Digital microfluidic (DMF) systems, DMF cartridges, and methods involving integrated fiber optic sensing

Abstract

A cartridge for use with an instrument for performing fluid measurements, the cartridge comprising: a digital microfluidic substrate having a plurality of electrowetting electrodes operative to perform droplet operations on liquid droplets within a droplet operations gap; a top plate separated from the digital microfluidic substrate to form the droplet operations gap and having an opening for injecting liquid into the droplet operations gap; and a fiber assembly including an optical fiber probe having a sensing end that extends into the droplet operations gap and is positioned adjacent to one or more electrowetting electrodes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Patent Application No. 62 / 964,424, filed January 22, 2020, which is incorporated herein by reference. [Technical Field]

[0002] The present disclosure relates to digital microfluidic (DMF) systems, DMF cartridges, and methods of using integrated fiber optic sensing. [Background technology]

[0003] Digital microfluidic systems are useful for handling and manipulating droplets for various lab-on-a-chip applications. Optical sensing techniques are commonly used in digital microfluidic systems, for example, for the detection of analytes within droplets. Simple fiber optic sensors have been described for use in detecting light in digital microfluidic systems. However, there is a need in the art to enhance fiber optic sensing capabilities for performing complex analyses using digital microfluidics. Summary of the Invention [Problem to be solved by the invention]

[0004] The present disclosure provides cartridges for use with instruments for performing fluid measurements, instruments for operating the cartridges, and methods for making and using the cartridges. The present disclosure also includes fiber assemblies useful for assembling the cartridges of the present disclosure. [Means for solving the problem]

[0005] In one embodiment, the cartridge includes a digital microfluidic cartridge having a plurality of electrowetting electrodes operative to perform droplet operations on liquid droplets within the droplet operations gap. The cartridge can also include a fiber optic probe that extends into the droplet operations gap proximate to a set of two or more electrowetting electrodes, such that droplets located on any of the sets of two or more electrodes can contact the fiber optic probe.

[0006] In another embodiment, a cartridge includes a digital microfluidic substrate including a plurality of electrowetting electrodes operative to perform droplet operations on droplets of liquid within a droplet operations gap, and a top plate separated from the digital microfluidic substrate to form a droplet operations gap and having an opening for injecting liquid into the droplet operations gap. The cartridge also includes a fiber assembly having a fiber optic probe with a sensing end that extends into the droplet operations gap and is positioned proximate to one or more electrowetting electrodes.

[0007] In some embodiments, a fiber optic probe penetrating the droplet manipulation gap is positioned adjacent to a set of two or more electrowetting electrodes, and droplets located on any of the two or more sets of electrodes can contact the fiber optic probe. The probe can include a ligand. Droplets within the cartridge can be controlled to contact the fiber optic probe by the electrowetting electrodes. Low viscosity oil or other filler material (e.g., filler fluid) can be used to fill the gap around the droplet.

[0008] The present disclosure also provides a method of performing an assay. The method can include providing a fiber optic probe containing a ligand; providing a droplet having a volume of less than about 1000 nL and containing an analyte potentially having affinity for the ligand; contacting the droplet with the end of the probe; and oscillating the droplet without removing the droplet from contact with the probe. In some cases, the oscillating range is about 0.5 to about 15 Hz, or about 4 to about 10 Hz. In some cases, the volume of the droplet is less than about 900 nL, or less than about 800 nL, or less than about 700 nL, or less than about 600 nL, or less than about 500 nL, or less than about 400 nL. In some cases, the volume of the droplet is 200 to 400 nL.

[0009] In some cases, the fiber optic probes include multiple ligands and the droplets include multiple analytes. In some cases, the method includes providing multiple fiber optic probes and multiple droplets, contacting each of the multiple droplets with a corresponding fiber optic probe, and vibrating each of the multiple droplets in contact with the corresponding fiber optic probe. In another embodiment, a single droplet can be transported from one probe to another for a series of assays.

[0010] In some cases, the vibration is mediated by electrowetting electrodes. In some cases, the vibration is mediated by electrowetting electrodes within a droplet operations gap of a droplet actuator or electrowetting cartridge. In some cases, the fiber optic probe is aligned so that its end is adjacent to the droplet operations electrode. In some cases, the fiber optic probe is aligned so that its end is close to an edge of the droplet operations electrode. In some cases, the vibration is approximately perpendicular to a line extending along the length of the fiber optic probe. In some cases, the vibration is approximately coincident with a line extending along the length of the fiber optic probe. In some cases, the vibration is multidirectional. In some cases, the vibration is multidirectional in a plane parallel to a line extending along the length of the fiber optic probe. In some cases, the vibration is implemented using elongated droplets, e.g., 2X, 3X, 4X, or longer, where X is the number of electrowetting electrodes used to elongate the droplets.

[0011] In some cases, the assay is selected from the following: molecular library screening assays, binding kinetics assays, affinity determination assays, binding site mapping assays, competition analysis assays, specificity determination assays, and antibody binding characterization, and combinations of the above. In some cases, the assay generates a response curve. [Brief explanation of the drawings]

[0012] Having described the subject matter above in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale. [Figure 1] FIG. 1 illustrates a block diagram of an embodiment of a DMF system including integrated fiber optic sensing. [Figure 2]Figure 2A illustrates an embodiment of a DMF cartridge associated with a DMF device and also illustrates an embodiment of a fiber optic interface. Figure 2B illustrates an embodiment of a DMF cartridge associated with a DMF device and also illustrates an embodiment of a fiber optic interface. [Figure 3] FIG. 12 is a side view of a portion of an embodiment of a DMF cartridge of a DMF system with a fiber optic probe introduced into the gap from the top. [Figure 4] FIG. 12 is a side view of a portion of an embodiment of a DMF cartridge of a DMF system with a fiber optic probe introduced into the gap from below. [Figure 5] FIG. 12 is a side view of a portion of an embodiment of a DMF cartridge of a DMF system with a fiber optic probe introduced into the gap from the side. [Figure 6] FIG. 10 is a side view of a portion of an embodiment of a DMF cartridge of a DMF system, with a fiber optic probe introduced into the gap and optical sensing operation performed in reflection mode. [Figure 7A] 1 illustrates a side view of a portion of an embodiment of a DMF cartridge of a DMF system in which a sensor surface is provided at the tip of a fiber optic probe. [Figure 7B] 1 illustrates a side view of a portion of an embodiment of a DMF cartridge of a DMF system in which a sensor surface is provided at the tip of a fiber optic probe. [Figure 8] 1 illustrates a side view of a portion of an embodiment of a DMF cartridge of a DMF system in which one or more optical elements are provided at the tip of a fiber optic probe. [Figure 9] FIG. 1 illustrates a side view of an embodiment of a DMF cartridge of a DMF system including both a primary optical measurement device and a secondary optical measurement device. [Figure 10] 1 illustrates a flow diagram of an example method for using a DMF system and / or DMF cartridge that includes integrated fiber optic sensing. [Figure 11]1 illustrates an example of a sample droplet oscillating back and forth on a droplet manipulation electrode and in contact with a fiber optic probe. [Figure 12A] FIG. 1 illustrates a top perspective view of an exemplary instantiation of a DMF cartridge with integrated fiber sensing. [Figure 12B] FIG. 1 illustrates a bottom perspective view of an exemplary instantiation of a DMF cartridge with integrated fiber sensing. [Figure 13A] 1 illustrates an exploded top view of an exemplary instantiation of a DMF cartridge with integrated fiber sensing. [Figure 13B] 1 illustrates an exploded view from the bottom side of an exemplary instantiation of a DMF cartridge with integrated fiber sensing. [Figure 14A] 14A and 14B illustrate various embodiments of the fiber assembly of a DMF cartridge. [Figure 14B] 1 illustrates various embodiments of a fiber assembly for a DMF cartridge. [Figure 15] An example of a fiber sensing prototyping process is shown. [Figure 16] 1 shows electron micrographs of example nanoparticle sensor surfaces. [Figure 17] 17A and 17B show plots of exemplary test results for determining the affinity of Protein A and IgG using fiber optic-based surface plasmon resonance detection in a DMF device. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to a digital microfluidic (DMF) system, a DMF cartridge, and a method involving integrated fiber optic sensing.

