IMPROVEMENTS IN OR RELATING TO IMAGING MICRODROPLETS IN MICROFLUIDIC DEVICES - Patent application

Abstract

An apparatus comprising a microfluidic device and an optical imaging device, the microfluidic device comprising a microfluidic space configured to contain a plurality of microdroplets, a first light source configured to illuminate the microfluidic space, a voltage source for providing a voltage to the microfluidic space to generate a voltage across the microfluidic space, and a first modulator configured to generate a waveform signal to modulate an electric field applied across the microfluidic space, the first modulator configured to switch between an active state and an inactive state, where in the active state an electric field is applied across the microfluidic space to hold a plurality of microdroplets and in the inactive state no electric field is applied across the microfluidic space, the first modulator further configured to directly or indirectly control the first light source such that during the active state at least a portion of light is provided throughout the microfluidic device, the optical imaging device generates an image of at least a subset of the microdroplets, and the first modulator further configured to control the optical imaging device such that during the inactive state an image of at least a subset of the microfluidic space is generated.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to an apparatus for imaging microdroplets in microfluidic devices, and more particularly to an apparatus for imaging microdroplets in optoelectronic microfluidic devices whilst maintaining microdroplet control. [Background technology]

[0002] 2. Background of the Invention Droplet-based microfluidic systems aim to rapidly carry out a large number of reactions in parallel using small reagent volumes. Droplets in microfluidic devices can be controlled by different techniques, including the optical electrowetting-on-dielectric (oEWOD) effect, a well-known effect in which an electric field applied between a liquid and a substrate makes the liquid more wettable than its natural state.

[0003] Optically-based droplet manipulation techniques can pattern and reconfigure light to provide dynamic control over microdroplets without the use of complex control circuits within the device. Optically mediated electrowetting-on-dielectric (oEWOD) devices include microdroplets that change position through a microfluidic space defined by containing walls; e.g., a pair of parallel plates that sandwich the microfluidic space between them. At least one of the containing walls includes what will be referred to below as "virtual" electrowetting electrode locations, which are generated by selectively illuminating certain areas of a semiconductor embedded within the containing walls. By selective illumination of the layer with light from an independent light source controlled by an optical assembly, a virtual path of virtual electrowetting electrode locations can be temporarily generated along which the microdroplets can be moved. Thus, the conductive cells are omitted and permanent droplet receiving locations are abandoned for a homogeneous dielectric surface, on which droplet receiving locations are temporarily generated by selective and variable illumination of multiple points on the photoconductive layer, e.g., with a pixelated light source. This allows for highly extreme electrowetting fields that can move the microdroplets over the surface by induced capillary-type forces that are established everywhere on the dielectric layer, optionally in conjunction with a directional flow of droplets of a carrier fluid in which the microdroplets are dispersed, for example by emulsification. In other words, the carrier fluid contains the microdroplets.

[0004] Optical-based droplet manipulation techniques such as oEWOD have the potential to provide a flexible and high-performance droplet manipulation platform. Challenges arise in integrating appropriate detection technologies to image and measure microdroplets in microfluidic spaces. Light-based measurements such as fluorescence measurements are crucial tools for performing biological assays. However, the light required to image and measure microdroplets can overwhelm the light used to hold or manipulate the microdroplets, resulting in loss of microdroplet control. If not controlled or floated, microdroplets tend to drift within the microfluidic platform, which can further complicate obtaining high-quality images or measurements.

[0005] Thus, there is a need for an invention that can precisely control microdroplets in microfluidic devices using optically-based droplet manipulation techniques and that can obtain images and / or optical measurements without loss of microdroplet control. Summary of the Invention [Problem to be solved by the invention]

[0006] It is against this background that the present invention was born. [Means for solving the problem]

[0007] According to one aspect of the invention, there is provided an apparatus comprising a microfluidic device and an optical imaging device, the microfluidic device comprising: a microfluidic space configured to contain a plurality of microdroplets; a first light source for illuminating the microfluidic space; a voltage source for providing a voltage to the microfluidic device to generate an electric field across the microfluidic space; and a first modulator configured to generate a waveform signal to modulate the electric field applied across the microfluidic space, the first modulator configured to switch between an active state and an inactive state, wherein in the active state an electric field is applied across the microfluidic space and in the inactive state no electric field is applied across the microfluidic space, the first modulator further configured to directly or indirectly control the first light source such that during the active state at least a portion of light is provided throughout the microfluidic device to hold the plurality of microdroplets in the presence of the electric field; the optical imaging device generating images of at least a subset of the microdroplets, the first modulator further configured to control the optical imaging device such that during the inactive state an image of at least a subset of the microdroplets is generated.

[0008] The first modulator provides a waveform for modulating the electric field at the switching frequency of the system. In other words, the first modulator modulates the electric field to meet the optoelectronic requirements of the system in terms of switching between active and inactive states. In some embodiments, this switching can be 50 Hz. This frequency is very different from the A / C (alternating current) frequency of the electric field, which can be, for example, 1 kHz.

[0009] According to another aspect of the invention, there is provided an apparatus comprising a microfluidic device and an optical imaging device, the microfluidic device comprising: a microfluidic space configured to contain a plurality of microdroplets; a first light source for illuminating the microfluidic space; a voltage source for providing a voltage to the microfluidic space to generate an electric field across the microfluidic space; and a first modulator configured to generate a waveform signal to modulate the electric field applied across the microfluidic space, the first modulator configured to switch between an active state and an inactive state, wherein in the active state an electric field is applied across the microfluidic space to hold a plurality of microdroplets and in the inactive state no electric field is applied across the microfluidic space, the optical imaging device generating images of at least a subset of the microdroplets, the first modulator further configured to directly or indirectly control the first light source such that during the active state at least a portion of light is provided across the microfluidic device; and the first modulator further configured to control the optical imaging device such that during the inactive state an image of at least a subset of the microdroplets is generated.

[0010] The device according to the invention can be a microfluidic device, such as an optoelectronic microfluidic device. The device according to the invention allows a user to apply an electric field to the microfluidic space to hold at least a subset of the microdroplets in place. The application of an electric field is advantageous because it allows the microdroplets to be held in an array within the microfluidic space. This electric field can be an A / C field.

[0011] Additionally, microdroplets, or at least a subset of microdroplets, can be manipulated and / or controlled within the microfluidic space by providing a light source that provides light suitable for performing operations on the microdroplets during an active state, e.g., through an oEWOD. In some embodiments, the sustained light source can be used to manipulate the microdroplets, e.g., to merge or split the microdroplets using an oEWOD.

[0012] In the embodiment with two light sources, droplet control can be maintained by the first light source, which always performs the droplet manipulation and remains illuminated. During the inactive state, there is no need for this light source to hold the droplet and therefore it can remain on. In these cases, when the light is collected again from the sample, the light from the first light source can be filtered out before imaging or computerized out of the recorded image. Alternatively, during the inactive state, the first light source can be switched off, which in some cases eliminates the need for filtering, but in others the need remains. This is in contrast to the need for an imaging light source in a two light source system, for which there is a need to switch the light source on and off in anti-phase with the electric field. For the avoidance of doubt, the filtering can be achieved by one or more shutters, color filters, or polarisation.

[0013] The light from the light source can provide "holding illumination," which is suitable for the oEWOD to manipulate the microdroplets. In some embodiments, the light from the light source can provide imaging illumination, which can be used, for example, for illumination for fluorescence measurements.

[0014] "Holding illumination" as used within the context of the present invention should be understood to include any illumination used in combination with an electric field to hold, manipulate, and / or control at least a subset of the microdroplets during an active state. Thus, light and electric fields for illumination are necessary for the operation of the oEWOD. Manipulation of the microdroplets can include, but is not limited to, sorting, merging, splitting, and / or arranging the microdroplets, for example, in an array.

[0015] A subset of the microdroplets disclosed herein can include one or more microdroplets.

[0016] The first modulator may further be configured to directly or indirectly control the first light source to provide at least a portion of the light throughout the microfluidic space during the active state. Directly controlling the first light source may include the first modulator being configured to switch the light source between an on state and an off state. Indirectly controlling the first light source may include using a filter, which may be controlled by the controller, or alternatively the first modulator and / or the second modulator. The filter may include a spatial filter and may operate on the first light source to provide a portion of light throughout the microfluidic space suitable for holding and / or manipulating the microdroplets during the active state.

