WO2017148785A1

WO2017148785A1 - Concentration of nanoparticles and/or microparticles in flow conditions by dielectrophoresis

Info

Publication number: WO2017148785A1
Application number: PCT/EP2017/054130
Authority: WO
Inventors: Noemi ROZLOSNIK; Maria DIMAKI; Mark Holm OLSEN; Winnie Svendsen
Original assignee: Danmarks Tekniske Universitet
Priority date: 2016-03-01
Filing date: 2017-02-23
Publication date: 2017-09-08

Abstract

A device for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis is disclosed in this invention.

Description

Concentration of nanoparticles and/or microparticles in flow conditions by dielectrophoresis

The invention relates to a device for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis

Background

Current technologies do not allow the detection of the presence of viruses in water in real time because of the small concentrations. In fact, several days can pass before the presence of a virus is detected in the water by the classical laboratory methods. Usually, the expensive laboratory methods are used in case of outbreak of an enteric disease somewhere. Concentration of particles is therefore needed.

The greatest challenge when concentrating nanoparticles or microparticles is the fabrication of a device, which will work on an industrial scale. Injection moulding may be used, but as the channels and the waste outlets in such device normally are very small, there are certain limitations to this technique.

Although a great number of publications and patents exist using dielectrophoresis for manipulating micro meter sized particles, it is very rarely that particles with diameter under 100 nm are treated. The term sub micro meter usually refers to particles having sizes between 200 nm and 1 μηη in diameter. The random motion of the particles - which has an effect on the particle manipulation - starting to dominate over other forces in case of smaller particles below 100 nm. Therefore the manipulation of these nanoparticles is challenging with any external forces.

Moreover, in most cases the manipulation of the particles is conducted in static conditions, which does not allow further handling/analysis of the particles.

Description of the invention

Disclosed herein is a device for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis.

The device comprises an inlet configured for injection of a sample with a first volume comprising the nanoparticles and/or microparticles in a first concentration, and an outlet configured for collection of a second volume comprising the nanoparticles and/or microparticles in a second concentration.

The device further comprises a microfluidic channel system comprising one or more channels, and a set of electrodes comprising a first electrode and a second electrode, for providing an electrical field having an electrical gradient.

Each of the first and second electrodes comprises a primary portion extending in the flow direction between the inlet and the outlet, one or more secondary portions extending from the primary portion towards the centre of the channel with a first angle a between 10 to 70 degrees relative to the extending direction of the primary portion and one or more tertiary portions extending from each of the one or more secondary portions towards the centre of the channel with a second angle β, wherein β is at least 10 degrees larger than a and at tops 100 degrees relative to the extending direction of the primary portion.

Disclosed herein is also a method for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis, the method comprising:

• providing a sample with a first volume comprising nanoparticles and/or microparticles with a first concentration;

• injecting the sample in the inlet in a device as described above;

• obtaining liquid flow conditions of the nanoparticles and/or microparticles in the micro fluidic channels of the device transporting the nanoparticles and/or microparticles from the inlet towards the outlet thereby concentrating the nanoparticles and/or microparticles at the centre of the channel;

• collecting a second volume comprising the nanoparticles and/or

microparticles at the outlet of the device, the second volume comprising the nanoparticles and/or microparticles being concentrated in a second concentration.

Using the above device and method allows the user to manipulate nanoparticles (smaller thanl 00 nm in diameter) by dielectrophoretic forces in continuous flow in a microfluidic system. It may be obtained to concentrate nanopartides from a large volume of liquid into a smaller volume being less than 10% of original volume.

It is thereby possible to e.g. concentrate enteric viruses in water bodies. The device and method is therefore highly relevant for all water treatment plants, drinking water providers, industries providing water for agriculture etc. The device and the method facilitates a very fast detection of the viruses and a faster reaction time, thereby reducing the amount of infected people. By using the above device and method enough large electrical field gradient can be achieved in large area of the channels to overcome the random motion of the particles. This gradient also works for particle focusing in flow conditions.

Brief description of the drawings

Figure 1 shows an embodiment of a device for concentration of nanopartides and/or microparticles in liquid flow conditions by dielectrophoresis.

