Patent Application
20210331175
The presently disclosed subject matter generally relates to the detection of molecules, such as DNA, proteins, drugs, and the like, and more particularly to a plasmon resonance (PR) system and instrument, digital microfluidic (DMF) cartridge, and methods of using localized surface plasmon resonance (LSPR) and droplet operations for analysis of analytes.
In traditional assays, the protein or DNA arrays are flooded with a solution containing labeled target biomolecules, incubated overnight, rinsed, and then “read-out” using fluorescence detection methods. This is not only time-consuming but requires high sample concentrations. Direct, label-free detection techniques exist, such as surface plasmon resonance (SPR). However, these techniques exhibit lower sensitivity and throughput, thus making them unsuitable for the detection of very low concentrations of the target analyte. Specifically, SPR technology has certain drawbacks. For example, immunoassays using SPR technology can be expensive, may require complex microfluidics systems and high precision optics, may require complex assays, and is a niche technology with few specialists.
SPR technology can be incorporated in, for example, a DMF cartridge. In DMF, droplet operations in the DMF cartridge may occur in a bulk filler fluid (e.g., a low-viscosity oil, such as silicone oil or hexadecane filler fluid). In another example, droplet operations in the DMF cartridge may occur in air, and the droplets may have a thin oil coating (or oil shell) thereon. Further embodiments of this technology may use droplet operations in air without an oil shell.
A first aspect includes a cartridge for use with an instrument to perform measurement of a fluid. The cartridge includes a digital microfluidics (DMF) portion comprising a plurality of droplet actuators operative to perform droplet operations on a fluid droplet in the DMF portion and a reaction portion comprising sensor media that is disposed in relation to the plurality of droplet actuators, wherein the plurality of droplet actuators are operative to induce movement of the fluid droplet relative to the sensor media while in contact with the sensor media.
A number of feature refinements and additional features are applicable to the first aspect. These feature refinements and additional features may be used individually or in any combination. As such, each of the following features that will be discussed may be, but are not required to be, used with any other feature or combination of features of the first aspect.
For instance, in an example, the plurality of droplet actuators may comprise reaction electrodes. The plurality of reaction electrodes may perform droplet operations by electrowetting.
In an example, the sensor media of the cartridge may comprise surface plasmon resonance (SPR) sensor media. The SPR sensor media may be functionalized with a capture molecule to which a target molecule of an analyte fluid binds to change an optical signal of the SPR sensor media. The capture molecule may include a ligand immobilized on the SPR sensor medium that is sensitive to binding with the target molecule of the analyte fluid to change an optical property of the SPR sensor media resulting in the change of the optical signal of the SPR sensor media. The change of the optical properties may be a change in the optical signal resulting from light interacting with the SPR sensor media.
In an example, the cartridge may include an SPR sensor surface disposed in the reaction portion and in relation to the plurality of reaction electrodes. The SPR sensor media may be disposed on the SPR sensor surface, and the droplet may be contactingly engageable with the SPR sensor surface by operation of the plurality of reaction electrodes. The SPR sensor media may include one of nanosized structures distributed on the sensor surface or a continuous film comprising nano-sized features. The reaction portion may include a first substrate and second substrate disposed in spaced-apart relation to define a reaction chamber therebetween. The SPR sensor surface may be disposed at the first substrate, and the plurality of reaction electrodes may be disposed at the second substrate opposite the first substrate. Alternatively, the SPR sensor surface may be disposed at the first substrate, and the plurality of reaction electrodes may be disposed at the first substrate.
In an example, the SPR sensor surface may be disposed adjacent to a terminal portion of an optical member comprising at least one optical fiber. The optical member may extend away from one of the first or second substrate to dispose the SPR sensor surface within the reaction chamber. In this example, the optical member may include a first optical fiber on which the optical signal is transmitted from the SPR sensor surface. The optical member may include a second optical fiber on which light from an illumination source is provided to the SPR sensor surface. The optical member may be moveable relative to the first substrate to dispose the SPR sensor surface between an extended position in which the SPR sensor surface is disposed in the reaction chamber and a retracted position in which the SPR sensor surface is not disposed in the reaction chamber. In an example, the reaction chamber contains a filler media. The optical member may be retractable to reduce contact between the SPR sensor surface and the filler media.
In an example, the SPR sensor surface may be disposed between a first reaction electrode and a second reaction electrode. The first reaction electrode and the second reaction electrode may be alternately activated to induce oscillation of the droplet between the first reaction electrode and the second reaction electrode to induce the movement of the fluid droplet relative to the SPR sensor surface. The oscillation of the droplet between the first reaction electrode and the second reaction electrode may be linear. Alternatively, the SPR sensor surface may be disposed between three or more reaction electrodes. The three or more reaction electrodes may be alternately activated to induce oscillation of the droplet between the three or more reaction electrodes to induce the movement of the fluid droplet relative to the SPR sensor surface. In turn, the oscillation of the droplet between the first reaction electrode and the second reaction electrode may be circular.
In an example, the sensor media may include a plurality of sensor nanoparticles suspended in a sensor droplet disposed in the reaction portion. The fluid droplet may be merged with the sensor droplet to form a reacted droplet for measurement of the optical signal of the SPR sensor media in the reacted droplet. The movement induced by the plurality of droplet actuators may be operative to mix the reacted droplet.
In an example, each of the plurality of sensor nanoparticles is magnetically responsive. For instance, each of the plurality of sensor nanoparticles may include a magnetically responsive core. Alternatively, each of the plurality of sensor nanoparticles may include a magnetically responsive element tethered to the sensor nanoparticle. The magnetically responsive element may be physically or chemically coupled to the sensor nanoparticle.