[0014] The DMF system and DMF cartridge can include, for example, a fiber optic probe inserted directly into the droplet operations gap of the DMF cartridge. The DMF system can include, for example, a DMF cartridge, one or more illumination sources, one or more optical measurement devices, and a controller. The DMF cartridge can further include a fiber optic probe inserted directly into the droplet operations gap of the DMF cartridge.

[0015] In some embodiments, the DMF systems, DMF cartridges, and methods provide a fiber optic probe whose tip is inserted directly into the droplet operations gap of the DMF cartridge.

[0016] In some embodiments, the DMF systems, DMF cartridges, and methods may provide for a fiber optic probe to be inserted through the top, bottom, and / or sides of the DMF cartridge.

[0017] In some embodiments, the present disclosure provides a single fiber optic probe that can act as a conduit for light to and from a sensing region within a droplet operations gap of a DMF cartridge.

[0018] Droplets used in the assays of the present disclosure can be much smaller in volume than many conventional assays, for example, less than about 1000 nL, or less than about 900 nL, or less than about 800 nL, or less than about 700 nL, or less than 600 nL, or less than about 500 nL, or less than about 400 nL, or less than about 300 nL, or less than about 200 nL, or less than about 100 nL.

[0019] Droplets used in the SPR assays of the present disclosure can be much smaller in volume than many conventional SPR assays, for example, less than about 1000 nL, or less than about 900 nL, or less than about 800 nL, or less than about 700 nL, or less than 600 nL, or less than about 500 nL, or less than about 400 nL, or less than about 300 nL, or less than about 200 nL, or less than about 100 nL.

[0020] The droplets used in the SPR biomolecular interaction assays of the present disclosure can be much smaller in volume than many conventional SPR biomolecular interaction assays, for example, less than about 1000 nL, or less than about 900 nL, or less than about 800 nL, or less than about 700 nL, or less than 600 nL, or less than about 500 nL, or less than about 400 nL, or less than about 300 nL, or less than about 200 nL, or less than about 100 nL. Examples of SPR biomolecular interaction assays include molecular library screening assays, binding kinetics assays, affinity determination assays, binding site mapping assays, competition analysis assays, specificity determination assays, and antibody binding characterization.

[0021] In some embodiments, the present disclosure may provide one or more illumination sources for providing excitation light to a fiber optic probe and one or more optical measurement devices for receiving and processing emission light from the same fiber optic probe.

[0022] In some embodiments, the present disclosure may provide one or more illumination sources for providing excitation light using free-space optics and one or more fiber optic probes for receiving and processing the emission light. Similarly, in some embodiments, the present disclosure may provide one or more illumination sources for providing excitation light coupled to a fiber optic probe and one or more optical measurement devices for receiving and processing the emission light using free-space optics.

[0023] In some embodiments, the present disclosure provides a fiber optic probe inserted directly into the droplet operations gap of a DMF cartridge, where optical sensing operations can occur in reflection mode, transmission mode, or both reflection and transmission modes.

[0024] In some embodiments, the present disclosure provides other components and / or elements (eg, sensing layer, optical element) at the tip of the fiber optic probe within the droplet operations gap of the DMF cartridge.

[0025] Additionally, the present disclosure provides methods of using DMF systems and DMF cartridges when equipped with integrated fiber optic sensing.

[0026] FIG. 1 is a block diagram of an example DMF system 100 including integrated fiber optic sensing. The DMF system 100 can be, for example, a plasmon resonance (PR) system and / or a localized surface plasmon resonance (LSPR) system for the analysis of analytes. Analysis can refer, for example, to the detection, identification, quantification, or measurement of the analyte and / or its interactions with other substances, such as binding kinetics and thermodynamics. Exemplary analytes can include, but are not limited to, small molecules, proteins, peptides, atoms, and ions. For example, the DMF system 100 can be used to measure the binding kinetics of a ligand to a macromolecule, such as a receptor.

[0027] The DMF system 100 can also be configured as a reflectance interferometric sensor, such as biolayer interferometry (BLI) or monochromatic reflectometry (SCORE). These interferometric sensors use techniques such as chemical vapor deposition (CVD) or physical vapor deposition (PVD) to deposit common materials used for thin film coatings, such as zinc sulfide, titanium dioxide, magnesium fluoride, or silicon dioxide, onto the tip of an optical fiber, with an ultimate metal layer of a plasmonic metal, such as gold. For example, a ligand can be immobilized on the tip of the fiber. The presence or absence of an analyte that interacts with the ligand causes a wavelength shift or a change in the interference pattern of the light reflected from the sensor. Analysis can refer, for example, to the detection, identification, quantification, or measurement of an analyte and / or its interaction with other substances, such as binding kinetics. Exemplary analytes can include, but are not limited to, small molecules, proteins, peptides, atoms, and ions. For example, the DMF system 100 can be used to measure the binding kinetics of a ligand to a macromolecule, such as a receptor. In one analytical embodiment, the light reflected by a BLI fiber sensor exposed to an analyte can be compared to the light reflected by a BLI fiber sensor not exposed to an analyte.

[0028] The DMF system 100 can also be configured as a fluorescence measurement system. The sensor in this case can be an unmodified sensor, or a fluorophore can be immobilized on the tip of the fiber. In another example, the tip of the fiber can be coated with a coating that modifies the luminescence properties of the fluorophore by quenching or by energy transfer, such as Förster resonance energy transfer. One or more fluorophores can be used simultaneously in combination with one another. Such a setup allows for the measurement of fluorescence intensity and / or lifetime. The fluorescent sample can be either in solution or captured at the tip of the optical fiber. The fluorescence can be intrinsic to the sample or can be generated using a fluorescent substance, such as a small molecule, quantum dot, or the like, that binds to or otherwise interacts with the analyte. Analysis can refer, for example, to the detection, identification, quantification, or measurement of the analyte and / or its interaction with other substances, such as binding kinetics. Exemplary analytes can include, but are not limited to, small molecules, proteins, peptides, atoms, and ions. For example, the DMF system 100 can be used to measure the binding kinetics of a ligand to a macromolecule, such as a receptor.

[0029] The DMF system 100 features the integration of fiber-optic sensors with digital microfluidics. For example, the DMF system 100 may include a DMF cartridge 110. The DMF cartridge 110 may be a droplet actuator device that provides DMF functions for generally performing droplet operations, such as droplet merging, splitting, dispensing, and dilution. One application of these DMF functions is sample preparation. However, the DMF functions may also be used for other processes, such as waste removal or flushing between runs. The DMF cartridge 110 may include an onboard sensing region 158 that may be coupled to a fiber-optic probe 130. In the DMF cartridge 110, a single fiber-optic probe 130 may serve as a conduit for light to and from the onboard sensing region 158. The fiber-optic probe 130 may include a single optical fiber or a bundle of multiple optical fibers. Details of the DMF cartridge 110, the onboard sensing region 158, and the fiber-optic probe 130 are shown and described below with reference to Figures 3-9.

[0030] In various embodiments, the sensing end of the fiber optic probe 130 is aligned so that it is adjacent to the droplet operations electrode 120. In various embodiments, the end of the fiber optic probe 130 is aligned so that it is close to the edge of the droplet operations electrode 120. In various embodiments, the end of the fiber optic probe 130 is aligned so that it is in line with the edge of the droplet operations electrode 120.