Figure 2 shows a close-up of one of the channels in a device for concentration of nanopartides and/or microparticles in liquid flow conditions by dielectrophoresis according to the invention.

Figure 3 shows an alternative embodiment of the electrode design shown in figure 2.

Figure 4 shows an embodiment of an electrode design and figure 5 is a close-up of figure 4.

Figure 6 shows an alternative embodiment of the electrode design in a close-up.

Figures 7a-b show an optimisation of the electrode design using the two different electrode designs shown in figure 5 and 6.

Figures 8 and 9 show the micrograph of fluorescent polystyrene particles focused with 200 kHz DEP in a single channel device according to the invention. Description of preferred embodiments

An embodiment of a device 100 for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis is shown in figure 1. In one or more embodiments, the particles are nanoparticles.

In one or more embodiments, the particles are microparticles.

The device comprises an inlet 102 configured for injection of a sample 200 with a first volume comprising the nanoparticles and/or microparticles 202 in a first concentration, and an outlet 104 configured for collection of a second volume comprising the nanoparticles and/or microparticles 202 in a second concentration.

The device further comprises a microfluidic channel system 106 comprising one or more channels 106a, 106b, 106c, and a set of electrodes comprising a first electrode 302 and a second electrode 304, for providing an electrical field having an electrical gradient.

In figure 1 is shown a device with many channels whereof only three channels can be seen clearly. The device is designed for parallelization of hundreds to thousands of channels in order to increase the possible flow rates of the liquid. The only limitation to the number of parallel channels is the final device dimensions.

Each of the first and second electrodes 302, 304 comprises a primary portion 306', 306" extending in the flow direction marked with the arrow in figure 1 between the inlet 102 and the outlet 104. Each of the first and second electrodes 302, 304 further comprises one or more secondary portions 308', 308" extending from the primary portion 306', 306" towards the centre 108 of the channel 106, 106a, 106b, 106c. As can be seen in the close-up of one of the channels in figure 2, the one or more secondary portions 308', 308" extends from the primary portion 306', 306" towards the centre 108 of the channel 106 with a first angle a between the primary 106 and the secondary portion 108 of the electrode 302, 304. The first angle a is normally between 10 to 70 degrees relative to the extending direction of the primary portion. In one or more embodiments, the angle a may be the same for all the secondary portions 308' connected to the primary portion 306' of the first electrode 302.

Likewise, in one or more embodiments, the angle a may be the same for all the secondary portions 308" connected to the primary portion 306" of the second electrode 304.

In one or more embodiments, the angle a is different all the secondary portions 308' connected to the primary portion 306' of the first electrode 302 compared to all the secondary portions 308" connected to the primary portion 306" of the second electrode 304.

In one or more embodiments, the angle a varies between different secondary portions 308', 308" connected to the same primary portion 306', 306" of the first electrode 302 and/or the second electrode 304.

In one or more embodiments, the one or more secondary portions 308', 308" extends from the primary portion 306', 306" towards the centre 108 of the channel 106 with a first angle a between 20 to 70 degrees relative to the extending direction of the primary portion 306', 306".

In one or more embodiments, the first angle a is between 30 to 60 degrees, such as e.g. 30, 40, 45, 50, 55 degrees. In one or more embodiments, a is between 20 and 70 degrees.

In one or more embodiments, a is between 20 and 60 degrees.

In one or more embodiments, a is between 20 and 50 degrees.

In one or more embodiments, a is between 25 and 45 degrees. In one or more embodiments, a is between 25 and 40 degrees. In one or more embodiments, a is between 30 and 35 degrees. In one or more embodiments, a is between 27 and 32 degrees.

In one or more embodiments, each of the first and second electrodes 302, 304 comprises one or more tertiary portions 310', 310". This can be seen in figure 2, where tertiary portions 310', 310" are connected to the secondary portions 308', 308" and where the tertiary portions 310', 310" extends inwards towards the middle of the channel 108 .

More than one tertiary portion 310', 310" may extend from each of the secondary portions 308', 308" as shown for the secondary portion 308' of the first electrode 302 positioned furthest to the right in figure 2, where two tertiary portions 310' are extends from the secondary portion 308'.