In turn, the cartridge may also include a magnet that is selectively operable to act on the magnetically responsive sensor nanoparticles to immobilize the sensor nanoparticles in the reaction portion to dispose the plurality of nanoparticles in a restrained position relative to the magnet. The plurality of droplet actuators may be operative to move fluid away from the sensor nanoparticles when the magnetically responsive sensor nanoparticles are immobilized by the magnet in the restrained position and to move fluid into contact with the sensor nanoparticles when the magnetically responsive sensor nanoparticles are immobilized by the magnet in the restrained position.
In an example, the movement of the fluid droplet is at a rate greater than a sampling rate of an optical system measuring an optical signal of the sensor media.
In an example, the cartridge may include a plurality of droplet operation electrodes in the DMF portion that are operative to supply fluid to the plurality of droplet actuators. The cartridge may include a reservoir electrode in the DMF portion to receive and maintain the fluid in the DMF portion. The droplet operation may include at least one of droplet merging, droplet splitting, droplet dispensing, or droplet diluting.
In an example, the fluid droplet may include an analyte fluid droplet, and the movement of the analyte fluid droplet relative to the sensor media while in contact with the sensor media may be an effective diffusion rate of the analyte fluid droplet relative to the sensor media. The effective diffusion rate of the analyte fluid droplet may be higher than a binding rate of an analyte relative to the SPR sensor.
In an example, the cartridge includes an electrical contact in electrical communication with the plurality of droplet actuators. The electrical contact may be configured for interface with a controller for control of the plurality of droplet actuators. The cartridge may include a pluggable interface of the cartridge comprising the electrical contact. The pluggable interface may be physically and electrically engageable with an instrument to establish electrical communication between a controller of the instrument and the at least one electrode.
In an example, the reaction portion may be substantially transparent to an illumination source incident on the reaction portion on at least one side of the reaction portion to facilitate real-time optical measurement of the sensor media in a reflectance mode. Alternatively, the reaction portion may be substantially transparent to an illumination source incident on the reaction portion on opposite sides of the reaction portion to facilitate real-time optical measurement of the sensor media in a transmission mode.
In an example the fluid droplet may be an analyte fluid droplet, the SPR sensor media may be operable to detect analyte affinity of the analyte fluid droplet during the movement of the analyte fluid droplet relative to the sensor media, and the analyte affinity may be characterized by an analyte affinity value (KD). The KD may be determined based on an on-rate (KON) measured during an association phase of the analyte fluid at the SPR sensor and an off-rate (KOFF) measured during a dissociation phase of the analyte fluid at the SPR sensor.
Another aspect of the present disclosure includes a plasmon resonance (PR) system. The PR system includes a cartridge according to the first aspect, including any of the foregoing examples discussed in relation to the first aspect. The system also includes a PR instrument with which the cartridge is engageable. The PR instrument includes a controller in operative communication with the electrical contacts for control of the plurality of droplet actuators and an optical detection system operative to measure an optical signal of the sensor media.
A number of feature refinements and additional features are applicable to the second aspect. These feature refinements and additional features may be used individually or in any combination. As such, each of the following features that will be discussed may be, but are not required to be, used with any other feature or combination of features of the second aspect.
In an example, the optical detection system further comprises an illumination source operative to direct light incident to the sensor media and an optical measurement device that measures the optical signal of the sensor media.
In the PR system of the second aspect, the fluid droplet may be an analyte fluid droplet, and the controller is operative to detect a target molecule in the analyte fluid droplet based on the optical signal of the sensor media in the presence of the analyte fluid droplet while in motion relative to the sensor media. The controller may be operative to measure binding events of the target molecule in the analyte fluid droplet in real time based on the optical signal of the sensor media in while the analyte fluid droplet is in motion relative to the sensor media.
Accordingly, the controller may be operative to determine a quantitative measurement of analyte affinity comprising an analyte affinity value (KD). The KD may be determined based on an on-rate (KON) measured during an association phase of the sensor media and an off-rate (KOFF) measured during a dissociation phase of the sensor media. The fluid droplet in the reaction portion may be an analyte fluid droplet during the association phase, and the fluid in the reaction portion may be a buffer solution fluid (e.g., a buffer solution fluid droplet) during the dissociation phase.
A third aspect includes a method of operation of a cartridge for measurement of an analyte fluid. The method includes contacting sensor media in a reaction portion of the cartridge with an analyte fluid droplet. The method also includes inducing movement of the analyte fluid droplet with respect to the sensor media while maintaining the analyte fluid droplet in contact with the sensor media. The inducing includes operation of a plurality of droplet actuators disposed relative to the sensor media. The method also includes generating a first optical signal at the sensor media during the movement of the analyte fluid droplet relative to the sensor media.
A number of feature refinements and additional features are applicable to the third aspect. These feature refinements and additional features may be used individually or in any combination. As such, each of the following features that will be discussed may be, but are not required to be, used with any other feature or combination of features of the third aspect.
For instance, in an example, the first optical signal may be an association signal corresponding to an association phase of the sensor media in the presence of the analyte fluid droplet. The method may also include determining an on-rate (KON) of the analyte fluid droplet based on the association signal. The determining the KON may include fitting an association curve to the association signal.
In an example, the method also includes moving the analyte fluid droplet from the reaction portion such that the analyte fluid droplet is no longer in contacting engagement with the sensor media. In turn, the method may include introducing a buffer solution fluid droplet to the reaction portion. The buffer solution may be in contacting engagement with the sensor media. The method may also include inducing movement of the buffer fluid droplet with respect to the sensor media while maintaining the buffer fluid droplet in contact with the sensor media. The inducing may include the operation of the plurality of droplet actuators disposed relative to the sensor media. The method may also include generating a second optical signal at the sensor media during the movement of the buffer fluid droplet relative to the sensor media. The second optical signal may be a dissociation signal corresponding to a dissociation phase of the sensor media in the presence of the buffer fluid droplet. In turn, the method may include determining an off-rate (KOFF) of the analyte fluid based on the dissociation signal. The determining the KOFF may include fitting a dissociation curve to the dissociation signal. In an example, the method of the third aspect may include calculating an analyte affinity value (KD) based on the KON and the KOFF. KD may be the quotient of KON and KOFF.
