Microfluidic devices and methods for sampling and analyzing cells using optical forces and raman spectroscopy

Abstract

Microfluidic devices and methods for sampling and analyzing cells using optical forces and Raman spectroscopy are disclosed. Provided herein are devices and methods for automated analysis of one or more samples in a single well or multi-well plate or vessel, wherein the automated analysis process includes flow and hydrodynamic, electrodynamic, and optical forces for analysis and sorting of samples, wherein the samples include liquids or particles in microfluidic channels, and wherein the devices include component assemblies capable of processing the samples for analytical evaluation of the instruments based on the fluids and / or particles. Microfluidic structures (channels, "T" junctions, "Y" junctions, branched "Y" junctions, wells, and weirs) are described for facilitating sample interactions and observation, sample analysis, sorting, or partitioning. Detection can be accomplished using spectroscopic methods, including but not limited to Raman spectroscopy of individual cells and bulk cell samples (cell populations; a few individuals to hundreds or thousands of cells).

Description

[0001] This application is a divisional application of Chinese Patent Application 202080077794.5, filed on September 9, 2020, entitled "Microfluidic Apparatus and Method for Sampling and Analyzing Cells Using Optical Power and Raman Spectroscopy". Technical Field

[0002] Specific embodiments of this disclosure relate to apparatuses and methods for automated nano / micro / millimeter fluid sampling from one or more containers. The containers may range from a single well or vial to multiple well plates. The apparatuses provided herein are further capable of mixing the contents of individual wells, illuminating cell populations sampled in or from wells using light for spectroscopic studies, microfluidically mixing or separating particle or cell samples for analysis using hydrodynamics, optical forces, acoustic forces, magnetic forces, or electrodynamics, and monitoring particle-based processes using optical or Raman spectroscopy. The apparatuses and methods described herein further include the use of Raman spectroscopy, as well as optical and hydrodynamic forces combined with electrodynamics, for cell analysis and sorting. Background Technology

[0003] The increasing use of automated systems for sample storage, processing, and analysis has spurred extensive research in the field of automation. Current sampling systems include robotic arms capable of moving samples from storage areas to loading areas for sampling, magnetic or mechanical stirring components that keep samples suspended, and heating or cooling zones that regulate the temperature of sample storage or analysis.

[0004] Existing technology devices include automated sampling equipment covering a wide range of analytical research areas (e.g., U.S. Patent No. 4,713,974 used in conjunction with liquid chromatography), but they are not suitable for biological cell analysis using laser force cytology (LFC), Raman spectroscopy, or other photodynamic and / or hydrodynamic and / or electrodynamic measurements, such as cell manipulation involving capture and sorting, due to the lack of accurate and consistent nanoliter flow rate control, adequate sample mixing, and reliable temperature control for sample processing.

[0005] U.S. Patent No. 4,816,730 to Wilhelm et al. describes the use of a device for handling and moving multiple objects, comprising a robotic arm capable of vertical, horizontal, and rotational movement, and a gripping mechanism driven by an electrically controlled stepper motor for holding samples. U.S. Patent No. 6,872,362 to While Schmidt et al. further describes the use of a powered automated sampler with vials, adapted to include a magnetic stirring rod driven by various methods of altering the magnetic field around the vials. While such prior art describes various methods for moving and mixing samples, these methods are not suitable for biological cell analysis using LFC instruments. What is needed is improved equipment capable of storing, mixing, and sampling in vessels ranging from single-well to multi-well plates, while maintaining the integrity of biological cells through adequate mixing and temperature maintenance using pneumatically based non-contact mixing and temperature-controlled single-well or multi-well plates.

[0006] Automated well plate stacking or retrieval systems with mechanical mechanisms can be used to achieve vertical or horizontal sequential stacking or retrieval (CN204136215U, US20040206419A1), loading and unloading using cassettes (US Patent No. 9744535), or to describe the simultaneous stacking or retrieval of entire batches of plates back to a storage tank or tower (US Patent No. 6086319). Furthermore, previous designs enabled well plates to be loaded or removed from storage towers in a random (non-sequential) manner, regardless of their stacking order (US Patent No. 7670555). What is needed is a system capable of specifically and automatically detecting, selecting, stacking, or retrieving desired well plates into or from storage towers in a non-sequential manner using a magnetic interface, and capable of well plate culture that can be used with multiplex analysis methods for automated sampling of such well plates.