[0031] DMF system 100 may further include a controller 150, a DMF interface 152, an illumination source 154, and an optical measurement device 156. Controller 150 may be electrically coupled to various hardware components of DMF system 100, such as DMF cartridge 110, illumination source 154, and optical measurement device 156. For example, controller 150 may be electrically coupled to DMF cartridge 110 via DMF interface 152, where DMF interface 152 may be, for example, a pluggable interface for mechanically and electrically connecting to DMF cartridge 110. DMF cartridge 110, controller 150, DMF interface 152, illumination source 154, and optical measurement device 156 together form DMF apparatus 105.

[0032] Controller 150 can be, for example, a general-purpose computer, a special-purpose computer, a personal computer, a microprocessor, or other programmable data processing device. Controller 150 provides processing capabilities, such as storing, interpreting, and / or executing software instructions, and helps control the overall operation of DMF system 100. Controller 150 can be configured and programmed to control data and / or power aspects of these devices. For example, controller 150 controls droplet operations within DMF cartridge 110 by activating / deactivating electrodes. In general, controller 150 can be used for any function of DMF system 100. For example, controller 150 can be used to authenticate DMF cartridge 110 in a manner similar to how printer manufacturers check their own brand of ink cartridges; controller 150 can be used to verify that DMF cartridge 110 has not passed its expiration date; controller 150 can be used to confirm the cleanliness of DMF cartridge 110 by executing protocols for that purpose; and so on.

[0033] The controller 150 may include one or more input interfaces that connect the processing unit to input devices. The input interfaces allow a user of the DMF system 100 to communicate commands to the processor. One exemplary such command is the execution of program code. The input devices may take the form of a keyboard, a mouse device, a voice-activated system, a touchscreen, and / or other suitable devices known to those skilled in the art.

[0034] In some embodiments, controller 150 may include one or more output interfaces, such as a graphical user interface (GUI), that connect the processing unit to an output device, allowing DMF system 100 to communicate the results of various processing operations, such as experimental results, to a user. Software instructions may be stored in a memory unit of controller 150, which may include conventional semiconductor random access memory (RAM) or other forms of memory known in the art, and / or the software instructions may be stored in the form of program code on one or more computer-readable storage media, such as a hard drive, USB drive, read / write CD-ROM, DVD, tape drive, flash drive, optical drive, etc. These instructions may be executed in response to a user's interaction with DMF system 100 via an input device.

[0035] In some embodiments, DMF cartridge 110 can include capacitive feedback sensing, i.e., a signal from a capacitive sensor that can detect the position and volume of a droplet. Furthermore, in other embodiments, instead of or in addition to capacitive feedback sensing, DMF cartridge 110 can include a camera that provides optical measurements of the droplet's position and volume, which can trigger controller 150 to reroute the droplet to the appropriate location.

[0036] In some embodiments, the DMF cartridge 110 can include a heating zone (not shown). It will be appreciated that various sample preparation steps and assays benefit from temperature control. As described in U.S. Pat. No. 8,658,111, incorporated herein by reference, thermal control can generally be achieved in three ways: (1) thermal control of the entire DMF cartridge 110; (2) thermal control of a region of the DMF cartridge 110 using a heater in contact with or proximate to the controlled region; and (3) thermal control of a region of the DMF cartridge 110 or the entire DMF cartridge 110 using a heater integrated into the DMF cartridge 110 (e.g., within a substrate containing the electrode paths or array and / or within a top substrate of the DMF cartridge 110, if present). Combinations of the above approaches are also possible. Examples of heating techniques include heater bars attached to an instrument adjacent to the cartridge and heaters integrated into the cartridge itself.

[0037] In the integrated heater approach, temperature zones can be created and controlled using thermal control elements integrated directly into the DMF cartridge 110. Thermal control elements (heating and / or cooling) can be integrated into the lower and / or upper substrates (if present) of the DMF cartridge 110, as well as the lower and / or upper surfaces of either substrate, or integrated within the structure of either substrate, or located between the substrates. In some cases, the thermal control elements can be electronically coupled to and controlled by the controller 150. Thermal zones can be created using separate heating elements and thus function as separate thermal zones within the DMF cartridge 110. This arrangement allows multiple steps in an analysis, such as sample preparation and thermal cycling, requiring different temperatures to be performed simultaneously at different temperatures in different thermal zones of the DMF cartridge 110. For example, using droplet manipulation via electrowetting, droplets can be physically transported or shuttled between thermal zones of different fixed temperatures to perform thermal cycling for amplification reactions.

[0038] In one embodiment, the heaters in the thermal zones can be formed using thin conductive films. Examples of suitable thin films include Pt heater wire and transparent indium tin oxide (ITO). In one embodiment, metal (e.g., copper) vias in the substrate of the DMF cartridge 110 are used.

[0039] Temperature regulation thermocouples may also be used to control the temperature of the thermal zones. Temperature probes may be used to measure the temperature of the thermal zones and provide the temperature measurements to the controller, allowing the controller to precisely control the temperature of the associated thermal zone.

[0040] The DMF device 105 can be connected to a network. For example, the controller 150 can communicate with a networked computer 160 via a network 162. The networked computer 160 can be, for example, any centralized server or cloud server. The network 162 can be, for example, a local area network (LAN) or a wide area network (WAN) for connecting to the Internet.

[0041] In the DMF system 100, the illumination source 154 and optical measurement device 156 can be positioned relative to the on-board sensing region 158 of the DMF cartridge 110 and / or the fiber optic probe 130. The illumination source 154 can be a light source in the visible range (wavelengths 400-800 nm), such as, but not limited to, a white light-emitting diode (LED), a halogen bulb, an arc lamp, an incandescent lamp, and a laser. The illumination source 154 is not limited to a white light source. The illumination source 154 can be any color light useful in the DMF system 100. The illumination source 154 provides excitation light 132 to the on-board sensing region 158 of the DMF cartridge 110.

[0042] The optical measurement device 156 can be, for example, any optical transducer device used to obtain light intensity readings. The optical measurement device 156 can be, for example, a charge-coupled device, a photodetector, a spectrometer, a photodiode array, a camera, or any combination thereof. Furthermore, the DMF system 100 is not limited to only one illumination source 154 and one optical measurement device 156. The DMF system 100 can include multiple illumination sources 154 and / or multiple optical measurement devices 156 to support any detection operations in the DMF system 100 and / or the DMF cartridge 110. The optical measurement device 156 receives and processes the emission light 134 from the onboard sensing region 158 of the DMF cartridge 110. Thus, the single fiber optic probe 130 of the DMF cartridge 110 can serve as a conduit for both the excitation light 132 to the onboard sensing region 158 and the emission light 134 from the onboard sensing region 158.

[0043] Components of the DMF system 100 and / or DMF device 105 can be optically coupled to and decoupled from the on-board sensing region 158 of the DMF cartridge 110 and / or the fiber optic probe 130. This optical coupling / decoupling can be, for example, a fiber optic connector, a fiber optic coupler, and / or a free-space optical coupler.

[0044] Conventional methods for measuring optical stimuli from DMF devices use free-space optics to capture the stimuli. The limitations of free-space optics in this context are primarily due to their relatively poor performance as a result of stray light and optical loss. While the performance degradation can often be overcome by using optical components such as lenses, filters, and other similar optical components, these components add cost to the system. Compared to conventional methods, the integration of fiber optic sensors with DMF offers several advantages. For example, advantages of DMF system 100 featuring the integration of fiber optic-based sensors with digital microfluidics may include, but are not limited to, enabling localized optical interrogation techniques, providing a low-cost method for achieving high optical performance (e.g., reduced component count due to low cost), limiting optical loss within the system for improved signal, and limiting the collection of stray light for low noise.