[0017] In some embodiments, the filter can be a color filter. The color filter can be arranged with one light source or with two light sources, and the color filter can be interchangeable. Generally, the color filter is not modulated by the modulator. In the two light source embodiment, if the color filtering is placed in the imaging path, the oEWOD light source can remain active throughout without adversely affecting the imaging light.

[0018] In some embodiments, the filter can be a spatial filter such as a digital mirror device (DMD). The spatial filter can be modulated by a first or second modulator. When using a single light source, the filter can have several vibration modes and vibrate at several different frequencies.

[0019] In some embodiments, the first modulator may be further configured to control an optical imaging device to generate an image of at least a subset of the micro-droplets during the inactive state.

[0020] In some embodiments, the first modulator is further configured to directly or indirectly control the first light source such that during the inactive state, the first light source provides imaging illumination for imaging the microdroplets, or at least a subset of the microdroplets. In some embodiments, the first modulator is further configured to directly or indirectly control the first light source such that during the inactive state, the first light source provides light suitable for fluorescence onto at least a subset of the microdroplets.

[0021] In some embodiments, the image can be a fluorescence image, or a fluorescence resonance energy transfer image, or a bright field image, or a chemiluminescence image. The image can be a single image captured during a single switching inactive interval. However, in some embodiments, the image can be integrated over a time interval of 0.25s, 0.5s, or 1s. Thus, if the first modulator applies switching modulation to the A / C field at 50Hz, a 1s time integration includes light collected from 50 modulation cycles. This integration method minimizes the effect of read noise and allows a larger signal to noise ratio to be achieved, which is important for many imaging techniques, such as fluorescence imaging.

[0022] In some embodiments, the controller, the first modulator, and / or the second modulator may be further configured to control a filter such that the light directed to at least a subset of the microdroplets during the inactive state is suitable for imaging. In some embodiments, the filter may be configured to filter at least a portion of the light from the first light source such that the microfluidic space is illuminated with different wavelengths of light during the active state and the inactive state. In some embodiments, the filter may be a color filter.

[0023] The above filters allow a single light source to be used for both holding illumination and imaging illumination during active and inactive states, which can be beneficial because such filters are simple, efficient, and cost-effective for a user to operate. When a single light source is used to hold the microdroplets and image at least a subset of the microdroplets, the single light source is spatially modulated.

[0024] In some embodiments, particularly in single light source configurations, the first light source is continuously on during active and inactive states, and the filter can operate on the first light source to switch between a holding pattern and an imaging illumination pattern.

[0025] In some embodiments, the first modulator is further configured to directly or indirectly control the first light source, such that the first light source does not provide light to at least a subset of the microdroplets during an inactive state. In some embodiments, the controller controls a filter, such that light is not provided to the microdroplets during an inactive state. In some embodiments, the first modulator controls the first light source, such that the first light source is in an "off" state during an inactive state. This may be advantageous when the optical imaging device is configured to generate, for example, chemiluminescence images. In some embodiments, the first light source may be a dual wavelength light source, and controlling the optical imaging device to generate an image of at least a subset of the microdroplets may include switching from a wavelength suitable for holding the microdroplets to a different specific wavelength suitable for imaging. By switching between different wavelengths for manipulation and imaging illumination, there is a spectral distinction that may facilitate detection and / or analysis of contents within at least a subset of the microdroplets.

[0026] It is generally known that the electric field is not modulated, and the holding illumination and the imaging illumination are present simultaneously, and the intensity of the imaging illumination in the inactive state can be low enough so as not to interfere with the illumination set to hold and / or manipulate the microdroplets. However, due to the weak intensity of the imaging illumination, this may require long image acquisition times, limiting the efficiency of the overall process. In addition, during the inactive state, the microdroplets will stray while they are not held by the electric field. Thus, long acquisition periods may be detrimental to maintaining control of the microdroplets in the microfluidic space.

[0027] By modulating the electric field between active and inactive states and by directly or indirectly controlling the first light source, the device of the present invention avoids the simultaneous application of the electric field and the imaging illumination to the microfluidic space. This prevents the light used to capture an image of at least a subset of the microdroplets from overwhelming the light used to hold at least a subset of the microdroplets, resulting in undesirable photoelectric effects such as oEWOD. This therefore means that there is no limitation on the wavelength range or intensity of the imaging illumination that can be utilized when using the device of the present invention. This is in contrast to other techniques for imaging microdroplets in photoelectric devices, which exclude certain wavelengths and limit the intensity so as not to overwhelm the illumination used to hold the microdroplets. A modulator can be provided to generate a waveform signal to modulate the electric field applied across the microfluidic space. The modulator can be in an active state and an inactive state, in which an electric field is applied across the microfluidic space to allow control of each microdroplet, or at least a subset of the microdroplets, and in which no electric field is applied across the microfluidic space. In the inactive state, the microdroplets are not held by the electric field for a short period of time. The first modulator can be switched between the active and inactive states for a specific time interval. During the inactive state of the first modulator, images of at least a subset of the microdroplets in the microfluidic space can be captured.

[0028] To account for response times of the modulator, the filter, the illumination source, and non-instantaneous transitions between active and inactive states of the modulator, there may be periods between active and inactive states during which an electric field may be present and no imaging illumination may be present on the microdroplet. Careful synchronization to account for transition times can minimize or eliminate the effects of these transition periods.

[0029] Another advantage of the present invention is that modulation between active and inactive states allows at least a subset of the microdroplets to return to their optimal holding position when they become lost during the inactive state, e.g., in the inactive state, no electric field is applied to the microfluidic space and the microdroplets are not held, and thus the microdroplets may become lost due to diffusion, Brownian motion, or under the flow of the carrier medium.

[0030] When imaging illumination is applied to at least a subset of the microdroplets during the active state, the microdroplets may be urged along an illumination gradient, which arises from non-uniformities in the illumination, which exerts optical electrowetting forces on the droplets, thus accelerating the displacement of the droplets from their retained positions.

[0031] For example, when capturing chemiluminescence images, failure to expose at least a subset of the microdroplets to imaging illumination during the inactive state may cause the microdroplets to passively drift away from their optimal holding positions, provided that modulation occurs in time sufficient to prevent the microdroplets from drifting beyond the control of the holding illumination, and once the modulator switches the microfluidic device to the active state and the microfluidic space is illuminated with holding illumination, the microdroplets may return to their positions before they drifted.

[0032] In some embodiments, a controller may be provided to monitor various parameters, for example, voltages during an experiment and / or the controller may monitor other structures of the device of the invention, such as filters. The controller may be a computer, microprocessor, or microcontroller.

[0033] In some embodiments, the modulator can be switched to an additional state, in which there is no electric field applied to the microfluidic space and there is no illumination of the microdroplets by the light source. In some embodiments, adding a filter to the light source during the inactive state may not be sufficient to allow the optical imaging device to image at least a subset of the microdroplets. In some embodiments, the light source may need to be in an "off" state to capture an image with the optical imaging device. This may be particularly advantageous, for example, during chemiluminescence imaging.

[0034] In some embodiments, the first modulator can dynamically switch between the active state, the inactive state, and the additional states in any given order, for example, the first modulator can directly or indirectly control the first light source, such that the first light source can be used for oEWOD control of the microdroplets in the active state, or for fluorescent imaging of the microdroplets in the inactive state, and can return to oEWOD control of the microdroplets in the active state, and then return to fluorescent imaging of the microdroplets in the additional state.

[0035] In some embodiments, the first modulator may be a function generator, or may be a digital switch, or may be an analog switch.

[0036] In some embodiments, the voltage source may be an AC voltage source, hi some embodiments, the voltage source may be any voltage source suitable for generating an electric field across a microfluidic space.

[0037] In some embodiments, the voltage source can provide a voltage of 1-200V during an active state to generate an electric field across the microfluidic space. In some embodiments, the voltage source can provide a voltage of greater than 1V, greater than 10V, greater than 20V, greater than 30V, greater than 40V, greater than 50V, greater than 60V, greater than 70V, greater than 80V, greater than 90V, greater than 100V, greater than 110V, greater than 120V, greater than 130V, greater than 140V, greater than 150V, greater than 160V, greater than 170V, greater than 180V, or greater than 190V during an active state. In some embodiments, the voltage source may provide a voltage during an active state of less than 200V, less than 190V, less than 180V, less than 170V, less than 160V, less than 150V, less than 140V, less than 130V, less than 120V, less than 110V, less than 100V, less than 90V, less than 80V, less than 70V, less than 60V, less than 50V, less than 40V, less than 30V, less than 20V, or less than 10V. In some embodiments, the voltage source may provide a voltage between 5V and 20V.

