Plasmon resonance (PR) system and instrument, digital microfluidic (DMF) cartridge, and method of using localized surface plasmon resonance (LSPR) for analysis of analyte

Abstract

To provide immunoassays using SPR technology which is inexpensive and does not require complex assays.SOLUTION: A plasmon resonance (PR) system and instrument, a digital microfluidic (DMF) cartridge, and a method of using localized surface plasmon resonance (LSPR) and droplet operations for analysis of an analyte are disclosed. For example, a PR system is provided which may include a DMF cartridge which may support both fixed LSPR sensing capability and in-solution LSPR sensing capability for analysis of the analyte. The DMF cartridge may include an electrode arrangement for performing droplet operations, wherein the droplet operations may be used for performing fixed LSPR sensing operations and in-solution LSPR sensing operations. Further, a method of using droplet operations in the DMF cartridge to perform fixed LSPR sensing operations and / or in-solution LSPR sensing operations is provided.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) The subject matter of this application is “PLASMON RESONANCE (PR) SYSTEM,” filed on September 6, 2018. AND INSTRUMENT, DIGITAL MICROFLUIDIC (DMF) CARTRIDGE, AND METHODS OF USING LOCAL US Provisional Patent No. 5,333,363, entitled “Luminescent Plasmon Resonance (LSPR) for Analysis of Analytes.” Application No. 62 / 727,934 and “PLASMON RESONANCE (PR)” filed on May 29, 2019 SYSTEM AND INSTRUMENT, DIGITAL MICROFLUIDIC (DMF) CARTRIDGE, AND METHODS OF USI NG LOCALIZED SURFACE PLASMON RESONANCE (LSPR) FOR ANALYSIS OF ANALYTES” This application is related to and claims priority from U.S. Provisional Patent Application No. 62 / 854,103, which is hereby incorporated by reference in its entirety. No. 6,393,636, which are incorporated herein by reference in their entirety. (Technical field)

[0002] The presently disclosed subject matter generally relates to detection of molecules, such as DNA, proteins, drugs, etc. More specifically, plasmon resonance (PR) systems and instruments, digital microscopy DMF cartridges, as well as localized surface plasmon resonance (LS) for the analysis of analytes. PR) and droplet manipulation. [Background technology]

[0003] In conventional assays, protein or DNA arrays are prepared containing labeled target biomolecules. The solution is injected, incubated overnight, rinsed, and then a fluorescent detection method is used to "read out" the cells. This is not only time consuming but also requires high sample concentration. Direct label-free detection techniques exist, such as selective plasmon resonance (SPR). The method has low sensitivity and throughput, making it unsuitable for detecting very low concentrations of target analytes Specifically, SPR technology has some drawbacks. For example, Such immunoassays are expensive and require complex microfluidic systems and high-precision optics. may require complex assays and is a niche technology with few experts .

[0004] SPR technology can be incorporated, for example, into a DMF cartridge. The droplet manipulation in the groove is carried out by using bulking fluids (e.g., silicone oil and hexadecane filling fluid). In another example, droplet manipulation in a DMF cartridge can occur with empty It can occur in the air, where the droplets have a thin oil coating (or oil shell) on them. Further embodiments of this technique may use droplet manipulation in air without an oil shell. do. Summary of the Invention

[0005] A first aspect provides a cartridge for use with an instrument to perform fluid measurements. The cartridge is operable to perform droplet operations on the fluid droplets in the DMF portion. A digital microfluidic (DMF) portion having a plurality of droplet actuators for detecting droplets of liquid; a reaction portion having a sensor medium disposed relative to the droplet actuator; The droplet actuator of the present invention is adapted to move a fluid relative to the sensor medium while in contact with the sensor medium. It acts to induce movement of the droplets.

[0006] Many improvements and additions to the first aspect are possible. The additional features may be used individually or in any combination. Each may be used with other features or combinations of features of the first aspect, but is not required. There is none.

[0007] For example, in one example, a plurality of droplet actuators may include reaction electrodes. The poles can perform droplet manipulation by electrowetting.

[0008] In one example, the sensor medium of the cartridge is a surface plasmon resonance (SPR) sensor medium. The SPR sensor medium may include a body in which target molecules in the sample fluid bind to the SPR sensor medium. The capture molecules can be functionalized with capture molecules that alter the optical signal of the body. The capture molecules react with target molecules in the sample fluid. It is highly sensitive to the binding of The SPR sensor medium may include a ligand immobilized thereon that alters the optical signal. The change can be a change in the optical signal due to light interacting with the SPR sensor medium.

[0009] In one example, the cartridge is disposed in the reaction section and is configured to include an SPR cell in association with a plurality of reaction electrodes. The SPR sensor medium may be disposed on the SPR sensor surface, and the liquid may include a The droplets can be engaged in contact with the SPR sensor surface by the action of a plurality of reaction electrodes. The SPR sensor medium is one of the nano-sized structures distributed on the sensor surface, or The reaction portion may include a continuous film having a first spaced apart feature. and a second substrate defining a reaction chamber therebetween. The surface may be disposed on a first substrate, and the plurality of reaction electrodes may be disposed on a second substrate opposite the first substrate. Alternatively, the SPR sensor surface may be disposed on a first substrate, with multiple counter electrodes. The reaction electrode can be disposed on a first substrate.

[0010] In one example, the SPR sensor surface is an optical member comprising at least one optical fiber. The optical member may be disposed adjacent to the end portion. The optical member may extend beyond the reaction chamber to position the SPR sensor surface within the reaction chamber. The optical member may include a first optical fiber through which the optical signal is transmitted from the SPR sensor surface. may include a second optical fiber through which light from the illumination source is provided to the SPR sensor surface. The optical member is disposed at an extended position where the SPR sensor surface is disposed within the reaction chamber, and at an extended position where the SPR sensor surface is disposed within the reaction chamber. Place the SPR sensor surface during retraction, where the sensor surface is not placed within the reaction chamber. In one example, the reaction chamber can be movable relative to the first substrate to fill the reaction chamber. The optical elements are arranged to reduce contact between the SPR sensor surface and the filling medium. It may be possible to store the

[0011] In one example, the SPR sensor surface is disposed between a first reaction electrode and a second reaction electrode. The first reaction electrode and the second reaction electrode are alternately activated to obtain a first reaction electrode and a second reaction electrode. Induce the vibration of the droplet between the second reaction electrode and the movement of the fluid droplet against the SPR sensor surface. The oscillation of the droplet between the first and second reaction electrodes can be linear. Alternatively, the SPR sensor surface can be disposed between three or more reaction electrodes. The reaction electrodes are alternately activated to induce droplet oscillation among three or more reaction electrodes, and the SPR sensor The movement of the fluid droplet relative to the sensor surface can then be induced. The oscillation of the droplet between the poles may be circular.

[0012] In one example, the sensor medium comprises a plurality of sensors suspended in a sensor droplet disposed in a reaction portion. The fluid droplet may include a sensor nanoparticle. The fluid droplet may assimilate with the sensor droplet to detect SPR in the reaction droplet. A reaction droplet may be formed for measuring an optical signal of the sensor medium. The motion induced by the actuator can operate to mix the reaction droplets.

[0013] In one example, each of the plurality of sensor nanoparticles is magnetically responsive. Each of the sensor nanoparticles may include a magnetically responsive core. Each of the nanoparticles can include a magnetic response element tethered to a sensor nanoparticle. The element may be physically or chemically bound to the sensor nanoparticles.

[0014] The cartridge then also acts on the magnetically responsive sensor nanoparticles to The molecules are immobilized in the reaction area, and multiple nanoparticles are placed in the inhibition position relative to the magnet. The droplet actuator may also include a magnetically responsive sensor. The fluid is directed away from the sensor nanoparticles when the nanoparticles are immobilized in the inhibiting position by a magnet. The magnetic sensor nanoparticles are immobilized at the inhibition position by the magnet. It may sometimes be operable to move the fluid into contact with the sensor nanoparticles.

[0015] In one example, the movement of the fluid droplet is reflected by the sample of an optical system that measures the optical signal of the sensor medium. This is faster than the ring rate.

[0016] In one example, the cartridge is operable to supply fluid to a plurality of droplet actuators. The cartridge may include a plurality of droplet manipulation electrodes in the DMF portion. A reservoir electrode may be included in the DMF portion to receive and hold the droplet. may include at least one of assimilation of the droplets, division of the droplets, distribution of the droplets, or dilution of the droplets. do.

[0017] In one example, the fluid droplet may include an analyte fluid droplet, which is detected while in contact with the sensor medium. The movement of the analyte fluid droplet relative to the sensor medium is determined by the effective diffusion of the analyte fluid droplet relative to the sensor medium. The effective diffusion rate of the analyte fluid droplet can be determined by the binding rate of the analyte to the SPR sensor. It can be faster than 100 times faster.

[0018] In one example, the cartridge includes electrical contacts in electrical communication with a plurality of droplet actuators. The electrical contacts interface with a controller for controlling the plurality of droplet actuators. The cartridge may comprise a pluggable interface for the cartridge that includes electrical contacts. The pluggable interface may include a controller and a A device that is physically and electrically connected to the device to establish electrical communication between at least one electrode. It may be possible to combine

[0019] In one example, the reaction moiety facilitates real-time optical measurement of the sensor medium in reflectance mode. For ease of understanding, the illumination source incident on the reaction portion on at least one side of the reaction portion is substantially Alternatively, the reactive moiety can be transparent to the real-time flow of the sensor medium in a transmission mode. To facilitate optical measurement of the The transparent material may be substantially transparent.

[0020] In one example, the fluid droplets can be analyte fluid droplets, and the SPR sensor medium can be and operable to detect the analyte affinity of the analyte fluid droplet during movement of the analyte fluid droplet relative to the The analyte affinity is the analyte affinity value (K D ) can be characterized by K D The SPR Center The on-rate (K ON ) and SPR sensor The off-rate (K OFF ) may be determined based on the

[0021] Another aspect of the present disclosure includes a plasmon resonance (PR) system. The PR system includes a first A cartridge according to the first aspect, including any of the examples discussed above in relation to the first aspect. The system also includes a PR device with which the cartridge is engageable. The PR device includes: A controller in operative communication with the electrical contacts for controlling the plurality of droplet actuators. and an optical detection system operative to measure an optical signal of the sensor medium.

[0022] Several 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. Each of the following features may be used in conjunction with other features or combinations of features of the second aspect. It is possible, but not required.

[0023] In one example, the optical detection system may include an illuminator that operates to direct light incident on the sensor medium. It further comprises a light source and an optical measurement device that measures the optical signal of the sensor medium.

[0024] In the PR system of the second embodiment, the fluid droplets may be analyte fluid droplets, and the controller on the optical signal of the sensor medium in the presence of an analyte fluid droplet while moving relative to the sensor medium. The controller is operable to detect target molecules in the analyte fluid droplet based on the analyte While the fluid droplet is moving relative to the sensor medium, the analyte is detected based on the optical signal of the sensor medium. It may operate to measure binding events of target molecules in fluidic droplets in real time.

[0025] Therefore, the controller determines the analyte affinity value (K D Quantitative measurement of analyte affinity with K D is the onlay measured during the assembly phase of the sensor medium. T(K ON ) and the off-rate (K OFF ) based on The fluid droplets in the reaction zone may be analyte fluid droplets during the association phase, and the reaction zone may be The second fluid may be a buffer fluid (e.g., a buffer fluid droplet) during the dissociation stage.

[0026] A third aspect includes a method of operating a cartridge for measuring an analyte fluid, the method comprising: , involving contacting a sensor medium in a reaction portion of the cartridge with a fluid droplet of the analyte. The method also includes subjecting a fluid droplet of the analyte to a sensor medium while the fluid droplet is in contact with the sensor medium. The method includes inducing the movement of a fluid droplet of the analyte disposed relative to the sensor medium. The method also includes actuating a plurality of droplet actuators to detect an analyte relative to the sensor medium. generating a first optical signal in the sensor medium during movement of the fluid droplet.

[0027] Some improvements and additional features are applicable to the third aspect. The improvements and additional features may be used individually or in any combination. Each of the following features described may be used in conjunction with other features or combinations of features of the third aspect. can be used, but is not required.

[0028] For example, in one example, the first optical signal may be indicative of association of a sensor medium in the presence of a fluid droplet of an analyte. The method may also include determining the presence or absence of a fluid of the analyte based on the association signal. Droplet on-rate (K ON ). ON The determination of the association curve This may include adapting the signal.

[0029] In one example, the method also includes detecting when the analyte fluid droplet is no longer in contact with and engaging the sensor medium. The method also includes removing the analyte fluid droplet from the reaction portion such that the analyte fluid droplet is The method may include introducing a liquid fluid droplet into the reaction portion. The buffer solution is in contact with the sensor medium. This method may also involve combining the buffer fluid droplet with the sensor medium while keeping the buffer fluid droplet in contact with the sensor medium. The sensor may include inducing a movement of the buffer fluid droplet relative to the sensor medium. The method may also include operating a plurality of droplet actuators disposed relative to the medium. generating a second optical signal at the sensor medium during movement of the buffer fluid droplet relative to the sensor medium; The second optical signal may include a signal corresponding to a dissociation stage of the sensor medium in the presence of the buffer fluid droplet. The method then comprises determining whether or not to off-rate the analyte fluid based on the dissociation signal. T(K OFF ). OFF To determine the dissociation rate, a dissociation curve is fitted to the dissociation signal. In one example, the method of the third embodiment may include: ON and K. OFF Based on the specimen Affinity value (K D ). K D is K ON and K. OFF It can be a quotient of.

[0030] In another example, the sensor medium can be a plurality of sensor nanoparticles disposed in the reaction portion. A plurality of sensor nanoparticles may be disposed in the sensor droplet. In this regard, the method In addition, the sample fluid droplet and the sensor are merged to measure the optical signal of the sensor medium in the reaction droplet. The method may also include forming a reaction droplet for determining the concentration of the reaction mixture.

[0031] In one example, each of the plurality of sensor nanoparticles can be magnetically responsive. The method includes activating a magnet to place the nanoparticles in a restraining position relative to the magnet to detect the sensor nanoparticles. The nanoparticles may be immobilized in the reaction area. The device may be maintained in a restrained position relative to the magnet while the device is in motion.

