate or polyoxyethylenesorbitan monolaurate at a concentration of > 0,001 % volume per volume in order to reduce non-specific binding of objects to the inner wall, particularly to the surface of the inner wall. A further aspect of the invention relates to a device for manipulating objects, particularly micro- or nano-objects in a fluid, comprising a channel, particularly a microfluidic or nanofluidic channel, wherein the channel has a maximal cross-sectional extension (emax) perpendicular to a longitudinal direction of the channel, and the channel comprises at least one constriction with a minimal cross-sectional extension (emin) perpendicular to the longitudinal direction of the channel, wherein the minimal cross-sectional extension (emin) is smaller than the maximal cross-sectional extension (emax), particularly wherein the cross- sectional area at the constriction is smaller than the cross-sectional area at both sides adjacent to the constriction, a first electrode and a second electrode, wherein an electric field in the channel can be generated by means of a voltage applied between the first electrode and the second electrode, wherein the first electrode and the second electrode are positioned, particularly embedded, in the channel along the longitudinal direction of the channel at opposite sides of the constriction.

In certain embodiments, at least a part of an inner wall of the channel is adapted to exhibit a surface charge when brought into contact with a fluid, particularly water and/ or an aqueous solution, particularly wherein the inner wall of the channel comprises S1O2.

In certain embodiments, the maximal cross-sectional extension (emax) is between 10 nm and 200 μηι. In certain embodiments, the device comprises a first constriction, a second constriction and a third electrode, wherein the first electrode is positioned between the first constriction and the second constriction, and wherein the second electrode is positioned at the opposite side of the first constriction in relation to the first electrode, and wherein the third electrode is positioned at the opposite side of the second constriction in relation to the first electrode.

In certain embodiments, the channel comprises a first segment comprising a first

constriction, wherein the first electrode is positioned adjacent to the first constriction, a second segment comprising a second constriction, wherein the second electrode is positioned adjacent to the second constriction, a third segment comprising a third

constriction, a third electrode, which is positioned adjacent to the third constriction, a junction, wherein the first segment, the second segment, and the third segment are connected by the junction, such that a flow connection between the first segment, the second segment, and the third segment is established, a fourth electrode, which is positioned at the junction.

In certain embodiments, at least a part of an inner wall of the channel comprises a passivation agent, particularly bovine serum albumin or polyethylene glycol.

A further aspect of the invention relates to a system for manipulating objects, particularly micro- and/or nano-objects, comprising at least two devices for manipulating objects, particularly micro- and/or nano-objects according to the invention, wherein each device comprises a channel having a respective maximal cross-sectional extension (emaxn), and wherein each channel comprises a constriction with a respective minimal cross-sectional extension (eminn), wherein the respective minimal cross-sectional extension (eminn) is smaller than the respective maximal cross-sectional extension (emaxn), and wherein each device comprises at least a first electrode and a second electrode, wherein an electric field in the respective channel of the respective device can be provided by means of a voltage applied between the respective first electrode and the respective second electrode.

A further aspect of the invention relates to a method for fabricating a device for manipulating objects, particularly micro- and/or nano-objects, comprising the steps of providing a surface, particularly a silicon wafer, providing a first layer, particularly comprising S1O2, on the surface, particularly by depositing the first layer on the surface, more particularly by plasma- enhanced chemical vapour deposition, low pressure chemical vapour deposition, or thermal oxidation of silicon, generating at least one recess in the first layer, particularly by generating a pattern on the first layer by means of a first electron beam lithography process or photolithography process, and etching the at least one recess from the first layer at locations patterned by the first electron beam lithography process or photolithography process, depositing a metal layer onto the first layer at the location of the at least one recess, particularly by vapour deposition, more particularly by electron beam evaporation, depositing a second layer, particularly comprising S1O2, onto the first layer and/ or onto the metal layer, generating at least one recess in the second layer, particularly by generating a pattern on the second layer by means of a second electron beam lithography process or photolithography process, and etching the at least one recess from the second layer at locations patterned by the second electron beam lithography or photolithography process.

In certain embodiments, a third layer, particularly comprising SiC and/ or of a thickness < 10 nm, is deposited onto the second layer.

A further aspect of the invention relates to a method for manipulating objects, particularly micro- and/or nano-objects, comprising the steps of providing at least one object, particularly a micro- and/or nano-object in a fluid in a channel of a device according to the present invention, providing a voltage between the first electrode and a second electrode of the device, moving and/or accelerating the at least one object and/ or restricting the movement of the at least one object by means of the electric field.

In certain embodiments, an alternating voltage is provided between the electrodes, which results in an alternating electric field in the channel, such that movement of the at least one object through the constriction is prevented by means of a dielectrophoretic force acting on the at least one object as a result of the alternating electric field.

In certain embodiments, a surface charge on at least a part of an inner wall of the channel is provided, such that a repulsive force between the inner wall of the channel and the object is generated, such that a movement of the object through the constriction is restricted by means of the repulsive force, particularly at an ionic strength of the fluid of < 0,5 mM.

In certain embodiments, direct current voltage between the electrodes is provided, wherein an electrostatic force on the object is generated, such that the object is moved through the constriction.

In certain embodiments, the objects have a size between 1 nm and 100 μηη. In certain embodiments, at least two selected objects, particularly nanoobjects, are brought into contact and/or contained at a location by means of a device for manipulating objects, particularly micro- and/or nano-objects or a system for manipulating objects, particularly micro- and/or nano-objects according to the present invention, wherein particularly a chemical reaction, more particularly a biochemical reaction, is triggered between the at least two selected objects.

In certain embodiments, the path of movement of at least one object is controlled by applying a voltage to the electrodes of the respective devices comprised in the system for

manipulating objects.

In certain embodiments, at least a part of an inner wall of the channel is coated with a passivation agent, particularly bovine serum albumin or polyethylene glycol, and/or wherein the fluid comprises a surfactant, particularly Tween-20 at a concentration of > 0,001 % volume per volume in order to reduce non-specific binding of objects to the inner wall.

The invention is further described by means of figures and examples hereafter, which are meant to illustrate the invention, but not limit its scope.