[0045] In the DMF system 100, the fiber optic probe 130 is integrated into the DMF cartridge 110 such that the tip of the fiber optic probe 130 resides directly in the droplet manipulation gap of the DMF cartridge 110 (see FIGS. 3-9). In this manner, the tip of the fiber optic probe 130 can directly interact with droplets being processed in the DMF cartridge 110. The tip of the fiber optic probe 130 thus forms an on-board sensing region 158, where the DMF cartridge 110 can transfer droplets to and from the on-board sensing region 158. The DMF system 100 and / or the DMF cartridge 110 are not limited to only one fiber optic probe 130 and one on-board sensing region 158. This is merely exemplary. The DMF system 100 and / or the DMF cartridge 110 may include any number of fiber optic probes 130 and / or on-board sensing regions 158. Additionally, one or more fiber optic probes 130 may be provided in the form of a fiber assembly 125 (see Figures 14A and 14B). Further details of an embodiment of a fiber optic probe 130 in the droplet operations gap of a DMF cartridge are shown and described below with respect to Figures 3-9.

[0046] 2A and 2B illustrate an embodiment of a DMF cartridge 110 associated with a DMF device 105 showing an example of an optical fiber interface. In this example, the DMF cartridge 110 can be designed to drop into a corresponding DMF device 105, such as the DMF device 105. In this example, the DMF device 105 includes a recessed area 164 for receiving the DMF cartridge 110. That is, the DMF cartridge 110 is sized to fit into the recessed area 164 of the DMF device 105. In this manner, the DMF cartridge 110 can be fluidically, optically, and / or electrically coupled to the DMF device 105. This embodiment is illustrative of various embodiments in which coupling the cartridge to the device automatically aligns the optical fiber elements of the cartridge with the optical fiber elements of the device. In one embodiment (not shown), alignment can be enhanced by posts or openings in the DMF fiber interface mating with corresponding posts or openings in the device, and various similar techniques that will be apparent to one skilled in the art.

[0047] For example, with regard to optical coupling, the fiber optic probe 130 of the DMF cartridge 110 can include a single optical fiber or a bundle of multiple optical fibers. Furthermore, the optical fiber can be multimode or single-mode, or a combination of the two (having multiple cores and / or cladding layers). The DMF cartridge 110 can include one or more interfaces to allow one or more optical fibers to be coupled to the DMF instrument 105. The optical interfaces can be, for example, fiber optic connectors, fiber optic couplers, and / or free-space optical couplers. For example, FIGS. 2A and 2B show that when the DMF cartridge 110 is loaded into the recessed region 164 of the DMF instrument 105, the outer end of the fiber optic probe 130 within the DMF cartridge 110 can be approximately aligned with, for example, one or more optical fibers 166 leading to the illumination source 154 and / or the optical measurement device 156. The system and cartridge can include alignment elements to ensure that the optical connections between the fiber optic elements of the instrument and the cartridge are optically coupled when the DMF cartridge is coupled to the instrument. For example, when DMF cartridge 110 is inserted into DMF device 105, DMF device 105 can automatically perform an alignment step to maximize coupling efficiency between the fibers in DMF cartridge 110 and the fibers in DMF device 105. It will be appreciated that a wide variety of mechanisms are possible for electrically and optically coupling the cartridge to the device.

[0048] 3, 4, and 5 show side views of an example of a portion of the DMF cartridge 110 of the DMF system 100 shown in FIG. 1, with fiber optic probes 130 introduced into the gap from the top, bottom, and side, respectively. The DMF cartridge 110 can include a lower substrate 112 and an upper substrate 114 separated by a droplet operations gap 116. The droplet operations gap 116 can be filled with a filler fluid, such as a gas or liquid, that is sufficiently immiscible with the droplets and does not substantially interfere with the desired analytical process. In one embodiment, the filler fluid is a low-viscosity oil, such as silicone oil or hexadecane. Additionally, an arrangement of droplet operations electrodes 120 (e.g., electrowetting electrodes) can be provided on the lower substrate 112. The DMF cartridge 110 can include optional lines or paths for the droplet operations electrodes 120.

[0049] In one embodiment, the lower substrate 112 can be made of a material that is substantially transparent to white light (or any colored light). For example, the lower substrate 112 can be formed of glass, plastic, or a class of polymers known as thermoplastic elastomers (TPEs). In another example, the lower substrate 112 can be a substantially transparent printed circuit board (PCB) or a PCB that includes holes or openings that allow light transmission. Similar to the lower substrate 112, the upper substrate 114 can be made of a material that is substantially transparent to white light (or any colored light). For example, the upper substrate 114 can be formed of glass, plastic, or TPE. Additionally, the inner surface of the upper substrate 114 can be coated with a conductive layer 118, such as a transparent conductive layer (e.g., indium tin oxide (ITO), poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS)), or other similar transparent or non-transparent (e.g., opaque) conductive coating. In other embodiments, not all regions of the DMF cartridge 110 need include a substantially transparent substrate and / or coating or layer. For example, the substrate and / or coating or layer may not be transparent, translucent, and / or opaque, except for the detection region. In some cases, such a substrate would be inappropriate for a free-space optical probe, which may require a transparent layer. This is an advantage of the present disclosure because it allows for the use of fiber optic probes that do not have transparency, thereby allowing for greater flexibility in the selection of substrates and manufacturing techniques.

[0050] The terms "top," "bottom," "over," "under," "in," and "on" are used throughout the specification to refer to the relative positions of components of a DMF cartridge, such as the relative positions of the upper and lower substrates of a DMF cartridge. It will be understood that a DMF cartridge will function regardless of its orientation in space.

[0051] In the DMF cartridge 110, the droplet operations gap 116 can be a space for processing any liquid of interest via droplet operations, such as, but not limited to, liquid reagents, buffers, sample fluids, etc. The height of the gap can be, for example, several hundred microns. The droplet operations electrodes 120 can be used to perform droplet operations via electrowetting. "Droplet operations" refers to any droplet operations on a digital microfluidic device or cartridge. Droplet operations can include, for example, loading a droplet into a digital microfluidic device, dispensing one or more droplets from a source droplet, splitting, separating, or dividing a droplet into two or more droplets, transporting a droplet from one location to another in any direction, merging or combining two or more droplets into a single droplet, droplet dilution, droplet mixing, droplet agitation, droplet deformation, holding a droplet in place, droplet incubation, droplet heating, droplet evaporation, droplet cooling, droplet discarding, droplet transport from the DMF cartridge 110, other droplet operations described herein, and / or any combination of the operations described above. Additionally, a temperature control element (not shown), such as a Peltier heat pump, can be used in combination with the DMF cartridge 110 to control the temperature of processes occurring in the droplet operations gap 116.

[0052] 3, 4, and 5 illustrate DMF cartridge 110 as manipulating droplets via electrowetting (e.g., using droplet manipulation electrodes 120), this is merely exemplary. In other embodiments, droplets can be handled and manipulated within DMF cartridge 110 via other methods, such as, but not limited to, optical methods, magnetic methods, thermocapillary methods, surface acoustic wave methods, and other electrical methods such as dielectrophoresis, and any combination thereof.