[0032] In one example, the sensor medium can be disposed adjacent to an end portion of the movable member. a movable member relative to a reaction chamber defined in the reaction portion between an extended position and a retracted position; The sensor medium may be disposed within the reaction chamber in an extended position. , can be removed from the reaction chamber in the retracted position. The reaction chamber can contain a loading medium. The method then includes storing the movable member in a stored position, and depositing a fluid droplet on the reaction portion after storing. introducing a pressure sensor to displace the fill medium from an area adjacent to the plurality of droplet actuators; and and then advancing the movable member to position the sensor medium within the fluid droplet. and,

[0033] In one example, the method includes engaging the cartridge with the instrument. , while maintaining the fluid droplet in contact with the sensor medium, measuring a signal from the sensor medium while the sensor medium is moving relative to the sensor. The medium can be an SPR sensor medium, and the signal is an optical signal of the SPR sensor medium. The method then includes providing light from a light source of the instrument that is incident on the SPR sensor medium. The measuring step may include measuring an optical signal of the SPR sensor medium with an optical measuring device of the instrument. This may include determining

[0034] In one example, the method also includes controlling a controller of the instrument and a plurality of droplet actuators of the DMF portion. The DMF section is connected to the power supply and the power supply is connected to the power supply. The induced movement of the fluid may be in response to control of a plurality of droplet actuators in the DMF portion. In one example, during the first period, the fluid may be a buffer fluid droplet, and the measurement may be performed by a sensor. The baseline as the buffer fluid moves relative to the sensor media while maintaining contact with the media The method also includes disposing the analyte fluid droplet in the reaction zone for a second period of time and recording a light signal. Measuring the association phase of the analyte fluid droplets during a second period of time can include introducing the analyte fluid droplets into the second period of time. and capturing an association signal corresponding to the effective spreading of the analyte fluid droplet relative to the sensor medium. The diffusion rate can be higher than the binding rate of the analyte fluid droplets to the sensor medium. is the on-rate of the analyte fluid droplet based on the association signal (K ON ) determining K ON Determining may include fitting an association curve to the association signal.

[0035] In a further example, the method further comprises moving the analyte fluid droplet away from the sensor medium for a third period of time. and introducing a buffer fluid droplet into the reaction portion at a time period, wherein the measurement is performed at a time period of The method includes capturing a dissociation signal corresponding to a dissociation stage of the analyte in a sample solution. Based on the off-rate of the analyte fluid (K OFF ). OFF Decision may include fitting a dissociation curve to the dissociation signal. ON and K. O FF Based on the analyte affinity value (K D ) D is K ON and K. OFF In business with could be.

[0036] The method also includes, during a fourth period, providing a regeneration buffer fluid droplet to the reaction portion to regenerate the This may include contacting a droplet of buffer fluid with the sensor medium to regenerate the sensor medium. The method of claim 1 further comprises functionalizing the sensor medium by contacting the sensor medium with a functionalizing fluid droplet comprising a ligand; The method may include binding a ligand to the sensor medium. Prior to functionalization, the sensor medium is activated by contacting an activation fluid droplet with the sensor medium. The method may include activating the

[0037] This Summary presents a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is provided to introduce the main features or essential features of the claimed subject matter. It is not intended to be specific and is not intended to be used to limit the scope of the claimed subject matter. It is not intended to

[0038] Other implementations are also described and listed herein. [Brief description of the drawings]

[0039] Having thus described the presently disclosed subject matter in general terms, the following drawings are not necessarily to scale. Please refer to the attached drawings, which are not drawn to scale. [Figure 1] FIG. 1 shows a block diagram of an embodiment of the presently disclosed PR system including a DMF cartridge that includes an LSPR sensing mechanism for analysis of an analyte. [Diagram 2] FIG. 2 shows a perspective view of an example of a DMF cartridge of the PR system shown in FIG. [Diagram 3] FIG. 1 shows a schematic diagram of an embodiment of a LSPR sensor in a presently disclosed DMF cartridge for analysis of an analyte. [Figure 4] FIG. 1 shows a schematic diagram of an embodiment of a LSPR sensor in a presently disclosed DMF cartridge for analysis of an analyte. [Figure 5A] 3A-3C show cross-sectional views of a portion of the embodiment of the DMF cartridge shown in FIG. 2 and examples of different configurations of LSPR sensors in relation to droplet manipulation electrodes. [Figure 5B] 3A-3C show cross-sectional views of a portion of the embodiment of the DMF cartridge shown in FIG. 2 and examples of different configurations of LSPR sensors in relation to droplet manipulation electrodes. [Figure 5C] 3A-3C show cross-sectional views of a portion of the embodiment of the DMF cartridge shown in FIG. 2 and examples of different configurations of LSPR sensors in relation to droplet manipulation electrodes. [Figure 6] 1 shows a top view of an example of a single LSPR sensor in association with multiple droplet manipulation electrodes, which is an example of a fixed LSPR sensing embodiment of the presently disclosed PR system and DMF cartridge. [Figure 7] 1 shows a top view of an example of a single LSPR sensor in association with multiple droplet manipulation electrodes, which is an example of a fixed LSPR sensing embodiment of the presently disclosed PR system and DMF cartridge. [Figure 8] 1 shows a top view of an embodiment of multiple LSPR sensors associated with multiple droplet manipulation electrodes, which is another example of fixed LSPR sensing of the presently disclosed PR system and DMF cartridge. [Figure 9] 1 shows a top view of an embodiment of multiple LSPR sensors associated with multiple droplet manipulation electrodes, which is another example of fixed LSPR sensing of the presently disclosed PR system and DMF cartridge. [Figure 10] 10A-10B show side views of an embodiment using a fiber optic probe and LSPR sensor for fixed LSPR sensing in a DMF cartridge. [Figure 11] 11A-11B show side views of another embodiment using a fiber optic probe and LSPR sensor for fixed LSPR sensing in a DMF cartridge. [Figure 12] 12A-12B show side views of yet another embodiment using a fiber optic probe and LSPR sensor for fixed LSPR sensing in a DMF cartridge. [Figure 13]FIG. 13A shows a side view of an example of a fiber optic probe and LSPR sensor provided through the bottom substrate of a DMF cartridge. FIG. 13B shows a side view of an example of a fiber optic probe and LSPR sensor provided through the side of a DMF cartridge and between the bottom substrate and the top substrate. FIG. 13C shows a side view of an example of using robotics to move a fiber optic probe and LSPR sensor into and out of a DMF cartridge. FIG. 13D shows a side view of an example of using robotics to move a fiber optic probe and LSPR sensor into and out of a DMF cartridge. [Figure 14A] 13A-13C show examples of different arrangements of multiple LSPR sensors for processing multiple droplets. [Figure 14B] 13A-13C show examples of different arrangements of multiple LSPR sensors for processing multiple droplets. [Figure 14C] 13A-13C show examples of different arrangements of multiple LSPR sensors for processing multiple droplets. [Figure 15] 13A-13C show examples of different arrangements of multiple LSPR sensors for processing multiple droplets. [Figure 16] 13A-13C show examples of different arrangements of multiple LSPR sensors for processing multiple droplets. [Figure 17] FIG. 1 shows a flow diagram of an example method of using a DMF cartridge, immobilized LSPR sensing process, and droplet manipulation for the analysis of an analyte. [Figure 18] FIG. 1 shows a block diagram of an example optical system of the presently disclosed PR system, including a DMF cartridge and an LSPR sensing mechanism for analysis of an analyte. [Figure 19] 1 shows a side view of a portion of a DMF cartridge and an embodiment of a process for LSPR sensing in solution. [Figure 20] 1 shows a side view of a portion of a DMF cartridge and an embodiment of a process for LSPR sensing in solution. [Figure 21]21A and 21B show side views of embodiments of magnetically responsive nanoparticles that can be used in in-solution LSPR sensing of the presently disclosed PR system and DMF cartridge. [Figure 22] FIG. 1 shows a flow diagram of an embodiment of an in-solution process for functionalizing magnetically responsive nanoparticles using droplet manipulation. [Diagram 23] 23A-23F show schematic diagrams of certain steps of the in-solution process shown in 22. [Figure 24] 24A-24D show an embodiment of an in-solution LSPR sensing process in a DMF cartridge, where droplet operations occur in air. [Diagram 25] 25A-25D show another embodiment of an in-solution LSPR sensing process in a DMF cartridge where droplet operations are performed in air with oil-covered droplets. [Figure 26] 26A-26D show yet another embodiment of an in-solution LSPR sensing process in a DMF cartridge, where droplet operations occur in oil (or filler fluid). [Figure 27] FIG. 1 shows a flow diagram of an embodiment of a method using a DMF cartridge, an in-solution LSPR sensing process, and droplet manipulation for the analysis of an analyte. [Figure 28] FIG. 1 shows a side view of an embodiment of an optical detection system operating in transmission mode in conjunction with fixed LSPR sensing in a DMF cartridge. [Figure 29] FIG. 1 shows a side view of an embodiment of an optical detection system operating in reflectance mode in conjunction with fixed LSPR sensing in a DMF cartridge. [Diagram 30] FIG. 1 shows a side view of an embodiment of an optical detection system operating in transmission mode in connection with in-solution LSPR sensing in a DMF cartridge. [Diagram 31]FIG. 1 shows a side view of an embodiment of an optical detection system operating in reflectance mode in connection with in-solution LSPR sensing in a DMF cartridge. [Diagram 32] FIG. 1 shows a side view of an embodiment of a DMF cartridge including an optical aperture for use with an optical detection system. [Diagram 33] 1 shows an example plot of SPR response versus time for first dropping 1% glycerol in DI water, then dropping 2% glycerol in DI water. [Diagram 34] 1 shows an example of a plot of the peak positions for carboxyl gold fiber optic activation, protein A binding, ethanolamine blocking, and IgG binding. [Diagram 35] 13A-13C are example plots of Protein A / IgG experimental data from both a DMF instrument and OpenSPR, showing increased sensitivity and reduced variance. [Diagram 36] 1 shows an example plot showing the fitting of binding kinetics to DMF Protein A data. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The subject matter of the present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which: Some, but not all, embodiments are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and may be modified in any way without departing from the spirit and scope of the present invention. The embodiments described should not be construed as being limited to the embodiments described. Instead, these embodiments This disclosure is provided so as to satisfy applicable legal requirements. Numerous modifications and other embodiments of the disclosed subject matter are apparent from the foregoing description and the associated drawings. It will be readily apparent to one of ordinary skill in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented herein. Thus, the presently disclosed subject matter is not intended to be limiting unless specifically disclosed. The embodiments are not intended to be limiting, and modifications and other embodiments are within the scope of the appended claims. It should be understood that all such inclusions are intended.

[0041] In some embodiments, the presently disclosed subject matter relates to plasmon resonance (PR) systems and mechanisms. A digital microfluidic (DMF) cartridge and a localization table for the analysis of samples. Methods using surface plasmon resonance (LSPR) and droplet manipulation may be provided.

[0042] In one embodiment, the PR system can be a LSPR system. The system includes a DMF cartridge that further includes LSPR sensing capabilities for the analysis of analytes. DMF cartridges are generally used for assimilation, division, dispensing, dilution, transport, and other types of One of these DMF features can be used to facilitate droplet manipulation of the The application of may be sample preparation. Further, the DMF cartridge may be used for (1) e.g. Detection of specific molecules (e.g., target analytes) and / or chemicals in a sample; and 2) On-rate information, off-rate information, affinity information, avidity, aggregation, specificity information, Structural changes, thermodynamic parameters, and / or other data / information related to the molecule under investigation LSPR sensing means for analysis of analytes, such as real-time measurement of binding events to extract Another application of this embodiment may include investigating interactions between drugs and biomolecules. and / or to study the chemical nature of the polymer.

[0043] In some embodiments, the LSPR sensing capabilities of the DMF cartridge can be achieved using droplet manipulation. In other embodiments, the DMF card may include a fixed LSPR sensing operation that may be performed using a DMF card. The LSPR sensing capabilities of the trellis may include LSPR sensing in solution, which may involve the droplet itself. The LSPR sensing process can take place in and using droplet manipulation. In some embodiments, the LSPR sensing capability of the DMF cartridge can be used to process multiple droplets. The present invention may include various arrangements of multiple LSPR sensors for detecting a signal.

[0044] Some of the presently disclosed PR systems, PR devices, DMF cartridges, and methods In some embodiments, the diffusion or flow rate of the fluid at the LSPR sensor is faster than the binding rate. It is possible that LSPR sensing measures binding kinetics and is mediated by slow diffusion or flow kinetics. DMF cartridges allow for the use of droplet manipulation to, for example, The droplets are oscillated back and forth or in a circular motion to generate a flow in the stationary LSPR sensing process, and and / or generating a mixture during the LSPR sensing process in the solution, thereby generating a LSPR sensor. In another embodiment, the LSPR sensing process comprises: For example, LSPs for droplets to generate flows and / or to generate mixing. This may include vibrating the R sensor itself. The system, PR instrument, DMF cartridge, and method are described below. A continuous unidirectional flow of may not be necessary. Instead, the sample or analyte droplets may be , which can be rapidly manipulated with LSPR sensing elements using droplet manipulation to generate a recirculating flow.

[0045] The PR instrument in the PR system consists of a DMF cartridge, an optical detection system, and a control The optical detection system may include, for example, an illumination source and a and an optical measurement device. In some embodiments, the optical detection system In another embodiment, the optical detection system may operate in a reflection mode. The controller controls the droplets by activating / deactivating electrodes in the DMF cartridge. The controller may also be provided to control the overall operation of the PR system. Furthermore, droplet manipulation in a DMF cartridge can be used to control immobilized LSPR sensing. Methods for performing sampling and / or in-solution LSPR sensing operations may be provided.

[0046] Some of the presently disclosed PR systems, PR devices, DMF cartridges, and methods In some embodiments, the bulking fluid (e.g., silicone oil or hexadecane-filled fluid) is When oil (such as oil from the body) is present and / or when droplets covered with oil are present, fixed LS LSPR sensing and / or LSPR sensing in solution can be used to prevent oil contamination of the LSPR sensing element. can be designed to be minimal.

[0047] In some embodiments, the presently disclosed PR systems, PR devices, DMF carts, ridge, and the method comprises: D Value, K ON Value, and and / or K OFF can be used to determine the value of K D The value is the affinity between the analyte and the ligand. is a quantitative measure of ON The value indicates the dynamic on-rate of binding, K OFF The value is the dynamic option of the binding. Indicates the freight.

[0048] In some embodiments, the immobilized LSPR sensor has nanostructures immobilized thereon. The nanostructures may include a surface having a nanoparticle size of 100 nm, where the nanostructures may be functionalized with capture molecules such as ligands. The sample droplet, with the target analyte suspended in it, is then brought into contact with the immobilized LSPR sensor. The target analyte may be a binding partner with the capture molecule of the immobilized LSPR sensor. In addition, optical detection systems can be used to obtain real-time kinetic measurements of the binding process (e.g., For example, the association stage (i.e., K ON value), dissociation phase (i.e., K OFF value), and analyte affinity (i.e., K D value).

[0049] In some embodiments, the LSPR sensing process in solution comprises: (1) detecting a nanostructure at its where the nanostructures are functionalized with capture molecules such as ligands, L (2) a target analyte is suspended therein, where the target analyte is in an SPR droplet (3) a sample droplet that can be a binding partner for the capture molecule of the capture molecule (such as a ligand); Droplet manipulation is used to allow binding to occur between the target analyte in the assimilated droplets (e.g., ELISA). (4) a process of assimilating (and / or mixing) the LSPR droplets with the sample droplets using light A biological detection system can be used to obtain real-time kinetic measurements of the binding process (e.g., The association phase (i.e., K ON value), dissociation phase (i.e., K OFF value), and analyte affinity ( Nawaki, K D and obtaining a value of the

[0050] In some embodiments, the LSPR sensing process is carried out in a solution by suspending the LSPR droplet. The nanostructure can be a magnetically responsive nanostructure.