Figure 1 shows a partially cutaway perspective view of an embodiment of the device (valve) for manipulating objects;

Figure 2 shows a particle free energy potential for closed and open states of the device for manipulating objects according to the invention operated in DC mode;

Figure 3 shows a particle free energy potential for closed, intermediate, and open states of the device for manipulating objects according to the invention operated in AC mode;

Figure 4 shows a sectional view of a further embodiment of the device for manipulating objects, comprising three electrodes and two constrictions;

Figure 5 shows a sectional view of a further embodiment of the device for manipulating objects, comprising three channels connected at a junction, four electrodes and three constrictions; Figure 6 shows a sequence of movement of an object through a device for manipulating objects according to the invention;

Figure 7 shows particle free energy potentials of the object moving through the device in the sequence depicted in Fig. 6; Figure 8 shows electrode voltages applied to the electrodes of the device for manipulating objects according to the invention during the sequence depicted in Fig. 6;

Figure 9 shows a sequence of movement of an object in a device for manipulating objects with three channels connected at a junction;

Figure 10 shows electrode voltages applied to the electrodes of the device for manipulating objects according to the invention during the sequence depicted in Fig. 9; Figure 1 1 shows lateral displacement data of polysterene bead in AC mode;

Figure 12 shows lateral displacement data of adenovirus in AC mode;

Figure 13 shows investigated nanoscale species and corresponding conditions; Figure 14 shows guiding, confining and releasing gold particles in DC mode in a trap-in-channel configuration;

Figure 15 shows particle free energy potentials of the gold particles moving through the device in the sequence depicted in Fig. 14;

Figure 16 shows electrode voltages applied to the electrodes of the device for manipulating objects according to the invention during the sequence depicted in Fig. 14;

Figure 17 shows guiding, confining and releasing gold particles in DC mode in a trap-in-junction configuration;

Figure 18 shows electrode voltages applied to the electrodes of the device for manipulating objects according to the invention during the sequence depicted in Fig. 17;

Figure 19 shows a interferometric scattering microscopy setup for imaging objects in a device according to the invention;

Figure 20 shows a fluorescent microscopy setup for imaging objects in a device according to the invention; Figure 21-22 show schematics of a fluidic chip comprising a device according to the invention;

Figure 23 shows a schematic of an example of a fabrication sequence of the device according to the invention;

Figure 24 shows a cyclic voltammogram of nanoelectrode materials used in a device according to the invention;

Figure 25 shows free energy and position probability density in AC mode;

Figure 26 shows a temperature distribution in the nanovalve from Joule heating;

Figure 27 shows guiding, confining and releasing lipid vesicles by means of a device according to the invention.

Figure 1 depicts a partially cutaway perspective view of a device 1 according to the present invention. The device 1 comprises a channel 10 delimited by inner walls 19 for conducting a fluid, for example water or an aqueous solution, wherein the channel 10 extends along a longitudinal direction L. In this particular example, the channel 10 has a rectangular cross- section identified by a depth h and a width w, wherein in this case the depth h is the maximal cross-sectional extension e_max. However, in other examples, the width w may be the maximal cross-sectional extension e_max. For example, the channel 10 may have a width w of 500 nm and a depth h of 300 nm. Furthermore, the device 1 comprises a first electrode (or nanoelectrode) 20, and a second electrode (or nanoelectrode) 21 , which are arranged in the channel 10 and embedded into an inner wall 19 of the channel 10. The electrodes 20,21 comprise a flat, plate-like geometry and extend along a plane formed by the longitudinal direction L and the direction of the width w. Figure 1 further shows a voltage source 24, which is electrically connected to the electrodes 20,21 , such that a voltage can be applied between the electrodes 20,21. The voltage source 24 may be an alternating current (AC) source or a direct current (DC) source. In particular, the electrodes 20,21 have an electrode length p of about 400 nm along the longitudinal direction L.

The device 10 further comprises a step 50, positioned between the first electrode 20 and the second electrode 21 , such that a constriction 14 is formed at the position of the step 50, wherein the channel 10 becomes shallower at the constriction 14 with a constriction depth g (wherein for example the constriction depth g is 180-220 nm, particularly 200 nm). The step 50 comprises a step length s along the longitudinal direction L, wherein particularly the step length s is about 500 nm.

To manipulate smaller objects, a certain device 10 comprises a step 50, positioned between the first electrode 20 and the second electrode 21 , such that a constriction 14 is formed at the position of the step 50, wherein the channel 10 becomes shallower and narrower at the constriction 14 with a constriction depth g (wherein for example the constriction depth g is 20-180 nm, particularly 40 nm) and constriction width 20-300 nm, particularly 70 nm.

When a voltage is provided between the first electrode 20 and the second electrode 21 , a resulting electric field is provided in the channel 10. Due to the electric field, the potential energy distribution of an object 30 in the channel 10 is altered, such that the object 30 is moved or accelerated in the channel 10, or such that the movement of the object 30 in the channel is restricted, that is such that the object 30 moves only in a part of the channel and cannot enter certain other parts of the channel.

When an external voltage is imposed to the electrodes 20,21 , the free energy potential of the object 30 is regulated to non-intrusively regulate its motion by the configuration of the electric field alone. For example, in case a direct voltage is provided between the first and the second electrode 20,21 (DC mode), the object 30 can be moved across the constriction 14 due to a double layer interaction of the object 30 with the surface of the inner wall 19. Alternatively, if an alternating voltage is provided between the first and the second electrode 20,21 (AC mode), movement of the object 30 across the constriction 14 can be prevented by means of a dielectrophoretic force acting on the object 30.

In particular, whether the device 1 is operated in DC or AC mode depends on the kind of object 30 to be manipulated in the channel 10. Whereas metal objects 30 can be manipulated in the DC mode, the AC can only manipulate polarizable objects 30. Furthermore, the choice of DC or AC mode depends on the ionic strength of the fluid in the channel 10. At ionic strengths above 0,5 mM, the AC mode is preferentially used, since the double layer interaction necessary for DC mode operation is weakened by high ionic strengths.

An object 30, for example a nano-object moving along a trajectory 31 in the channel 10 is also depicted in Figure 1. The depicted trajectory 31 was obtained by interference scattering (iSCAT) microscopy of a single adenovirus (-90 nm in diameter).

To give a quantitative example of the function of the device 1 in DC mode, the solid curve in Fig. 2 shows the calculated free energy potential distribution in the device 1 for a charged 100 nm (diameter) gold particle in solution with ionic strength of 0,05 mM. The dashed curve in Fig. 2 shows the open state of the valve in DC mode, in which an electrode potential of E_DC=-12mV^m is applied to the first and second electrodes 20,21 , wherein the electrode potential bends the free energy potential profile of the object 30 (or particle), decreases the barrier (AF_b = 17 k_bT → ~k_bT) and actuates the object 30 (or nano-object), such that the object 30 moves past the constriction 14.