The one or more secondary portions 308', 308" has a first end 307', 307" connected to the primary portion 306', 306" and a second end 309', 309" opposite the first end 307', 307". As can be seen in the figures, at least one of the tertiary portions 310', 310" on each of the one or more secondary portions 308', 308" extends from a position on the secondary portions 308', 308" located between the first end 307', 307" and the opposite second end 309', 309" on the secondary portion 308', 308".

The tertiary portions 310', 310" extend from the secondary portions 308', 308" towards the centre of the channel 108 with a second angle β relative to the extending direction of the primary portion 306', 306".

In one or more embodiments, the one or more tertiary portions 310', 310" extend from the secondary portion(s) 308', 308" towards the centre of the channel 108 with a second angle β between 80 to 100 degrees relative to the extending direction of the primary portion 306', 306". The second angle β is normally at least 10 degrees larger than a and at tops 100 degrees relative to the extending direction of the primary portion. In one or more embodiments, the second angle β is at least 30 degrees, or at least 40 degrees, or at least 50 degrees, or at least 60 degrees, or at least 70 degrees, or at least 80 degrees or at least 85 degrees.

In one or more embodiments, the second angle β is between 55 and 100 degrees, or between 60 and 100 degrees, or between 70 and 100 degrees, or between 80 and 100 degrees. In one or more embodiments, the second angle β is between 85 and 95 degrees, such as 90 degrees.

In one or more embodiments, the one or more tertiary portions 310' of the first electrode 302 are arranged offset/displaced relative to the one or more tertiary portions 310" of the second electrode 304.

By offset/displaced relative to is meant that the tertiary portions 310' on the first electrode 302 extending towards the centre of the channel 108 will not be able to touch the tertiary portions 310" on the second electrode 304. An example of such a design is shown in figures 2-5.

Figure 4 and the enlargement in figure 5 shows a channel 106 where the secondary portions 308', 308" can be seen on the first and the second electrode. The electrodes shown in figure 4 are typically positioned in a ~ 40 μηη x 20 μηη microfluidic channel.

In one or more embodiments, the one or more tertiary portions 310' of the first electrode 302 are arranged straight opposite the one or more tertiary portions 310" of the second electrode 304. Thus, if the tertiary portions 310', 310" extend far enough towards the middle of the channel 108, they will touch each other. An example of such an arrangement is shown in figure 6. In one or more embodiments, the one or more tertiary portions 310' of the first electrode 302 are arranged with a distance d relative to the one or more tertiary portions 310" of the second electrode 304. In one or more embodiments, the one or more tertiary portions 310' of the first electrode 302 are positioned at offset positions relativity to the one or more tertiary portions 310" of the second electrode 304, wherein the one or more tertiary portions 310', 310" on both the first electrode 302 and the second electrode 304 are at least partly overlapping the centre of the channel 108. An example of such a configuration is shown in figure 3.

In one or more embodiments, the distance d(Ti.i-Ti.₂) between any pair of two consecutive tertiary portions 310' in the flow direction on the first electrode 302 is the same. This is illustrated in figure 5.

In one or more embodiments, the distance d(T₂.₁-T₂.2) between any pair of two consecutive tertiary portions 310" in the flow direction on the second electrode 304 is the same. This is illustrated in figure 5. In one or more embodiments, the distance d(T- _A-T- _A) between any pair of a tertiary portion on the first electrode and the consecutive tertiary portion on the second electrode in the flow direction is the same.

The nanoparticles and/or microparticles are displaced due to a large electrical gradient produced by the electrodes. In this manner, the particles can be directed towards the centre of the channel.

In one or more embodiments, the electrical gradient is controlled by turning on and/or off an AC electrical field between the first and the second electrode.

In some embodiments the voltage of the electrical field is in a range of 2 V to 20 V.

In some embodiments the frequency of the electrical field is in a range of 10 kHz to 10 MHz. In one or more embodiments, the first volume is larger than the second volume, and the second concentration is higher than the first concentration. This allows the user to up-concentrate nanoparticles in 10 times smaller volume of liquid comparing the volume at the inlet with that at the outlet of the device.

In some embodiments, the second concentration is about two, three, four, five, six, seven, eight, nine, or ten times higher than the first concentration. In one or more embodiments, the nanoparticles and/or microparticles are nanoparticles having a diameter smaller than 100 nm.