[0051] FIG. 1 includes a DMF cartridge that may include an LSPR sensing mechanism for the analysis of an analyte. 1 shows a block diagram of an embodiment of the presently disclosed PR system 100. The PR system 100 may be a LSPR system, where the PR system 100 comprises: A DMF cartridge 110 is included that further includes LSPR sensing capability for analysis of an analyte. In the PR system 100 for the analysis of an analyte, the analysis may include, for example, detection, identification, Quantifying or measuring, and / or determining the interaction of an analyte with other substances, such as binding kinetics Exemplary analytes include small molecules, proteins, peptides, antibodies, nucleic acids, atoms, ions, etc. The PR system 100 may include, but is not limited to, polyaniline, polymers, and the like. can be used to measure the binding kinetics of a ligand to a macromolecule such as a receptor.

[0052] The DMF cartridge 110 generally performs a variety of functions including droplet assimilation, splitting, dispensing, dilution, etc. One use of these DMF functions is for sample preparation. However, the DMF function can be used for other processes such as waste removal. The DMF cartridge 110 of the system 100 may be, for example, disposable and / or reusable. An example of a DMF cartridge 110 is shown in detail in FIG. As shown and described below with reference to:

[0053] Additionally, the DMF cartridge 110 may include a LSPR sensor 112. The detection 112 may include (1) detecting, for example, a particular molecule (e.g., a target analyte) in a sample and / or (2) detecting a specific molecule (e.g., a target analyte) in the sample. and / or (2) to provide on-rate information, off-rate information, and / or to measure real-time binding events to extract affinity information, etc. The LSPR sensing 112 can be used, for example, for fixed LSPR sensing and and / or any solution-based LSPR sensing process. In addition, fixed LSPR sensors The LSPR sensing in solution and oil contamination of the LSPR sensing element was minimized. The details of fixed LSPR sensing are described below with reference to Figures 5A through 18. For details of LSPR sensing in solution, see Figures 19 to 27. These are presented and explained below.

[0054] The discussion presented herein relates to SPR sensors (e.g., LS in certain embodiments). PR sensors), but instead of SPR or LSPR sensors, or In addition, it is contemplated that other sensors may be used. Sensor options include electronic, electrochemical, mechanical, or Other suitable sensor types may be included, such as biolayer interferometric or piezoelectric sensors. Any sensor may be used. In this regard, the sample / sensor interaction is described in the present specification. The interaction between the analyte and the sensor using the DMF performance described herein is generally consistent with any suitable The present invention may be applicable to the analysis of analytes using appropriate sensors.

[0055] The PR system 100 includes a controller 150, a DMF interface 152, an illumination source, 154, an optical measurement device 156, and a thermal control mechanism 158. The detector 150 includes the DMF cartridge 110, the illumination source 154, and the optical measurement device 155. 6. The PR system 100 may be electrically coupled to various hardware components of the PR system 100, such as the PR system 100 shown in FIG. In particular, the controller 150 receives a DMF cartridge via a DMF interface 152. The DMF interface 152 may be electrically coupled to the DMF card 110, for example. 1. A pluggable interface for mechanically and electrically connecting to the cartridge 110. Both include a DMF cartridge 110, a controller 150, and a DMF interface. The sensor 152, the illumination source 154, and the optical measurement device 156 may comprise the PR instrument 105. .

[0056] The controller 150 may be, for example, a general purpose computer, a special purpose computer, a personal computer, or the like. It may be a computer, microprocessor, or other programmable data processing device. The controller 150 stores, interprets, and / or executes software instructions. It may also provide processing functions such as controlling the overall operation of the PR system 100. The software instructions are accessible by the controller 150 for execution of the instructions. The controller 150 may include a machine-readable code stored in a non-transitory memory. configured and programmed to control data and / or power aspects of the device For example, with respect to the DMF cartridge 110, the controller 150 may activate the electrodes. In general, the controller 150 may control the droplet operations by activating / deactivating the droplets. The controller 150 may be used for any function of the PR system 100. For example, the controller 150 may DM in a manner similar to how printer manufacturers check branded ink cartridges. The DMF cartridge 110 may be used to authenticate the DMF cartridge. This can be used to ensure that the controller 110 is not out of date. The 150 can read and write DMF cartridges by implementing a specific protocol for that purpose. The present invention can be used to check the cleanliness of the edge 110, etc.

[0057] Additionally, in some embodiments, the DMF cartridge 110 may include a volumetric feedback For example, the signal may detect the position, velocity, and size of the droplet. It may be generated or detected by a capacitive sensor. Instead of volume feedback sensing, the DMF cartridge 110 can measure the droplet position, velocity, and The camera or other optical device may provide optical measurements of the lens, lens diameter, and size. This may trigger the controller 150 to reroute the droplet to the appropriate location. The feedback optimizes the droplet actuation speed and notifies the droplet operation completion. This can be used to create a closed loop control system to verify.

[0058] If necessary, the PR device 105 may be connected to a network. For example, the controller 1 50 may communicate with a network computer 160 via a network 162. The network computer 160 may be, for example, any centralized server or cloud server. The network 162 may be, for example, a local It can be a Local Area Network (LAN) or a Wide Area Network (WAN). .

[0059] In the PR system 100, the illumination source 154 and the optical measurement device 156 are LSPR sensing 112 (e.g., fixed LSPR sensing and / or soluble LSPR sensing) of the cartridge 110 The illumination source 154 may be, for example, a white light emitting diode. LEDs, halogen bulbs, arc lamps, incandescent bulbs, lasers, etc. The illumination source 154 may be a light source in the visible range (400-800 nm), but is not limited to: The illumination source 154 is not limited to a white light source. It can be any color light useful in the PR system 100. An optical measurement device 156 may be used to obtain readings of the LSPR light intensity. The optical measurement device 156 may be, for example, a charge-coupled device, a photodetector, a spectrometer, a photodiode, or a In addition, the PR system 1 may be a junction array, a diode array, or any combination thereof. 00 is not limited to only one illumination source 154 and one optical measurement device 156 . The LSPR system 100 may include multiple illumination sources to support multiple LSPR sensing elements. 154 and / or a plurality of optical measurement devices 156. The thermal control mechanism 158 may include: This can be any mechanism for controlling the operating temperature of the DMF cartridge 110. Examples of the mechanism 158 may include Peltier elements and resistive heaters.

[0060] FIG. 2 is a perspective view of an example of the DMF cartridge 110 of the PR system 100 shown in FIG. FIG. 1 shows a schematic diagram of a DMF cartridge 110 that may include a lower substrate 116 and an upper substrate 118. In one example, the lower substrate 116 is made of glass, plastic, or a thermoplastic elastomer. Materials that are virtually transparent to white light, such as the class of polymers known as thermoplastic elastomers (TPEs) In another example, the lower substrate 116 may be substantially transparent or may be light-transmitting. The substrate may be a printed circuit board (PCB) that includes holes or openings that allow for Additionally, a set of electrical contacts 138 may be provided at one or both ends of the lower substrate 116. Point 138 connects, for example, to a DMF interface 152, which in turn connects to a controller 150. Like the lower substrate 116, the upper substrate 118 may be made of glass, plastic, or other suitable material. The display may be formed of a material that is substantially transparent to white light, such as a plastic or TPE. The inner surface of the upper substrate 118 is made of indium tin oxide (ITO), poly(3,4-ethylene Dioxythiophene)polystyrenesulfonate (PEDOT:PSS), or other similar In another embodiment, the transparent conductive layer may be coated with a transparent conductive layer such as a transparent conductive coating of All areas of the DMF cartridge 110 are substantially transparent substrates and / or coatings. For example, the substrate and / or the coating or layer need not be included. may be transparent, translucent, and / or opaque, except for the detection area.

[0061] "top", "bottom", "over", "under", "middle" The terms "in" and "on" refer to the top and bottom substrates of the DMF cartridge. Used throughout the description to refer to the relative positions of components on a DMF cartridge, such as the relative positions of the It is understood that the DMF cartridge will function regardless of its orientation in space. sea ​​bream.

[0062] In the DMF cartridge 110, the gap between the lower substrate 116 and the upper substrate 118 The chamber may define a reaction (or assay) chamber 122. The assay chamber 122 is a chamber for delivering a liquid of interest, i.e., a liquid reagent, a buffer solution, etc., via droplet manipulation. , a lower substrate 11 for handling liquids such as, but not limited to, sample droplets, 6 and the upper substrate 118. Thus, the electrode configuration 124 can be The electrode configuration 12 may be provided on the lower substrate 116 within a (electrode or assay) chamber 122. 4 includes, for example, a droplet manipulation electrode 126 (e.g., an electrowetting electrode) and a relay For example, the electrode configuration 124 may include any number of reservoir electrodes 128. This may include any line or path of the droplet operations electrodes 126 in relation to the server electrodes 128.

[0063] The electrode configuration 124 is used to perform droplet manipulation via electrowetting. "Droplet manipulation" refers to the manipulation of droplets on a DMF device or cartridge. Droplet manipulation can include, for example, loading droplets onto a DMF device; Dispensing a droplet; splitting, separating, or dividing a droplet into two or more droplets; transporting two or more droplets into one droplet in any direction; or combining; diluting the droplets; mixing the droplets; agitating the droplets and; deforming the droplet; holding the droplet in place; and incubating the droplet. heating the droplets; vaporizing the droplets; cooling the droplets; disposing the droplets transporting droplets from the droplet actuator; and / or any combination of the above. a) A Peltier heat source is used to control the temperature of the process occurring within the chamber 122. A temperature control element (not shown), such as a pump, is used in combination with the DMF cartridge 110. It is possible.

[0064] Furthermore, FIG. 2 shows a method for manufacturing a droplet by electrowetting (for example, a droplet manipulation electrode 126 The DMF cartridge 110 is described as manipulating droplets (using are merely exemplary. In other embodiments, the droplets may be captured by optical methods, magnetic methods, thermocapillary methods, etc. Such as, but not limited to, the Lee method, the surface acoustic wave method, and any combination thereof. The DMF cartridge 110 may be manipulated through other methods not described above.

[0065] For example, an example of LSPR sensing 112 may be implemented with respect to a line or path of droplet manipulation electrodes 126. In one example, referring to detail A of FIG. 2, LSPR sensing 112 is performed using a DMF Associated with the droplet operations electrode 126 within or near the droplet operations gap of the cartridge 110 The LSPR sensor 136 may include an integrated LSPR sensor 136. The LSPR sensor 136 may include: For example, to detect specific molecules (e.g., target analytes) and / or chemicals in a sample. and / or (2) on-rate information, off-rate information, and / or affinity Used for analysis of samples, such as to measure binding events in real time to extract information. It is possible.

[0066] As described herein, "localized surface plasmon resonance (LSPR)" refers to a method for detecting surface plasmon resonance (SPR) in nanoparticles. Using either nanostructure-based or nanostructure-based transducers, This means observing binding events in a system that uses nanoparticle-based transducers, for example. The nanostructures may include metal nanoparticles of various dimensions ranging from about 1 nm to about 1000 nm. Structure-based transducers have nano-sized features (e.g., nano-sized protrusions, Some nanoparticle-based or nanostructure-based gold films may contain nanoparticles, holes, ridges, lines, etc. The diagnostic assays described herein are "label-free."

[0067] LSPR is a phenomenon associated with noble metal nanoparticles and is characterized by sharp spectral absorbance and scattering peaks. , producing a strong electromagnetic near-field enhancement. These spectral peaks are The spectral peak is the refractive index in the immediate vicinity of the nanoparticle surface. When a chemical target binds near the surface of a metal nanoparticle, The spectral peak shift occurs due to the change in refractive index due to the complex medium. It can be used to determine the concentration of a particular target. Alternatively, the peaks in the spectrum The shift can be detected through a change in absorption at a given wavelength.

[0068] The LSPR sensor may include metal nanoparticles, for example on a flat surface or a microstructured surface. The nanoparticles work by immobilizing specific capture molecules, which can be antibodies, on a solid support. The sample of interest is flowed over the top of the metal nanoparticles, and the target The target chemicals are bound to the respective capture molecules, and the overall spectral peak of the sensor is The shift occurs depending on the concentration of the chemical target on the capture molecule. The transducer works by flowing the sample vertically over a surface. Reflectance or transmission absorbance spectroscopy may be used to determine the concentration of the compound. The analysis by can be performed using an LSPR sensor. More details on an example of an LSPR sensor is shown and described below with reference to FIGS. 3 and 4.

[0069] 3 and 4 show a presently disclosed DMF cartridge 110 for the analysis of analytes. 1 shows a schematic diagram of an embodiment of an LSPR sensor 136. In general, LSPRs measure However, LSPR can be used with labels to enhance the signal. The basic structure of the assay can be a collection of individual nanostructures distributed on a surface, such as a LS A sensor chip (e.g., For example, LSPR sensors136) or nano-sized features are formed. Then, the two bonds One of the partners can be immobilized on the surface of the sensor. "Analyte" can refer to a binding partner that can be immobilized on the surface of a sensor. It can also refer to the flow of a solution over a ligand on the surface of a cell. When exposed to light, the optical properties of the sensor's surface change, which can be measured in real time.

[0070] In this example, the LSPR sensor 136 is mounted on a glass, plastic, or TPE substrate. The substrate 210 may include any substantially transparent or opaque substrate. When used in a reflecting mode configuration, the substrate 210 may be substantially transparent. When used in a 3D mode configuration, the LSPR sensor layer 212 may be opaque. 10. The LSPR sensor layer 212 may be provided on the substrate 10, for example. The LSPR sensor layer 212 may be a gold film containing specific nanostructures. The capture molecules 214 may be functionalized on the surface of the LSPR sensor layer 212. In one example, the capture molecules 214 may be functionalized on the surface of the LSPR sensor layer 212. In this example, the ligand is one of the two binding partners. and the other binding partner may be a target analyte 216, where target analyte 21 6 flows in solution over the capture molecule 214, as shown in FIG. 4 shows a target analyte 216 binding to a capture molecule 214. This binding is called a binding event. It can be called.

[0071] Referring again to FIG. 3, the adsorption of the LSPR sensor layer 212 before a binding event occurs. A plot 218 is provided showing the luminosity peaks. For example, plot 218 shows the luminosity peaks of the target detection The peak position before the body 216 binds to the capture molecule 210 in the LSPR sensor 136 or Here, as shown in FIG. 4, the intensity of the target analyte 216 bound to the capture molecule 214 is shown. Binding-induced changes in peak position or intensity can be observed in real time. For example, the peak position before binding (i.e., plot 218) is converted to the peak position after binding (i.e., In general, the LSPR sensor 13 In 6, when an analyte binds to the surface, a resonant peak of light is generated, measurable in real time. Shift to higher wavelengths.