In Figure 3, the simulated free energy potential profiles of the object 30, wherein the object 30 is a 100 nm diameter polystyrene bead, for three root mean square (rms) AC voltages (V_AC) applied between the two first and second electrodes 20,21 are shown. For the largest potential V_AC = V_ACi (corresponding to an electric field at the constriction (E_AC= 850 mV^m) at 10 MHz (solid curve) the object 30 experiences a potential barrier substantially higher than its thermal energy. This corresponds to a closed valve and the object 30 is prevented from going through the constriction 14 of the channel 10. In contrast, for V_AC = 0 V (dashed line) the potential barrier is not present. Here, the valve is open and the object 30 can move through the constriction 14. The dash-dotted line depicts an intermediate state (E_AC= 625 Γπν/μηΊ), in which the object 30 has a lower probability of going through the constriction 14.

Figure 4 depicts a device 1 comprising a first electrode 20, a second electrode 21 , and a third electrode 22, which are embedded into a channel wall 19 along the longitudinal direction L of the channel 10. The device 1 further comprises a first step 50a and a second step 50b, resulting in first constriction 15 of the channel 10 positioned between the first electrode 20 and the second electrode 21 , and a further constriction 15a of the channel 10 positioned between the second electrode 21 and the third electrode 22 along the longitudinal direction L.

When a voltage is provided between the first electrode 20 and the second electrode 21 , the device 1 acts as a valve at the position of the first constriction 15. That is, in particular when a voltage is provided between the first electrode 20 and the second electrode 21 is applied, the resulting electric field can move or accelerate an object 30 in the channel 10 across the first constriction 15 (for example when a direct voltage is applied in DC mode), or prevent the movement of the object 30 across the first constriction 15 (for example when an alternating voltage is applied in AC mode). Likewise, when a voltage is provided between the first electrode 20 and the third electrode 22, the resulting electric field can move or accelerate an object 30 in the channel 10 across the further constriction 15a (for example when a direct voltage is applied in DC mode), or prevent the movement of the object 30 across the further constriction 15a (for example when an alternating voltage is applied in AC mode).

Furthermore, when the object 30 is provided at the position of the second electrode 21 , the object can be trapped at this location if both the valve formed by the first electrode 20, the first constriction 15 and the second electrode 21 is in its closed state, and if the valve formed by the first electrode 20, the further constriction 15a and the third electrode 22 is in its closed state.

In particular, while a single valve can regulate the passage of an individual object 30, connecting two such valves in series allows to guide an object 30 to the interspace between the two valves and confine it in this region (Fig. 4). In such a trap-in-channel system, the central electrode (first electrode 20) may be shared between the two valves. Alternatively, two valves having two separate electrodes each may be connected in series to form a trap- in-channel system. For example, in the device shown in Fig. 4, the first electrode 20 and the second electrode 21 can be connected to the same potential, whereas the third electrode 22 can be connected to the ground potential.

Another embodiment of the device 1 as shown in Fig. 5 employs three joined valves, particularly nano-valves, joined in a Y-shaped configuration by a junction 18 of three channels 1 1 ,12,13 (or channel branches). The device 1 comprises a first channel 1 1 comprising a first electrode 20, also designated L, and a first constriction 15, a second channel 12 comprising a second electrode 21 , also designated R, and a second constriction 16, and a third channel 13 comprising a third constriction 17 and a third electrode 22, also designated E. At the junction 18 of the first channel 1 1 , the second channel 12 and the third channel 13, a fourth electrode 23, also designated C, is positioned. The electrodes 20,21 ,22 are embedded into inner walls 19 of the respective channels 1 1 ,12,13, whereas the fourth electrode 23 is embedded into an inner wall 19 of the junction 18.

In the embodiment shown in Fig. 5A, the first constriction 15, second constriction 16, and third constriction 17 are shallower than the respective channel 1 1 ,12,13 only in one dimension, for example the depth h (see Fig. 1 ), whereas in the embodiment shown in Figure 5B, the first constriction 15, second constriction 16, and third constriction 17 are shallower and narrower than the respective channel 1 1 ,12,13 in two dimensions, for example the depth h and the width w (see Fig. 1 ).

When a voltage is provided between the first electrode 20 and the fourth electrode 23, the first channel 1 1 can act as a valve due to the resulting electric field in the first channel 1 1 at the first constriction 15; when a voltage is provided between the second electrode 21 and the fourth electrode 23, the second channel 12 can act as a valve due to the resulting electric field in the second channel 12 at the second constriction 16; and/ or when a voltage is provided between the third electrode 22 and the fourth electrode 23, the third channel 13 can act as a valve due to the resulting electric field in the third channel 13 at the third constriction 17. These valves can be operated in DC mode, where an applied direct voltage leads to an open state of the respective valve, and/ or in AC mode, where an applied alternating voltage leads to a closed state of the respective valve.

In particular, the so constructed three valves can be independently operated to open and closed states to enable entrance of an object 30 from a selected channel 1 1 ,12,13, confinement at the central junction 18 (or in the region of the junction 18) and on-demand release to a selected exit channel 1 1 ,12,13. Such a trap-in -junction device 1 can be used for sorting, mixing or combining objects 30, particularly nano-objects, at the single entity level.

In Figure 6 (in conjunction with Figures 7 and 8), guiding, confining and releasing of an adenovirus (object 30) in buffer solution (0,2X PBS, n₀ = 32,54 mM) in a trap-in-channel structure (two devices 1 a, 1 b for manipulating an object in serial flow connection with each other or nanovalves) is shown. Adenovirus particles were manipulated in AC mode

(V_Ac=1 ,75 V, E_AC « 800 mV /μτη). Since the ionic strength largely (almost by two orders of magnitude) exceeds the limit for the DC mode (n₀ < ~0,5 mM), the AC mode with its higher operating ionic strength range was employed. Figure 6 further shows trajectories 31 of the object 30 in a channel 10 containing a fluid.

The free energy diagrams depicted in Figure 7 resulting from numerical simulations show the device 1 a, 1 b (nanovalve) states for each step depicted in Figure 6. First, in frame (I) (Fig. 6A, corresponding free energy diagram shown in Fig. 7A), the object 30 is prevented from going through the left device 1 a due to the presence of an energy barrier induced by the AC electric field. In frame (II) (Fig. 6B, corresponding free energy diagram shown in Fig. 7B), the energy barrier of the left device 1 a is removed and the object 30 moves freely to the interspace 2 between the two devices 1 a, 1 b. In frame (III) (Fig. 6C, corresponding free energy diagram shown in Fig. 7C), the energy barrier of the left device 1 a is restored and the object 30 is confined between the two devices 1 a, 1 b. Upon removing the energy barrier of the right nanovalve in frame (IV) (Fig. 6D, corresponding free energy diagram shown in Fig. 7D) the object 30 moves freely through the right device 1 b and is released to the channel 10.