In another example, the sensor media may be a plurality of sensor nanoparticles disposed in the reaction portion. The plurality of sensor nanoparticles may be disposed in a sensor droplet. In this regard, the method may also include merging the analyte fluid droplet and the sensor to form a reacted droplet for measurement of the optical signal of the sensor media in the reacted droplet.
In an example, each of the plurality of sensor nanoparticles may be magnetically responsive. In turn, the method may include activating a magnet to dispose the nanoparticles in a restrained position relative to the magnet to immobilize the sensor nanoparticles in the reaction portion. The nanoparticles may be maintained in the restrained position relative to the magnet during moving of a droplet relative to the reaction portion.
In an example, the sensor media may be disposed adjacent to a terminal portion of a moveable member. The method may include moving the moveable member relative to a reaction chamber defined in the reaction portion between an extended position and a retracted position. The sensor media may be disposed in the reaction chamber in the extended position and is removed from the reaction chamber in the retreated position. The reaction chamber may include a filler media. In turn, the method may include retracting the moveable member to the retracted position, introducing a fluid droplet to the reaction portion after the retracting to displace the filler media from an area adjacent to the plurality of droplet actuators, and advancing the moveable member after the introducing to the extended position to dispose the sensor media in the fluid droplet.
In an example, the method includes engaging the cartridge with an instrument. The method may also include measuring a signal from the sensor media while the fluid droplet is moved relative to the sensor media while maintaining the fluid droplet in contact with the sensor media. The sensor media may be SPR sensor media, and the signal may be an optical signal of the SPR sensor media. In turn, the method may include providing light from a light source of the instrument incident to the SPR sensor media. The measuring may include measuring the optical signal of the SPR sensor media at an optical measurement device of the instrument.
In an example, the method may also include establishing electrical communication between a controller of the instrument and the plurality of droplet actuators of the DMF portion and controlling the plurality of droplet actuators of the DMF portion. The inducing movement of the fluid may be in response to the controlling of the plurality of droplet actuators of the DMF portion. In an example, in a first period, the fluid may be a buffer fluid droplet, and the measuring comprises recording a baseline optical signal as the buffer fluid is moved relative to the sensor media while maintaining contact with the sensor media. The method may also include introducing an analyte fluid droplet to the reaction portion in a second period. The measuring may include capturing an association signal corresponding to an association phase of the analyte fluid droplet in the second period. An effective diffusion rate of the analyte fluid droplet relative to the sensor media may be higher than a binding rate of the analyte fluid droplet relative to the sensor media. The method may include determining an on-rate (KON) of the analyte fluid droplet based on the association signal. The determining the KON may include fitting an association curve to the association signal.
In a further example, the method may include moving the analyte fluid droplet away from the sensor media and introducing a buffer fluid droplet to the reaction portion in a third period, wherein the measuring comprises capturing a dissociation signal corresponding to a dissociation phase of the analyte in the third period. The method may further include determining an off-rate (KOFF) of the analyte fluid based on the dissociation signal. The determining the KOFF may include fitting a dissociation curve to the dissociation signal. The method may include calculating an analyte affinity value (KD) based on the KON and the KOFF. KD may be the quotient of KON and KOFF.
The method may also include supplying, in a fourth period, a regeneration buffer solution fluid droplet to the reaction portion and contacting the regeneration buffer solution fluid droplet with the sensor media to regenerate the sensor media. The method may include functionalizing the sensor media by contacting a functionalization fluid droplet comprising ligands to bind the ligands to the sensor media. The method may also include activating the sensor media by contacting an activation fluid droplet with the sensor media prior to the functionalizing of the sensor media.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
Other implementations are also described and recited herein.
Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein:
The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the presently disclosed subject matter are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; instead, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
In some embodiments, the presently disclosed subject matter may provide a plasmon resonance (PR) system and instrument, digital microfluidic (DMF) cartridge, and methods of using localized surface plasmon resonance (LSPR) and droplet operations for analysis of analytes.
In one embodiment, the PR system can be an LSPR system, wherein the LSPR system includes a DMF cartridge that further includes LSPR sensing capability for analysis of analytes. The DMF cartridge can be used to facilitate DMF capabilities generally for merging, splitting, dispensing, diluting, transporting, and other types of droplet operations. One application of these DMF capabilities may be for sample preparation. Further, the DMF cartridge may include LSPR sensing means for (1) detecting, for example, certain molecules (e.g., target analytes) and/or chemicals in the sample, and (2) analysis of analytes, such as for measuring binding events in real time to extract ON-rate information, OFF-rate information, affinity information, avidity, aggregation, specificity information, conformation changes, thermodynamic parameters, and/or other data/information related to the molecules under study. Another application of this embodiment may be to explore the interactions between drugs and biomolecules or to study the chemical properties of polymers.
In some embodiments, the LSPR sensing capability of the DMF cartridge may include fixed LSPR sensing operations that can be performed using droplet operations. In other embodiments, the LSPR sensing capability of the DMF cartridge may include in-solution LSPR sensing, which is LSPR sensing processes that can occur in the droplets themselves and using droplet operations. In some embodiments, the LSPR sensing capability of the DMF cartridge may include various arrangements of multiple LSPR sensors for processing multiple droplets.