[0072] Referring again to FIGS. 1-4, the presently disclosed PR system 100, In the instrument 105, and in the DMF cartridge 110, LSPR sensing 112 (e.g. , fixed LSPR sensors 136 and / or LSPR sensing in solution) The diffusion or flux rate may be faster than the binding rate, thereby limiting LSPR sensing. can measure the binding rate and ensure that it is not limited by slow diffusion or flow rates. In the MF cartridge 110, droplet manipulation can be used to move droplets back and forth or in a circular motion, for example. to generate a flow at the LSPR sensor and / or to generate a LSPR sensor in solution. The mixing process can generate mixing, thereby ensuring the flux of molecules to the surface of the LSPR sensor. Therefore, the presently disclosed PR system 100, PR device 105, and DM F cartridge 110 does not require continuous flow of sample over the LSPR sensor Alternatively, sample droplets can be rapidly manipulated onto the LSPR sensor using droplet manipulation. It can be done.

[0073] In the PR system 100, the PR device 105, and the DMF cartridge 110 of the present disclosure, Using droplet manipulation, droplets are rapidly moved back and forth in a linear or circular manner on the DMF surface. By moving the droplets, a flow can be artificially created. If the beam moves faster than the grating, the artifacts caused by this movement are almost invisible to the optical measurement. In another example, if a droplet spreads over multiple electrodes, ,Movementum is changed without moving the droplet from the sensor position, avoiding,movement artifacts. The following Figures 5A to 26D show the continuous flow of a sample at a LSPR sensor spot. Rather than using a liquid phase transition metal, the presently disclosed technique may utilize droplet manipulation to generate a stream. A fixed LSPR of the PR system 100, the PR device 105, and the DMF cartridge 110. Examples of both in-solution and in-solution LSPR sensing are presented and discussed.

[0074] (Fixed LSPR sensing in DMF) 5A, 5B, and 5C are embodiments of the DMF cartridge 110 shown in FIG. 1 shows a cross-sectional view of a portion of a droplet manipulation electrode 126 in accordance with a second embodiment of the present invention; FIG. 5A, to which reference is made, shows an example of a configuration in which the upper substrate 11 of the DMF cartridge 110 is 8. For example, the LSPR sensor 136 is a DM F. In the upper substrate 118 of the cartridge 110, the droplet operation electrodes 126 are arranged in an arbitrary manner. A sample (or specimen) droplet 140 can be placed on the opposite side of the lower substrate 116. The sample droplet 140 may be placed in the gap between the working electrode 126 and the upper substrate 118. The sample droplet 140 may be transported along the droplet operations electrodes 126 via droplet operations. The sample droplet 140 may contain any target analyte 216. The sample droplet 140 is contacted with the LSPR sensor 136. When activated, the illumination source 154 and optical measurement device 156 of the PR system 100 detects the LSPR The target analyte 2 in the sample droplet 140 is coupled to a capture molecule 214 (e.g., a ligand) of the sensor 136. 16 Real-time kinetic measurements (e.g., of the association phase (i.e., K ON value), dissociation stage (i.e. Wachi, K OFF value), and analyte affinity (i.e., K D can be used to obtain the .

[0075] Referring now to FIG. 5B, the lower substrate 116 of the DMF cartridge 110 is 1 is an example of an LPSR detection spot that is in a different layer or plane than the steering electrode 126. For example, LSPR sensor 136 is located within lower substrate 116 and at certain droplet manipulation electrodes 126. It can be placed on top.

[0076] Referring now to FIG. 5C, the lower substrate 116 of the DMF cartridge 110 is 1 is an example of an LPSR detection spot that is in the same layer or plane as the droplet manipulation electrode 126. For example, , the LSPR sensor 136 is a line between and having two droplet manipulation electrodes 126 It can be installed along

[0077] In the example shown in FIGS. 5A, 5B, and 5C, the lower substrate 11 of the DMF cartridge 110 6 and / or any LSPRs located in and / or near the upper substrate 118. The sensor 136 may be an example of LSPR sensing 112. In particular, the lower substrate 116 and and / or any LSPR sensor located in and / or near the top substrate 118. 136 may be an example of a fixed LSPR sensing 112 in a DMF cartridge 110. Additionally, fixed LSPR sensors136 measure binding kinetics and are not limited by slow diffusion or flow rates. To help increase the likelihood of detecting a signal that is not limited by the LSPR sensor 136, e.g., This is used in combination with the rapid movement of the sample droplet 140 when it is on the PR sensor 136. and this rapid movement is achieved via droplet manipulation.

[0078] In the examples shown in FIGS. 5A, 5B, and 5C, the position of the LSPR sensor 136 is The droplet operation electrodes 126 may be located approximately at the boundary between the droplet operation electrodes 126. Next, the two droplet operation electrodes 126 and the Using droplet manipulation, a sample droplet 140 is rapidly moved back and forth between two droplet manipulation electrodes 126. The LSPR sensor 136 may be moved back and forth rapidly across the surface of the LSPR sensor 136. Further, the sample droplet 140 may be, for example, sampled by a sampling radar of an optical measurement system. This allows the optical In one example, the optical sampling rate is about 4 Hz (about 2 50 ms), the sample droplets 140 are ejected at a rate faster than about 4 Hz (droplet manipulation The droplet may move back and forth between the two droplet manipulation electrodes 126 (via the

[0079] Additionally, moving the sample droplet 140 may: (1) cause the surface of the LSPR sensor 136 to (2) helps increase the flux of molecules to the surface, and (3) increases the binding rate without diffusion or flow limitations. 136 DMF cartridges help improve the likelihood of In this paper, droplet manipulation is used to generate LSPs, for example by oscillating the droplet back and forth or in a circular motion. Generate flow with R sensor and / or generate mixing for LSPR sensing process in solution In another embodiment, the LSPR sensor may be configured to selectively oxidize the LSPR molecules, thereby helping to increase the flux of molecules to the surface of the LSPR sensor. Alternatively, the sample droplet 140 may spread across two or more droplet operations electrodes 126. The actuation electrode 126 can be actuated at a rate faster than the bulk droplet can move away from the sensor location. , which may agitate the liquid and promote the flux of molecules as well. A more detailed example of using manipulation to capture real-time dynamic measurements of a sample droplet is shown in Fig. 5A through 17, as shown and described below.

[0080] 6 and 7 show a single LSPR sensor 13 associated with multiple droplet manipulation electrodes 126. 6 shows a plan view of an embodiment of the presently disclosed PR system 100 and D An example of a fixed LSPR sensing 112 in a MF cartridge 110. Now referring to FIG. Then, a single LSPR sensor 136 can be configured in a 2×2 configuration, for example, droplet manipulation electrode 126A, The four droplet manipulation electrodes 126 are arranged in 126B, 126C, and 126D. For example, the LSPR sensor 136 may be located approximately in the middle of a 2×2 configuration of the droplet manipulation electrodes 126. Additionally, the area of ​​the LSPR sensor 136 may be positioned such that it minimizes interference with the movement of the droplet. In order to minimize the size of the droplets, the area of ​​each droplet may be relatively small compared to the area of ​​the droplet manipulation electrodes 126. It is possible.

[0081] In operation, and referring to FIG. 7, each of the droplet operations electrodes 126 is activated one at a time. For example, droplet operations electrode 126A may be activated, followed by droplet operations electrode 126B, Then droplet operations electrode 126C, and then droplet operations electrode 126D, may be activated. Thus, the sample droplet 140 containing the target analyte 216 therein is liquid (using droplet operations) The droplet may be moved in a circular motion around the 2×2 configuration of droplet manipulation electrodes 126. The droplet 140 moves (using droplet manipulation) in a circular fashion around the surface of the LSPR sensor 136. In this example, the bottom area of ​​the sample droplet 140 may be It may be approximately equal to or greater than the base area (i.e., area). Thus, the sample droplet 140 is moved relative to which droplet manipulation electrode 126 it is placed on. Again, the LSPR sensor 136 may be contacted without the need for, for example, If the sample droplet 140 is moved back and forth rapidly at a rate faster than the sampling rate, (1 ) the flux of molecules to the surface of the LSPR sensor 136 may be increased; and (2) slow diffusion or flow rates. Without the limitations of Regarding the movement of the electrodes, for example, if the droplet has a base area about twice that of the unit electrode, By activating diagonal electrodes simultaneously, up-down and / or left-right movement is possible.

[0082] 8 and 9 show an embodiment of multiple LSPR sensors 136 associated with multiple droplet manipulation electrodes. 1 shows a plan view of an embodiment of the presently disclosed PR system 100 and DMF camera. 1 is another example of a fixed LSPR sensing cartridge 110. This configuration of the sensor 136 may allow for multiple measurements of sample droplets 140. Referring now to FIG. For reference, a 2×2 configuration of droplet operations electrodes 126, e.g., droplet operations electrodes 126A, 126 In addition, droplet operation electrodes 126A, 126B, 126C, and 126D may be provided. 26C, 126D are associated with corresponding LSPR sensors 136A, 136B, 136 For example, the LSPR sensor 136 may have a Again, the area of ​​each LSPR sensor 136 may be approximately centered. To minimize interference with the droplet manipulation electrodes, the area of ​​the droplet manipulation electrodes 126 is relatively small compared to the area of ​​the droplet manipulation electrodes 126. It's okay.

[0083] In operation, and referring now to FIG. 9, each of the droplet operations electrodes 126 is For example, droplet operations electrode 126A may be activated, followed by droplet operations electrode 126B. Electrode 126B, then droplet operations electrode 126C, and then droplet operations electrode 126D are activated. Thus, the sample droplet 140 containing the target analyte 216 therein is (by performing droplet operations ) to move in a circular fashion around the 2×2 configuration of droplet operations electrodes 126. , the sample droplet 140 is transferred (using droplet manipulation) from one LSPR sensor 136 to the next For example, LSPR sensor 136A, then LSPR sensor 136B, then L 13C, and then to the LSPR sensor 136D. Again, this requires sampling at a rate faster than the sampling rate of the optical measurement system, for example. Rapidly moving the droplet 140 back and forth induces (1) the attachment of molecules to the surface of the LSPR sensor 136. (2) the binding kinetics can be increased to the LSPR sensor without slow diffusion or flow rate limitations; The likelihood of being measured at 136 may increase.

[0084] With respect to the droplet patterns described above with respect to FIGS. 6 through 9, the droplet motion pattern is a circular The motion pattern is not limited to the above. Other motion patterns are possible. For example, the motion of a droplet The motion pattern can be X-shaped or random. In another example, a particular Certain droplet manipulation electrodes 126 may simply be pulsed on and off to accommodate certain LSPR sensors. The droplets may be agitated at 136.

[0085] 10A and 10B show fixed LSPR sensing 112 in a DMF cartridge 110. An embodiment using a fiber optic probe in combination with a LSPR sensor 136 for In this example, the LSPR sensor 136 is connected to a fiber optic probe 17. The fiber optic probe 170 can be mechanically and optically coupled to the tip of the LSP. The R sensor 136 is disposed between the droplet manipulation electrode 126 (on the lower substrate 116) and the upper substrate 118. The upper substrate 118 may be provided with a spacer 118a through which the optical measurement may extend into the gap. When the measurement system is operating in transmission mode, the fiber optic probe 170 is connected to the optical measurement device. The illumination source 154 may be coupled to a device 156, the illumination source 154 being disposed on the opposite side of the LSPR sensor 136. In another example, if the optical measurement system is operating in reflection mode, a fiber optic probe Lobe 170 may actually contain at least two optical fibers, at least the first The fiber couples to an illumination source 154 and at least another fiber couples to an optical measurement device 15 6. Alternatively, one fiber can be used for both illumination and readout. In this embodiment, the LSPR sensor 136 is can be constructed as follows.

[0086] 10A and 10B show a fiber optic probe 170 and a LSPR sensor 13. The sample droplet 140 is then inserted into the fiber optic probe 170 so that the sample droplet 140 can project into the fiber optic probe 170. 1 illustrates an embodiment of a process for transporting a sample droplet 140. Again, the sample droplet 140 is , which can be rapidly transferred via droplet manipulation to increase the flux of molecules to the surface of the LSPR sensor136. For example, the fiber optic probe 170 and the LSPR sensor 136 may be 6, 7, 8 and 9, the fixed LSPR sensing 112 is described above. It may be implemented in the following configuration.

[0087] In the process shown in Figures 10A and 10B, droplet operations can occur in air. For example, the gap between the lower substrate 116 and the upper substrate 118 may be filled with air. However, in other embodiments, the droplet operations in the DMF cartridge 110 are carried out in air. may occur, and the droplets may have a thin oil coating (or oil shell) on them. In yet another embodiment, the droplet operations in the DMF cartridge 110 are carried out using a bulking fluid (e.g. For example, low viscosity oils such as silicone oil or hexadecane fill fluids. For example, the gap between the lower substrate 116 and the upper substrate 118 is filled with a fill fluid. The disadvantage of having oil in the DMF cartridge is that it can cause oil However, in the PR system 100, the optical fiber The bar probe 170 and LSPR sensor 136 detect oil contamination at the LSPR sensing element. Mechanisms may be provided to minimize or eliminate altogether.

[0088] For example, referring now to FIGS. 11A and 11B, a sample droplet 140 may have thereon The sample droplet 140 may have an oil shell (or coating) 145 on it. When the fiber optic probe 170 is transported to the fiber optic probe 170, the LSPR sensor The sensor 136 passes briefly through the oil shell 145 and then into the water of the sample droplet 140. Since the surface of the LSPR sensor 136 is generally hydrophilic, the LSPR There is a tendency for a layer of water to exist on the surface of the R sensor 136, which resists oil contamination and makes it unreliable. In addition, this configuration can be used to facilitate the reading of LSPR sensors. The exposure time of the cellulose acetate-136 to the oil phase can be minimized.

[0089] As a further embodiment, now referring to FIGS. 12A and 12B, the lower substrate 11 The gap between the substrate 116 and the upper substrate 118 may be filled with a fill fluid 146. 6 can be a low viscosity oil such as silicone oil or hexadecane filling fluid Thus, droplet operations within the DMF cartridge 110 can occur in oil. When the sample droplet 140 is transported to the fiber optic probe 170, The lobe 170 and LSPR sensor 136 are fully oiled in the DMF cartridge 110 The LSPR sensor is then transferred from the aqueous environment to the completely aqueous environment of the sample droplet 140. Since the surface of the LSPR sensor 136 is generally hydrophilic, a water layer exists on the surface of the LSPR sensor 136. This helps to resist oil contamination and promotes reliable readings.

[0090] In another embodiment, the fiber optic probe 170 and the DMF cartridge 110 include: For example, the fiber optic probe 170 may be arranged for relative movement therebetween. is selectively displaced from the DMF cartridge 110 to expose the fiber optic probe 170. 13C and 13D. In this regard, the DMF cartridge between the lower substrate 116 and the upper substrate 118 While an oil environment is present within 110, the fiber optic probe 170 is inserted into the DMF cartridge 11. However, with the droplet in place, the lower substrate 116 and the upper substrate When an aqueous environment is present between the plate 118 and the fiber optic probe 170, the LSPR sensor The sensor 136 can be moved to a position such that it can be placed in the aqueous environment established by the droplet. In this regard, oil contamination of the LSPR sensor136 was observed when the optical fiber probe was used during droplet manipulation. This can be minimized by moving the head 170 out of position, in which case the DMF The area occupied by the fiber optic probe 170 when in the inserted position relative to the cartridge 110. However, the droplets are in place and the DMF cartridge 11 The area occupied by the fiber optic probe 170 when in the insertion position relative to 0 is an aqueous environment. The inclusion of the oil pressure sensor 136 causes the LSPR sensor 136 to experience little or no oil contact. In addition, a plurality of fiber optic probes 170 may be inserted into the 0 can be selectively introduced in this manner to provide new probes and / or This may allow the use of probes with different types of sensors.