Figure 8A-D shows an example of electrode potentials applied to achieve the sequence of events (I) to (IV) shown in Figure 6A-D. The curve shown in Figure 8B represents the potential applied to the first electrode 20 of the first device 1 a and the second device 1 b, the curve depicted in Figure 8A represents the potential applied to the second electrode 21 of the first device 1 a, and the curve shown in Figure 8C represents the potential applied to the second electrode 21 of the second device 1 b.

In case the above-described sequence of events ((I) to (IV)) is performed using a device 1 according to the embodiment shown in Figure 4, the first device 1 a and the second device 1 b share a common first electrode 20 (also termed central electrode C). For example the potential depicted in Figure 8B can be applied to this common first electrode 20, whereas the potential depicted in Figure 8A is applied to the second electrode 21 of the device 1 (also termed left electrode L), and the potential shown in Figure 8C is applied to the third electrode 22 of the device 1 (also termed right electrode R) in order to achieve the sequence of events.

The applied voltage was V_Ac=1 ,75 V. A value of E_AC « -800 την/μτη was obtained from simulation for the given applied voltage along the constriction of the nanovalve. The electrode potentials during the steps (I) to (IV) are summarized in Table 1.

Figure 9 shows trajectories 31 of objects 30 in a device 1 according to the embodiment shown in Figure 5 (trap-in-junction structure) comprising a first, a second and a third channel 1 1 ,12,13 connected by a junction 18, demonstrating the capabilities of the trap-in-junction structure to load an object 30 (for example a virus) from the lower branch (third channel 13) into the junction 18 (center), confine it there and release the object 30 on demand to a selected channel 1 1 ,12 (branch).

Figure 9 represents the trajectories 31 of two sequential object 30 (virus) handling events, where the respective objects 30 are first trapped in the junction 18 and then released on- demand to the top-left (first) channel 1 1 (Figure 9A, sequences l-IV) or to the top-right (second) channel 12 (Figure 9B, sequences V-VIII) obtained in AC mode by switching energy barriers (V_AC= 1 ,75 V, E_AC « 800 mV/μτη) on and off.

Figure 10 shows an example of electrode potentials applied to achieve the sequence of events (I) to (IV) and (V) to (VIII) shown in Figure 9. Therein L designates the potential applied to the first electrode 20 in the first channel 1 1 , R designates the potential applied to the second electrode 21 in the second channel 12, E designates the potential applied to the third electrode 22 in the third channel 13, and C designates the potential applied to the fourth electrode 23 at the junction 18. The applied voltage was V_Ac=1 ,75 V. A value of E_AC « -800 την/μτη was obtained from simulation for the given applied voltage along the constriction of the nanovalve. The electrode potentials during the steps (I) to (VIII) are summarized in Table 2.

Figures 1 1 and 12 show scatter plots of lateral (x-y) positions and probability density p(x) . The data shown in Figure 1 1 were obtained from a 100 nm (diameter) fluorescent polystyrene bead in 0,1 X PBS at V_AC = 2,0 V {E_AC « 900 mV/μηι, closed symbols) and 1X PBS at V_Ac = 3,0 V (E_AC « 800 mV/μτη, open symbols) measured at 200 fps in a trap-in- channel structure. In order to quantitatively investigate the probability distribution of the lateral particle displacement and free energy profile in a nanovalve, the position of a highly fluorescent 100 nm polystyrene bead, which allowed measurements at 200 frames per second (fps) acquisition rate, was recorded. Figure 1 1 shows the scatter plot of the positions obtained for 0,1 X (n₀ = 16,27 mM, V_Ac = 2 V, closed symbols) and 1X (n₀ = 162,7 mM, V_AC = 3 V, open symbols) PBS concentration measured in a trap-in-channel structure. Figure 1 1 right diagram also shows the particle probability density and free energy profile (dots) calculated from the scatter plot and corrected for the finite exposure time. The simulation results (solid lines) are fitted to the measurements using Re(CM) as a fitting parameter. The mean lateral displacement along the channel increases when going from 0,1 X to 1X PBS concentration. This can be explained by the smaller absolute value of Re(CM) at 1X PBS concentration, which originates from the enhanced conduction of the double layer on the surface of the bead at higher ionic strengths. From these fitted simulations we obtained AF_b = 17k_bT and AF_b = 10k_bT for 0,1 X PBS and 1X PBS cases. These values of the free energy barriers can be independently calculated by inserting the measured residence times of the trapped particles into Kramer's relation (see Methods). For 0,1X PBS and 1 X PBS concentrations, we repeatedly measured residence times of the order of several tens of minutes and tens of seconds, respectively. These values correspond to energy barriers which are in good agreement with the fitted simulation results mentioned above. The data depicted in Figure 12 were obtained from an adenovirus in 0,2X PBS at V_Ac = 1 ,75 V (E_AC « 800 την/μτη, open symbols) and V_Ac = 2,25 (E_AC « 1000 mV/μτη, closed symbols) measured at 28 fps in a trap-in-channel structure. Probability density p(x) and free energy potential AF(x) obtained from scatter plot (dots) and simulation (solid line) fitted to measurements using the CM-factor as a fitting parameter. AF(x) is corrected for the finite exposure time.

In Figure 12, the measurements of an adenovirus in the trap-in-channel structure are presented. We can observe a clear increase in the confinement when going from 1 ,75 V_AC (open symbols) to 2,25 V_AC (closed symbols). The observed trend is the result of an increase in the free energy barrier for higher applied voltages and is in agreement with theoretical simulations. According to Equation (1 ), the height of the energy barrier is approximately proportional to the square of the applied AC voltage. The respective residence times for 0,2X PBS and 1 X PBS concentrations at 2 VAC were of the order of several minutes and several tens of seconds. Using Kramer's law we estimate AF_b = 13 k_bT and AF_b = 10 k_bT for the cases of 0,2X and 1 X PBS concentrations, respectively (see Example 1 ).

In Figure 13, nano-object types investigated are shown as a function of particle zeta potential ψ and electrolyte ionic strength n₀. The operational range of the DC mode for 100 nm particle is shown in delimited by a dashed line. Crossing the DC mode boundary (AF_b « 7,5 k_bT) from left to the right (n₀ increasing) the topographically induced free energy potential barrier vanishes. In the AC mode, the nanovalve can be operated at higher ionic strengths and when surface-passivation agents are employed. DC mode fails for both these conditions. For lipid vesicles and adenoviruses the channel-walls were passivated with Bovine Serum Albumin (BSA) and Polyethylene Glycol (PEG) to avoid sticking.