In some embodiments of the presently disclosed PR system, PR instrument, DMF cartridge, and method, the diffusion or flow rate of the fluid at the LSPR sensor may be faster than the binding rate, thereby increasing the likelihood that the LSPR sensing is measuring the binding rate and is not limited by a slow diffusion or flow rate. In the DMF cartridge, droplet operations may be used to, for example, oscillate a droplet back and forth or in circular motion to generate flow at the fixed LSPR sensing processes and/or to generate mixing in the in-solution LSPR sensing processes and thereby increasing the flux of the molecules to the surface of the LSPR sensor. In other embodiments, the LSPR sensing processes may include vibrating the LSPR sensor itself with respect to, for example, a droplet to generate flow and/or to generate mixing. Accordingly, in the presently disclosed PR system, PR instrument, DMF cartridge, and method, continuous uni-directional flow of the sample at the LSPR sensor may not be required. Instead, a sample or analyte droplet may be manipulated rapidly at the LSPR sensing elements using droplet operations to generate a recirculating flow.
A PR instrument of the PR system may include the DMF cartridge, an optical detection system, and a controller. The optical detection system may include, for example, an illumination source and an optical measurement device in relation to the LSPR sensing elements. In some embodiments, the optical detection system may operate in transmission mode while in other embodiments, the optical detection system may operate in reflection mode. The controller may be provided for controlling the droplet manipulation by activating/deactivating electrodes in the DMF cartridge. The controller also may manage the overall operations of the PR system. Additionally, methods of using droplet operations in the DMF cartridge to perform fixed LSPR sensing operations and/or in-solution LSPR sensing operations may be provided.
In some embodiments of the presently disclosed PR system, PR instrument, DMF cartridge, and method, when bulk filler fluid (e.g., oil, such as silicone oil or hexadecane filler fluid) is present and/or when oil-covered droplets are present, the fixed LSPR sensing and/or the in-solution LSPR sensing may be designed to minimize oil contamination at the LSPR sensing elements.
In some embodiments, the presently disclosed PR system, PR instrument, DMF cartridge, and method can be used to determine the KD value, the KON value, and/or the KOFF value of the analyte sample with an immobilized ligand, wherein the KD value is a quantitative measurement of affinity between the analyte and ligand, the KON value indicates the kinetic ON-rate of binding, and the KOFF value indicates the kinetic OFF-rate of binding.
In some embodiments, the fixed LSPR sensors may include a surface with nanostructures immobilized thereon and wherein the nanostructures can be functionalized with capture molecules, such as ligands. Then, a sample droplet that has the target analyte suspended therein may come into contact with the fixed LSPR sensor and wherein the target analyte can be a binding partner with the capture molecules of the fixed LSPR sensor. Then, the optical detection system may be used to capture the real-time kinetic measurements (e.g., the association phase (i.e., KON value), the dissociation phase (i.e., KOFF value), and the analyte affinity (i.e., KD value)) of the binding process.
In some embodiments, the in-solution LSPR sensing processes may include (1) an LSPR droplet that has nanostructures suspended therein and wherein the nanostructures are functionalized with capture molecules, such as ligands, (2) a sample droplet that has the target analyte suspended therein and wherein the target analyte can be a binding partner with the capture molecules in the LSPR droplet, (3) a process of merging (and/or mixing) the LSPR droplet with the sample droplet using droplet operations such that binding can occur between the capture molecules (e.g., ligands) and the target analyte in the merged droplet, and (4) using the optical detection system, a process of capturing the real-time kinetic measurements (e.g., the association phase (i.e., KON value), the dissociation phase (i.e., KOFF value), and the analyte affinity (i.e., KD value)) of the binding process.
In some embodiments, the nanostructures suspended in the LSPR droplet of the in-solution LSPR sensing processes may be magnetically responsive nanostructures.
DMF cartridge 110 may facilitate DMF capabilities generally for fluidic actuation including droplet merging, splitting, dispensing, diluting, and the like. One application of these DMF capabilities may be sample preparation. However, the DMF capabilities may be used for other processes, such as waste removal. DMF cartridge 110 of PR system 100 can be provided, for example, as a disposable and/or reusable cartridge. More details of an example of DMF cartridge 110 are shown and described hereinbelow with reference to
Further, DMF cartridge 110 may include LSPR sensing 112. LSPR sensing 112 may be used for (1) detecting, for example, certain molecules (e.g., target analytes) and/or chemicals in the sample, and/or (2) for analysis of analytes, such as for measuring binding events in real time to extract ON-rate information, OFF-rate information, and/or affinity information. LSPR sensing 112 can be, for example, fixed LSPR sensing and/or any in-solution LSPR sensing processes. Additionally, the fixed LSPR sensing and the in-solution LSPR sensing may be designed to minimize oil contamination at the LSPR sensing elements. More details of fixed LSPR sensing are shown and described hereinbelow with reference to
While the discussion presented herein may involve use of an SPR sensor (e.g., an LSPR sensor in certain embodiments), it is contemplated that other sensors can also be used in place of or in addition to the SPR or LSPR sensor. Such alternative or additional sensor options may include electronic sensors, electrochemical sensors, mechanical sensors, or other appropriate sensor types. For example, sensors may be used, such as biolayer interferometry or piezoelectric sensors. In this regard, the interaction between an analyte and a sensor using the DMF capabilities described herein for sample/sensor interaction may generally be applicable for analysis of an analyte using any appropriate sensor.