[0091] Referring again to FIGS. 10A-12B, the fiber optic probe 170 and the L The SPR sensor 136 may be replaceable. For example, once the assay is complete, the optical fiber Remove the bar probe 170 and the LSPR sensor 136 and install a new fiber optic probe. The probe 170 and the LSPR sensor 136 are attached to the upper substrate 11 of the DMF cartridge 110. It can be reinserted through 8.

[0092] In the fixed LSPR sensing process shown in FIG. 5A to FIG. 12B, sample droplet 1 The analyte solution at 40 may be dispensed as needed to avoid accumulation of analyte in the sample droplet 140. If necessary, the buffer solution may be replaced (via droplet operations).

[0093] A fiber optic probe (e.g., a fiber optic probe) for the DMF cartridge 110 The configuration of the probe 170 is not limited to those shown in, for example, Figures 10A to 12B. For example, FIGS. 13A to 13D show a configuration for fixed LSPR sensing in a DMF cartridge 110. FIG. 1 shows a side view of yet another configuration of a fiber optic probe and LSPR sensor for detecting LSPR signals. In the example, FIG. 13A shows the bottom of the DMF cartridge 110 instead of through the top substrate 118. A fiber optic probe 170 is shown inserted through the substrate 116. FIG. 13B shows a fiber optic probe 170 having a LSPR sensor 136. Insert the cartridge 110 from the side into the gap between the upper substrate 118 and the lower substrate 116. In this example, as shown in Figs. 10A to 12B, The bar probes 170 are aligned with the plane of the upper substrate 118 rather than perpendicular to the plane of the upper substrate 118. The advantage of this configuration of the fiber optic probe 170 is that it It is possible to provide only minimal disturbance to the fluid function.

[0094] In yet another example, FIGS. 13C and 13D show a method for detecting a DM using robotics 174. In the gap between the lower substrate 116 and the upper substrate 118 of the F cartridge 110 The optical fiber probe 170 is shown moving or retracting from the tip. In this manner, the fiber optic probe 170 is movable. In another embodiment of the probe 170, a vibration action is applied to the fiber optic probe 170 to aid in mixing. It is possible.

[0095] In yet another example, a robotics system (not shown) can be used to deliver the LSPR sensor A fiber optic probe 170 equipped with 136 is inserted into the DMF cartridge 110 to detect a specific liquid. For example, a robotics system may be used to move the LSPR sensor 1 to a droplet position. 36 is attached to the lower substrate 11 of the DMF cartridge 110. 6 and / or through the top substrate 118 and / or from the side of the DMF cartridge 110 from the substrate 116 to the gap between the lower substrate 116 and the upper substrate 118. The fiber optic probe 170 is, for example, mechanically coupled within the DMF cartridge 110. Can be electrically and / or magnetically moved to different sample lanes and / or locations .

[0096] In some embodiments, the LSPR sensing capability of the DMF cartridge 110 includes a plurality of Various arrangements of multiple LSPR sensors 136 for processing droplets may be included, examples of which are shown in FIG. 14A, 14B, 14C, 15, and 16. For example, FIG. 4A, for example, a plurality of fiber optic probes 170 and an LSPR sensor. A sensor configuration 240 is provided that includes an arrangement of 136 different LSPR sensors 1 36 characteristics (e.g., immobilization chemistry) while the sample droplets 140 are substantially the same. In another example, referring now to FIG. 14B, for example, a plurality of optical fiber plates may be used. A sensor configuration 242 is provided that includes an arrangement of lobes 170 and LSPR sensors 136. , the characteristics of the different LSPR sensors 136 are substantially the same, but the sample droplets 140 In yet another example, referring to FIG. 14C, for example, A sensor configuration 244 is provided that includes an arrangement of a probe 170 and an LSPR sensor 136. The characteristics of the LSPR sensors 136 that are the same as those of the LSPR sensors 136 are shown in a single sample across all of the LSPR sensors 136. The amount of the droplet 140 may be varied to process the droplet 140.

[0097] In yet another example, and referring now to FIG. 15, a sensor configuration 246 is provided. The sensor arrangement 246 may be, for example, a single sensor array that may be disposed across the DMF cartridge 110. The optical fiber 176 includes a plurality of detection spots (e.g., a plurality of detection spots). In one example, the detection spot includes a spectrally resolved LSPR sensor 136. In another example, the nanostructures can be resolved by having different nanostructures at each spot, such as , an optical fiber 1 equipped with multiple detection spots (e.g., multiple LSPR sensors 136) 76 can be imaged from above with a camera and then spatially resolved using image processing. In one example, each detection spot (e.g., each LSPR sensor 136) is individually addressable. The electrodes 126 may be aligned to each other so that each detection The spots (e.g., each LSPR sensor 136) are separated for individual functionalization / sensing. This method allows multiplexing of multiple LSPR sensors 136. This method also integrates the optical fiber with the DMF cartridge 110. This overcomes certain limitations imposed by the physical space available to

[0098] In yet another example, and referring now to FIG. 16, a sensor configuration 248 is provided. The sensor arrangement 248 includes a detection spot (e.g., , LSPR sensor 136). In 248, instead of performing reflection mode measurements, the signal was transmitted through a U-shaped bent optical fiber 176. Bending can enable several useful optical effects: .

[0099] FIG. 17 illustrates a DMF cartridge 110 for analysis of an analyte, a fixed LSPR sensing process, and 3 shows a flow diagram of an embodiment of a method 300 for using a liquid droplet manipulation system and a droplet manipulation method. The method 300 includes the steps of: However, the reactive surface groups are not limited to, for example, COOH, streptavidin, These may include vidin, thiol, protein A, nitrilotriacetic acid (NTA), etc. 0 may include, but is not limited to, the following steps:

[0100] In step 310, a PR system 100 including a DMF cartridge 110 is provided. where a fixed LSPR sensing process (e.g., a fixed LSPR sensor 136) can be used for the analysis of a sample.

[0101] In step 315, the immobilized LSPR sensor 136 can be activated using droplet manipulation. For example, “activation” can be achieved by the addition of a COOH functional surface coating on the LSPR sensor. The present invention may include an amine coupling step in which the amine is converted to an active ester. Droplets of activation buffer with EDC / NHS are inserted into the specific immobilized LSPR using droplet manipulation. The EDC can be transported to the sensor 136. NHS is N-hydroxysuccinimide. EDC / NHS is The activation buffer droplet having the ED may be present on the immobilized LSPR sensor 136 for a period of time. The C / NHS solution reacts with the COOH sites on the immobilized LSPR sensor136 and releases them. This converts the amine groups on the support into active functional groups that can be covalently bonded to any amine groups on the support. The LSPR sensor 136 may be activated.

[0102] In step 320, the immobilized LSPR sensor 136 is functionalized using droplet manipulation. For example, a buffer droplet having a ligand therein can be manipulated using droplet manipulation to obtain a specific The buffer droplets with the ligand can be transported to the immobilized LSPR sensor 136. The LSPR sensor 136 may then be placed in a fixed position for a period of time. 136 may be functionalized.

[0103] In step 325, the immobilized LSPR sensor 136 is deactivated using droplet operations. Inactivation converts any remaining active binding sites on the LSPR sensor 136 into inactive sites. For example, a "blocking" solution such as ethanolamine may be used to convert may be used to react with any remaining COOH sites and inactivate them. For example, Using droplet manipulation, droplets of ethanolamine were then used to capture specific immobilized LSPR sensors. The ethanolamine droplets can be transported to the immobilized LSPR sensor 136 at regular intervals. In doing so, the fixed LSPR sensor 136 can be deactivated.

[0104] In step 330, the assay protocol is The sensing process may be performed using a fixed LSPR sensing process. The sensor readings may be taken in real time. For example, the illumination source 154 and the optical A measuring device 156 is used to measure the LSPR signal from a particular fixed LSPR sensor 136. The data can be captured in real time while the assay protocol is being performed. A pulled droplet (e.g., sample droplet 140) is subjected to droplet manipulation to identify a specific immobilized LSPR sensor. The assay protocol is then carried out, and readings of the association phase can be recorded. The buffer droplets can then be transported to the immobilized LSPR sensor 136 using droplet manipulation. The dissociation step can then be read off and the dissociation step readings recorded. Using droplet manipulation, a volume of 1000 s of the LSPR sensor 136 (3x that of the LSPR sensor 136) can be delivered to the fixed LSPR sensor 136 and the This is usually performed for at least three analyte concentrations to determine the kinetics. Generally, the DMF procedure in this step automatically performs the dilutions required for the analysis. Execute lawfully.

[0105] In step 335, the sensor data from the fixed LSPR sensor 136 may be processed. K of the elephant specimen ON Value, K OFF value, and K D For example, the PR system 100 may determine The controller 150 is used to measure the sensor data from the fixed LSPR sensor 136. Fit a binding model to the data and use regression to identify the analyte of interest that best represents the experimental data K ON Value, K OFF value, and K D This can be done by finding the value of For example, using a data set containing at least three analyte concentrations as described in step 330, This can be achieved.

[0106] FIG. 1 illustrates the fixed LSPR sensing process in a DMF cartridge 110. Referring again to FIG. 17 from 5A, the rapid motion of the droplet on the fixed LSPR sensor 136 In this case, the association phase data (i.e., K ON value) and dissociation stage Floor data (i.e., K OFFvalue), but in this case the affinity data of the analyte (i.e. Ri, K D This can be useful to obtain the

[0107] FIG. 18 is a block diagram of an example optical system 400 of the presently disclosed PR system 100. In another example of a reflection mode configuration, the presently disclosed PR system 10 The optical system 400 of this embodiment has the illumination source and the reflected light sharing a single optical fiber. For example, the optical system 400 includes an LED light source 410, a y-coupler that The optical system 400 also includes a spectrometer 412, and a Y-coupler 414. A gold nanoparticle (AuNP) chip was optically coupled to the cascade 418 via an optical fiber 422. The fiber sensor 416 further includes a gold nanotube 420. The P-chip 420 is provided in association with a DMF device, such as the DMF cartridge 110. For example, the fiber sensor 416 is connected to a DMF cartridge via an AuNP chip 420. The fiber sensor 416 is optically coupled to the optical fiber 110 via an SMA connector. It is optically coupled to the Y coupler 414 via 418 .

[0108] In the optical system 400, a Y coupler 414 couples an LED light source 410 (illumination source) and The reflected light from the AuNP tip 420 can share a single optical fiber 422. The advantage of this example is that the illumination source (e.g., LED light source 410) and the sensor (e.g., , spectrometer 412) is coupled to an activated fiber tip (e.g., AuNP tip 420) For example, a Y coupler 4 with a 90:10 split 14, the 10% side was connected to an LED light source 410 and the 90% side was connected to a spectrometer 412 The LED light source 410 then passes through a Y coupler 414 to , a fiber-sector with gold nanoparticles (e.g., AuNP tip 420) in a DMF device. This light is reflected by the layer of nanoparticles and certain wavelengths are absorbed according to the SPR signal. The reflected light then passes through a Y-coupler 414 to a spectrometer 412 for analysis. .

[0109] (LSPR sensing in solution in DMF) "LSPR sensing in solution" refers to any LSPR sensing process that occurs in the droplet itself. This can refer to any LSPR sensing process occurring in a solution, such as a liquid phase. FIG. 20 shows an embodiment of a general solution-based LSPR sensing process, and FIG. 21A to FIG. 7 shows a specific embodiment of the LSPR sensing process in solution.

[0110] 19 and 20 are side views of a portion of an embodiment of a DMF cartridge 110; As an example of LSPR sensing in solution, we have demonstrated a general LSPR sensing process in solution. In this example, LSPR droplets 142 are provided in a DMF cartridge 110. The LSPR droplet 142 may be a water droplet containing a plurality of nanoparticles 232 suspended therein. The nanoparticles 232 may comprise a hydrophilic droplet, as described above with reference to FIGS. Each of the nanoparticles 232 may be a metal nanoparticle, such as gold and / or silver nanoparticles. It is functionalized with a capture molecule 214 (e.g., a ligand) to create the LSPR effect. In the LSPR sensor 136, the nanoparticles 232 can be immobilized on a surface. However, here, the nanoparticles 232 may not be immobilized on a surface, but rather The nanoparticles 232 may be suspended in a solution, such as suspended in a LSPR droplet 142. .

[0111] FIG. 19 shows the LSPR droplet 142 along with a separate sample droplet 140. In either case, the sample droplet 140 is an aqueous solution containing a plurality of target analytes 216 suspended therein. In this example, the target within the sample droplet 140 may be a droplet (e.g., a droplet of an analyte solution). The analyte 216 binds to a molecule 214 (e.g., a ligand) on a nanoparticle 232 within the LSPR droplet 142. Referring now to FIG. 20, this general In the typical in-solution LSPR sensing process, the LSPR droplet 142 and the sample droplet 1 40 can be assimilated and / or mixed using droplet manipulation, thereby reacting with the LSPR In doing so, the target analyte 216 reacts with the LSPR of the nanoparticle 232 to form a droplet 143. For effect, the nanoparticles 232 may be bound to capture molecules 214 (not shown) on the nanoparticles 232. .

[0112] Generally, in this process, the flux of molecules to the surface of the nanoparticles 232 is achieved using droplet manipulation. For example, droplet manipulation can be used to rapidly mix droplets together. Then, a rapid circular motion (as shown in Figures 7 and 9) over the droplet manipulation electrode 126 and and / or rapid back and forth oscillatory movements (as shown in Figure 5). In doing so, a binding event can be promoted in the reaction LSPR droplet 143. Binding of the analyte in the SPR droplet 143 causes a shift in the LSPR wavelength. The optical measurement system of the PR system 100 is used to measure the capture molecules 232 on the nanoparticles 232. 14 (not shown) to measure the dynamics of the target analyte 216 (e.g., association phase) (i.e., K ON value), dissociation phase (i.e., K OFF value), and analyte affinity (i.e. Chi, K D You can get the value of

[0113] 21A and 21B show the presently disclosed PR system 100 and DMF card. Magnetically responsive nanotubes that can be used in the in-solution LSPR sensing process of the transistor 110 A side view of an embodiment of a particle 250 is shown. "Magnetic responsive" means responsive to a magnetic field. A "magnetically responsive nanoparticle" may include or comprise a magnetically responsive material. Examples of magnetic materials include, for example, paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, cobalt, as well as Fe3O4, B aFe 12 O 19 These include metal oxides such as CoO, NiO, Mn2O3, Cr2O3, and CoMnP. However, the present invention is not limited to the above.