Figure 13 summarizes the broad range of cases where the effectiveness of our nanovalving platform could be successfully demonstrated. These include handling of different natural and artificial nanoscopic entities with varying sizes and surface charges under various electrolytic strengths and channel wall chemistry. The operational range for the facile DC mode shown is limited to n₀ <~ 0,5 mM and highly charged particles. In contrast, the more advanced AC mode is capable of handling a diverse range of nano-objects with varying charges in electrolytes of ionic strengths up to physiological conditions, n₀ = 162,7 mM. successful valving was demonstrated at three orders of magnitude higher ionic strengths compared to the DC mode along with an insensitivity to channel surface chemistry. According to equation (1 ), the free energy potential barrier in the AC mode is proportional to R³ and, therefore, it rapidly decreases as the particle size becomes smaller. However, \E_AC \² in the same relation, scales approximately with 1/L⁴, where L is the characteristic cross-sectional dimension of the channel constriction. Therefore, the drop of the free energy potential barrier for very small particles can be compensated by shrinking the cross-sectional dimensions of the channel constriction. We estimate that we can downscale the constriction cross-section to control the motion of objects with sizes of approximately one order of magnitude smaller than reported here, before approaching nanofabrication resolution constraints and inherent dielectric and electrohydrodynamic limitations observed in dielectrophoretic manipulations of particle agglomerates. Our thermodynamic simulations (see Example 1 and Figure 26) also show that the temperature increase due to Joule heating in the current design is negligibly small (< 0,2 K), ensuring that biological species will not be harmed. The trajectories 31 of a 100 nm spherical polystyrene bead (object 30) in 0,1 X physiological buffer solution (phosphate-buffered saline, PBS; n₀ = 16,27 mM, more than 30 times higher than the threshold manageable for the DC mode) are shown in Figure 14 to 16.

Figure 14 shows a trajectory 31 of a spherical 100 nm (diameter) gold particle (object 30) guided in DC mode in a trap-in-channel configuration at n₀ = 0,05 mM (V_DC=-3,5 V, E_DC « -12 την/μτη). The particle is (I) stopped before the energy barrier induced by the left device 1 a (nanovalve, (A), (II) moved through the constriction 14 of the left first device 1 a (valve) by modulating the free energy potential (B), (III) confined in the interspace 2 (C), and (IV) released through the right second device 1 b (nanovalve, D). When the free energy potential is bent to move the particle through the left valve 1 a (II), the energy barrier of the right valve 1 b remains sufficiently high (AF_b~8k_bT) to stop the particle.

Figure 15 shows the corresponding simulated free energy potentials, and the applied electrode voltages are shown in Figure 16 and table 3. The applied voltage was V_DC= -3,5 V. From simulation E_DC « -12 mV/μτη was obtained for the given applied voltage along the constriction 14 of the nanovalve. Figure 17 shows a trajectory 31 of a 60 nm (in diameter) gold particle (object 30) moved in a trap-in-junction device 1 in DC mode in a sequence (l-VI) at n₀ = 0,05 mM. The sequence of motion occurred along the path bottom-center-left-center-right-center-bottom. The gold particle was confined in the trap-in-junction structure at single entity level, released to a selected branch and brought back to the trap. This sequence was repeated for each branch with the same particle. The applied voltage was V_DC= -4,0 V. E_DC « -50 την/μτη was obtained from simulation. For the data shown in Figures 14 to 17, V_DC was applied for a time period of 20 ms. This time period was selected to be sufficiently long, so that the particle can overcome the reduced energy barrier AF_b~k_bT of the open valve and move through the valve. The corresponding electrode voltages are shown in Figure 18.

in Figure 16.

Furthermore, the AC mode was employed to handle lipid vesicles (100 nm in diameter) in a trap-in-channel structure. The measurement results are shown in Figure 27. A lipid vesicle (100 nm in diameter) was guided in AC mode in buffer solution 0,1X PBS (n₀ = 16,27 mM) within a trap-in-channel structure (V_AC=2 V, E_AC « 900 την/μτη). Lipid vesicle (I) comes from right and is stopped before the energy barrier of the right nanovalve, (II) moves through the right nanovalve (energy barrier of right valve removed), (III) is confined within the interspace (right energy barrier restored) and (IV) moves through the left nanovalve (left energy barrier removed). Trajectories overlaid on top of SEM images and time-trace diagrams show electrode voltages for each step.

The presented methodology and its demonstrated use for on-demand handling, confining and guiding of nanoscale species with single entity specificity may be used for the realization of such electrokinetic nanovalves, as basic building blocks in nanofluidics. Independent actuation of a group of such valves can enable simultaneous, yet individual handling of multiple entities in applications ranging from in-situ chemical or biochemical synthesis to precise drug delivery with lab on chip compatibility.

Example 1 - Method for manipulating objects

Preparation of Natural and Synthetic Particles: Gold nanoparticles (60 and 100 nm in diameter, British Biocell International), gold nanorods (50 x 95 nm, Nanopartz) and fluorescent carboxylate modified polystyrene beads (40 nm and 100 nm in diameter,

FluoSpheres 505/515, ThermoFischer Scientific) were washed by centrifugation (800g, 5 minutes) and re-dispersion in deionized (Dl) water (> 18 MQcrm) to reduce ionic strength of solution and remove contaminants (gold particles required three and PS beads one washing cycles). Solutions of fluorescent beads were also redispersed in 0,1X and 1X PBS. Lipid vesicles (-100 nm in diameter) prepared by extrusion and tagged with Atto 532 fluorophores were dialyzed and diluted in Dl water (>18 MQcrm). Lipid vesicles were also re- dispersed in PBS solution of 0,1X and 1X concentration.

The adenovirus culture (~ 80 nm in diameter) labeled with Alexa Fluor 488 was diluted in PBS to 0,2X and 1X buffer concentration with the addition of 0,02 Vol% polyethylene glycol sorbitan monolaurate or polyoxyethylenesorbitan monolaurate (Tween 20; detergents do not destroy virus particles as shown by immunoprecipitation reactions with anti-fiber antibodies). The handlings of the viruses, including loading them into nanochannel chips and cuvettes for zeta potential measurements, sealing the loaded devices, and autoclaving of the used devices, were carefully performed in a biosafety level 2 classified biolab. Zeta potential was measured by means of dynamic light scattering in a Malvern Zetasizer.

Interferometric Scattering (iSCAT) and Fluorescence Microscopy: Gold nanoparticles and nanorods were imaged by the interferometric scattering (iSCAT) technique at 1 kHz in a home-built inverted microscope equipped with high numerical aperture (1 ,3 NA) objective, a diode pumped green laser (532 nm) for excitation, and a CMOS camera (MV-1024-160-CL, PhotonFocus) for imaging. A schematic of the setup is shown in Figure 19 and 20.