PR system 100 may further include a controller 150, a DMF interface 152, an illumination source 154, an optical measurement device 156, and thermal control mechanisms 158. Controller 150 may be electrically coupled to the various hardware components of PR system 100, such as to DMF cartridge 110, illumination source 154, and an optical measurement device 156. In particular, controller 150 may be electrically coupled to DMF cartridge 110 via DMF interface 152, wherein DMF interface 152 may be, for example, a pluggable interface for connecting mechanically and electrically to DMF cartridge 110. Together, DMF cartridge 110, controller 150, DMF interface 152, illumination source 154, and optical measurement device 156 may comprise a PR instrument 105.
Controller 150 may, for example, be a general-purpose computer, special purpose computer, personal computer, microprocessor, or other programmable data processing apparatus. Controller 150 may provide processing capabilities, such as storing, interpreting, and/or executing software instructions, as well as controlling the overall operations of PR system 100. The software instructions may comprise machine readable code stored in non-transitory memory that is accessible by the controller 150 for the execution of the instructions. Controller 150 may be configured and programmed to control data and/or power aspects of these devices. For example, with respect to DMF cartridge 110, controller 150 may control droplet manipulation by activating/deactivating electrodes. Generally, controller 150 can be used for any functions of the PR system 100. For example, controller 150 can be used to authenticate the DMF cartridge 110 in a fashion similar to how printer manufacturers check for their branded ink cartridges, controller 150 can be used to verify that the DMF cartridge 110 is not expired, controller 150 can be used to confirm the cleanliness of the DMF cartridge 110 by running a certain protocol for that purpose, and so on.
Additionally, in some embodiments, DMF cartridge 110 may include capacitive feedback sensing. For example, a signal may be generated or detected by a capacitive sensor that can detect droplet position, velocity, and size. Further, in other embodiments, instead of capacitive feedback sensing, DMF cartridge 110 may include a camera or other optical device to provide an optical measurement of the droplet position, velocity, and size, which can trigger controller 150 to re-route the droplets at appropriate positions. The feedback can be used to create a closed-loop control system to optimize droplet actuation rate and verify droplet operations are completed successfully.
Optionally, PR instrument 105 can be connected to a network. For example, controller 150 may be in communication with a networked computer 160 via a network 162. Networked computer 160 can be, for example, any centralized server or cloud server. Network 162 can be, for example, a local area network (LAN) or wide area network (WAN) for connecting to the internet.
In PR system 100, illumination source 154 and optical measurement device 156 may be arranged with respect to LSPR sensing 112 (e.g., fixed LSPR sensing and/or in-solution LSPR sensing) of DMF cartridge 110. The illumination source 154 may be, for example, a light source for the visible range (400-800 nm), such as, but not limited to, a white light-emitting diode (LED), a halogen bulb, an arc lamp, an incandescent lamp, lasers, and the like. Illumination source 154 is not limited to a white light source. Illumination source 154 may be any color light that is useful in PR system 100. Optical measurement device 156 may be used to obtain LSPR light intensity readings. Optical measurement device 156 may be, for example, a charge coupled device, a photodetector, a spectrometer, a photodiode array, or any combinations thereof. Further, PR system 100 is not limited to one illumination source 154 and one optical measurement device 156 only. PR system 100 may include multiple illumination sources 154 and/or multiple optical measurement devices 156 to support multiple LSPR sensing elements. Thermal control mechanisms 158 may be any mechanisms for controlling the operating temperature of DMF cartridge 110. Examples of thermal control mechanisms 158 may include Peltier elements and resistive heaters.
The terms “top,” “bottom,” “over,” “under,” “in,” and “on” are used throughout the description with reference to the relative positions of components of the DMF cartridge, such as relative positions of top and bottom substrates of the DMF cartridge. It will be appreciated that the DMF cartridge is functional regardless of its orientation in space.
In DMF cartridge 110, a gap between the bottom substrate 116 and top substrate 118 may define a reaction (or assay) chamber 122. For example, reaction (or assay) chamber 122 may comprise a space between the bottom substrate 116 and top substrate 118 for processing any liquids of interest via droplet operations; liquids, such as, but not limited to, liquid reagents, buffer solution, sample droplets, and the like. Accordingly, an electrode arrangement 124 may be provided atop bottom substrate 116 in reaction (or assay) chamber 122. Electrode arrangement 124 may include, for example, any arrangement of droplet operations electrodes 126 (e.g., electrowetting electrodes) and reservoir electrodes 128. For example, electrode arrangement 124 may include any lines or paths of droplet operations electrodes 126 in relation to any number of reservoir electrodes 128.
Electrode arrangement 124 may be used for performing droplet operations via electrowetting. “Droplet operation” means any manipulation of a droplet on a DMF device or cartridge. A droplet operation may, for example, include: loading a droplet into the DMF 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; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; retaining a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; cooling a droplet; disposing of a droplet; transporting a droplet out of a droplet actuator; other droplet operations described herein; and/or any combination of the foregoing. Further, for controlling the temperature of processes occurring in reaction (or assay) chamber 122, a temperature control element (not shown), such as a Peltier heat pump, can be used in combination with DMF cartridge 110.
Further, while
By way of example, an instance of LSPR sensing 112 is shown with respect to a line or path of droplet operations electrodes 126. In one example and referring now to Detail A of
As described herein, “localized surface plasmon resonance (LSPR)” means using nanoparticle-based or nanostructure-based transducers to monitor binding events in real time without additional labels. For example, nanoparticle-based transducers may include metal nanoparticles from about 1 nm to about 1000 nm in various dimensions. For example, nanostructure-based transducers may include gold films that include nano-sized features (e.g., nano-sized bumps, posts, holes, ridges, lines, and the like.) Some nanoparticle-based or nanostructure-based diagnostic assays are “label-free.”