[0053] Referring now to FIG. 3, the DMF cartridge 110 of the DMF system 100 is shown, with a fiber optic probe 130 introduced into the droplet operations gap 116 from above (e.g., through the upper substrate 114, approximately perpendicular to the plane of the DMF cartridge 110). A sample droplet 140 resides within the droplet operations gap 116 and on a specific droplet operations electrode 120. The tip of the fiber optic probe 130 may interact directly within / with the sample droplet 140, thereby forming an on-board sensing region 158. The DMF cartridge 110 can be used to transfer droplets to and from the on-board sensing region 158 via droplet operations. Integrating the fiber optic probe 130 directly within the droplet operations gap 116 and in the path of the sample droplet 140 enables localized optical interrogation techniques. FIG. 3 shows excitation light 132 being delivered to the sample droplet 140 via the fiber optic probe 130. Additionally, emitted light 134 from sample droplet 140 exits through fiber optic probe 130. Again, fiber optic probe 130 may include a single optical fiber or a bundle of multiple optical fibers.

[0054] Referring now to FIG. 4, the DMF cartridge 110 of the DMF system 100 is shown with a fiber optic probe 130 introduced into the droplet operations gap 116 from below (e.g., through the lower substrate 112, approximately perpendicular to the plane of the DMF cartridge 110).

[0055] Referring now to FIG. 5 , the DMF cartridge 110 of the DMF system 100 is shown with a fiber optic probe 130 introduced into the droplet operations gap 116 from the side (e.g., between the lower substrate 112 and the upper substrate 114, approximately parallel to the plane of the DMF cartridge 110).

[0056] 6, which is a side view of an example DMF cartridge 110 of DMF system 100 with fiber optic probe 130 installed in droplet operations gap 116 and optical sensing operations performed in reflection mode. While FIG. 6 illustrates the configuration of fiber optic probe 130 shown in FIG. 5, the configurations of fiber optic probe 130 shown in FIGS. 4 and 5 are equally applicable for operation in reflection mode.

[0057] 6 shows excitation light 132 being delivered from illumination source 154 to sample droplet 140 via fiber optic probe 130. Furthermore, emission light 134 from sample droplet 140 exits fiber optic probe 130 to optical measurement device 156. This configuration of DMF system 100 and DMF cartridge 110 provides fiber optics integrated with the DMF cartridge in a manner that enables reflectance sensing of optical properties of a liquid (e.g., the optical properties of sample droplet 140). Fiber optic probe 130 is part of DMF cartridge 110, which also contains other components necessary for digital microfluidics.

[0058] Again, the illumination source 154 can be one or more light sources. The light sources can be monochromatic or polychromatic. These light sources include, but are not limited to, light-emitting diodes (LEDs), lasers, incandescent light sources, fluorescent light sources, or any combination thereof. The light sources can be modulated to allow for very rapid intensity adjustment. The light source can also include one or more sensors, such as a photodetector, to adjust brightness. The light source can also contain additional filters to ensure the quality of the incident light. Furthermore, the light source can be a combination of multiple individual light-emitting elements. These elements can be active at the same or different wavelengths.

[0059] Again, the optical measurement device 156 can be one or more optical sensors. The optical measurement device 156 can be, for example, a narrowband sensor or a broadband sensor. The optical measurement device 156 converts the optical signal into an electronic signal that can be further processed. The optical measurement device 156 can also use multiple sensors sensitive to different portions of the optical spectrum. This can be in the form of a spectrometer or multiple individual sensors sensitized to different portions of the electromagnetic spectrum. The optical measurement device 156 can contain additional optical filters necessary to ensure best performance. The optical measurement device 156 can include additional components that enable control of the illumination source 154 using closed-loop control, such as a correlator.

[0060] During operation, excitation light 132 from an illumination source 154 passes through a fiber optic probe 130. The fiber optic probe 130 directs the incident light to the droplet being investigated (e.g., sample droplet 140). Emission light 134 is reflected back from the droplet along the same core of the fiber or along a different core, depending on the system configuration. The fiber optic probe 130 can have a single or multiple cores. These cores can be, for example, single-mode or multimode (or any combination of the two). This reflected emission light 134 is coupled into an optical measurement device 156.

[0061] The configuration of the fiber optic probe 130 in the DMF cartridge 110 shown in Figures 3, 4 and 5, where the optical sensing operation is performed in reflection mode, allows for specific techniques for droplet characterization, such as, but not limited to: (1) Reflectance spectroscopy, such as infrared spectroscopy; (2) Variable path length spectroscopy (3) absorbance spectroscopy, such as that used in enzyme-linked immunosorbent assay (ELISA) readings and polymerase chain reaction (PCR) readings; (4) Raman spectroscopy, (5) photoelectron spectroscopy, such as laser-induced breakdown spectroscopy and atomic emission spectroscopy; (6) Fluorescence spectroscopy, such as used in ELISA readings and PCR readings; (7) Turbidity measurement, (8) time-resolved spectroscopy, such as photon correlation spectroscopy and fluorescence correlation spectroscopy; (9) Microscale thermophoresis, Enables the use of

[0062] FIG. 7A is a side view of an example DMF cartridge 110 of DMF system 100, in which sensor surface 142 is provided at the tip of fiber optic probe 130 within droplet operations gap 116.

[0063] Various techniques are known to be useful for fabricating sensors using optical fibers. In one embodiment, the present disclosure utilizes the technique described in Non-Patent Document 1 (Jeong, Hyeon-Ho & Erdene, Norov & Lee, Seung Ki & Jeong, Dae & Park, Jae-Hyoung. (2011) Fabrication of fiber-optic localized surface plasmon resonance sensor and its application to detect antibody-antigen reaction of interferon-gamma. Optical Engineering. 50. 124405-124405. 10.1117 / 1.3662418.). See also Non-Patent Document 2 (Proll, G., Markovic, G., Steinle, L., & Gauglitz, G. (2009). Reflectometric Interference Spectroscopy. Methods in Molecular Biology: Biosensors and Biodetection, 503, 167-178. doi: 10.1007 / 978-1-60327-567-5_8.). The entire disclosures of Jeonge et al. and Proll et al. are incorporated herein by reference.

[0064] The sensor surface 142 can be, for example, an optical layer capable of converting a property of the droplet or an analyte in the droplet into an optical response, such as the presence of the analyte, temperature, a change in temperature, a change in a property of the analyte or solution, etc.

[0065] In one example, the sensor surface 142 may use an interference filter, which may be deposited onto the fiber using vacuum deposition techniques, allowing measurement of binding to the surface using monochromatic intensity or phase measurements or polychromatic spectroscopic measurements.

[0066] An example of a technique for producing filters useful in the fiber optic probes of the present disclosure is described in Non-Patent Document 3 (Proll G., Markovic G., Steinle L., Gauglitz G. (2009) Reflectometric Interference Spectroscopy. In: Rasooly A., Herold KE (eds) Biosensors and Biodetection. Methods in Molecular Biology). TM , vol. 503. Humana Press), the entire disclosure of which is incorporated herein by reference.

[0067] In another example, the sensor surface 142 may be an interference filter or optical material that can be deposited using wet chemistry. The optical material may include, but is not limited to, metal nanoparticles. Properties such as surface plasmon resonance or surface-enhanced Raman spectroscopy may be used to analyze the droplets and / or analytes within the droplets. Other examples of the sensor surface 142 may include the deposition of nanostructured surfaces using lithographic techniques. Example materials include metals (e.g., plasmonic nanoparticles), glass (e.g., diffractive elements), and plastics (e.g., nanoimprinted diffractive elements). Additionally, the sensor surface 142 may be in the form of dielectric microspheres used to focus the excitation light 132 into a smaller area.

[0068] The optical sensor surface 142 may include a layer involved in binding of the analyte from solution to the surface for an enhanced signal. For example, this layer can be deposited on the optical sensor surface 142. This layer can also be used without the optical sensor surface 142 as a layer for concentrating the analyte at the tip of the fiber to enhance the optical signal from the analyte. In one example, the layer is a gel matrix that promotes an increase in binding sites. In another example, the layer is a porous material that promotes an increase in the surface concentration of the analyte. Various chemical enhancements for increasing binding sites (e.g., hydrophobic sites) or adding specific binding sites (e.g., antibodies / antigens or aptamers / analytes) are known to those skilled in the art. The porous material may, in some cases, be organic in nature, such as polymer brushes, gels, nanoparticles, or other high-surface-area structures. These organic porous media may be synthetic polymers such as polydimethylsiloxane, divinylbenzene, polyethylene glycol, etc. In another embodiment, the porous media may be biologically derived, such as cellulose, chitin, collagen, etc.