In Figure 19, an Interferometric scattering (ISCAT) setup 60 for imaging metallic particles is shown. The beam of a diode pumped laser 61 (532 nm) was deflected by an acousto-optical deflector 62 (AOD) to scan the field of view through a 100X, 1 ,3 numerical aperture (NA) oil immersion objective focusing the light at the particle/sample 63 plane. A CMOS camera 64 (MV-1024-160-CL, PhotonFocus) measured the collected interferometric light from the particle and the Si0₂/Si interface (reflected light).

Figure 20 depicts a fluorescence setup 60 for imaging fluorescent beads, lipid vesicles, and viruses in standard wide field configuration comprising a 450 nm first laser 61 a (for exciting fluorescent beads and viruses) and a 532 nm second laser 61 b (for exciting lipid vesicles). The fluorescent light was collected by a 100X 1 ,4NA oil immersion objective and measured by a scientific CMOS camera 64 (Andor Zyla 4.2 sCMOS). Long-pass filters (cut-off at 500 nm for 450 nm excitation and 550 nm for 532 nm excitation) were used in the detection path to remove the excitation light.

Figures 21 and 22 show the design of a fluidic chip comprising of four fluidic ports 71 , two micro-channels 72 each connecting two fluidic ports 71 , an array of nano-channels 73 connecting the two micro-channel 72 branches, and electrical connections to electrodes 20 in the fluidic ports 71 , the micro-channels 72 and the nano-channels 73 (nano-electrodes 74). Figure 22 shows the nano-channel designs of the trap-in-channel and trap-in-junction structure. Scale bars show 10 μηη. For the DC mode (low ionic strength), the particles (objects 30) were driven into the micro-channel 71 by an evaporation induced flow from fluid ports 71 or/and by an electric field induced by an externally applied voltage on the electrodes 20 (see Figure 21 ). Within the nano-channels 73 the particles were moved by an electric field induced by applying an external voltage on the nano-electrodes 74 or on the electrodes 20 at the micro-channels 72. For the AC mode (high ionic strength), the particles (objects 30) were driven into the micro-channel 71 by an evaporation induced flow from fluid ports 71 (see Figure 21 ) or/and by diffusion. Within the nano-channels 73 the particle motion was based on diffusion.

Example 2 - Fabrication process of the device for manipulating objects

The fabrication process employs additive (depositions), subtractive (etchings) and pattern transfer techniques such as electron beam lithography (EBL) and photolithography (PL) to form channel and electrode structures. For EBL a Vistec 100kV system and for PL a Karl Suss mask aligner MA/BA6 were used for exposing the resist. Each fabrication step was verified by optical microscopy, scanning electron microscopy (SEM) and profilometry. If needed in addition atomic force microscopy (AFM) for channel dimension characterization and ellipsometry for film thickness measurements were employed.

Therein, a first layer 41 (for example an initial Si0₂ layer) was deposited on a clean surface 40 (for example a Si wafer; Figure 23a). Recesses 45 (for example trenches) were etched (Figure 23b) to accommodate the metal layer 44 giving rise to the metal nanoelectrodes 20 deposited by evaporation (Figure 23c). A second layer 42 (for example of Si0₂) was grown on the top facet (Figure 23d) in which the recesses 45 resulting in the channels 10 were etched with an initial depth (Figure 23e). As shown in Figure 23f, a subsequent dry etch process deepened the channels down to the nanoelectrodes 20 (metal layer 44) and regions in the channel excluded from the etching, i.e. masked by the resist, formed the steps. Figure 23g shows deposition of a 2 nm thin third layer 43 (for example Si0₂ layer) for electrochemical passivation of the nanoelectrodes 20. In the last step, as shown in Figure 23h, the channels 10 were closed and sealed by anodic bonding of a top wall structure 46 (for example a glass slide) to the top facet of the structure.

First, on a double side polished 400 μηη thick Silicon wafer (orientation 100, p-doped, r = 1..30 Qcrm) a 100 nm thick sputtered tungsten layer was deposited which was patterned by EBL in ARN 7520.17 resist. Dry etching with SF₆ gas chemistry formed precise EBL markers. Next, an initial layer of -300 nm Si0₂ was deposited (Fig. 23a) using plasma enhanced chemical vapor deposition (PECVD). For masking trenches in the Si0₂ layer, a CSAR resist was spin-coated and patterned by EBL (Fig. 23b). The trenches were dry etched by reactive ion etching (RIE) in CHF₃/Ar gas chemistry down to a depth of 70 nm. These trenches will accommodate thin metal wires deposited in (Fig. 23c), which serve as nanoelectrodes 20 in the channel 10 and connecting wires. A P MM A/ MM A resist layer is exposed by EBL to form a mask for the following lift-off process to deposit the metal wires in the trenches. A Cr/Au/Cr metal layer stack is deposited by ebeam evaporation and outside the trenches the metal layer was lifted off by etching the underlying resist mask. The total thickness of the metal layer stack was adjusted to match precisely the depth of the trenches, resulting in a smooth top facet.

As shown in Fig. 23d, a seed layer of 20 nm Si0₂was deposited by atomic layer deposition (ALD) which ensures a conformal deposition of the Si0₂ layer over the previously deposited metal wires. PECVD was employed to increase the commenced Si0₂ layer to a total thickness of 300 nm, i.e. 280 nm were deposited by PECVD. For forming the channels (Fig. 23e) a CSAR mask was written by EBL and the channels 10 were dry etched to a depth of 180 - 220 nm using RIE with CHF₃/Ar gas chemistry. This initial channel depth was varied and adjusted to obtain devices with the desired step height (constriction 14). To form the steps (Fig. 23f), the channels were again patterned in CSAR resist using EBL, but the mask excluded the zones in the channels 10 where steps had to be formed. Dry etching using RIE with CHF₃/Ar gas chemistry deepened the channels excluding the step zones to a total depth of 300 nm, leaving steps in the channel as shown by (Fig. 23f).

As shown in Fig. 23g, a 2 nm thick Si0₂ layer was deposited by ALD to form an

electrochemically protective layer over the nanoelectrodes (thickness of layer is exaggerated in Figure 23). This protective layer increased markedly the inertness of the nanoelectrodes and in addition the nanoelectrodes have the same Si0₂ surface chemistry as the channel walls.