LSPR is a phenomenon associated with noble metal nanoparticles that creates sharp spectral absorbance and scattering peaks and produces strong electromagnetic near-field enhancements. These spectral peaks may be monitored using absorbance spectroscopy. The spectral peak changes with refractive index changes in the immediate vicinity of the nanoparticle surface. When chemical targets are bound near the surface of a metal nanoparticle, a shift in the spectral peak occurs due to changes in the local refractive index. This may be used to determine the concentration of a specific target in a complex medium. Alternatively, the spectral peak shift may be detected through a change in absorption at a given wavelength.
LSPR sensors operate through the immobilization of metal nanoparticles onto a solid support that may include, for example, a flat surface or a microstructured surface. The nanoparticles may be functionalized with specific capture molecules, which may be an antibody. The sample of interest may be flowed over the top of the metal nanoparticles, the target chemicals of interest bind to their respective capture molecules, and the overall spectral peak of the sensor shifts according to the concentration of the chemical target on the capture molecules. LSPR sensors with nanoparticles on planar surfaces operate by flowing the sample longitudinally over the surface. In order to measure this shift, reflectance or transmission absorbance spectroscopy may be employed. Further, analysis via “intensity or colorimetric methods” may be performed using LSPR sensors. More details of examples of LSPR sensors are shown and described hereinbelow with reference to
In this example, LSPR sensor 136 may include a substantially transparent or opaque substrate 210, such as glass, plastic, or TPE substrate. That is, substrate 210 may be substantially transparent when used in a transmission mode configuration. By contrast, substrate 210 may be opaque when used in a reflection mode configuration. An LSPR sensor layer 212 may be provided atop substrate 210. LSPR sensor layer 212 may be, for example, a gold film that includes certain nanostructures that create an LSPR effect. LSPR sensor layer 212 may be functionalized with one or more capture molecules 214. In one example, capture molecules 214 may comprise ligands that are immobilized on the surface of LSPR sensor layer 212. In this example, the ligands may comprise one of two binding partners, the other binding partner may be a target analyte 216, wherein the target analyte 216 flows in solution over the capture molecules 214 as shown in
Referring now again to
With reference now again to
In the presently disclosed PR system 100, PR instrument 105, and DMF cartridge 110, flow can be artificially created by using droplet operations to move the droplet rapidly around on the DMF surface, either back and forth in a line or in a circular method. If the droplet is moved faster than the optical sampling rate, it is likely that artifacts from this movement will have little to no effects on the optical measurements. In another example, if the droplet spans multiple electrodes, the momentum may be changed without moving the droplet off the sensor location and thus avoiding movement artifacts.
Referring now to
Referring now to
In the examples shown in
In the examples shown in
Further, moving sample droplet 140 (1) helps increase the flux of the molecules to the surface of LSPR sensor 136 and (2) helps improve the likelihood that the binding rate is being measured at LSPR sensor 136 without limitation of diffusion or flow rate. In the DMF cartridge, droplet operations may be used to, for example, oscillate a droplet back and forth or in circular motion to generate flow at the LSPR sensor and/or to generate mixing in the in-solution LSPR sensing processes and thereby help increase flux of the molecules to the surface of the LSPR sensor. In another embodiment, sample droplet 140 can span two or more droplet operations electrodes 126. Then, droplet operations electrodes 126 can be actuated at a rate faster than the bulk droplet can move off of the sensor location, agitating the liquid and similarly promoting flux of the molecules. More details of examples of using fixed LSPR sensing 112 and droplet operations to capture the real-time kinetic measurements of a sample droplet are shown and described hereinbelow with reference to
In operation and referring now to
In operation and referring now to
With respect to the droplet patterns described hereinabove with respect to
In the process shown in
For example and referring now to
Further to the embodiment and referring now to
In another embodiment, the fiber optic probe 170 and the DMF cartridge 110 may be disposed for relative movement therebetween. For instance, the fiber optic probe 170 may be selectively displaced from the DMF cartridge 110 to remove the fiber optic probe 170 from between the bottom substrate 116 and the top substrate 118 (see
With reference now again to
In the fixed LSPR sensing processes shown in
The configuration of the fiber optic probe (e.g., the fiber optic probe 170) with respect to DMF cartridge 110 is not limited to those shown, for example, in
In yet another example,
In still another example, a robotics system (not shown) may be used to move fiber optic probes 170 with LSPR sensors 136 to specific droplet locations within DMF cartridge 110. For example, the robotics system can be used to move fiber optic probes 170 with LSPR sensors 136 through the bottom substrate 116 and/or top substrate 118 of DMF cartridge 110 and/or into the gap between the bottom substrate 116 and top substrate 118 from the side of DMF cartridge 110. Generally, fiber optic probes 170 can be moved, for example, mechanically, electrically, and/or magnetically within DMF cartridge 110 to different sample lanes and/or locations.
In some embodiments, the LSPR sensing capability of DMF cartridge 110 may include various arrangements of multiple LSPR sensors 136 for processing multiple droplets, examples of which are shown in
In yet another example and referring now to
In yet another example and referring now to
At a step 310, PR system 100 may be provided that includes DMF cartridge 110, wherein fixed LSPR sensing processes (e.g., fixed LSPR sensors 136) can be used for the analysis of analytes.
At a step 315, the fixed LSPR sensors 136 can be activated using droplet operations. For example, “activation” may include an amine coupling step in which the COOH functional surface coating on the LSPR sensors 136 is converted into an active ester. For example, an activation buffer droplet that has EDC/NHS therein may be transported using droplet operations to a certain fixed LSPR sensor 136. EDC is 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide. NHS is N-hydroxysuccinimide. The activation buffer droplet with the EDC/NHS therein may reside at the fixed LSPR sensor 136 for a period of time. The EDC/NHS solution reacts with the COOH sites on the fixed LSPR sensor 136 and turns them into active functional groups that can covalently bind to any amine group on the ligand. In so doing, the fixed LSPR sensor 136 may be activated.