[0114] In one example, referring now to FIG. 21A, the magnetically responsive nanoparticles 250 are The magnetically responsive core element 252 may be formed by embedded nanoparticles 232. 252 can be, for example, a magnetically responsive particle. In some embodiments, the nanoparticles 232 may include two or more magnetically-responsive core elements 252 .

[0115] In another example, referring now to FIG. 21B, the separate magnetically responsive elements 254 may be nanoparticles. 232 to form magnetically responsive nanoparticles 250. For example, 54 are magnetically responsive particles that are linked to the nanoparticles 232 using physical or chemical bonds. In one example, the magnetically responsive element 254 can be attached to the nanoparticles 232 using a linker. Additionally, more than one magnetically responsive element 254 may be coupled to the nanoparticle 232, and The LSPR sensor in solution can bind two or more nanoparticles 232 to one magnetic response element. An example of using magnetically responsive nanoparticles 250 for labeling is described below with reference to FIGS. 19 through 27. is shown and explained.

[0116] FIG. 22 illustrates a solution process for functionalizing magnetically responsive nanoparticles 250 using droplet manipulation. 5 shows a flow diagram of an embodiment of the process 500. As an example, the in-solution process 500 is The process is described for a DMF cartridge 110 of the same type. However, the reactive surface groups are For example, COOH, streptavidin, and NTA may be included. 23A through 23F diagrammatically illustrate certain steps of an embodiment of an in-solution process 500. 23A through 23F are references to specific steps of the process 500 in solution. The in-solution process 500 may include, but is not limited to, the following steps: do not have.

[0117] In step 510, a LSPR sensor in a solution using droplet operations is used for the analysis of an analyte. A PR system 100 is provided that includes a DMF cartridge 110 that supports the printing process. It can be done.

[0118] In step 515, processes in solution and droplet manipulation are used to produce magnetically responsive nanoparticles. For example, the “activation” can be the activation of the COOH on the magnetically responsive nanoparticle 250. The method includes an amine coupling step in which the functional surface coating is converted to an active ester. For example, referring now to FIG. 23A, nanoparticle droplets 180 are deposited on a DMF cartridge. The nanoparticle droplets 180 may be provided in a container 110 having a COOH coating thereon. The magnetically responsive nanoparticles 250 may be contained in the solution. Next, referring now to FIG. Activation buffer droplet 182 may be provided in DMF cartridge 110. 182 contains EDC / NHS. Now, referring now to FIG. 23B, nanoparticle droplet 1 80 and the activation buffer droplet 182 may be combined or assimilated using droplet manipulation. In doing so, the nanoparticle droplets 180 may be activated. For example, 180 and the activation buffer droplet 182 for a period of time. The magnetically responsive nanoparticles 250 in the nanoparticle droplet 180 may be activated. The molecule 250 was prepared for ligand functionalization.

[0119] At step 520, processes in solution and droplet manipulation are used to produce magnetically responsive nanoparticles. 250 may be washed. For example, referring now to FIG. 23C, a magnet (e.g., a permanent magnet or A magnet, which may be an electromagnet, may be used to align the magnetically responsive nanoparticles 250 with a particular droplet manipulation device. The nanoparticle droplets 180 may then be immobilized on the working electrode 126. The magnetically responsive nanoparticles 250 are transferred using droplet manipulation and fixed on the top of the droplet manipulation electrode 126. The term "immobilization" in relation to magnetically responsive nanoparticles refers to the ability of the nanoparticles to remain immobilized. This may mean that the molecule is substantially constrained to a position above the droplet manipulation electrode in the DMF cartridge. As a result, the forces acting on the nanoparticles resulting from the fluid flow relative to the nanoparticles are shifted from the restraining position to the Next, referring to Figures 23C and 23D, The buffer solution droplet 144 is then transferred to the immobilized magnetically responsive nanoparticles 250 using droplet manipulation. A buffer droplet 140 may be delivered to the site of immobilization as needed to wash the structure. The magnetically responsive nanoparticles 250 may be passed multiple times over the magnetically responsive nanoparticles 250 .

[0120] In step 525, the process and droplet manipulation in the solution produces magnetically responsive nanoparticles 2 50. Referring now to FIG. 23E, magnetically responsive The nanoparticles 250 can be released into the buffer solution droplet 144. The buffer solution droplet 144 can then be, for example, The ligand droplet 182 may be combined or assimilated with the ligand droplet 18 2 is provided with a capture molecule or ligand 214. In this way, the LSPR droplet 142 For example, a buffer solution droplet 14 may be formed in which the magnetically responsive nanoparticles 250 are functionalized. 4 (having magnetically responsive nanoparticles 250 therein) and assimilate the ligand droplet 182 for a period of time. By this, the ligand 214 is attached to the magnetically responsive nanoparticle 250 and the LSPR droplet 1 42. A specific washing procedure then occurs by exchanging multiple buffer drops. possible.

[0121] In step 530, the process and droplet manipulation in the solution produces magnetically responsive nanoparticles 2 50. The deactivation can be used to deactivate the magnetically responsive nanoparticles within the LSPR droplet 142. This can be done to convert the remaining active binding sites on the particles 250 to inactive sites. For example, , using a "blocking" solution such as ethanolamine to react with the remaining COOH sites, For example, a droplet of ethanolamine can be combined with the LSPR droplet 142 to inactivate them. For example, the LSPR droplet 142 (within which the magnetic responsive nanoparticles are By assimilating the LSP particles (having 250 particles) with ethanolamine droplets for a certain period of time, R droplet 142 may then be inactivated. Cleaning operations may occur.

[0122] 24A, 24B, 24C, and 24D; 25A, 25B, 25C, and FIG. 25D; FIG. 26A, FIG. 26B, FIG. 26C, and FIG. 26D show magnetically responsive nanoparticles and liquid LSPR sensing process in solution in DMF cartridge 110 using droplet manipulation For example, Figures 24A, 24B, 24C, and 24D show droplet manipulation embodiments. The LSPR sensing process in solution in the DMF cartridge 110 can occur in air. 25A, 25B, 25C, and 25D show the DMF cartridge process. The LSPR sensing process in solution in the edge 110 is shown, and the droplet operation is performed on a liquid covered with oil. This can occur in the air with droplets. 1 illustrates the LSPR sensing process in solution in a DMF cartridge 110, where: Droplet operations can occur in oil (or filler fluid).

[0123] Now referring to FIG. 24A, the LSPR droplets 142 are presented to the DMF cartridge 110. The LSPR droplets 142 can be prepared, for example, according to the in-solution process 500 shown in FIG. Further, a sample droplet 140 having a target analyte 216 therein is provided. Now referring to FIG. 24B, the LSPR droplet 142 and the sample droplet 140 may , can be combined or assimilated and rapidly mixed using droplet manipulation. For example, droplets can be , which are rapidly mixed using droplet manipulation and rapid circular motion on the droplet manipulation electrode (Figure 7 and Figure 8). 9) and / or rapid back and forth oscillatory movements (as shown in FIG. 5) In doing so, the binding event affects the magnetically responsive nanoparticles 250 within the LSPR droplet 142. This can occur, which is now referred to as a reactive LSPR droplet 143.

[0124] In this process, the data depletion effect (i.e., the bulk phase is bound to the surface) To avoid the concentration of the analyte in the sample droplet 140 decreasing, the sample droplet 140 is replaced with a new sample droplet. Alternatively, the replacement procedure may involve periodically replacing the droplets 140 or their contents. This can be triggered through real-time observation of some measurable property of the container. For example, Now referring to FIG. 24C, this can be done quickly using a magnet, which is magnetically responsive. Immobilize the nanoparticles 250, resulting in a new analyte droplet (e.g., sample droplet 140). For example, a magnet 172 may be used to attach the magnetically responsive nanoparticles 250 to a particular droplet manipulation electrode 172. 26. The reactive LSPR droplet 143 may then be immobilized in a liquid. The magnetically responsive nanoparticles 250 can be transported using droplet manipulation and fixed onto the droplet manipulation electrodes 126. Now, referring to both FIG. 24C and FIG. 24D, The sample droplet 140 is then subjected to droplet manipulation to separate the immobilized magnetically responsive nanoparticles 250 from the sample droplet 140. The magnetically responsive nanoparticles 250 can then be released back into suspension. During the process, optical measurements are performed in real time to provide affinity and association phase data. It can be obtained.

[0125] Now referring again to both FIG. 24C and FIG. 24D, once the set time has elapsed The sample droplet 140 can then be replaced with a buffer droplet 144 and optical measurements taken in real time. , dissociation phase data can be acquired. For example, again using magnet 172, a magnetically responsive nanoparticle can be Nanoparticles 250 can be immobilized in reaction LSPR droplets 143 on specific droplet manipulation electrodes 126. The reactive LSPR droplets 143 can then be transported using droplet manipulation to attach magnetically responsive nanoparticles. The particles 250 may remain immobilized atop the droplet operations electrode 126. Next, the buffer droplet 1 44 was delivered to the site of immobilized magnetically responsive nanoparticles 250 using droplet manipulation. The magnetically responsive nanoparticles 250 are then released back into suspension and the dissociation stage is measured. Multiple buffer drops may be exchanged to prevent analyte accumulation.

[0126] The process shown in Figures 25A, 25B, 25C, and 25D involves the droplets being covered with oil. 24A, except that the droplets are not oil shells (e.g., droplets having an oil shell 145). 24B, 24C, and 24D. In this example, when the magnetically responsive nanoparticles 250 are immobilized, they react with the oil. The nanoparticles are then briefly passed through a 145 mL filter and then resuspended in the aqueous portion of the droplet. Since the surface of the nanoparticle 250 is generally hydrophilic, a water layer is formed on the surface of the magnetically responsive nanoparticle 250. tends to be present, which helps resist oil contamination and promotes reliable readings. Further, using this configuration, the time during which the magnetically responsive nanoparticles 250 are exposed to the oil phase can be determined. can be minimized.

[0127] Furthermore, the process illustrated in Figures 26A, 26B, 26C, and 26D 24A, 24B, except that operation may occur in oil (e.g., fill fluid 146). , and the process may be substantially the same as that described above with reference to FIG. 24C and FIG. 24D. In this example, once the magnetically responsive nanoparticles 250 are immobilized, they are transferred to the DMF cartridge. The transition from the completely oily environment of the ridge 110 to the completely aqueous environment of the droplet occurs again with the magnetic field. Since the surface of the magnetically responsive nanoparticles 250 is generally hydrophilic, the surface of the magnetically responsive nanoparticles 250 There tends to be a water layer on top, which resists oil contamination and promotes reliable readings. It is useful.

[0128] Further, in the process shown in FIGS. 23 to 26D, a magnet 172 (e.g., a permanent magnet) The position of the magnet 172 is not limited to the vicinity of the lower substrate 116. , near the top substrate 118, or in other locations, such as on the sides or edges of the DMF cartridge 110. The device may be located at a location.

[0129] FIG. 27 illustrates the DMF cartridge 110, the in-solution LSPR sensing process, and the detection of analytes. 6 shows a flow diagram of an embodiment of a method 600 for using droplet manipulation for analysis. The method 600 may include a process for a COOH-based DMF cartridge 110. However, reactive surface groups can be, for example, COOH, streptavidin, and NTA The method 600 may include the steps of FIG. 24A, FIG. 24B, FIG. 24C, and FIG. 24D; 25B, 25C and 25D; and FIG. 26A, 26B, 26C and 26D. The method 600 may include the following steps: The present invention is not limited to these.

[0130] In step 610, a LSPR sensor in solution is used to analyze an analyte using droplet operations. A PR system 100 is provided that includes a DMF cartridge 110 that supports the printing process. It can be done.

[0131] In step 615, droplet manipulation is performed to analyze an analyte using LSPR sensing in solution. For example, the DMF cartridge 110 may be used to prepare LSPR droplets. For example, the LSPR droplet 142 may be subjected to an in-solution process 500 as shown and described with reference to FIG. It can be functionalized according to

[0132] At step 620, the method 600 comprises dissolving the functionalized LSPR droplets and the sample droplets in a D Initiate the assay protocol by delivering liquid to the MF cartridge and then performing droplet manipulation may include combining or assimilating the LSPR droplets and the sample droplets using For example, referring now to FIG. 24A, LSPR droplets 142 are placed in a DMF cartridge 1 10. Additionally, a sample droplet 140 having a target analyte 216 therein may be provided. Referring now to FIG. 24B, the LSPR droplet 142 and the sample droplet 140 can be combined or assimilated and rapidly mixed using droplet operations. The droplets are rapidly mixed using droplet manipulation, with rapid circular motion and / or can affect the rapid back and forth oscillatory motion. In doing so, the binding event occurs in the LSPR droplet14 This occurs with magnetically responsive nanoparticles 250 within 2, which are now referred to as responsive LSPR droplets 143.

[0133] In step 625, the method 600 avoids depletion effects and collects affinity data and association To obtain stepwise data, the sample droplets were periodically replaced with new ones. For example, see FIG. 24C herein. When checking the data, periodically (e.g., every few seconds), the sample is updated to reduce the data exhaustion effect. It may be necessary to replace the pulled droplet 140 with a new sample droplet 140. For example, 72 to attach magnetically responsive nanoparticles 250 to the reaction LS on a specific droplet manipulation electrode 126. The reaction LSPR droplet 143 may then be immobilized using droplet manipulation. The magnetically responsive nanoparticles 250 are transported and remain immobilized on the droplet manipulation electrode 126. Now, referring to both FIG. 24C and FIG. 24D, the sample droplet 140 is , which can be transported to the site of the immobilized magnetically responsive nanoparticles 250 using droplet manipulation. The magnetically responsive nanoparticles 250 may then be released back into suspension. During this process, Measurements can be performed in real time to obtain affinity and association phase data.

[0134] In step 630, the method 600 periodically replaces the sample droplet with a buffer droplet. The assay protocol may be continued by performing the above steps to capture dissociation phase data. 24C and 24D, after a given time has passed or a signal has been observed, When it is time to exchange the liquid, the sample droplet 140 may be replaced with a buffer droplet 144, and the light Optical measurements can be taken in real time to obtain dissociation phase data. 2, the magnetically responsive nanoparticles 250 are reacted with the reaction LS on a specific droplet manipulation electrode 126. The reaction LSPR droplet 143 may then be immobilized using droplet manipulation. The magnetically responsive nanoparticles 250 are then transferred to the droplet manipulation electrode 126, leaving the nanoparticles immobilized thereon. The buffer solution droplet 144 is then transferred to the immobilized magnetically responsive nanoparticles using droplet manipulation. The magnetically responsive nanoparticles 250 can then be released back into suspension. Again, a buffer droplet 140 may be immobilized as needed to wash the structure. The magnetically responsive nanoparticles 250 can be passed over the nanoparticles multiple times.

[0135] In step 635, the method 600 detects different concentrations in the sample droplet 140. Repeat steps 620, 625, and 630 using the same number of The assay protocol may be continued by repeating the procedure. This is usually done to perform kinetic analysis. The assay is performed for at least three analyte concentrations that can be used to perform the assay.