Before closing the channels 10, the following micro-structures had to be fabricated: (1 ) 4 dedicated holes were etched through the silicon layer by deep RIE (Bosch process), providing the fluidic access (i.e. ports) from the backside of the device to the channel system on the front side. (2) pads for electric interface contacts and (3) electrodes in the fluidic ports were deposited by metal evaporation and lift off. In all these steps, PL was employed for the pattern-transfer. Last, the entire channel system was closed and sealed (Fig. 23h) by anodic bonding of a 200 μηη thick glass slide to the top facet of the Si0₂ structure. This transparent glass substrate provided optical access to the interior of the channels for observing and tracking of the nano- objects with an inverted microscope. A thin glass slide was selected to enable the use of high numerical aperture objectives (1 ,4 NA) in the imaging system. Design and Electrochemical Characterization of Nanoelectrodes: The top surface of the nanoelectrodes, which consist of a Cr film with a 2 nm thin layer of Si0₂, was passivated to reduce electrode degradation (i.e. to avoid corrosion and deposition), suppress hysteresis in the l-V curve and retard water electrolysis. Such a thin insulation layer is known to increase the electrode-electrolyte electric contact resistance. Figure 24 shows the cyclic voltammogram (l-V measurements) of a Si0₂ passivated Cr film electrode (material layer stack: Cr/Au/Cr, 25/25/20 nm) in different electrolytes. Cyclic voltammetry was performed at a voltage sweep rate of 50 mV/s using a three electrode cell incorporating a platinum counter, an Hg/Hg₂S0₄ reference electrode, and a SP300 potentiostat (BioLogic Science Instruments, Grenoble). I-V measurements of the Cr-film electrodes (material layer stack Cr/Au/Cr, 25/25/20 nm) passivated by a 2 nm thick Si0₂ layer were measured in Dl water D and in 1 mM KCI aqueous solution K. The IV relation J(V) = ae^bv— ce^~dv fitted to the Dl water measurements is shown by the dashed line. For reference, the l-V measurements of Au electrodes Au and Cr electrodes Cr measured in Dl water are also given.

The Si0₂ passivated electrodes have negligible hysteresis as shown in Fig. 24 and water electrolysis was retarded up to a potential difference of -2 V. The onset of electrolysis imposes essentially an upper limit for the applied potentials in DC and AC mode. In the AC mode (10 MHz) our experiments did not show any nascent bubble formation due to water electrolysis for voltages up to -5 VAC at 1X PBS concentration. Similarly no nascent bubble formation was observed in the DC mode, in the range 3 - 5 V DC of applied potentials.

Correction for Finite Exposure Time: Figure 25 shows the simulated free energy potential and position probability distribution for a 100 nm sized particle (polystyrene bead) along a trap structure at a distance of 70 nm from the channel upper wall. By fitting the

experimentally determined probability density to the probability density function obtained from simulation using the real part of the CM factor as a fit parameter, the effective real part of the CM value can be obtained. The experimentally determined probability density has to be corrected for the finite exposure time before the fitting (see section correction for finite exposure time). This effective real part of the CM-factor can be employed to update and, hence, increase the precision of the simulated free energy potential profiles and the estimated energy barrier height.

The free energy potential landscape for a 100 nm sized polystyrene particle in the trap-in- channel structure shown in Fig. 6 was obtained by a 2D COMSOL simulation of the electric field (f=10 MHz, c₀=0,1X PBS and CM=-0,18). Dimensions were h = 300 nm, s = 500 nm, g = 200 nm and e = 400 nm (see Figure 1 ). The free energy profile AF(x) and position probability density p(x) are shown for a particle moving along the channel at 70 nm distance from the upper wall (shown by a solid line in the top panel).

Joule Heating in the Nanovalve: The effect of joule heating was investigated by a simulation (COMSOL) of the temperature distribution in the cross section of the nanovalve using a heat source Q_h=5- 10¹² W/m³ in the electrolyte. The selected value for the heat source represents the worst-case scenario at 1X PBS concentration (σ = 2,3 Sm and |E_AC|=1 ν/μηη. Figure 26 shows the obtained temperature distribution. The resulting maximum temperature increase in the channel is < 0,2 K. This negligible rise in temperature ensures that biological species investigated in the proposed system will not be harmed. The temperature increase was calculated using COMSOL Multiphysics for a channel (width = 500 nm, height = 300 nm shown by the black rectangle) embedded in a 10 μηη thick Si0₂ layer 300 nm above a Si0₂/Si interface. A heat source of Q_h=5- 10¹² W/m³ was considered in the electrolyte. The temperature distribution is shown across the channel cross section and in the Si0₂ layer. The Si0₂/Si interface was set to 0 K assuming perfect thermal conduction in the Si, and the Si0₂ boundaries were defined as thermal insulators.

List of reference signs

Device for manipulating objectsa First device for manipulating objectsb Second device for manipulating objects

Interspace

0 Channel

1 First channel

2 Second channel

3 Third channel

4 Constriction

5 First constriction

5a Further constriction

6 Second constriction

7 Third constriction

8 Junction

9 Inner wall

0, L Electrode, first electrode

1 , R Second electrode

2, E Third electrode

3, C Fourth electrode

4 Voltage source

0 Object

1 Trajectory

0 Surface

1 First layer

2 Second layer

3 Third layer

4 Metal layer

5 Recess

6 Top wall structure

0 Step

0a First step

0b Second step

0 Microscope setup

1 Laser

1a First laser

1 b Second laser

2 Acousto-optical deflector

3 Sample 64 Camera

65 Controller

70 Fluidic chip

71 Fluidic port

72 Micro-channel

73 Nano-channel

74 Nano-electrode

L Longitudinal direction

^max Maximal cross-sectional extension

^min Minimal cross-sectional extension g Constriction depth

h depth

P Electrode length

s Step length

w Width

Au Gold electrode

Cr Chromium electrode

D Dl water

K KCI solution

Claims

1. Device (1 ) for manipulating objects (30), particularly micro- or nano-objects in a fluid, comprising:

• a channel (10), having a maximal cross-sectional extension (e_max) perpendicular to a longitudinal direction (L) of the channel (10), wherein the channel (10) comprises at least one constriction (14) with a minimal cross-sectional extension (e_min) perpendicular to the longitudinal direction (L) of the channel (10), wherein the minimal cross-sectional extension (e_min) is smaller than the maximal cross- sectional extension (e_max),

• a first electrode (20) and a second electrode (21 ), wherein the first electrode (20) and the second electrode (21 ) are adapted to generate an electric field in the channel (10) by means of a voltage applied between the first electrode (20) and the second electrode (21 ), characterized in that the first electrode (20) and the second electrode (21 ) are positioned, in the channel (10) along the longitudinal direction (L) of the channel (10) at opposite sides of the constriction (14).