At a step 320, the fixed LSPR sensors 136 may be functionalized using droplet operations. For example, a buffer droplet that has ligands therein may be transported using droplet operations to a certain fixed LSPR sensor 136. The buffer droplet with the ligands therein may reside at the fixed LSPR sensor 136 for a period of time. In so doing, the fixed LSPR sensor 136 may be functionalized.
At a step 325, the fixed LSPR sensors 136 may be deactivated using droplet operations. Deactivation may be performed to convert any remaining active binding sites on the LSPR sensors 136 into non-active sites. For example, a “blocking” solution, such as ethanolamine, may be used to react with any remaining COOH site and deactivate them. For example, a droplet of ethanolamine may be transported using droplet operations to a certain fixed LSPR sensor 136. The ethanolamine droplet may reside at the fixed LSPR sensor 136 for a period of time. In so doing, the fixed LSPR sensor 136 may be deactivated.
At a step 330, the assay protocol may be performed using droplet operations in DMF cartridge 110 and using the fixed LSPR sensing processes. Additionally, sensor readings may be captured in real time. For example, using illumination source 154 and optical measurement device 156, the LSPR signal from a certain fixed LSPR sensor 136 may be captured in real time while running the assay protocol. For example, an assay protocol may be performed in which the sample droplet (e.g., sample droplet 140) is transported using droplet operations to a certain fixed LSPR sensor 136 and readings for the association phase may be recorded. Next, a buffer droplet may be transported using droplet operations to the fixed LSPR sensor 136 and readings for the dissociation phase may be recorded. Next, a different concentration of the analyte (usually 3× the previous one) can be transported using droplet operations the fixed LSPR sensor 136 and the above may be repeated. This is typically done for at least three analyte concentrations, which may facilitate performing the kinetic analysis. Generally, DMF operations of this step will autonomously perform the dilutions required for analysis.
At a step 335, the sensor data from the fixed LSPR sensor 136 may be processed, and the KON value, KOFF value, and KD value of the analyte of interest may be determined. For example, using controller 150 of PR system 100, the sensor data from the fixed LSPR sensor 136 may be processed by fitting a binding model to the data and using regression to find the KON value, KOFF value, and KD value of the analyte of interest that best represents the experimental data. This can be accomplished using a data set that includes, for example, the at least three analyte concentrations described in step 330.
Referring now again to
In optical system 400, Y-coupler 414 allows LED light source 410 (the illumination source) and reflected light from AuNP tip 420 to share a single optical fiber 422. The advantage of this example is that the illumination source (e.g., LED light source 410) and sensor (e.g., spectrometer 412) are inherently aligned to the activated fiber tip (e.g., AuNP tip 420). For example, a Y-coupler 414 with a 90:10 split can be used with the 10%-side connected to LED light source 410 and the 90%-side connected to spectrometer 412. LED light source 410 then passes through Y-coupler 414 to fiber sensor 416 with gold nanoparticles (e.g., AuNP tip 420) inside the DMF device. This light reflects off the layer of nanoparticles, and certain wavelengths are absorbed according to the SPR signal. The reflected light is then passed through Y-coupler 414 to spectrometer 412 for analysis.
“In-solution LSPR sensing” may refer to any LSPR sensing processes that occur in solution, such as any LSPR sensing processes that occur in the droplets themselves. Below,
Generally, in this process, the flux of the molecules to the surface of the nanoparticles 232 can be created by rapidly mixing the droplets using droplet operations. For example, droplet operations may be used to affect a rapid circular motion (like shown in
In one example and referring now to
In another example and referring now to
At a step 510, PR system 100 may be provided that includes DMF cartridge 110 that supports in-solution LSPR sensing processes that use droplet operations for analysis of analytes.
At a step 515, in-solution processes and droplet operations may be used for activating magnetically responsive nanoparticles 250. For example, “activation” may comprise an amine coupling step in which the COOH functional surface coating on the magnetically responsive nanoparticles 250 is converted into an active ester. For example and referring now to
At a step 520, in-solution processes and droplet operations may be used for washing magnetically responsive nanoparticles 250. For example and referring now to
At a step 525, in-solution processes and droplet operations may be used for functionalizing magnetically responsive nanoparticles 250. Next and referring now to
At a step 530, in-solution processes and droplet operations may be used for deactivating magnetically responsive nanoparticles 250. Deactivation may be performed to convert any remaining active binding sites on the magnetically responsive nanoparticles 250 in LSPR droplet 142 into non-active sites. For example, a “blocking” solution, such as ethanolamine, may be used to react with any remaining COOH sites and deactivate them. For example, a droplet of ethanolamine may be combined or merged with LSPR droplet 142. For example, by merging the LSPR droplet 142 (with the magnetically responsive nanoparticles 250 therein) and the ethanolamine droplet for a period of time, the LSPR droplet 142 may be deactivated. Then, certain washing operations may occur by exchanging multiple buffer droplets.
With reference now to
In this process, the sample droplet 140 may need to be swapped out periodically with a fresh sample droplet 140 in order to avoid depletion effects in the data (i.e., the concentration of an analyte in the bulk phase decreasing because it is binding to the surface). Alternatively, the swapping out procedure could be triggered through real-time monitoring of some measurable property of the droplet or its contents. For example and referring now to
Referring now again to both
The process shown in
Further, the process shown in
Further, in the processes shown in
At a step 610, PR system 100 may be provided that includes DMF cartridge 110 that supports in-solution LSPR sensing processes that use droplet operations for analysis of analytes.