In one or more embodiments, the nanoparticles and/or microparticles have a diameter between 100 nm - 20 μηη.

In one or more embodiments, the nanoparticles and/or microparticles have a diameter between 500 nm - 20 μηη.

The device may in one or more embodiments have a dimensions of 5-7 cm in a first direction and 3-6 cm in a second direction. The first direction is parallel to the flow direction thus being the length of the device. The second direction is perpendicular to the first direction and thus defines the width of the device. An example of a dives has a length of 6.5 cm and a width of 4.5 cm. The one or more channels in the device may in one or more embodiments have a length l_channei and a width w_channel, wherein l_channei is between 5-10 times larger than

^c annel-

The channels may have a length of 3-5 cm such as e.g. 4 cm, a width of 30-50 μηη, such as e.g. 40 μηι, and a height of 15-30 μηι, such as e.g. 20 or 25 μηι.

The one or more tertiary portions may in one or more embodiments have a width Wtertiary, wherein Wchannei is between 5-60 times larger than w_te!tia!y. Alternatively, w_channe, is between 20-60 times larger than w_tertiary, or 20-50 times larger than w_tertiary, or 10-40 times larger than w_tertiary, or 5-30 times larger than w_tertiary, or 5- 20 times larger than w_tertiary, or 5-10 times larger than w_tertiary, or 7-15 times larger than w_tertiary.

In one or more examples, the channels are 40 μηη wide and the one or more tertiary portions have a w_tertiary of 4μη-ι. In one or more embodiments, the shortest distance between the primary portions

306', 306" of the first electrode 302 and the second electrode 304 is in a range of 1 - 2 μηι.

The shape of the electrodes 302, 304 may be optimized by numerical simulations using e.g. COMSOL or Matlab in order to guide the particles 202 in the channels 106 towards to the centre 108 of the channel 106. The particles in the center of the channel may be collected in a small volume outlet, where only approximately 10% of the total fluid volume will be collected. The remaining 90% of the fluid flow will be removed as waste. In figure 1 , the waste outlet channels have been omitted for clarity.

Figure 7 shows the optimisation of electrode design using the two different electrode designs shown in figure 5 and 6. In design 1 as shown in figure 5, the tertiary electrodes on respectively the first and the second electrode are positioned in a displaced configuration opposite one another, whereas in design 2 as shown in figure 6, the tertiary electrodes on respectively the first and the second electrode are positioned directly opposite one another.

Figures 7a-b show the predicted deflection after the particles has flown 500 μηη in the channel length. The simulation is done for particles that are starting in the middle of the channel in terms of height.

Design 1 (the displaced configuration) is marked dY1 and design 2 (the directly opposite configuration) is marked dY2 in figure 7a showing the displacement of the particles towards the channel centre along the width of the channel after the particles has flown 500 μηι in the channel length. Figure 7a shows that the particles are pushed towards the channel middle as indicated by the arrows 312, 312' indicate the direction of movement. The particles are pushed towards the middle by the amount read in the y axis (the displacement) after 500 μηη channel length travel. Figure 7a shows the results for particles starting at a height 10 μηη above the channel bottom surface.

In figure 7b showing the displacement of the particles towards the channel bottom surface from the middle of the channel along the height after the particles has flown 500 μηη in the channel length, design 1 is marked dZ1 and design 2 is marked dZ2. Figure 7b shows that while travelling along the channel length, the particles are attracted more and more towards the bottom of the channel. Particles directly above the middle of the channel (along the width), i.e. at 0 μηη in particle starting position, experience the most vertical displacement.

From figure 7a and 7b it can be seen that the particles are pushed towards the middle of the channel in regards to the width (figure 7a) and towards the bottom of the channel in terms of height (figure 7b). This facilities an efficient concentration of particles along the bottom-middle of the channel.

Figures 8 and 9 show the micrograph of fluorescent polystyrene particles focused with 200 kHz DEP in a single channel device according to the invention. The polystyrene particles in the measurements shown in figure 8 has a size of 47 nm and a flow speed of 2 μΙ/h is used. The polystyrene particles in the measurements shown in figure 9 has a size of 85 nm and a flow speed of 10 μΙ/h is used. Figure 8a and 9a show the particle distribution when no voltage is applied, whereas figures 8b and 9b show the particle distribution when a 20 V voltage is applied over the first and second electrode.