[0136] In step 640, the LSPR droplet 142 of the LSPR sensing process in solution The sensor data of K may be processed, and ON Value, K OFF Value, K D Value and / or object search The affinity of the body is determined, for example, using the controller 150 of the PR system 100. The sensor data from the LSPR droplets142 were then analyzed by fitting a binding model to the data and performing regression Use the K of the analysis of the analytes of interest that best represents the experimental data.ON Value, K OFF Value, K D Value, and / or This can be done, for example, by finding affinities in step 635. This can be achieved using a data set that includes at least the three analyte concentrations listed. do.

[0137] Method 6 Using LSPR Sensing Process and Droplet Manipulation in Solution for the Analysis of Analytes 00 features can be used in a number of (e.g. This may include the fact that droplets (e.g., hundreds) can be processed simultaneously. In addition, the DMF cartridge The in-solution LSPR sensing process in the chip 110 can be performed for multiple analytes and / or concentrations. According to the in-solution process 500 of FIG. Thus, the functionalized LSPR droplet 142 can be divided into multiple droplets and processed separately. This is because it is possible.

[0138] Further, another feature of the in-solution process 500 of FIG. 22 and the method 600 of FIG. The chemical preparation of may be carried out using the in-solution process 500 of FIG. 22, followed by method 6 of FIG. Dynamic measurements can be performed while the magnetically responsive nanoparticles 250 are immobilized using It can be something like that.

[0139] Additionally, another feature of the in-solution process 500 of FIG. 22 and the method 600 of FIG. 27 is The exact same device can be used for various analyses with only sample / activation changes. could be.

[0140] Further, another feature of the in-solution process 500 of FIG. 22 and the method 600 of FIG. The design may facilitate autonomous optimization of the immobilization chemistry for a particular analyte.

[0141] Further, another feature of the in-solution process 500 of FIG. 22 and the method 600 of FIG. This may be because the nanoparticles 250 in the DMF cartridge 110 can be easily washed. This can facilitate surface chemistry and ligand immobilization to be performed directly on the nanostructures. Features allow for processing of complex samples such as blood.

[0142] Here again we describe the LSPR sensing process in solution in the DMF cartridge 110. 19 to 27, the rapid movement of the droplets may not exist. In this case, the association phase data (i.e., K ON value) and dissociation phase data (i.e., K OFF value) Although it is not necessary to obtain the affinity data for the analyte (i.e., K D Get the value In addition, K ON and K. OFF is estimated via an additional equation that models diffusion. It can be measured.

[0143] Referring again to FIGS. 1 through 27, various configurations of the optical measurement system of the PR system 100 are shown. For example, Figures 28 to 32 show examples of different configurations of the optical measurement system. For example, FIG. 28 shows a fixed LSPR sensor 13 in a DMF cartridge 110. 6. An illumination source 154 and an optical measurement device configured to operate in a transmission mode in conjunction with the In this example, a lower substrate 116, an upper substrate 118, and a droplet manipulation electrode 156 are shown. 26 is substantially optically transparent or has an optically transparent window within the sensing area. It may contain wheat flour.

[0144] FIG. 29 shows a reflectance model for a fixed LSPR sensor 136 in a DMF cartridge 110. The illumination source 154 and the optical measurement device 156 are shown configured to operate in a reflection mode. When operating in the irradiated mode, the DMF cartridge 110 is In this regard, the optical measurement system may be configured to allow optical observation from a given side. Depending on which side of the DMF cartridge 110 the system is located on, either the top substrate 118 or Either the lower substrate 116 is optically transparent or has an optically transparent window. In particular, the portion of the substrate adjacent to the optical measurement system may be optically transparent. It is possible, and the opposite side does not have to be optically transparent when operated in reflective mode. Thus, in the embodiment shown in FIG. 29, the upper substrate 118 is substantially optically transparent. Although it may be clear, the lower substrate 116 (not shown) and the droplet manipulation electrodes 126 are substantially It is not necessarily optically transparent.

[0145] FIG. 30 illustrates the LSPR sensing process in solution within the DMF cartridge 110. 1 shows an illumination source 154 and an optical measurement device 156 configured to operate in a transmit mode. In this example, the lower substrate 116, the upper substrate 118, and the droplet manipulation electrode 126 are substantially It is optically transparent.

[0146] FIG. 31 illustrates the reaction in relation to the in-solution LSPR sensing process in the DMF cartridge 110. 1 shows an illumination source 154 and an optical measurement device 156 configured to operate in a projection mode. As noted above, when operating in reflection mode, the substrate adjacent to the optical measurement device 156 The optical measuring device 156 may be optically transparent, but the opposite side of the optical measuring device 156 may not be optically transparent. In the embodiment shown in FIG. 31, the upper substrate 118 is substantially optically transparent. Although it is possible to use a liquid droplet manipulation electrode 126, the lower substrate 116 (not shown) and the liquid droplet manipulation electrode 126 are substantially optically It may be transparent.

[0147] FIG. 32 illustrates an optical aperture in relation to the illumination source 154 and / or the optical measurement device 156. 1 shows an example of a DMF cartridge 110 that may include an optical aperture 148. In this example, may be at one of the droplet operations electrodes 126, the droplet operations electrodes 126 being substantially optically transparent. It doesn't have to be this way. EXAMPLES

[0148] FIG. 33 shows the results first for a drop of 1% glycerol in DI water, followed by a drop of 2% glycerol in DI water. 7 shows an example plot 700 of SPR response versus time for a droplet of 100% glycerol. , plot 700 shows an SPR response curve 710. In plot 700, The LSPR sensor on the fiber chip with H functional groups was first washed with deionized water (0-33 s), 1% glycerol in deionized water (33s-66s), deionized water (66s-103s), 2% glycerol (103s-140s) and finally exposed to deionized water again. The experiment was carried out in an oil environment.

[0149] This experiment proves two things: (1) LSPR-active gold nanoparticles at the tip of an optical fiber The sensor measured the refractive index from water to both 1% and 2% glycerol. Moreover, the signal difference between 1% and 2% is linear as expected. ) The oil environment (>30-fold refractive index shift of 2% glycerol) does not interfere with the measurement.

[0150] Previously, LSPR sensors were typically hydrophilic, but retained a layer of water to protect the sensor from the oily environment. It was noted that the 2-second exposure to oily medium between each solution change The effect of this exposure is not documented in the data. This is because the interaction distance of the PR sensor is short.

[0151] A proof-of-concept experiment was performed for the activation of LSPR optical fibers with ligands. In the demonstration experiment, a COOH surface LSPR sensor on a fiber was embedded in a DMF device and ligated The bond immobilization procedure was carried out. This process, described below, involves the immobilization of COOH groups with EDC+NHS. The surface is activated, protein A is attached to the sensor surface, and unreacted surface sites are passivated. and finally human IgG that is bound to Protein A for validation. The structure was constructed by immobilizing a ligand on a substrate.

[0152] In this experiment, an optical fiber with a COOH surface LSPR sensor at its tip was attached to the DMF membrane from the side. The DMF device then executes the command to place the fiber tip in the Each step is performed in accordance with the chemical It consisted of dispensing 600 nL of the substance and transferring that amount to the fiber tip. Exposure time During this time, the device vibrated the liquid at a rate of 4 Hz. The following steps were performed: (1) Condition the sensor surface with phosphate-buffered saline (PBS) for 10 min; (2) washing the sensor surface with 10 mM hydrochloric acid (HCl) for 2 min; (3) PBS for 5 minutes; (4) Dispense 300 nL of EDC and 300 nL of NHS into the DMF device, mix the products, and apply them to the sensor. is exposed to the mixture for 5 minutes, (5) PBS for 3.5 minutes; (6) 20 μg / mL protein A for 5 min; (7) Rinse off excess Protein A with PBS for 2 minutes. (8) Block unreacted binding sites with ethanolamine for 5 min; (9) PBS for 6 minutes; (10) 5 min exposure to 100 nM human IgG to verify Protein A binding; (11) PBS for 8 min.

[0153] The results of this experiment are shown in Figure 34. Activation and protease activity of carboxyl-gold optical fibers. Peak positions of IgG binding, blocking with ethanolamine, and IgG binding. Plot 800 shows the binding steps (taps) where each droplet exchange shows a conventional binding curve. The results show that curve 810 works as expected for the following antibodies: protein A, ethanolamine, and IgG. The platform was validated as a method to automate SPR sample mixing and analysis. can be.

[0154] Another proof-of-concept experiment was carried out to activate LSPR optical fibers with ligands. In a proof-of-concept experiment, a Protein A-activated LSPR sensor was inserted into a DMF device and reassembled. A series of concentrations of analyte were introduced with an intermediate step between them, allowing the binding rate of the analyte to be monitored. It is determinable.

[0155] In this experiment, an optical fiber with a protein A-activated LSPR sensor at its tip was The DMF device then executes the command to The chip was then exposed to the next sample. In this experiment, each step was 600 nL pulsed at 10 Hz. The sample was constructed as follows: (1) Condition the sensor surface with PBS for 10 min; (2) 11 nM human IgG sample for 2.5 min; (3) Measure the release kinetics in PBS for 12.5 min; (4) regenerating the surface with glycine / HCl, pH 2.0, for 2.5 min; (5) 33 nM IgG human IgG sample for 2.5 minutes; (6) Measure the release kinetics in PBS for 12.5 min; (7) regenerating the surface with glycine / HCl, pH 2.0, for 2.5 min; (8) 100 nM IgG human IgG sample for 2.5 minutes; (9) Measure the release kinetics in PBS for 12.5 min.

[0156] The results of this experiment are shown in Figures 35 and 36. Referring now to Figure 35, the DMF instrument and Plots of Protein A / IgG experimental data from both OpenSPR and OpenSPR are shown. The results of this study show improved sensitivity and reduced variance. For analysis, the data were imported into the standard analysis software Tracedrawer (Figure 35). (See plot 900). This shows an excellent fit parameter, 1.36x10 5 M -1 s -1 Measured The association constant (K ON ), 1.17x10 -4 s -1 The measured dissociation constant (K OFF ) and conventional equipment The measured analyte affinity (K) of 0.86 nM was predicted based on the same assay performed. D ) I did.

[0157] Referring now to Figure 36, fitting of binding kinetics to DMF Protein A data 36 is a plot 1000 showing the results of the DMF-based tests. The results were compared to the OpenSPR analytical device, which uses a more conventional flow cell fluidic system. In comparison, the DMF device exhibits higher sensitivity and significantly reduced dispersion. During conventional flow cell-based SPR analysis, the signal is distributed across the buffer / sample interface. When a sample moves to or from the sensor surface, it usually , adjacent to the buffer solution, which is the interface through which the sample spreads. This allows the leading and trailing edges of the sample to In the DMF device, the sample volume is proportional to the concentration gradient. The dispersion is measured because the sample droplets can be exchanged without exposure to the buffer front. This lack of dispersion results in faster results because there is no change in the unbound reference signal. This allows for more accurate analysis of the synthesis chemistry and improved dynamic fitting accuracy.

[0158] In accordance with long-standing patent law treatises, the terms "a," "an," and "the" are used throughout the scope of a patent claim. As used herein, including the range, refers to "one or more." Thus, for example, A reference to "the subject" refers to a plurality of subjects, unless the context clearly indicates the contrary (e.g., plural subjects). Includes the title.

[0159] Throughout this specification and the claims, the terms "comprise" and "comprises" are used. ) and "comprising" are used interchangeably unless the context otherwise requires. Similarly, the term "include" and its subtext are used in a non-exclusive sense. A legal variation is when the enumeration of an item in a list is a substitution or addition to the listed items. The list is intended to be non-limiting so as not to exclude other similar items that may be used.

[0160] For purposes of this specification and the appended claims, unless expressly stated otherwise, Any amounts, sizes, dimensions, proportions, shapes, compositions, parameters, All numbers expressing percentages, quantities, properties, and other numerical values ​​are expressed using the word "about." Even if the term "about" is not explicitly stated with a value, amount, or range, and therefore, unless otherwise indicated to the contrary, Unless expressly stated, the numerical parameters set forth in the following specification and attached claims are not necessarily precise. Measurements need not be precise or exact, but may be subject to tolerances, conversion factors, rounding, measurement errors, and other factors that are within the scope of the subject matter of this disclosure. and other factors known to those skilled in the art depending on the desired properties sought to be obtained by They may be approximate and / or larger or smaller as necessary to reflect the For example, the term "about" when referring to a value can, in some embodiments, refer to a value that is ±100 from a particular amount. %, in some embodiments ±50%, in some embodiments ±20%, In some embodiments, ±10%, in some embodiments, ±5%, in some embodiments, ±1%, In some embodiments, the variation is ±0.5%, and in some embodiments, ±0.1%. It may be meant to encompass such modifications as are necessary to carry out the disclosed methods or to It is suitable for use in the compositions shown.

[0161] Additionally, when the term "about" is used in connection with one or more numerical values ​​or numerical ranges, When a range is stated, it refers to all such numbers inclusively and to the entire range of numbers stated. It should be understood that by extending the upper and lower boundaries you change the range. The enumeration of a range of values ​​by a number includes all numbers within that range, e.g., including their decimals. (For example, an enumeration of 1-5 includes 1, 2, 3, 4, 5, and their decimals, e.g. , 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range. do.

[0162] The foregoing subject matter has been described in some detail by way of illustration and example for clarity of understanding. However, it will be apparent to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims. I want to be understood.

Claims

1. 1. A cartridge for use with an instrument for performing a measurement of a fluid, comprising: A plurality of droplet actuators operative to perform droplet operations on the fluid droplets in the DMF portion. a digital microfluidic (DMF) portion including a computer; a reaction portion including a sensor medium disposed relative to the plurality of droplet actuators; while in contact with the sensor medium, operative to induce movement of the fluid droplet relative to the sensor medium; cartridge.

2. 10. The cartridge of claim 1, wherein the plurality of droplet actuators comprises a plurality of reaction electrodes. ridge.

3. The plurality of reaction electrodes perform droplet manipulation by electrowetting. Item 3. The cartridge according to item 2.

4. 4. The sensor medium of claim 3, wherein the sensor medium comprises a surface plasmon resonance (SPR) sensor medium. The cartridge described in

5. The SPR sensor medium is configured such that target molecules in a sample fluid bind to the SPR sensor medium. The cartridge of claim 4 , functionalized with capture molecules that alter the optical signal.

6. The capture molecules are sensitive to binding with target molecules in the sample fluid and are capable of binding to the SPR sensor. changing the optical properties of the SPR sensor medium to result in a change in the optical signal of the SPR sensor medium. The cartridge according to claim 5, further comprising a ligand immobilized on an SPR sensor medium. J.

7. The change in the optical property is a change in the optical signal resulting from light interacting with the SPR sensor medium. The cartridge of claim 6 comprising a change.

8. the reaction portion comprises an SPR sensor surface disposed in association with a plurality of reaction electrodes; An SPR sensor medium is disposed on the SPR sensor surface, and the droplets are 8. The method of claim 7, wherein the reaction electrode is operable to contact and engage the SPR sensor surface. The cartridge described.