At a step 615, droplet operations may be used in DMF cartridge 110 to prepare LSPR droplets for analysis of analytes using in-solution LSPR sensing. For example, LSPR droplets 142 may be functionalized according to in-solution process 500 shown and described with reference to
At a step 620, the method 600 may include beginning assay protocol by providing the functionalized LSPR droplet and the sample droplet in the DMF cartridge and then using droplet operations to combine or merge the LSPR droplet and the sample droplet. For example and referring now to
At a step 625, the method 600 may comprise continuing the assay protocol by periodically swapping out the sample droplet with a fresh sample droplet in order to avoid depletion effects and to capture the affinity data and the association phase data. For example and referring now to
At a step 630, the method 600 may continue assay protocol by periodically swapping out the sample droplet with a buffer droplet and capture the dissociation phase data. For example and referring now again to both
At a step 635, the method 600 may continue assay protocol by repeating steps 620, 625, and 630 using different concentrations of analyte (usually 3× the previous one) in sample droplets 140. This is typically done for at least three analyte concentrations, which may be used to perform the kinetic analysis.
At a step 640, the sensor data from the LSPR droplets 142 of the in-solution LSPR sensing processes may be processed, and the KON value, KOFF value, KD value, and/or affinity of the analyte of interest is determined. For example, using controller 150 of PR system 100, the sensor data from the LSPR droplets 142 may be processed by fitting a binding model to the data and using a regression to find the KON value, KOFF value, KD value, and/or affinity of the analyte of interest that best represents the experimental data. This can be accomplished using a data set that includes, for example, the at least three analyte concentrations described in step 635.
A feature of method 600 that uses in-solution LSPR sensing processes and droplet operations for analysis of analytes may include the fact large numbers (e.g., hundreds) of droplets can be processed simultaneously using PR system 100 and DMF cartridge 110. Additionally, the in-solution LSPR sensing processes in DMF cartridge 110 may allow one to easily test against multiple analytes and/or concentrations with high consistency because LSPR droplets 142 functionalized according to in-solution process 500 of
Further, another feature of the in-solution process 500 of
Further, another feature of in-solution process 500 of
Further, another feature of in-solution process 500 of
Further, another feature of in-solution process 500 of
With reference now again to
Referring again to
This experiment proves 2 points: (1) that the optical fiber has LSPR active gold nanoparticles on its tip. The sensor can detect the refractive index shift from water to both 1% and 2% glycerol. Furthermore, the difference in signal between 1% and 2% is linear as expected; and (2) that the oil environment (a refractive index shift over 30× higher than 2% glycerol) does not interfere with the measurements.
Previously, the point has been made that the generally hydrophilic LSPR sensor retains an aqueous layer that protects the sensor against the oil environment. Despite a 2-second exposure to the oil medium in between each solution exchange, no effect of this exposure is noted in the data. This is due to the short-interaction distance of the LSPR sensor being within the retained aqueous layer.
A proof of concept experiment for the activation of the LSPR optical fiber with ligand was performed. In this proof of principle experiment, a COOH-surfaced LSPR sensor on a fiber was embedded into a DMF device, and a ligand immobilization procedure was performed. This process, described below, consisted of activation of the COOH surface with EDC+NHS, attachment of Protein A to this sensor surface, the introduction of ethanolamine to passivate unreacted surface sites and finally the introduction of human IgG that binds to Protein A to verify the ligand immobilization.
In this experiment, an optical fiber with a COOH-surfaced LSPR sensor on its tip was inserted from the side into a DMF device. The DMF device then performed commands to expose the fiber tip to the following samples. Each step, unless specified otherwise, consisted of dispensing 600 nL of the chemical in question and moving that volume to the fiber tip. During the exposure time, the device oscillated the liquid at a rate of 4 Hz. The following procedure was performed:
The results of this experiment are shown in
Another proof of concept experiment for the activation of the LSPR optical fiber with ligand was performed. In this proof of principle experiment, a Protein A-activated LSPR sensor was inserted into a DMF device and introduced to a series of concentrations of analyte with a regeneration step in-between. This allows for the determination of the analyte binding kinetics.
In this experiment, an optical fiber with a Protein A activated LSPR sensor on its tip was inserted from the side into a DMF device. The DMF device then performed commands to expose the fiber tip to the following samples. In this experiment, each step consisted of a 600 nL sample that was oscillated at 10 Hz. The following procedure was run:
The results of this experiment are shown in
With reference now to
Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, a reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.
Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include,” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments ±100%, in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.
Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.
This application is a § 371 national phase filing of PCT/IB2019/057540 entitled “PLASMON RESONANCE (PR) SYSTEM AND INSTRUMENT, DIGITAL MICROFLUIDIC (DMF) CARTRIDGE, AND METHODS OF USING LOCALIZED SURFACE PLASMON RESONANCE (LSPR) FOR ANALYSIS OF ANALYTES” filed on 6 Sep. 2019, which is related to and claims benefit of priority to U.S. Provisional Patent App. No. 62/727,934, entitled “PLASMON RESONANCE (PR) SYSTEM AND INSTRUMENT, DIGITAL MICROFLUIDIC (DMF) CARTRIDGE, AND METHODS OF USING LOCALIZED SURFACE PLASMON RESONANCE (LSPR) FOR ANALYSIS OF ANALYTES” filed on 6 Sep. 2018 and U.S. Provisional Patent App. No. 62/854,103, entitled “PLASMON RESONANCE (PR) SYSTEM AND INSTRUMENT, DIGITAL MICROFLUIDIC (DMF) CARTRIDGE, AND METHODS OF USING LOCALIZED SURFACE PLASMON RESONANCE (LSPR) FOR ANALYSIS OF ANALYTES” filed on 29 May 2019, the entire disclosures of each of the foregoing is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2019/057540 | 9/6/2019 | WO | 00 |