As can clearly be seen in figures 8 and 9, the particles are directly towards the centre of the channel when a voltage is applied over the electrodes. The particles are thereby clearly concentrated in the centre of the channel. Figures 8c and d show the distribution of particles in figure 8a and b, respectively.

The currently known technologies do not allow the detection of the presence of viruses in water in real time because of the small concentrations. In fact, several days can pass before the presence of a virus is detected in the water by the classical laboratory methods. Usually, the expensive laboratory methods are used in case of outbreak of an enteric disease somewhere.

The device disclosed herein can be used for concentration of viruses, extracellurlar vesicles (exosomes or microvesicles), bacteria, parasites, cells, foreign micro- and nanoparticles. Thus, in one or more embodiments, the nanoparticles and/or microparticles are one or more of the following:

- viruses

- extracellular vesicles, e.g. exosomes or microvesicles

- foreign microparticles

- foreign nanoparticles

- bacteria

- parasites

- cells.

The device can for example be used in connection with all water treatment plants, drinking water providers, industries providing water for agriculture etc., where it is absolutely essential to be able to detect e.g. virus or bacteria in a fast an efficient manner, which normally requires an efficient concentration of the particles.

Disclosed herein is therefore also a method for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis. The method comprises the steps of:

• injecting the sample in the inlet in a device 100 as described above;

• obtaining liquid flow conditions of the nanoparticles and/or microparticles in the micro fluidic channels of the device transporting the nanoparticles and/or microparticles from the inlet towards the outlet thereby concentrating the nanopartides and/or microparticles at the centre of the channel;

• collecting a second volume comprising the nanopartides and/or

microparticles at the outlet of the device, the second volume comprising the nanopartides and/or microparticles being concentrated in a second concentration.

The device may however also be used (with minor modifications) for water purification, e.g. by removing the viruses/bacteria, foreign bodies, from drinking water, and for other medical/industrial particle concentration.

Alternatively, the device can be used for concentrating nanopartides (for example viruses or other pathogens) in other fluids than water. Other fluids includes among others urine, and blood plasma.

Yet alternatively, the device may be used for collecting and concentrating nanopartides from air for further analysis.

The described method is unique because it is designed for and works for manipulating nanopartides (< 100 nm in diameter) with large electrical field gradient over a large area under liquid flow conditions resulting in up-concentration of nanopartides in 10 times smaller volume of the liquid which has not yet been demonstrated to the best of our knowledge.

References

100 device for concentration of nanoparticles and/or microparticles

102 inlet

104 outlet

106 microfluidic channel system

106a channel

106b channel

106c channel

108 centre of the channel

200 sample

202 nanoparticles and/or microparticles

302 first electrode

304 second electrode

306' primary portion of the first electrode

306" primary portion of the second electrode

307' first end of the secondary portion of the first electrode

307" first end of the secondary portion of the second electrode

308' secondary portion of the first electrode

308" secondary portion of the second electrode

309' second end of the secondary portion of the first electrode

309" second end of the secondary portion of the second electrode

310' tertiary portion of the first electrode

310" tertiary portion of the second electrode

312 direction of movement

312' direction of movement

a angle between the secondary portion and the primary portion of the

electrode

β angle between the tertiary portion and the primary portion of the electrode

Claims

1. Device for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis, the device comprises:

- an inlet configured for injection of a sample with a first volume

comprising the nanoparticles and/or microparticles in a first concentration;

- an outlet configured for collection of a second volume comprising the nanoparticles and/or microparticles in a second concentration;

- a microfluidic channel system comprising one or more channels;

- a set of electrodes comprising a first electrode and a second electrode, for providing an electrical field having an electrical gradient; wherein each of the first and second electrodes comprises:

- a primary portion extending in the flow direction between the inlet and the outlet;

- one or more secondary portions extending from the primary portion towards the centre of the channel with a first angle a between 10 to 70 degrees relative to the extending direction of the primary portion;

- one or more tertiary portions extending from each of the one or more secondary portions towards the centre of the channel with a second angle β, wherein β is at least 10 degrees larger than a and at tops 100 degrees relative to the extending direction of the primary portion.