2. Device (1 ) for manipulating objects (30) according to claim 1 , wherein at least a part the surface of an inner wall (19) of the channel (10) is adapted to exhibit a surface charge when brought into contact with a fluid, particularly water and/ or an aqueous solution, particularly wherein the surface of the inner wall (19) of the channel (10) comprises Si0₂.

3. Device (1 ) for manipulating objects (30) according to claim 1 or 2, wherein the

maximal cross-sectional extension (e_max) is between 10 nm and 200 μηη.

4. Device (1 ) for manipulating objects (30) according to any of the preceding claims, wherein the device (1 ) comprises a first constriction (15), a further constriction (15a) and a third electrode (22), wherein the first electrode (20) is positioned between the first constriction (15) and the further constriction (15a), and wherein the second electrode (21 ) is positioned at the opposite side of the first constriction (15) in relation to the first electrode (20), and wherein the third electrode (22) is positioned at the opposite side of the further constriction (15a) in relation to the first electrode (20).

5. Device (1 ) for manipulating objects (30) according to any of the preceding claims, wherein the device (1 ) comprises • a first channel (1 1 ) comprising a first constriction (15), wherein the first electrode (20) is positioned adjacent to the first constriction (15),

• a second channel (12) comprising a second constriction (16), wherein the second electrode (21 ) is positioned adjacent to the second constriction (16), · a third channel (13) comprising a third constriction (17),

• a third electrode (22), which is positioned adjacent to the third constriction (17),

• a junction (18), wherein the first channel (1 1 ), the second channel (12), and the third channel (13) are connected by the junction (18), such that a flow connection between the first channel (1 1 ), the second channel (12), and the third channel (13) is established,

• a fourth electrode (23), which is positioned at the junction (18).

6. Device (1 ) for manipulating objects (30) according to any of the preceding claims, wherein at least a part of the surface of an inner wall (19) of the channel (10) comprises a passivation agent, particularly bovine serum albumin or polyethylene glycol.

7. System for manipulating objects (30), particularly micro- and/or nano-objects,

comprising at least two devices (1 ) for manipulating objects (30) according to any of the claims 1 to 6, wherein the respective channels (10) of the at least two devices are in flow connection with each other, and wherein an electric field in the respective channel (10) of the respective device (1 ) can be provided by means of a voltage applied between the respective first electrode (20) and the respective second electrode (21 ).

8. Method for fabricating a device (1 ) for manipulating objects (30), particularly micro- and/or nano-objects, according to any of the claims 1 to 6, comprising the steps of

• providing a surface (40),

• providing a first layer (41 ) on the surface (40), particularly by depositing the first layer (41 ) on the surface (40),

• generating at least one recess (45) in the first layer (41 ), particularly by generating a pattern on said first layer (41 ) by means of a first electron beam lithography process or photolithography process, and etching the at least one recess (45) into the first layer (41 ) at locations patterned by the first electron beam lithography process or photolithography process,

• providing at least one electrode (20,21 ) by depositing a metal layer (44) onto the first layer (41 ) at the location of the at least one recess (45), · depositing a second layer (42) onto the first layer (41 ) and/ or onto the metal layer

(44),

• providing at least one constriction (14) by generating at least one recess (45) in the second layer (42), particularly by generating a pattern on said second layer (42) by means of a second electron beam lithography process or photolithography process, and etching the at least one recess (45) into the second layer (42).

9. Method for manipulating objects (30), particularly micro- and/or nano-objects,

comprising the steps of

• providing at least one object (30) in a fluid in a channel (10) of a device (1 )

according to any of the claims 1 to 6 or a system according to claim 7, · providing a voltage between the electrodes (20,21 ) of the device (1 ) or system,

• moving and/or accelerating the at least one object (30) and/ or restricting the movement of the at least one object (30) by means of an electric field generated by the electrodes (20,21 ).

10. Method for manipulating objects (30) according to claim 9, wherein an alternating voltage is provided between the electrodes (20,21 ), which results in an alternating electric field in the at least one channel (10), such that movement of the at least one object (30) through the at least one constriction (14) is prevented by means of a dielectrophoretic force acting on the at least one object (30) as a result of the alternating electric field.

1 1. Method for manipulating objects (30) according to claim 9 or 10, wherein a surface charge on at least a part of the surface of an inner wall (19) of the at least one channel (10) is provided, such that a repulsive force between the surface of the inner wall (19) of the respective channel (10) and the object (30) is generated, such that a movement of the object (30) through the respective constriction (14) is restricted by means of the repulsive force, particularly at an ionic strength of the fluid of < 0,5 mM.

12. Method for manipulating objects (30) according to claim 1 1 , wherein direct current voltage between the electrodes (21 ,22) is provided, and wherein an electrostatic force on the object (30) is generated, such that the object (30) is moved through the respective constriction (14).

13. Method for manipulating objects (30) according to any of the claims 9 to 12, wherein the object (30) is a particle from a conductive material, particularly a gold particle, or a particle from a dielectric material, particularly a polysterene bead, or a biological particle, more particularly a prokaryotic or eukaryotic cell, a bacterium, a protein, a complex comprising proteins, most particularly an exosome, an organelle, most particularly an endosome, a DNA molecule, an RNA molecule, a virus, a

bacteriophage, a lipid vesicle, a combination thereof, a part thereof, or a combination of parts thereof.

14. Method for manipulating objects (30) according to any of the claims 9 to 13, wherein the object (30) has a maximum extension between 1 nm and 100 μηη.

15. Method for manipulating objects (30) according to any of the claims 9 to 14, wherein at least two selected objects (30), are brought into contact with each other and/or positioned at nearly the same location by means of a device (1 ) for manipulating objects (30) according to any of the claims 1 to 6, or a system for manipulating objects (30) according to claim 7, wherein particularly a chemical reaction, more particularly a biochemical reaction, is triggered between the at least two selected objects (30).

16. Method for manipulating objects (30) according to any of the claims 9 to 15, wherein the path of movement of at least one object (30) is controlled by applying a voltage to the electrodes (21 ,22) comprised in the device (1 ) or system for manipulating objects (30).

17. Method for manipulating objects (30) according to any of the claims 9 to 16, wherein at least a part of the surface of an inner wall (19) of the at least one channel (10) is coated with a passivation agent, particularly bovine serum albumin or polyethylene glycol, and/or wherein the fluid comprises a surfactant, particularly polyethylene glycol sorbitan monolaurate or polyoxyethylenesorbitan monolaurate at a

concentration of > 0,001 % volume per volume in order to reduce non-specific binding of objects (30) to the surface of the inner wall (19).