[0038] In some embodiments, the voltage supplied to the microfluidic space in the inactive state may be less than 1 V. In embodiments, the voltage supplied to the microfluidic space in the inactive state may be 0 V.

[0039] In some embodiments, the waveform signal generated by the modulator can be a square wave. In some embodiments, the modulator can generate any waveform signal with varying voltage. In some embodiments, a waveform with fast transitions, such as a square wave or a top-hat wave, is advantageous because it prevents overlap between active and inactive states. Overlap can result in the imaging illumination overpowering the light illumination to retain at least a subset of the microdroplets, which can result in loss of microdroplet control.

[0040] In some embodiments, the microfluidic device may be in the active state, the inactive state, and / or the additional state for equal lengths of time.

[0041] In some embodiments, the microfluidic device may be in an active state for 90% of the time and in an inactive or additional state for 10% of the time.

[0042] In some embodiments, the microfluidic device may be in an active state for 10% of the time and in an inactive or additional state for 90% of the time.

[0043] The time spent between the active state and the inactive or additional state can be divided to achieve a balance between holding and imaging the microdroplets so that the microdroplets remain well controlled. The duty cycle between the active state and the inactive or additional state allows at least a subset of the microdroplets to return to their optimal holding position when they have strayed. This duty cycle depends on the speed at which the microdroplets stray from their optimal holding position. Microdroplets that stray at a higher speed require a longer active state to regain control of the microdroplets, whereas microdroplets that stray at a lower speed can be well controlled by a shorter duty cycle of the active state.

[0044] The speed at which the microdroplets drift depends on several factors, including but not limited to the intensity of the operating light source, the length of time the device is inactive, the size of the microdroplets, the shape of the microdroplets, the compression of the microdroplets, and / or the viscosity of the carrier phase the microdroplets are dispersed in. In some embodiments, the speed at which the microdroplets drift from their optimal position can be reduced by, for example, coating the microfluidic space with a frictional coating, cooling the microdroplets, compressing the microdroplets, and / or using a more viscous carrier phase.

[0045] In some embodiments, when acquiring chemiluminescence images during the inactive or additional state, a longer duty cycle of the inactive or additional state may be preferred, such as a duty cycle of 10:90 or 1:99. Ideally, there is no electric field applied to the microfluidic space and no imaging illumination applied during acquisition of the chemiluminescence image. It may be beneficial to have a longer inactive or additional state to maximize the time during which chemiluminescence signals can be detected from at least a subset of the microdroplets. However, since the microdroplets are not held during the inactive or additional state, they will passively drift from their optimal holding position, and thus modulating the active and inactive or additional states allows control over the microdroplets to be maintained.

[0046] In some preferred embodiments, the waveform signal may have a frequency range of 0.5 to 5000 Hz. In some embodiments, the waveform signal may have a frequency of more than 0.5 Hz, more than 10 Hz, more than 50 Hz, more than 100 Hz, more than 250 Hz, more than 500 Hz, more than 750 Hz, more than 1000 Hz, more than 1250 Hz, more than 1500 Hz, more than 1750 Hz, more than 2000 Hz, more than 2250 Hz, more than 2500 Hz, more than 2750 Hz, more than 3000 Hz, more than 3250 Hz, more than 3500 Hz, more than 3750 Hz, more than 4000 Hz, more than 4250 Hz, more than 4500 Hz, or more than 4750 Hz. In some embodiments, the waveform signal can have a frequency of less than 5000Hz, 4750Hz, 4500Hz, 4250Hz, 4000Hz, 3750Hz, 3500Hz, 3250Hz, 3000Hz, 2750Hz, 2500Hz, 2250Hz, 2000Hz, 1750Hz, 1500Hz, 1250Hz, 1000Hz, 750Hz, 500Hz, 250Hz, 100Hz, 50Hz, or 10Hz. Preferably, the range is 4Hz to 50Hz.

[0047] In some embodiments, the waveform signal modulation frequency can be selected depending on the speed at which the microdroplet strays from its optimal holding position.

[0048] In some embodiments, a higher modulation frequency may result in reduced microdroplet control. This may result in illumination of at least a subset of the microdroplets by illumination configured to hold and / or manipulate the microdroplets not occurring for a sufficient length of time to maintain microdroplet control.

[0049] In some embodiments, a particular modulation frequency can provide improved microdroplet control, for example, in an embodiment where the modulation frequency matches a 1 kHz AC voltage source frequency, the sustain strength at a modulation frequency of 2.5 kHz can be greater than the sustain strength at a modulation frequency of 1 kHz.

[0050] The minimum modulation frequency depends on the speed at which the droplets wander: the time the device spends in the inactive or additional state must be less than the time it takes for the droplets to wander beyond a position at which they can return to their optimal holding position in the active state.

[0051] In some embodiments, the microfluidic space can be configured to contain a plurality of microdroplets, and the device is suitable for holding and / or manipulating and imaging the microdroplets at the single microdroplet level, and the device is also suitable for holding and / or manipulating and imaging at least a subset of the plurality of microdroplets.

[0052] In some embodiments, at least one microdroplet can contain a biological entity. In some embodiments, the device can be used to image at least a subset of the microdroplets as part of a biological sample. In some embodiments, the device can be used to perform fluorescence measurements as part of a biological assay.

[0053] In some preferred embodiments, the biological entity may be a cell, a virus, a protein sample, an antibody sample, a functionalized microbead or an enzyme.

[0054] In some preferred embodiments, at least one microdroplet may contain a fluorescent entity. The fluorescent entity may be a fluorescent dye or a fluorescent bead. The fluorescent entity may be attached to a biological entity, such as a cell, present in the microdroplet. The biological entity, such as a cell, may be stained with a fluorescent dye.

[0055] In some embodiments, a single light source can be used during the active state and during the inactive state to illuminate at least a subset of the microdroplets. In some embodiments, the device can further include a second light source. In some embodiments, a first light source can provide microdroplet-holding illumination during the active state, and a second light source can provide imaging illumination during the inactive state. A device with two light sources is advantageous because it can reduce losses. A single light source may be required to pass through a spatial light modulator, which has associated losses. This may require filtering the single light source using the spatial light modulator, which creates additional undesirable losses. Thus, using multiple light sources has a higher efficiency.

[0056] In some embodiments, imaging during the inactive state can be performed without an illumination source and light emitted by the sample contained within the microdroplet can be detected. These embodiments are particularly suitable for imaging samples that are chemiluminescent, phosphorescent, or bioluminescent.

[0057] In some embodiments, the apparatus may further comprise a filter to filter at least a portion of the light from the first and / or second light sources.

[0058] In some embodiments, a controller can be provided to control the filter, which during an inactive state allows imaging illumination provided by the first and / or second light sources to be directed throughout the microfluidic space.

[0059] In some embodiments, the apparatus may further include a second modulator configured to generate a second waveform signal for modulating light from a second light source. The second modulator may modulate light from the second light source such that the light is modulated out of phase with the first modulator and the modulated electric field such that the electric field and the imaging illumination are not simultaneously applied to the microfluidic space.

[0060] In some embodiments, a second modulator is provided to control a second light source, whereby the second light source is configured to provide imaging illumination to at least a subset of the microdroplets during the inactive state. In some embodiments, the first and / or second modulators may be further configured to control the second light source, whereby the second light source is configured to provide imaging illumination to at least a subset of the microdroplets during the inactive state. In some embodiments, the first and / or second modulators may be further configured to control the second light source, whereby the second light source is configured to provide light suitable for fluorescence to at least a subset of the microdroplets during the inactive state.

[0061] In some embodiments using two light sources, a first light source can illuminate at least a subset of the microdroplets continuously during both active and inactive states. A second light source is configured to provide imaging illumination to at least a subset of the microdroplets, and the second light source is modulated by a second modulator, such that imaging illumination is provided only during the inactive state. For example, the second modulator can modulate the light source and switch the light source between an on state and an off state. The controller can be configured to modulate or monitor the first and second modulators out of phase with each other, to prevent the second light source and the electric field from being simultaneously active and the imaging illumination from interfering with the manipulation of the microdroplets, resulting in a loss of droplet control.

[0062] In some embodiments using two light sources, the controller or the first and / or second modulators control a filter so that at least a subset of the microdroplets can be illuminated with different wavelengths for imaging and retention, and the two illuminations are spatially distinct. In some embodiments, a filter is added to the light source for retention illumination so that during an inactive state, the light source for retention illumination does not illuminate the microdroplets and does not interfere with the imaging light illumination.