[0069] Alternatively, the porous media can be inorganic in nature, such as nanostructured carbon, silica, titania, etc. The porous media can be any combination of these materials and can take advantage of the unique chemical properties of the analytes to increase their surface concentration.

[0070] In another example, the sensor surface 142 can be an LSPR sensor layer functionalized with one or more capture molecules. The LSPR sensor layer can be composed of metal nanostructures and / or multilayer nanostructures in which one of the layers is metal. Examples of metals commonly used in LSPR sensors include gold, silver, platinum, palladium, and copper nanoparticles. In one example, the capture molecule is a ligand immobilized on the surface of the LSPR sensor layer. For example, a protein, antibody, antigen, or aptamer as the ligand can be attached to the metal nanoparticle sensor layer using carboxyl, NTA, or streptavidin surface attachment chemistries. In this example, the ligand is one of two binding partners, and the other binding partner is the target analyte 144 in the sample droplet 140.

[0071] In another example, the sensor surface 142 can be a pH-sensitive optical layer, such as fluorescein contained in a gel matrix. The optical properties of these pH-sensitive gels are related to the pH of the environment in which they occur. As a result, the optical signal readout can change based on the pH of the sample droplet 140.

[0072] In another example, the sensor surface 142 can even be an LSPR sensor coated with a heat-sensitive layer, such as a thermochromic coating, or a passivation layer, such as silicon oxide. The optical properties of these sensors are strongly correlated with the temperature of the environment in which they are placed. As a result, the optical signal can change as a function of the temperature of the sample droplet 140.

[0073] The configuration shown in FIG. 7A, where the sensor surface 142 resides at the tip of the fiber optic probe 130 within the DMF cartridge 110, allows for certain characterization techniques, including, but not limited to: (1) Monochromatic reflectometry, (2) Reflectance interferometry, such as biolayer interferometry and monochromatic reflectometry (SCORE); (3) Surface-enhanced Raman spectroscopy, (4) Surface plasmon resonance, (5) Diffraction optical measurements, (6) solid-phase microextraction (combined with optical readout); (7) Temperature measurement; This makes it possible.

[0074] 7B is a side view of an example DMF cartridge 110 of DMF system 100 that includes a nanoparticle sensor surface 143 at the tip of fiber optic probe 130. Details of an exemplary nanoparticle sensor surface 143 are provided below with reference to FIG.

[0075] 8, which is a side view of an embodiment of the DMF cartridge 110 of the DMF system 100, one or more optical elements 148 are provided at the tip of the fiber optic probe 130 within the droplet operations gap 116. The one or more optical elements 148 may be provided alone or in addition to the sensor surface 142 shown in FIG. 7. The one or more optical elements 148 may be used to more effectively couple light into and / or out of the sample droplet 140 and / or sensor surface 142. The one or more optical elements 148 may include, but are not limited to: (1) Tapered optical fibers, such as conical tapers; (2) U-shaped optical fiber, (3) Lenses such as hemispherical lenses, ball lenses, GRIN lenses, and aspherical lenses; (4) Prisms such as ATR prisms and Kretschmann prisms; (5) Angled polishes such as side polishes and wedge polishes; (6) diffusion factors; (7) Reflective cavities such as Fabry-Perot etalons and transflection cavities; may include:

[0076] 9 is a side view of an embodiment of a DMF cartridge 110 of a DMF system 100 configured substantially similarly to that shown in FIG. 6, except for the addition of a second illumination source 154′ and a second optical measurement device 156′. In this embodiment, the illumination source 154′ and / or the optical measurement device 156′ may be used independently or in combination with a fiber optic probe 130 coupled to the illumination source 154 and the optical measurement device 156.

[0077] Similar to optical measurement device 156, optical measurement device 156' may be in the form of an optical measurement device such as a photodetector, camera, spectrometer, or hyperspectral imager. Optical measurement device 156' may perform time-resolved measurements. Additionally, input from optical measurement device 156' may be used to control illumination source 154 and / or illumination source 154'.

[0078] The presence of the second illumination source 154' and the second optical measurement device 156' enables various modes of operation. In one example, using the first illumination source 154 and the first optical measurement device 156, the optical sensing operation may be performed entirely in a reflection mode via the fiber optic probe 130, i.e., using the fiber optic probe 130 for both illumination and collection. In another example, using the first illumination source 154 and the second optical measurement device 156', the optical sensing operation may be performed in a transmission mode, i.e., using the fiber optic probe 130 and the first illumination source 154 to illuminate, but using the external second optical measurement device 156' to collect. In yet another example, using the second illumination source 154' and the first optical measurement device 156, the optical sensing operation may again be performed in a transmission mode, i.e., using the external second illumination source 154' to illuminate, but using the fiber optic probe 130 and the first optical measurement device 156 to collect.

[0079] 10, there is shown a flow diagram of an example method 200 for using the DMF system 100 and / or DMF cartridge 110 with integrated fiber optic sensing. The method 200 may include, but is not limited to, the following steps:

[0080] In step 210, a DMF system and / or DMF cartridge including integrated RI sensing is prepared. For example, a DMF system 100 and / or DMF cartridge 110 including a fiber optic probe 130 integrated in a droplet operations gap 116 of the DMF cartridge 110 is prepared, for example, as shown in FIGS.

[0081] The droplets to be processed are transported to the sensing region of the DMF cartridge in step 215. For example, referring now to Figures 3-9, the sample droplets 140 to be processed can be transported using droplet operations to the on-board sensing region 158 of the DMF cartridge 110, where the on-board sensing region 158 is at the tip of the fiber optic probe 130.

[0082] In step 220, an optical sensing operation is performed within the DMF cartridge using integrated fiber optic sensing. For example, referring now to FIGS. 6-9, an optical sensing operation is performed in the DMF cartridge 110 using an integrated fiber optic probe 130 in the droplet operations gap 116, illumination source 154, and optical measurement device 156 of the DMF cartridge 110. During operation, excitation light 132 from the illumination source 154 passes through the fiber optic probe 130 toward and into the sample droplet 140 being interrogated. Emission light 134 is then reflected from the sample droplet 140 along the fiber optic probe 130 back to the optical measurement device 156, which captures an optical reading for processing. In one example, the optical sensing operation within the DMF cartridge 110 can occur in a reflection mode, as shown in FIGS. 6, 7, and 8. In another embodiment, optical sensing operation within DMF cartridge 110 can occur in a reflective mode, a transmissive mode, or both a reflective and a transmissive mode, as shown and described in FIG.

[0083] In certain embodiments, it may be useful to vibrate the droplet while it is in contact with the sensor. For example, in one embodiment, the first and second reaction electrodes are alternately activated to induce vibration of the droplet between the first and second reaction electrodes, inducing movement of the droplet relative to the SPR sensor surface. In one embodiment, the vibration of the droplet between the first and second reaction electrodes is linear. In one embodiment, the vibration is in the range of about 0.5 to about 15 Hz. In another embodiment, the vibration is in the range of about 4 to about 10 Hz.

[0084] In another embodiment, the SPR sensor surface is disposed between three or more reaction electrodes, and the three or more reaction electrodes are alternately activated to induce oscillation of the droplet between the three or more reaction electrodes and induce movement of the droplet relative to the SPR sensor surface. In another embodiment, the oscillation of the droplet between the three or more reaction electrodes is circular.