Device according to claim 1 , wherein each of the one or more secondary portions has a first end connected to the primary portion and a second end opposite the first end, and wherein at least one of the tertiary portions on each of the one or more secondary portions extends from a position on the secondary portions located between the first end and the opposite second end on the secondary portion.

Device according to any of the preceding claims, wherein a is between 20 and 70 degrees, or between 20 and 60 degrees, or between 20 and 50 degrees, or between 25 and 45 degrees, or between 25 and 40 degrees, or between 30 and 35 degrees, or between 27 and 32 degrees, or 30 degrees.

Device according to any of the preceding claims, wherein β is at least 30 degrees, or at least 40 degrees, or at least 50 degrees, or at least 60 degrees, or at least 70 degrees, or at least 80 degrees or at least 85 degrees, or 90 degrees.

Device according to any of the preceding claims, wherein β is between 55 and 100 degrees, or between 60 and 100 degrees, or between 70 and 100 degrees, or between 80 and 100 degrees.

Device according to any of the preceding claims, wherein the distance d(T ₁- T_{1 2}) between any pair of two consecutive tertiary portions in the flow direction on the first electrode is the same.

Device according to any of the preceding claims, wherein the distance d(T_{2 1}- T2.2) between any pair of two consecutive tertiary portions in the flow direction on the second electrode is the same.

Device according to any of the preceding claims, wherein the distance d(Ti.i- T1.1) between any pair of a tertiary portion on the first electrode and the consecutive tertiary portion on the second electrode in the flow direction is the same.

Device according to any of the preceding claims, wherein the one or more tertiary portions of the first electrode are arranged offset/displaced relative to the one or more tertiary portions of the second electrode.

10. Device according to any of the preceding claims, wherein the one or more

tertiary portions of the first electrode are arranged with a distance relative to the one or more tertiary portions of the second electrode.

1 1 . Device according to any of the preceding claims, wherein the one or more

tertiary portions of the first electrode are positioned at offset positions relativity to the one or more tertiary portions of the second electrode, wherein the one or more tertiary portions on both the first electrode and the second electrode are at least partly overlapping the centre of the channel.

Device according to any of the claims 1 -8, wherein the one or more tertiary portions of the first electrode are arranged straight opposite the one or more tertiary portions of the second electrode.

Device according to any of the preceding claims, wherein the one or more channels has a length l_channei and a width w_channel, wherein l_channei is between 5-10 times larger than w_channel.

14. Device according to any of the preceding claims, wherein the one or more

channels has a width w_channei and the one or more tertiary portions has a width Wtertiary, wherein Wchannei is between 5-60 times larger than

15. Device according to any of the preceding claims, wherein the nanoparticles and/or microparticles are displaced due to a large electrical gradient produced by the electrodes.

16. Device according to any of the preceding claims, wherein the electrical gradient is controlled by turning on and/or off an AC electrical field between the first and the second electrode.

17. Device according to any of the preceding claims, wherein the first volume is larger than the second volume, and wherein the second concentration is higher than the first concentration.

18. Device according to any of the preceding claims, wherein the nanoparticles and/or microparticles are one or more of the following:

- viruses

- extracellular vesicles, e.g. exosomes or microvesicles

- foreign microparticles

- foreign nanoparticles

- bacteria - parasites

- cells.

19. Device according to any of the preceding claims, wherein the nanoparticles and/or microparticles are nanoparticles having a diameter smaller than 100 nm.

20. Device according to any of the preceding claims, wherein the dimensions of the device is 5-7 cm in a first direction and 3-6 cm in a second direction.

21 . Method for concentration of nanoparticles and/or microparticles in liquid flow conditions by dielectrophoresis, the method comprising:

- providing a sample with a first volume comprising nanoparticles and/or microparticles with a first concentration;

- injecting the sample in the inlet in a device according to any of claims 1 -

20;

- obtaining liquid flow conditions of the nanoparticles and/or microparticles in the micro fluidic channels of the device transporting the nanoparticles and/or microparticles from the inlet towards the outlet thereby concentrating the nanoparticles and/or microparticles at the centre of the channel;

- collecting a second volume comprising the nanoparticles and/or