[0063] In some embodiments, the apparatus further comprises: a first light source; a second light source; a first modulator; and a second modulator, where the first light source is configured to provide illumination light for manipulating at least a subset of the microdroplets, the second light source configured to provide imaging illumination to at least a subset of the microdroplets, the first modulator configured to generate a first waveform signal to modulate an electric field applied across the microfluidic space, and the second modulator configured to generate a second waveform signal to modulate the illumination of at least a subset of the microdroplets by the second light source, the first modulator configured to switch between an active state and an inactive state, where in the active state an electric field is applied across the microfluidic space to hold at least a subset of the microdroplets, and where in the inactive state no electric field is applied across the microfluidic space, and the first and second modulators are further configured to directly or indirectly control the second light source, whereby the second light source provides imaging illumination to at least a subset of the microdroplets during the inactive state.

[0064] In some embodiments, the second modulator can be, but is not limited to, a direct electrical modulator with a function generator, a digital or analog switch, a chopper, an electro-optical modulator, an acousto-optical modulator, or a shutter. In some embodiments, the second modulator can be implemented as a direct LED (light emitting diode) or laser modulation. In some embodiments, the second modulator can be a spatial light modulator, such as a digital micromirror device (DMD), a liquid crystal spatial light modulator (SLM). In some embodiments, it may be preferable to use an acousto-optical or electro-optical modulator when the second illumination source is a laser. An acousto-optical modulator can operate over a fixed frequency range and can be used to modulate light of a fixed wavelength. An electro-optical modulator can also operate over a fixed frequency range and can be tuned for each wavelength of light.

[0065] In some embodiments it may be preferred to use a chopper as the second modulator. Choppers are suitable for modulating all wavelengths of light. Choppers may be suitable for use in mid-range frequency modulation, for example 100Hz to 1000Hz.

[0066] In some embodiments, direct LED or laser modulation may be preferred, which may be suitable for modulation up to the MHz frequency range.

[0067] In some embodiments, it may be preferable to use a second modulator that is a shutter. The shutter is suitable for realizing a well-defined square wave, which is advantageous for preventing any overlap of the electric field applied across the microfluidic space with the imaging illumination of the microdroplet. The shutter is suitable for use with all wavelengths of light. The shutter may have a lower maximum frequency than the other modulations.

[0068] In some embodiments, the first and / or second light source may be an LED, or may be a laser, or may be a lamp.

[0069] In some embodiments, the laser beam used as the light source can be spatially and temporally coherent. The laser beam can be a collimated beam, since it has high coherence and low divergence. The light provided by the collimated light beam can be used to illuminate the microfluidic space in which the microdroplets are located. In some embodiments, the laser beam is used to achieve a higher signal-to-noise ratio than can be achieved with a non-coherent light source. This can be advantageous, for example, when imaging using raster scanning.

[0070] In some embodiments, it may be beneficial to use an LED or lamp as the light source. In one embodiment, a white LED can be used as the light source because it can provide a high power output that can be filtered to selectively target a narrow wavelength band, for example, for fluorescence measurements. In some embodiments, a colored LED can be used as the light source. In some embodiments, a lamp, such as a xenon lamp, can be filtered to provide narrow band illumination for optical measurements.

[0071] In some embodiments, the microfluidic device may be in an active state before the first modulator switches between active and inactive states. In some embodiments, the microfluidic device may be used to perform manipulations of the microdroplets before it is necessary to capture images of at least a subset of the microdroplets. Thus, the device may be in an active state to provide a constant electric field or A / C field to the microfluidic space before the first modulator begins to control modulation of the electric field applied across the microfluidic space.

[0072] In some embodiments, the microfluidic device may be an optical electrowetting-on-dielectric (oEWOD) device, an optical tweezers device, an opto-electronic tweezer (OET) device, or a dielectrophoresis (DEP) device.

[0073] In some preferred embodiments, a device for manipulating microdroplets using electrowetting is provided, the device comprising a first composite wall and a second composite wall, the first composite wall comprising: a first substrate; a first conductor layer on the substrate; and a first continuous dielectric layer on the photoactive layer having a thickness of less than 20 nm, and the second composite wall comprising: a second substrate; and a second conductor layer on the substrate.

[0074] In some embodiments, the second composite wall optionally comprises a second continuous dielectric layer on the second conductor layer having a thickness of less than 20 nm.

[0075] In some embodiments, the first composite wall further comprises a photoactive layer on the first conductive layer.

[0076] In some preferred embodiments, the microfluidic device may be an oEWOD device, the oEWOD structure comprising: a first composite wall; a second composite wall; an A / C power source; a first and second electromagnetic radiation source; and an operating means, the first composite wall comprising: a first substrate; a first conductor layer on the substrate, the first conductor layer having a thickness in the range of 70-250 nm; a photoactive layer on the conductor layer, the photoactive layer activated by electromagnetic radiation in a wavelength range of 400-850 nm and having a thickness in the range of 300-1500 nm; a first dielectric layer on the photoactive layer, the first dielectric layer having a thickness below 20 nm, such as 1 nm to 20 nm, which thickness may be in the range of 30-160 nm; the second composite wall comprising: a second substrate; a second conductor layer on the substrate, the second conductor layer having a thickness in the range of 70-250 nm; and optionally a second dielectric layer on the second conductor layer, the first dielectric layer having a thickness below 20 nm, such as 1 nm to 20 nm, which may be in the range of 30-160 nm. and a second dielectric layer having a thickness below 20 nm, such as between 20 nm and 20 nm, which thickness may be in the range of 30-160 nm, the exposed surfaces of the composite walls being spaced 20-180 μm apart to define a microfluidic space configured to contain a microdroplet, an A / C power source connected to the first and second conductor layers to provide a voltage between the first and second composite walls, the electromagnetic radiation of the electromagnetic radiation source having an energy higher than the band gap of the photoactive layer and configured to impinge on the photoactive layer and induce corresponding virtual electrowetting locations on the first dielectric layer, and the manipulation means is operable to manipulate the points at which the electromagnetic radiation impinges on the photoactive layer to change the location of the virtual electrowetting locations, thereby generating at least one electrowetting path along which the microdroplet can move. The first and second walls of the structure are transparent, and a microfluidic space is sandwiched between them.

[0077] In some embodiments, the first and / or second substrate may be transparent. The first and second conductor layers may be transparent.

[0078] The A / C power supply may be configured to provide a voltage between 0V and 100V across the microfluidic space bounded by the first and second composite walls and is connected to the first and second conductor layers. In some embodiments, the voltage provided may be between 0V and 50V, or between 0V and 10V. In some embodiments, the A / C power supply may be configured to provide a voltage greater than 0V, greater than 5V, greater than 10V, greater than 15V, greater than 20V, greater than 25V, greater than 30V, greater than 35V, greater than 40V, greater than 50V, greater than 60V, greater than 70V, greater than 80V, or greater than 90V, or the voltage may be less than 90V, less than 80V, less than 70V, less than 60V, less than 50V, less than 45V, less than 40V, less than 35V, less than 30V, less than 25V, less than 20V, less than 15V, less than 10V, or less than 5V.

[0079] The first and second substrates are suitably made of a material having mechanical strength, such as glass, silicon, metal, or industrial plastic. In some preferred embodiments, the substrates may have a certain degree of flexibility. In other preferred embodiments, the first and second substrates may have a thickness of 100 to 1500 μm, such as 500 μm or 1100 μm. In some preferred embodiments, the first substrate is made of silicon, quartz glass, and glass. In some preferred embodiments, the second substrate is made of one of quartz glass or glass.

[0080] The first and second conductor layers are disposed on one surface of the first and second substrates and typically have a thickness in the range of 70-250 nm, with 70-150 nm being preferred. At least one of the layers is made of a transparent conductive material such as indium tin oxide (ITO), a conductive material such as silver, or a very thin film of a conductive polymer such as PEDOT (poly(3,4-ethylenedioxythiophene)). The layers can be formed as a continuous sheet or as a series of discrete structures such as wires. Alternatively, the conductor layer can be a mesh of conductive material, with electromagnetic radiation being directed between the openings of the mesh.

[0081] The photoactive layer is suitably formed of a semiconductor material capable of generating localized regions of charge in response to stimulation by a source of electromagnetic radiation. Examples include a hydrogenated amorphous silicon layer having a thickness in the range of 300-1500 nm. In some preferred embodiments, the photoactive layer is activated by the use of visible light. The photoactive layer in the case of the first wall, and optionally the conductor layer in the case of the second wall, may be coated with a dielectric layer, typically in the range of 30 nm to 160 nm thick. The dielectric properties of this layer are >10 7 Preferred properties include a high dielectric strength in V / m, and a dielectric constant > 3. In some embodiments, the dielectric layer is selected from alumina, silica, hafnia, or a thin non-conductive polymer film.