[0085] In one embodiment, the set of electrodes is positioned relative to the sensing tip of the sensor such that vibrations caused by moving the droplet from one electrode to the next in the set keep the droplet in contact with the sensing tip of the sensor.

[0086] FIG. 11 shows an example of a 2X sample droplet 140 oscillating back and forth on the droplet operations electrode 120 and in contact with the fiber optic probe 130. In FIG. 11, the droplet is moving generally along the path of the droplet operations electrode 120 in a direction perpendicular to a line along the length of the fiber optic probe 130 (not shown). It will be understood that other types of movement, such as movement in a direction coinciding with a line along the length of the fiber optic probe 130 (not shown), and movement in various directions, are possible within the scope of this disclosure. The oscillation may also include elongation and contraction of the droplet. Furthermore, while the image shows a 2X elongation, it will be understood that the elongation may be 3X, 4X, or more (X = the number of activated electrodes used to elongate the droplet).

[0087] 12A and 12B show top and bottom perspective views, respectively, of an exemplary instantiation of a DMF cartridge 110 including integrated fiber sensing. For example, FIGS. 12A and 12B show an example of the top substrate 114, fiber assembly 125, and optical fiber core 146 of the DMF cartridge 110.

[0088] The fiber assembly 125 may be configured with a set of fibers extending from a first edge of the assembly and a set of fibers extending from a second edge of the assembly. When the fiber assembly 125 is assembled with the DMF cartridge 110, the droplet operations gap 116 between the upper substrate 114 and the lower substrate 112 is sealed, and the fiber optic probe extends into the gap. Ideally, the fibers extend sufficiently close to one or more electrowetting electrodes 120 that droplets interacting with the one or more electrowetting electrodes 120 contact the end of the fiber optic probe. For assays requiring droplet oscillation, the set of electrodes may be positioned relative to the sensing tip of the probe so that oscillation, caused by moving the droplet from one electrode in the set to the next, keeps the droplet in contact with the sensing tip of the sensor.

[0089] Figures 13A and 13B show top and bottom exploded views, respectively, of an embodiment of a DMF cartridge 110. The top substrate 114 includes openings for depositing buffers, reagents, and samples into the DMF cartridge 110. The openings are spaced apart to allow the use of a multichannel pipettor to introduce reagents, buffers, and samples into the DMF cartridge 110. The footprint of the DMF cartridge 110 can be optimized for use with instruments used with well plates. The bottom substrate 112 is a printed circuit board with electrodes patterned therein to control droplet actuation. The bottom substrate 112 has a hydrophobic coating to optimize the electrowetting forces of the DMF. The top substrate 114 is made from an injection-molded polymer such as polycarbonate, acrylic, or cyclic olefin copolymer. A conductive coating such as indium tin oxide is applied to the top substrate 114 to enhance its conductivity. The top substrate 114 is sealed to the bottom substrate 112 using an elastomeric adhesive such as silicone. The fibers are introduced into the cartridge through V-grooves in the top substrate 114, which aid in fiber alignment. The elastomeric adhesive also seals around the fibers as they enter the gap between the top substrate 114 and the bottom substrate 112. The fiber assembly 125 is secured to the top substrate 114 using either adhesives, fasteners, ultrasonic welding, heat staking, or other fastening methods.

[0090] 14A and 14B show various views of an embodiment of a fiber assembly 125 of the DMF cartridge 110. FIG. 14A shows an exploded view and a cross-sectional view of the fiber assembly 125. FIG. 14B shows a perspective view of the fiber assembly 125. The fiber assembly 125 can include two main components: a plurality of optical fibers 180 (i.e., for forming the fiber optic probe 130) and a fiber holder 182. The fiber holder 182 also includes two alignment holes 184, one at each end. To fabricate the fiber assembly 125, the optical fibers 180 are cut to length and glued to the fiber holder 182. The optical fibers 180 extend approximately 20 mm from one side (the sensor end) of the fiber holder 182 and approximately 5 mm from the opposite side (the instrument mating face 186). The excess fiber on the instrument side can be cut with a ruby ​​scribe to make it flush with the mating surface 186 of the instrument, and then polished. If the optical fiber 180 has been cleaved to length, the other end (the sensor end) is polished. The sensor end then undergoes further processing to form the fiber optic probe 130. For example, processing can include coating the tip of the sensor end with nanoparticles.

[0091] 1-14B, DMF system 100, DMF cartridge 110, and / or method 200 enable localized optical interrogation techniques using optical fibers (e.g., fiber optic probe 130) integrated directly into a disposable cartridge (e.g., DMF cartridge 110). Compared to conventional methods, the integration of fiber optic sensors and digital microfluidics in DMF system 100 enables localized optical interrogation techniques, providing a low-cost way to achieve high optical performance (e.g., reduced component count for lower cost), limiting optical losses within the system for improved signal, and limiting the collection of stray light for lower noise. [Example]

[0092] [Example 1] 15, which is an example of a process 300 for fabricating a sensor fiber. Process 300 may include, but is not limited to, the steps of: (1) stripping and cleaving fibers to a predetermined length; (2) fixing the fiber in a jig; (3) depositing a sensor on the tip of the fiber; and (4) testing the optical properties, fiber integrity, and coupling characteristics. This is one example of how to fabricate and prepare a fiber optic probe for insertion into the droplet manipulation gap of a DMF cartridge.

[0093] [Example 2] This experiment was conducted to determine the affinity of a common protein-protein interaction, Protein A, with IgG using fiber-optic surface plasmon resonance detection in a DMF device. The experiment demonstrated both the advantages of the autonomous processing and low volume of the DMF device, as well as the low background, limited components, and alignment advantages of fiber-optic detection.

[0094] [material] A 125 μm diameter optical fiber was cleaved and treated to deposit gold nanoparticles on the tip of the fiber. The nanoparticles were then surface-finished with carboxyl groups. Briefly, the optical fiber was thoroughly cleaned and oxidized. Exemplary oxidation methods include exposure to highly basic solutions such as sodium hydroxide, ozone, or oxygen plasma. Next, a self-assembled interface layer was deposited to bond the gold to the sensor. In this example, a mercaptosilane compound such as (3-mercaptopropyl)trimethoxysilane was used. Finally, the optical fiber was immersed in a solution containing gold nanoparticles of the desired size and shape. In this case, 10 nm diameter gold nanoparticles were used. After gold deposition, a thiol compound was used to reveal functional groups for future chemistry. Various compounds can be used; for example, a compound with a thiol group on one side and a hydroxyl group on the other side will reveal a carboxyl surface for future chemical reactions. 3-mercapto-1-propanol was used. For example, Figure 16 shows details of an example nanoparticle sensor surface 143 (see Figure 7B). FIG. 16 shows an electron micrograph of the resulting fiber optic probe tip, showing the fiber optic cladding 145 , fiber optic core 146 , and nanoparticle coating 147 .

[0095] After surface preparation, the sensor was inserted into the gap of the DMF cartridge so that the tip was aligned with the edge of the electrode. Each channel of collected data references a different fiber optic sensor.

[0096] A DMF cartridge was loaded with 2 centistokes of polydimethylsiloxane to prepare the oil environment for the DMF device. The wells of the DMF device were filled with 100 μL of pH 7.4 phosphate-buffered saline (PBS), 35 μL of ethanolamine, 35 μL of pH 1.5 glycine + hydrochloric acid (HCl), and 35 μL of Protein A (the ligand used). The cartridge also contained 8 μL of 10% glycerol in PBS and 16% glycerol in PBS for fiber sensitivity calibration, 8 μL of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 8 μL of N-hydroxysuccinimide (NHS) to activate the carboxyl surface of the sensor, and 8 μL of 900 nM IgG for analysis. However, only 2 μL is required for this protocol. All reagents contained 0.1% Tween-20 surfactant to stabilize the oil-water interface.