9. The SPR sensor medium comprises one of nano-sized structures distributed on the sensor surface; or a continuous film comprising nano-sized features. J.

10. The reaction portion includes a first substrate and a second substrate disposed at a distance from each other, The cartridge of claim 9 , defining a reaction chamber.

11. The SPR sensor surface is disposed on the first substrate, and the plurality of reaction electrodes are The cartridge of claim 10 , wherein the second substrate is disposed opposite the first substrate.

12. The SPR sensor surface is disposed on the first substrate, and the plurality of reaction electrodes are The cartridge of claim 10 disposed on a first substrate.

13. The SPR sensor surface is an end portion of an optical member comprising at least one optical fiber. The optical member is disposed adjacent to one of the first substrate or the second substrate.

2. The method of claim 1 , wherein the SPR sensor is disposed within the reaction chamber and extends away from the surface of the reaction chamber.

10. The cartridge according to claim 0.

14. The optical member includes a first optical fiber through which the optical signal is transmitted from the SPR sensor surface. The cartridge of claim 13 comprising a bar.

15. The optical member includes a second optical fiber through which light from an illumination source is provided to the SPR sensor surface. The cartridge of claim 14 comprising a fiber.

16. The optical member is movable relative to the first substrate, and the SPR sensor surface is The reaction chamber includes an extension position and an SPR sensor surface. and a storage position not disposed within said storage area. The cartridge described in

17. The cartridge of claim 13 , wherein the reaction chamber contains a loading medium.

18. The optical member is adapted to reduce contact between the SPR sensor surface and the loading medium.

20. The cartridge of claim 17, which is restorable in a

19. The SPR sensor surface is disposed between a first reaction electrode and a second reaction electrode. The cartridge according to claim 9.

20. The first reaction electrode and the second reaction electrode are alternately activated, and the first reaction potential is and inducing vibration of the droplet between the first electrode and the second reaction electrode against the SPR sensor surface.

20. The cartridge of claim 19, which induces movement of a fluid droplet.

21. The oscillation of the droplet between the first reaction electrode and the second reaction electrode is linear. Item 21. The cartridge according to item 20.

22. The SPR sensor surface is disposed between three or more reaction electrodes, and the three or more reaction electrodes are alternately activated to induce vibration of the droplet between the three or more reaction electrodes, and the SPR cell The cartridge of claim 9 , further comprising: a sensor adapted to induce movement of the fluid droplet relative to a sensor surface.

23. The cartridge of claim 22, wherein the oscillation of the droplet between the three or more reaction electrodes is circular. J.

24. The sensor medium includes a plurality of sensors suspended in a sensor droplet disposed in the reaction portion. The cartridge of claim 7 comprising a surface nanoparticle.

25. The fluid droplet is assimilated into a sensor droplet, and the SPR sensor medium in a reaction droplet is assimilated into a sensor droplet.

25. The cartridge of claim 24, which forms a reaction droplet for measuring an optical signal.

26. The motion induced by the plurality of droplet actuators is adapted to mix the reaction droplets.

26. The cartridge of claim 25, which operates as follows:

27. 27. The method of claim 26, wherein each of the plurality of sensor nanoparticles is magnetically responsive. Cartridge.

28. 28. The method of claim 27, wherein each of the plurality of sensor nanoparticles comprises a magnetically responsive core. Cartridge included.

29. Each of the plurality of sensor nanoparticles has a magnetic field coupled to the sensor nanoparticle.

28. The cartridge of claim 27 comprising an answering element.

30. the magnetic response element is physically or chemically bound to the sensor nanoparticles; 30. A cartridge according to claim 29.

31. The magnetically responsive sensor nanoparticles are immobilized on the reaction moiety by acting on the magnetically responsive sensor nanoparticles. and selectively operable to position the plurality of nanoparticles in an inhibiting position relative to a magnet.

28. The cartridge of claim 27, comprising a magnet.

32. The plurality of droplet actuators are configured to have the magnetically responsive sensor nanoparticles in the inhibited position. A magnetic field is generated between the sensor nanoparticles and the magnet to move the fluid away from the sensor nanoparticles when immobilized by the magnet. and the magnetically responsive sensor nanoparticles are immobilized by the magnet in the inhibiting position. and operable to move a fluid into contact with said sensor nanoparticles when the fluid is moved into said sensor nanoparticles.

32. The cartridge of claim 31.

33. The movement of the fluid droplet is monitored by a sampling optical system that measures the optical signal of the sensor medium. The cartridge of claim 1 , wherein the speed is faster than the grading rate.

34. A plurality of droplet actuators are provided in the DMF portion. The cartridge of claim 2 , further comprising a number of droplet operations electrodes.

35. a reservoir electrode in the DMF portion for receiving and maintaining fluid in the DMF portion; 35. The cartridge of claim 34, further comprising:

36. The droplet operation may include at least one of assimilation of droplets, division of droplets, dispensing of droplets, or dilution of droplets.

35. The cartridge of claim 34, comprising at least one

37. The fluid droplet comprises an analyte fluid droplet, the sensor medium being in contact with the fluid droplet. The movement of the analyte fluid droplet relative to the sensor medium determines the amount of analyte fluid droplet relative to the sensor medium. The cartridge of claim 1 , including an effective spreading rate of the droplets.

38. The effective diffusion rate of the analyte fluid droplets is determined by the binding rate of the analyte to the SPR sensor.

38. The cartridge of claim 37, wherein

39. further comprising electrical contacts in electrical communication with the plurality of droplet actuators, The point is to interface with a controller to control multiple droplet actuators. The cartridge of claim 1 .

40. a pluggable interface for said cartridge comprising said electrical contacts; The pluggable interface connects an instrument controller and the plurality of droplet actuators. a device that is physically and electrically engageable with the device to establish electrical communication between the device and the 40. The cartridge of claim 39.

41. The reactive moiety facilitates real-time optical measurement of the sensor medium in reflectance mode. In order to achieve this, the illumination source on at least one side of the reaction portion is substantially 2. The cartridge of claim 1, which is substantially transparent.

42. The reactive moiety facilitates real-time optical measurement of the sensor medium in a transmission mode. to be substantially transparent to an illumination source incident on the reaction portion on a side opposite the reaction portion.

2. The cartridge of claim 1 ,

43. The fluid droplet comprises an analyte fluid droplet, and the SPR sensor medium is detecting an analyte affinity of the analyte fluid droplet during said movement of the analyte fluid droplet relative to said operatively, wherein the analyte affinity is an affinity value for the analyte (K D ) characterized by The cartridge according to claim 7 .

44. The above K Ｄ is the on-rate measured during the association phase of the analyte fluid at the SPR sensor (K ＯＮ ) and the off-rate measured during the dissociation step of the analyte fluid at the SPR sensor. T (K ＯFF 44. The cartridge of claim 43, wherein the measurement is based on the

45. A cartridge according to claim 1; a PR device with which the cartridge is engageable; The PR equipment comprises: operatively operable with electrical contacts of the cartridge for controlling the plurality of droplet actuators. a controller for communicating with the an optical detection system operative to measure an optical signal of the sensor medium; Plasmon resonance (PR) system.

46. The optical detection system includes: an illumination source operative to direct light incident on said sensor medium; an optical measurement device for measuring the optical signal of the sensor medium; 46. ​​The PR system of claim 45, further comprising:

47. The fluid droplet comprises an analyte fluid droplet, and the controller is configured to: and detecting a front end of the sensor medium based on an optical signal of the sensor medium in the presence of the analyte fluid droplet while the sensor medium is moving in the forward direction.

46. ​​The PR system of claim 45, operative to detect target molecules in analyte fluid droplets. Hmm.

48. The controller may further comprise a controller for detecting a pre-existing sample fluid droplet while the sample fluid droplet is moving relative to the sensor medium. detecting the target in the analyte fluid droplet in real time based on the optical signal of the sensor medium; 48. The PR system of claim 47, operable to measure molecular binding events.

49. The controller determines an analyte affinity value (K Ｄ ) determining a quantitative measure of analyte affinity with 49. The PR system of claim 48, operative to:

50. The above K Ｄ is the on-rate (K ＯＮ ) and The off-rate (K ＯFF ) is determined based on The PR system of claim 49.

51. The fluid droplets in the reaction portion comprise analyte fluid droplets during the association step, 51. The PR system of claim 50, wherein the second fluid comprises a buffer fluid during the dissociation stage. 。

52. 1. A method of operating a cartridge for measuring a sample fluid, comprising: contacting a sensor medium in a reaction portion of the cartridge with a fluid droplet of an analyte; 、 While maintaining the analyte fluid droplet in contact with the sensor medium, and inducing movement of the analyte fluid droplet relative to a sensor medium disposed on the sensor medium. and operating a plurality of droplet actuators. During movement of the analyte fluid droplet relative to the sensor medium, a first generating an optical signal; A method comprising:

53. The first optical signal indicates an association stage of the sensor medium in the presence of the analyte fluid droplet.

53. The method of claim 52, comprising an association signal corresponding to:

54. The on-rate (K) of the analyte fluid droplets based on the association signal ON ) step of determining 、 54. The method of claim 53, further comprising:

55. K ＯＮ The step of determining comprises fitting a meeting curve to the meeting signal.

55. The method according to claim 54.

56. The analyte fluid droplet is prevented from contacting and engaging the sensor medium. from the reaction area; A droplet of buffer fluid is introduced into the reaction area, the buffer fluid contacting and engaging the sensor medium. The steps are aligned, While maintaining the buffer fluid droplet in contact with the sensor medium, and inducing a movement of the buffer fluid droplet relative to the sensor medium, the inducing and operating the plurality of droplet actuators positioned on the substrate. During movement of the buffer fluid droplet relative to the sensor medium, a second generating an optical signal; 56. The method of claim 55, further comprising:

57. The second optical signal corresponds to a dissociation stage of the sensor medium in the presence of the buffer fluid droplet.

57. The method of claim 56, comprising a corresponding dissociation signal.

58. The off-rate (K) of the analyte fluid is determined based on the dissociation signal. OFF ) determining the The method of claim 57, further comprising:

59. K ＯFF The step of determining comprises fitting a dissociation curve to the dissociation signal. Item 59. The method according to item 58.

60. The above K ON and the aforementioned K OFF calculating an analyte affinity value (KD) based on 60. The method of claim 59, further comprising:

61. K D is K ON and K. OFF 61. The method of claim 60, wherein the quotient is

62. The sensor medium comprises a plurality of sensor nanoparticles disposed in the reaction portion. The method of claim 52.

63. The plurality of sensor nanoparticles are disposed within a sensor droplet, and the method further comprises: The sensor medium in the reaction droplet is mixed with the sample fluid droplet and the sensor. forming a reaction droplet for measuring said optical signal of the body; 63. The method of claim 62, comprising:

64. 63. The method of claim 62, wherein each of the plurality of sensor nanoparticles is magnetically responsive. Law.

65. Activating a magnet to place the nanoparticles in a restraining position relative to the magnet; and 63. The method of claim 62, further comprising immobilizing nanoparticles to the reactive moiety. 。

66. The nanoparticles are maintained in a restrained position relative to the magnet during movement of the droplet relative to the reaction portion.

66. The method of claim 65, wherein the

67. The sensor medium is disposed adjacent the distal end portion of a movable member, and the method further comprises: The movable member is moved between the extended position and the retracted position by a reaction portion defined in the reaction portion. the sensor medium is moved relative to the reaction chamber in the extended position and removing the reaction chamber at the storage position.

53. The method of claim 52.

68. The reaction chamber comprises a loading medium, and the method further comprises: storing the movable member in the stored position; introducing a fluid droplet into the reaction portion after the contraction and adjacent to the plurality of droplet actuators; removing the packing medium from the area where the packing medium is to be packed; After being introduced into the extended position, the movable member is advanced to cause the sensor medium to form a fluid droplet.

68. The method of claim 67, further comprising the steps of: placing the

69. 53. The method of claim 52, further comprising the step of engaging the cartridge with an instrument. Law.

70. While the fluid droplet is in contact with the sensor medium, A step of measuring a signal from the sensor medium while moving relative to the sensor medium.

70. The method of claim 69, further comprising:

71. The sensor medium comprises an SPR sensor medium, and the signal is 71. The method of claim 70 comprising a body light signal.

72. providing light from a light source of the instrument incident on the SPR sensor medium. 、 wherein the measurement is performed by measuring the optical intensity of the SPR sensor medium with an optical measurement device of the instrument. measuring a signal; 72. The method of claim 71.

73. The engagement is Electrical connection between the controller of the device and the plurality of droplet actuators of the DMF portion. Establishing communication; controlling the plurality of droplet actuators of the DMF portion; Further comprising: wherein the induced movement of the fluid is controlled by the control of the plurality of droplet actuators in the DMF portion. Respond to the Lord, 70. The method of claim 69.

74. During a first period, the fluid comprises a buffer fluid droplet, and the measurement is performed by measuring the amount of the buffer fluid droplet in the sensor medium. The buffer is moved relative to the sensor medium while maintaining contact with the body.

74. The method of claim 73, comprising recording a baseline optical signal.

75. The method comprises: A droplet of analyte fluid is introduced into the reaction portion for a second period of time, wherein the measurement is capturing an association signal corresponding to an association phase of the analyte fluid droplets during a period of time.

75. The method of claim 74, comprising:

76. The effective diffusion rate of the analyte fluid droplet relative to the sensor medium is 76. The method of claim 75, wherein the binding rate of the analyte fluid droplets is faster than the binding rate of the analyte fluid droplets.

77. The on-rate (K) of the analyte fluid droplets based on the association signal ON ) determining 、 77. The method of claim 76, further comprising:

78. The above K ＯＮ determining comprises fitting a meeting curve to the meeting signal.

78. The method according to claim 77.

79. moving the analyte fluid droplet away from a sensor medium; introducing a buffer fluid droplet into the reaction portion for a third period of time; wherein said measuring corresponds to a dissociation phase of said analyte during said third period of time.

80. The method of claim 78, comprising capturing a dissociation signal.

80. The off-rate (K) of the analyte fluid is determined based on the dissociation signal. OFF ) 80. The method of claim 79, further comprising:

81. The above K ＯFF determining comprises fitting a melting curve to the melting signal.

81. The method according to claim 80.

82. K ON and K. OFF and based on the analyte affinity value (K D ) and 82. The method of claim 81, further comprising:

83. K D is K ON and K. OFF 83. The method of claim 82, wherein the quotient is

84. providing a regeneration buffer fluid droplet to the reaction portion during a fourth period of time; contacting the regeneration buffer fluid droplet with the sensor medium to regenerate the sensor medium; and 84. The method of claim 83, further comprising:

85. A functionalized fluid droplet having a ligand is contacted to bind the ligand to the sensor medium.

85. The method of claim 84, further comprising the step of functionalizing the sensor medium by combining the sensor medium with a substrate. The method described above.

86. Contacting an activation fluid droplet with the sensor medium prior to functionalization of the sensor medium.

86. The method of claim 85, further comprising activating the sensor medium by: Law.

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