[0082] In other preferred embodiments of these structures, at least the first dielectric layer, and preferably both dielectric layers, are coated with an anti-fouling layer to help establish the desired microdroplet / dispersion medium fluid / surface contact angles at the various tentative electrowetting locations, and additionally to help prevent the contents of the microdroplet from sticking to the surface and being reduced as the microdroplet moves through the chip. If the second wall does not include a second dielectric layer, a second anti-fouling layer can be applied directly onto the second conductor layer.

[0083] For optimal performance, the antifouling layers should help establish a microdroplet / dispersion medium fluid / surface contact angle that should be in the range of 50°-180° when measured at 25°C at the three-point air-liquid-surface interface. In some preferred embodiments, these layers have a thickness of less than 10 nm and are typically monolayers. In other preferred embodiments, these layers are composed of polymers of acrylate esters such as methyl methacrylate or its derivatives substituted with hydrophilic groups, e.g., alkoxysilyl. Either or both of these antifouling layers are hydrophobic to ensure optimal performance. In some preferred embodiments, an intervening layer of silica less than 20 nm thick can be inserted between the antifouling coating and the dielectric layer to provide a chemically compatible bridge.

[0084] The first and second dielectric layers, and thus the first and second walls, define a microfluidic space, which is at least 10 μm wide, preferably in the range of 20-180 μm wide, within which the microdroplets are contained. Before the microdroplets are contained, the microdroplets themselves preferably have a characteristic diameter, which is 10% larger, suitably 20% larger than the width of the microfluidic space. Thus, the microdroplets are subjected to compression upon entering the chip, resulting in enhanced electrowetting performance, for example due to better microdroplet merging ability. In some embodiments, the first and second dielectric layers can be coated with a hydrophobic coating, such as fluorosilane.

[0085] In some embodiments, the microfluidic space includes one or more spacers to hold the first and second walls apart by a predetermined amount. Spacer options include beads or pillars, ridges generated from an intermediate resist layer, which are generated by photopatterning. Alternatively, deposition materials such as silicon oxide or silicon nitride can be used to create the spacers. Alternatively, layers of film can be used to form the spacer layer, including flexible plastic films with or without adhesive coatings. Various spacer geometries can be used to form narrow channels, tapered channels, or partially closed channels defined by straight lines of pillars. With careful design, these spacers can be used to assist in the deformation of the microdroplets, after which the microdroplets can be split and operations can be performed on the deformed microdroplets. Similarly, these spacers can be used to physically separate zones of the chip to prevent cross-contamination between droplet constituents and direct the flow of droplets when loading the chip under hydraulic pressure. In some embodiments, the spacers can be, but are not limited to, blade-shaped structures, wedge structures, pillars, hydrophobic patches, narrow channels, or dimples on the surface.

[0086] An A / C power supply attached to the conductor layer can be used to bias the first and second walls to provide a potential difference between them; suitably this potential difference is in the range of 0-50 volts. These oEWOD structures are typically used with a second electromagnetic radiation source having a wavelength in the range of 400-850 nm, for example 550, 620 and 660 nm, and an energy above the band gap of the photoactive layer. The photoactive layer is suitably activated at a virtual electrowetting electrode location, where the incident intensity of the radiation used is between 0.01 and 0.2 Wcm. -2 is within the range.

[0087] When the electromagnetic radiation sources are pixelated, they are powered either directly or indirectly using a reflective screen such as a digital micromirror device (DMD) illuminated by LEDs or other lamps. This allows highly complex patterns of virtual electrowetting electrode positions to be rapidly created and destroyed on the first dielectric layer, thereby enabling the microfluid to be precisely directed along essentially arbitrary virtual paths using tightly controlled electrowetting forces. Such electrowetting paths can be viewed as consisting of a continuum of virtual electrowetting electrode positions on the first dielectric layer.

[0088] In some embodiments, the shape of the points where the electromagnetic radiation hits is determined by the shape of the pixelation of the first and / or second modulator. The points where the electromagnetic radiation source radiation hits on the photoactive layer can be of any convenient shape, including conventional circular or annular shapes. In some embodiments, the shape of these points is determined by the shape of the corresponding pixelation, while in other embodiments it corresponds in whole or in part to the shape of the microdroplets once they enter the microfluidic space. In one embodiment, the points where the electromagnetic radiation hits, and therefore the electrowetting electrode positions, can be crescent-shaped and oriented in the intended direction of travel of the microdroplets. The electrowetting electrode positions themselves are suitably smaller than the surface of the microdroplets that adhere to the first wall, providing a maximum electric field strength gradient across the contact line formed between the droplet and the surface dielectric.

[0089] The first and second dielectric layers may be composed of a single dielectric material or may be a composite of two or more dielectric materials. The dielectric layers may be made of, but are not limited to, Al2O3 and SiO2.

[0090] A structure can be provided between the first and second dielectric layers. The structure between the first and second dielectric layers can be made of, but is not limited to, epoxy, polymer, silicon, or glass, or a mixture or composite thereof, and has straight, angular, curved, or microstructured walls / surfaces. The structure between the first and second dielectric layers can be connected to the top and bottom composite walls to create a sealed microfluidic device, defining channels and regions within the device. The structure can occupy the gap between the two composite walls. Alternatively or in addition, the conductors and dielectrics can be deposited on a molded substrate that already has walls.

[0091] Some aspects of the method and apparatus of the present invention are suitable for application to optically active devices other than electrowetting devices, such as devices configured to manipulate microparticles by dielectrophoresis or optical tweezers. In such devices, functionally identical optical instruments are used to manipulate and examine cells or particles to generate virtual optical dielectrophoretic gradients. Microparticles as defined herein may refer to particles such as biological cells, microbeads made of materials including polystyrene and latex, hydrogels, magnetic microbeads or colloids. The mechanisms of dielectrophoresis and optical tweezers are currently well known in the art and can be readily implemented by one of ordinary skill in the art.

[0092] In some embodiments, the optical imaging device further comprises a detector configured to detect optical signals from at least a subset of the microdroplets. In some embodiments, the detector can be configured to detect fluorescent signals from the microdroplets. Fluorescence measurements are an essential tool for performing biological assays. In some embodiments, the detector can be configured to detect chemiluminescence signals from the microdroplets.

[0093] In some embodiments, the detector may further comprise a camera configured to capture images of at least a subset of the microdroplets. In some embodiments, the detector may comprise a camera, which may allow for capturing images during the inactive state or the additional state. In some embodiments, the device may also be used to move the microdroplets and record images on the moving microdroplets.

[0094] According to one aspect of the present invention, there is provided a species screened by the device, apparatus, or method disclosed herein.

[0095] According to one aspect of the present invention, a species selected by the device, apparatus, or method disclosed herein is provided.

[0096] According to one aspect of the present invention, there is provided a species sequestered by the device, apparatus, or method disclosed herein.

[0097] According to one aspect of the invention, there is provided a species produced by the device, apparatus, or method disclosed herein.

[0098] These species can be chemical, biochemical, or biological in nature.

[0099] For example, the present invention can provide agonists / antagonists to an entity identified by the screening, selection, and / or isolation methods disclosed herein. The present invention can provide agonists / antagonists to an entity identified by the screening, selection, and / or isolation methods disclosed herein for use in therapy. The entity can be chemical, biochemical, or biological in nature.

[0100] According to one aspect of the present invention, there is provided a method of using the devices, apparatus, methods, or species disclosed herein.

[0101] According to one aspect of the present invention, there is provided a method of use of the devices, apparatus, methods or species disclosed herein in therapy.

[0102] The present invention provides for the use of the devices, apparatus, methods, or species disclosed herein in making an article of manufacture, which may be chemical, biochemical, or biological in nature.

[0103] The method of use may be peptide synthesis. The method of use may be synthetic biology. The method of use may be cell line engineering or cell line development. The method of use may be cell therapy. The method of use may be drug discovery. The method of use may be antibody discovery.

[0104] According to one aspect of the invention there is provided a method of use of the devices, apparatus, methods or species disclosed herein in analysis.

[0105] The analysis can be a physical analysis, a chemical analysis, or a biological analysis.

[0106] The method of use may be intracellular imaging.The method of use may be high content imaging.

[0107] The above method of use may be diagnostic.