[0097] The droplet was vibrated while in contact with the probe, as shown in Figure 11. Using the electrowetting electrodes, the droplet was stretched to a 2X droplet and oscillated over three electrodes at 10 Hz for 10 min while maintaining contact with the probe.

[0098] [method] The entire experiment was carried out automatically with a DMF cartridge. First, the sensitivity of the sensor was calibrated with glycerol. This procedure consisted of the following: (1) Inject 700 nL of PBS for baseline; (2) Measure the refractive index shift of 700 nL of 10% glycerol. (3) Rinse the sensor with 700 nL of PBS. (4) Measure the refractive index shift of 700 nL of 16% glycerol, and (5) Rinse the sensor with 700 nL of PBS. It was something like that.

[0099] The above estimates the degree of signal shift that a known refractive index introduces. The cartridge then automatically measures the binding kinetics of the system. This involves the following steps. Note that for time optimization, many steps were performed in parallel with others. (1) conditioning the sensor with 700 nL of PBS; (2) washing the sensor with 700 nL of glycine-HCl; (3) rinsing the fiber with 700 nL PBS; (4) Mixing 350 nL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide with 350 nL of N-hydroxysuccinimide in each channel to activate the carboxyl surface of the sensor with the mixture; (5) rinsing the fiber with 700 nL PBS; (6) Inject 700 nL of Protein A solution into each sensor, thereby immobilizing Protein A on the sensor surface and serving as a ligand for the Protein A antibody. (7) rinsing the fiber with 700 nL PBS; (8) preparing an IgG sample by the following method: (a) Take 350 nL of 900 nM IgG and dilute with 700 nL of PBS. Mix the above mixture thoroughly and divide into two 700 nL mixtures to create 300 nM samples. (b) Take the remaining 350 nL from 8a and dilute with 700 nL of PBS. Similarly, mix the formulation and divide into 700 nL portions, reserving one for the 100 nM sample and further diluting with a 350 nL drop. (c) Repeat step 8b to make 33 nM, 11 nM, and 3.67 nM samples. Discard the remaining 350 nL drop. preparing an IgG sample by (9) 700 nL of IgG sample is introduced into the various sensors. The association of IgG with Protein A is measured in real time. (10) Rinse the fiber with 700 nL PBS. This is the step for measuring the dissociation of IgG from Protein A in real time.

[0100] [Results and Analysis] Using the above steps, response curves were generated for each IgG sample. These curves were first adjusted for sensitivity to the refractive index shift obtained from the glycerol correction and then adjusted for the amount of Protein A immobilized on the sensor measured during the experiment. The results are shown in plot 400 shown in FIG. 17A. Plot 400 shows response curves A, B, C, D, E, and F. Legend 410 indicates that response curve A corresponds to an affinity of 0 nM, response curve B corresponds to an affinity of 3.67 nM, response curve C corresponds to an affinity of 11 nM, response curve D corresponds to an affinity of 33 nM, response curve E corresponds to an affinity of 100 nM, and response curve F corresponds to an affinity of 3000 nM.

[0101] A 1:1 kinetic model was applied and fitted to this data. As shown in plot 405 shown in Figure 17B, this resulted in a very close correlation and a measured affinity of 2 nM, consistent with measurements of the same sample on other instruments. However, unlike other instruments, this entire experiment, including sample preparation, was performed autonomously on a disposable fluidic cartridge without the risk of cross-contamination with previous or subsequent samples. Furthermore, the experiment consumed only 350 nL of analyte solution, a dramatically reduced volume compared to other systems.

[0102] The present invention may be implemented using hardware, software, or a combination thereof, and may be implemented in one or more computer systems or other processing systems. In one aspect, the present invention contemplates one or more computer systems capable of performing the functions described herein.

[0103] Terms such as "preferably," "commonly," and "typically" are not used herein to limit the scope of the claimed embodiments or to imply that particular features are critical or essential to the structure or function of the claimed embodiments. These terms are intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure.

[0104] The term "substantially" is used herein to describe the degree of inherent uncertainty that can result from any quantitative comparison, value, measurement, or other representation, and to describe the extent to which a quantitative representation can vary from its stated reference without resulting in a change in the fundamental functionality of the object in question.

[0105] The terms "a," "an," and "the" as used in this application, including the claims, refer to "one or more." Thus, for example, reference to "a subject" includes a plurality of subjects unless the context clearly dictates otherwise (e.g., a plurality of subjects), etc.

[0106] The terms "comprise," "comprises," and "comprising" and "include," "includes," "including" are intended to be non-limiting, and the recitation of items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0107] All publications mentioned in the above specification are herein incorporated by reference.

[0108] Various modifications and variations of the disclosed methods, compositions, and uses of the invention will become apparent to those skilled in the art from a review of this disclosure and the claims, without departing from the scope and spirit of the invention. Although the invention has been disclosed herein in connection with specific preferred aspects or embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

Claims

1. 1. A digital microfluidic (DMF) cartridge comprising: a first substrate having one or more electrowetting electrodes for performing droplet operations; a second substrate offset from the first substrate; a droplet operations gap defined between the first substrate and the second substrate, the one or more electrowetting electrodes operative to perform droplet operations on liquid droplets within the droplet operations gap; a fiber optic probe disposed within the droplet operations gap to provide excitation light from a first illumination source to the droplet operations gap and emission light from the droplet operations gap to a first optical measurement device, the fiber optic probe configured to form a reflective interferometric sensor; A DMF cartridge comprising:

2. 10. The DMF cartridge of claim 1, wherein the fiber optic probe is positioned within the droplet operations gap adjacent to the one or more electrowetting electrodes such that the liquid droplet contacts the fiber optic probe.

3. 3. The DMF cartridge according to claim 1, wherein the optical fiber probe penetrates the first substrate and is positioned within the droplet operations gap.

4. 4. The DMF cartridge according to claim 1, wherein the optical fiber probe penetrates the second substrate and is positioned within the droplet operations gap.

5. A DMF cartridge as described in any one of claims 1 to 4, wherein the optical fiber probe extends into and is positioned within a droplet operations gap between the first substrate and the second substrate, approximately parallel to the first substrate and the second substrate.

6. A DMF cartridge according to any one of claims 1 to 5, wherein at least one of the first substrate or the second substrate has at least one opening for introducing liquid into the droplet operations gap.

7. 7. The DMF cartridge of claim 1, wherein the fiber optic probe comprises a ligand.

8. 8. The DMF cartridge of claim 1, wherein the fiber optic probe includes one or more optical elements on a sensor surface disposed at a distal surface portion of the fiber optic probe.

9. 9. The DMF cartridge of claim 1, wherein the droplet operations gap contains a filler material that is immiscible with the liquid droplets.

10. A DMF cartridge as described in any one of claims 1 to 9, wherein the first substrate and the second substrate comprise a material that is sufficiently transparent to allow excitation light from a second illumination source to pass through the first substrate to reach the droplet operations gap, and emission light from the droplet operations gap to pass through the second substrate to reach a second optical measurement device.

11. 1. A digital microfluidic (DMF) system comprising: A DMF cartridge according to any one of claims 1 to 10; one or more illumination sources that provide excitation light to the droplet manipulation gap of the DMF cartridge via a fiber optic probe; one or more optical measurement devices that receive and process light emitted from the droplet operations gap via the fiber optic probe; A DMF system comprising:

12. The DMF system of claim 11 further comprising: a controller in operative communication with the one or more electrowetting electrodes to control the performance of droplet operations by the one or more electrowetting electrodes; A DMF system comprising:

Images