[0108] The method of use may be a biological assay. The biological assay may be a high throughput screen. The biological assay may be an enzyme-linked immunosorbent assay (ELISA).

[0109] The method of use may be cell secretion.

[0110] The method of use may be QC (quality control) safety profiling.

[0111] The invention will now be further and more particularly described, by way of example only, with reference to the accompanying drawings in which: [Brief description of the drawings]

[0112] [Figure 1] FIG. 1 shows a schematic diagram of an apparatus according to the present invention. [Diagram 2] FIG. 1 shows a spatial filter applied across a microfluidic space. [Diagram 3] 3A and 3B are diagrams illustrating an oEWOD configuration in accordance with the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0113] Detailed Description The present invention discloses an apparatus herein that can be used to maintain control of one or more microdroplets using optoelectronic droplet manipulation techniques while acquiring images and / or optical measurements of one or more microdroplets.

[0114] 1, a microfluidic device 10 is provided that includes a microfluidic space 12 configured to contain at least one microdroplet 14, and a light source 16 for illuminating the microfluidic space 12. The microfluidic device 10 may be an optical electrowetting-on-dielectric (oEWOD) device, or may be an optical tweezers device, or may be an optoelectronic tweezers (OET) device, or may be a dielectrophoresis (DEP) device.

[0115] The microdroplets 14 may contain biological entities, such as cells or enzymes, and / or fluorescent entities, such as fluorescent dyes or fluorescent beads.

[0116] The light source 16 may be a halogen lamp, a laser, an LED, or any other suitable light source. The light source 16 may be a monochromatic LED, or preferably a white LED.

[0117] A voltage source supplies a voltage to the microfluidic device to generate an electric field across the microfluidic space 12. This voltage is applied across the microfluidic space for an extended period of time, as shown by the graphical representation 13 in FIG. 1. A modulator 22 is configured to generate a waveform signal 28 to modulate the electric field applied across the microfluidic space 12, such that there are active and inactive states, where an electric field is applied across the microfluidic space 12 in the active state and no electric field is applied across the microfluidic space 12 in the inactive state. During the active state, an electric field is generated across the microfluidic space 12, and illumination of the microfluidic space by the light source 16 generates an optoelectronic force, such as an oEWOD force. This optoelectronic force is used to control the microdroplets, which may be held in place and / or manipulated, for example, to merge, split, sort, and / or arrange in an array.

[0118] The modulator 22 may be a function generator, or may be a digital switch, or may be an analog switch. The waveform signal 28 may have a frequency range of 0.5-5000 Hz. The voltage source may be an A / C voltage source. The voltage source may provide a voltage of 1-200V to generate an electric field across the microfluidic space 12 during the active state. The voltage provided to the microfluidic space 12 during the inactive state may be less than 1V, and is preferably 0V.

[0119] The apparatus also includes a controller 20, which may be a computer, a microprocessor, or a microcontroller. The controller 20 may be configured to control a filter 32, such as a spatial filter. The filter 32 may be a color filter or a spatial filter. The controller 20 may be configured to control the first modulator 22, the second modulator 23, the filter 32, and / or the optical imaging device 18, so that an image of the microdroplet 14 may be captured during the inactive state. The filter 32 may be controlled such that it may be used to filter the light used to hold the microdroplet 14, so that illumination suitable for imaging is applied to the microdroplet 14 during the inactive state. This may include the controller 20 controlling the filter 32 so that no imaging illumination is applied to the microdroplet 14 during the inactive state, and an image of the microdroplet 14 is captured, for example, by chemiluminescence.

[0120] In addition, the first modulator 22 can be configured to switch to an additional state, in which there is no electric field applied to the microfluidic space 12 and the light source 16 is in an off state. This can be advantageous, for example, when performing chemiluminescence imaging. Figure 1 also shows that a second modulator 23 can be provided. The second modulator can be configured to directly or indirectly control a second light source 24, which can provide imaging illumination to at least a subset of the microdroplets.

[0121] 1, the optical imaging device 18 may further include an additional filter 25, one or more sets of mirrors 27, and one or more lenses 29, such as an objective lens 29, for focusing the light from the second light source 24 onto the microdroplet of interest. The optical imaging device 18 also includes a detector 31, such as a camera 31, for acquiring an image of the microdroplet, such as a fluorescent or chemiluminescent image.

[0122] The optical imaging device 18 generates an image of at least a subset of the microdroplets, which may include one microdroplet 14. The subset of microdroplets may include multiple microdroplets. The optical imaging device 18 may include a detector 31. The detector 31 may be configured to detect an optical signal from the microdroplets 14, such as a fluorescent signal or a chemiluminescent signal. The optical imaging device 18 may also include a camera, which may be used to capture an image of the microdroplets 14. The image may then be processed by a processor.

[0123] The device may include a single light source 16 that may be used to illuminate at least a subset of the microdroplets 14 during the inactive state. As shown in FIG. 1, the device may also include a second light source 24. When the device includes two light sources, the first light source 16 may provide microdroplet-holding illumination during the active state, and the second light source 24 may provide imaging illumination during the inactive state. The second light source 24 may be a lamp, a laser, an LED, or any other suitable light source. The second light source 24 may be a monochromatic LED, or preferably a white LED. The second light source 24 may be used to image the microdroplets 14 during the inactive state, and may be a light source suitable for fluorescent illumination, fluorescence resonance energy transfer illumination, bright field illumination, or any other illumination suitable for imaging the microdroplets 14.

[0124] When the device includes a second light source 24, the second light source 24 can be modulated using a second modulator 26. The second modulator 26 is configured to generate a second waveform signal 30 to modulate the light from the second light source 24. The second modulator 26 can be configured to modulate the second light source 24 out of phase with the modulation of the first light source 22 and the electric field. This causes the electric field and the imaging illumination of the microdroplets 14 to not be active at the same time, preventing the imaging illumination from interfering with the illumination used to hold the microdroplets 14. The second modulator 26 can be, but is not limited to, direct electrical modulation by a function generator, a digital or analog switch, a chopper, an electro-optical modulator, an acousto-optical modulator, or a shutter. The second modulator 26 can be a direct LED or laser modulator. The second modulator can be a spatial light modulator, such as a digital micromirror device (DMD) or a liquid crystal spatial light modulator (SLM).

[0125] As shown in FIG. 1, the graphical representation shows that the electric field 40 is modulated out of phase with the fluorescent light 41. This means that the electric field and the imaging illumination, such as the fluorescent illumination of the microdroplets 14, are not active at the same time, preventing the imaging illumination from interfering with the illumination used to hold the microdroplets 14. With reference to FIGS. 2A and 2B, an example of spatial modulation is shown. In some examples, a single light source is used to hold and image at least a subset of the microdroplets. In this example, a spatial filter is used to pattern the illumination in sync with the applied voltage to create active and inactive states, as shown in FIGS. 2A and 2B. The active state consists of the voltage being applied and a holding light pattern being applied, an example of which is shown in the image of FIG. 2A. The inactive state consists of the applied voltage being disabled and the illumination pattern being switched to blanket illumination, as shown in FIG. 2B, where the light illuminates the entire field when synchronized with the detector, which can then be used to generate an image.

[0126] With reference to FIG. 3A, a microfluidic device, in particular an oEWOD device 100, is provided. The oEWOD device 100 comprises, as shown in FIG. 3A: a first composite wall 102 made of a first substrate 104, a first transparent conductor layer 106 on the substrate 104, a photoactive layer 108 activated by electromagnetic radiation in the wavelength range 400-850 nm on the conductor layer 106, and a first dielectric layer 110 on the photoactive layer 108, where the first substrate 104 can be made of glass, the first conductor layer 106 has a thickness in the range of 70-250 nm, and the photoactive layer 108 has a thickness in the range of 300-1500 nm. The first dielectric layer 110 has a thickness below 20 nm. The lower limit of the thickness of this layer is determined, at least in part, by the method of providing such a thin layer, which must be continuous. However, theoretically it can have a thickness of 0.1 nm to 20 nm. The first conductor layer can be transparent.

[0127] The device 100 also comprises a second composite wall 112, which comprises: a second substrate 114 and a second conductor layer 116 on the substrate 114, which may be made of glass. The second conductor layer may be transparent. The second conductor layer 116 may have a thickness in the range of 70-250 nm. A second dielectric layer 118 may be present on the second conductor layer 116, which may have a thickness below 20 nm, such as 1 nm-20 nm, or may be 25-160 nm. The exposed surfaces of the composite wall 102 and the composite wall 112 are spaced 20-180 μm apart to define a microfluidic space 121 configured to contain a microdroplet 122.

[0128] The photoactive layer 108 may be made of amorphous silicon. The first and second conductors may be made of ITO.

[0129] An intermediate bonding layer 124 is provided on the first dielectric layer 110 and may also be provided on the second dielectric layer 118. The intermediate layer may have a thickness between 0.1 nm and 5 nm. The intermediate layer may have a thickness greater than 0.1 nm, greater than 0.25 nm, greater than 0.5 nm, greater than 0.75 nm, greater than 1 nm, greater than 1.5 nm, greater than 2 nm, greater than 2.5 nm, greater than 3 nm, greater than 3.5 nm, greater than 4 nm, or greater than 4.5 nm, or may have a thickness less than 5 nm, less than 4.5 nm, less than 4 nm, less than 3.5 nm, less than 3 nm, less than 2.5 nm, less than 2 nm, less than 1.5 nm, less than 1 nm, less than 0.75 nm, less than 0.5 nm, or less than 0.25 nm. The intermediate layer, e.g. silicon oxide, is provided on the first and / or second dielectric layers. The advantage of this intermediate layer is that it can be used as a tie layer for the antifouling or non-fouling layer, which can be hydrophobic. In some preferred embodiments, not shown in the accompanying drawings, the intermediate tie layer can be omitted. In such embodiments, the hydrophobic layer is applied directly to the dielectric layer.

[0130] A hydrophobic layer 126 is disposed on the intermediate bonding layer 124. An example of a hydrophobic layer can be fluorosilane or fluorosiloxane. The intermediate bonding layer 124 is optional and the channel walls 120 can be made of SU-8 (a type of negative photoresist) or can be part of a glass structure. The intermediate layer 124 is disposed between the dielectric layers 110, 118 and the hydrophobic layer 126.

[0131] As shown in Figure 3A, incident light 130 can be used to provide a light sprite pattern 131, where the incident light 130 provides light onto a portion of the microdroplet 122 to hold the microdroplet 122 in a stationary position within the microfluidic space 121. An oil carrier phase 134 can be provided to the microdroplet 122 through holes 136 in the device to provide key nutrients and key components to keep the contents within the microdroplet 122, such as one or more cells, alive and healthy. In some cases, phase 134 can provide key nutrients, medium, and contents for cell growth, survival, and / or proliferation.

[0132] The first and second substrates 104, 114 are made of a material with high mechanical strength, such as glass, metal, or industrial plastic. In some embodiments, the substrates can have some flexibility. In still other embodiments, the first and second substrates have a thickness of at least 100 μm. In some embodiments, the first and second substrates can have a thickness of more than 2500 μm, such as 3000, 3500, or 4000 μm. In some embodiments, the first and second substrates can have a thickness in the range of 100 to 2500 μm. In some embodiments, the first and second substrates can have a thickness of greater than 100 μm, greater than 200 μm, greater than 300 μm, greater than 400 μm, greater than 500 μm, greater than 600 μm, greater than 700 μm, greater than 800 μm, greater than 900 μm, greater than 1000 μm, greater than 1100 μm, greater than 1200 μm, greater than 1300 μm, greater than 1400 μm, greater than 1500 μm, greater than 1600 μm, greater than 1700 μm, greater than 1800 μm, greater than 1900 μm, greater than 2000 μm, greater than 2100 μm, greater than 2200 μm, greater than 2300 μm, or greater than 2400 μm. In some embodiments, the first and second substrates can have a thickness of less than 2500 μm, less than 2400 μm, less than 2300 μm, less than 2200 μm, less than 2100 μm, less than 2000 μm, less than 1900 μm, less than 1800 μm, less than 1700 μm, less than 1600 μm, less than 1500 μm, less than 1400 μm, less than 1300 μm, less than 1200 μm, less than 1100 μm, less than 1000 μm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm, less than 400 μm, less than 300 μm, or less than 200 μm. In some embodiments, the first substrate has a thickness of approximately 1100 μm and the second substrate has a thickness of 700 microns. In other embodiments, the first and second substrates have a thickness of 800 microns. In some embodiments, the first substrate is made of one of silicon, fused silica, and glass. In some embodiments, the second substrate is made of one of fused silica and / or glass. The glass may be, but is not limited to, soda-lime glass or float glass.

[0133] The first and second conductor layers 106, 116 are disposed on one surface of the first and second substrates 104, 114 and typically have a thickness in the range of 70-250 nm, with a thickness in the range of 70-150 nm being preferred. At least one of these layers is made of a very thin film of a transparent metal such as indium tin oxide (ITO), a conductive metal such as silver, or a conductive polymer such as PEDOT. These layers can be formed as a continuous sheet or as a series of discrete structures such as a number of wires. Alternatively, the conductor layer can be a mesh of conductive material, with electromagnetic radiation being directed between the openings of the mesh.

[0134] The photoactive layer 108 is formed of a semiconductor material capable of generating localized regions of charge in response to stimulation by a source of electromagnetic radiation. Examples include a hydrogenated amorphous silicon layer having a thickness in the range of 300-1500 nm. In some embodiments, the photoactive layer is activated by the use of visible light. The photoactive layer is activated by the use of visible light. The dielectric properties of this layer are >10 7 It preferably has a high dielectric strength (V / m) and a dielectric constant (dielectric constant) of > 3. In some embodiments, the dielectric layer is selected from alumina, silica, hafnia, or a non-conductive polymer thin film.

[0135] Alternatively, at least the first dielectric layer, and preferably both dielectric layers, can be coated with an anti-fouling layer to help establish desired microdroplet / dispersion medium fluid / surface contact angles at various hypothetical electrowetting electrode locations, the anti-fouling layer intended to additionally prevent the contents of the microdroplets from adhering to the surface and being reduced as the microdroplets move through the chip.

[0136] For optimal performance, the antifouling layers should help establish a surface contact angle of the microdroplet / dispersion medium with the fluid / surface, which should be in the range of 50°-180° when measured at the air-liquid-surface three-point interface at 25°C. In some embodiments, these layers have a thickness of less than 10 nm and are typically monolayers. Alternatively, these layers can be composed of polymers of acrylate esters such as methyl methacrylate or its derivatives substituted with hydrophilic groups, e.g., alkoxysilyl groups. Either or both of the antifouling layers are hydrophobic to ensure optimal performance. In some embodiments, an intervening layer of silica less than 20 nm thick can be inserted between the antifouling coating and the dielectric layer to provide a chemically compatible bridge.

[0137] The first and second dielectric layers, and thus the first and second walls, define a microfluidic space, which is at least 10 μm wide, and preferably in the range of 20-180 μm wide, within which the microdroplets are contained. Before the microdroplets are contained, the microdroplets themselves preferably have a characteristic diameter, which is 10% larger, or alternatively 20% larger than the width of the microfluidic space. Thus, the microdroplets are subjected to compression upon entering the chip, leading to deformation of the spherical microdroplets, resulting in enhanced electrowetting performance, e.g., due to better microdroplet merging ability. In some examples, the first and second dielectric layers can be coated with a hydrophobic coating, such as fluorosilane.

[0138] In some embodiments, the microfluidic space includes one or more spacers to hold the first and second walls apart by a predetermined amount. Spacer options include beads or pillars, ridges generated from an intermediate resist layer, which are generated by photopatterning. Alternatively, deposition materials such as silicon oxide or silicon nitride can be used to create the spacers. Alternatively, layers of film can be used to form the spacer layer, including flexible plastic films with or without adhesive coatings. Various spacer geometries can be used to form narrow channels defined by straight lines of pillars, tapered channels, or partially closed channels. With careful design, these spacers can be used to assist in the deformation of microdroplets, after which microdroplet splitting can be performed and operations can be performed on the deformed microdroplets. Similarly, these spacers can be used to physically separate zones of the chip to prevent cross-contamination between droplet constituents and direct the flow of droplets when loading the chip under hydraulic pressure.

[0139] An A / C power supply attached to the conductor layer can be used to bias the first and second walls to provide a potential difference between them; suitably this potential difference is in the range of 0 to 50 volts. These oEWOD structures are typically used with electromagnetic radiation sources having wavelengths in the range of 400 to 850 nm, e.g. 550 nm, 620 nm, and 660 nm, and energies above the band gap of the photoactive layer. The photoactive layer is suitably activated at a virtual electrowetting electrode location, where the incident intensity of the radiation used is 0.005 to 0.1 Wcm. -2 The electromagnetic radiation source is in the range of 0.005 to 1 Wcm -2 or 0.005 Wcm -2 Super, 0.0075Wcm -2 Super, 0.01Wcm -2 Super, 0.025Wcm -2 Super, 0.05Wcm -2 or 0.075Wcm -2In some embodiments, the electromagnetic radiation source can be greater than 0.1 Wcm -2 Less than 0.075Wcm -2 Less than 0.05Wcm -2 Less than 0.025Wcm -2 Less than 0.01Wcm -2 Less than 0.0075Wcm -2 Less than 0.005Wcm -2 Less than or equal to 0.0025Wcm -2 can be made to a level less than

[0140] When the electromagnetic radiation sources are pixelated, they are powered either directly or indirectly using a reflective screen such as a digital micromirror device (DMD) illuminated by LEDs or other lamps. This allows highly complex patterns of virtual electrowetting electrode positions to be rapidly created and destroyed on the first dielectric layer, thereby enabling the microfluid to be precisely directed along essentially arbitrary virtual paths using tightly controlled electrowetting forces. Such electrowetting paths can be viewed as consisting of a continuum of virtual electrowetting electrode positions on the first dielectric layer.

[0141] The first and second dielectric layers may be composed of a single dielectric material or may be a composite of two or more dielectric materials, including, but not limited to, Al2O3 and SiO2.

[0142] A structure can be provided between the first and second dielectric layers. The structure between the first and second dielectric layers can be made of, but is not limited to, epoxy, polymer, silicon or glass, or a mixture thereof, and has planar, angled, curved or microstructured walls / surfaces. The structure between the first and second dielectric layers can be connected to the top and bottom composite walls to create a sealed microfluidic device and define channels and regions within the device. The structure can occupy the gap between the two composite walls. Alternatively or in addition, the conductors and dielectrics can be deposited on a molded substrate that already has walls.

[0143] The oEWOD device 100 shown in Figure 3B provides an alternative oEWOD configuration. As shown in Figure 3B, the oEWOD device comprises: a first composite wall 102 made of a first substrate 104, a conductor layer 106 on the substrate 104, a photoactive layer 108, and a first dielectric layer 110 on the photoactive layer 108, where the first substrate 104 can be made of glass, the first conductor layer 106 has a thickness in the range of 70-250 nm, the photoactive layer 108 is activated by electromagnetic radiation in the wavelength range of 400-850 nm on the conductor layer 106, and the photoactive layer 108 has a thickness in the range of 300-1500 nm. The first dielectric layer 110 is formed as a continuous layer having a thickness of less than 20 nm.

[0144] The device 100 shown in FIG. 3B also comprises a second composite wall 112, which comprises: a second substrate 114 and a second conductor layer 116 on the substrate 114, which may be made of glass. The second conductor layer may be transparent. The second conductor layer 116 may have a thickness in the range of 70-250 nm. A second dielectric layer 118 may be present on the second conductor layer 116, the second dielectric layer 118 having a thickness of less than 20 nm. Like the first dielectric layer, the second dielectric layer must be continuous, and the practical lower limit for its thickness is determined by manufacturing constraints, but may be between 1 nm and 20 nm. The exposed surfaces of the first continuous dielectric layer 110 and the second continuous dielectric layer 118 are spaced 20-180 μm apart to define a microfluidic space 121 configured to contain a microdroplet 122.

[0145] 1B shows an alternative embodiment of the oEWOD device 100 in which the spacer layer is not formed of a separate material, but is formed as part of the structure within the first (active) substrate 104. The secondary layers of the oEWOD device, formed of the first conductor layer 106, the photoactive layer 108, the first dielectric layer 110, the intermediate bonding layer 124, and the hydrophobic layer 126, can partially or completely cover the walls of the spacer structure. An additional embodiment is an alternative configuration of the device 100 in which the spacer layer is formed by structuring the second (passive) substrate 114.

[0146] In some cases, spacers can be formed by structuring the first and / or second substrates 110, 114 together, or by using a combination of structures in the first and / or second substrates 104, 114 and an insert material such as the channel walls 120, as shown in FIG. 3A.

[0147] As shown in FIG. 3B, incident light 130 can be used to provide a light sprite pattern 131 where the incident light 130 illuminates a portion of the photoactive layer 108 to hold the microdroplets 122 in a stationary position within the microfluidic space 121. An oil carrier phase 134 can be provided to the microdroplets 122 through holes 136 in the device to replenish the macronutrients and macronutrients to keep the contents within the microdroplets 122, such as one or more cells, alive and healthy. In some cases, the oil phase 134 can provide macronutrients, one or more media, and contents for cell growth, survival, and / or proliferation.

[0148] Various additional aspects and embodiments of the present invention will be apparent to those skilled in the art in view of this disclosure.

[0149] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components with the other or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually set forth herein.

[0150] Unless otherwise indicated by context, the feature descriptions and definitions set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0151] Furthermore, while the present invention has been described by way of example with reference to certain embodiments, it will be understood by those skilled in the art that the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the present invention as defined in the appended claims.

Claims

1. An apparatus comprising a microfluidic device and an optical imaging device, wherein the microfluidic device comprises a microfluidic space configured to contain a plurality of microdroplets, a first light source for illuminating the microfluidic space, a voltage source for applying a voltage to the microfluidic space to generate an electric field between both ends of the microfluidic space, and a first modulator configured to generate a waveform signal to modulate the electric field applied between both ends of the fluid space, the first modulator is configured to switch between an active state and a non-active state, in the active state, an electric field is applied between both ends of the microfluidic space, and in the non-active state, no electric field is applied between both ends of the microfluidic space, the first modulator is further configured to directly or indirectly control the first light source, whereby, during the active state, a portion of the light from the first light source is supplied across the entire microfluidic space to hold the plurality of microdroplets in the presence of the electric field, the optical imaging device generates an image of at least a subset of the microdroplets, the first modulator is further configured to control the optical imaging device, whereby, during the non-active state, an image of at least a subset of the microdroplets is generated.

2. The apparatus according to claim 1, wherein the first modulator is further configured to directly or indirectly control the first light source, whereby the first light source provides imaging illumination for imaging at least a subset of the microdroplets during the non-active state.

3. The apparatus according to claim 1, wherein the image is a fluorescence image, or a fluorescence resonance energy transfer image, or a bright field image, or a chemiluminescence image.

4. The apparatus according to claim 1, wherein the waveform signal generated by the first modulator is a square wave.

5. The apparatus according to claim 1, wherein the microfluidic device is in the active state, the non-active state, and / or an additional state for equal time lengths.

6. The apparatus according to claim 1, wherein the microfluidic device is in the active state for 90% of one period and in the non-active state for 10% of the one period.

7. The apparatus according to claim 1, wherein the microfluidic device is in the active state for 10% of one period and in the non-active state for 90% of the one period.

8. The apparatus according to claim 1, wherein the waveform signal has a frequency range of 0.5 to 5000 Hz.

9. The apparatus according to claim 1, wherein at least one of the micro-droplets contains a biological entity, and the biological entity is a cell, a virus, or a protein.

10. The apparatus according to claim 1, wherein at least one of the micro-droplets contains a fluorescent entity.

11. The apparatus according to claim 1, wherein the first modulator is a function generator, a digital switch, or an analog switch.

12. The apparatus according to claim 1, further comprising a second light source.

13. The apparatus according to claim 12, further comprising a filter, wherein the filter filters at least a portion of the light from the first light source and / or the second light source.

14. A controller is provided, and the controller is configured to control the filter, whereby the filter can provide imaging illumination provided from the first light source and / or the second light source over the entire microfluidic space during the non-active state. The apparatus according to claim 13.

15. The apparatus according to claim 12, further comprising a second modulator configured to generate a second waveform signal and modulate the light from the second light source.

16. The first modulator or the second modulator is further configured to control the second light source, whereby the second light source is configured to apply imaging illumination to at least a subset of the microdroplets during the inactive state. The apparatus according to claim 12.

17. The apparatus according to claim 1, wherein the microfluidic device is in the active state before the first modulator switches between the active state and the inactive state.

18. The apparatus according to claim 1, wherein the microfluidic device is a dielectrophoresis (DEP) device, or an optoelectronic tweezer (OET) device, or an optical tweezer device, or a dielectrophoresis (DEP) device.

19. The apparatus according to claim 1, wherein the optical imaging device further comprises a detector configured to detect an optical signal from at least a subset of the microdroplets.

20. The apparatus according to claim 1, wherein the first light source and / or the second light source is an LED, or a laser, or a lamp.

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