[0007] We also need to be able to represent and analyze particles (e.g., biological cells) in ways that provide meaningful information for a range of applications. For example, we need to be able to analyze fluid samples by accurately assessing their composition. In some implementations, it is necessary to be able to monitor and control fluid flow so that particles in the fluid can be sorted and then selectively located for evaluation. In some implementations, the ability to sort particles in a fluid sample and orient them appropriately to represent their properties (i.e., internal organelles, surface proteins, receptors, nuclear changes, or other biophysical or biochemical markers) will contribute to the development of integrated medicine, such as gene or cell therapies. In other implementations, it is beneficial to be able to bring cells or particles into contact with each other (in the presence or absence of chemicals / biochemicals) for the purpose of representing and quantifying interactions (binding affinity, cell-killing ability, or other interactions). Furthermore, accurate particle characterization is also crucial for the development and design of novel biologics or cell or gene therapy products. Specifically, label-free measurements of the interactions between target cells (e.g., cancer cells—cell lines or primary cells) and effector cells (e.g., modified T cells—chimeric antigen receptor T cells (CAR-T), T cell receptor (TCR), etc.), or any other cell therapy products that destroy cancer, would be a significant step in replacing inadequate co-culture cell killing assays based on imaging or staining (antibody- or other types). Therefore, what we need is a device capable of improving the characterization and data collection of particles (e.g., cells and other chemical and biological entities).

[0008] In the cell therapy manufacturing cycle, measuring intrinsic properties using Raman spectroscopy and / or electrodynamics and / or photodynamics at multiple stages can be valuable. This characterization is particularly relevant to the manufacture of autologous CAR T cells. The first step is typically apheresis, during which white blood cells must be extracted from red blood cells and other components. Due to the differences in each patient's medical history and individuality, intrinsic measurements of the apheresis product are valuable, as this will be the starting material for the manufacturing process. This characterization can be accomplished using Raman spectroscopy and / or electrodynamics and / or photodynamics, and once sufficient data has been accumulated, it can be used to guide the manufacturing process or predict the chances of success. After apheresis, cells are typically engineered using viral vectors such as lentiviruses or retroviruses, but other methods exist, such as transposons, zinc finger nucleases, or regularly spaced clustered short palindromic repeats (CRISPR). The genetic modification process of patient cells is often difficult to monitor, thus potentially another valuable area for Raman spectroscopy or photodynamics. After the cells are engineered, they are scaled up to provide adequate doses to patients. This process is also difficult to monitor, requiring novel analytical tools to determine optimal process conditions. Due to the unstable nature of T cells, this process can vary from patient to patient and is not always possible to represent samples using antibody labeling. Therefore, this is another area where Raman spectroscopy or photodynamics can be used to monitor scaling-up steps to optimize and ensure the success of the process. Finally, to predict the clinical success of a treatment, the final product must be represented. Several methods exist, such as co-culture killing assays or measurements of certain cytokines, such as interleukin-2 (IL-2). To date, no specific key quality attributes have been identified that can accurately predict clinical or biomanufacturing success. Therefore, the measurement of product cells using Raman spectroscopy and / or electrodynamics and / or photodynamics (alone or in synergistic with target cells) provides another valuable area. The product must also undergo safety testing, including the absence of mycoplasma, bacteria, and viruses. Characterization of Raman spectroscopy and / or electrodynamics and / or photodynamics throughout the manufacturing process or in the final product can be used to test each of these exogenous agents in a rapid and label-free manner. Raman spectroscopy and / or electrodynamics and / or photodynamics can also be used to measure patient responses to cell or gene therapies by collecting blood or biopsy samples and analyzing phenotypic changes in cells resulting from cell therapy.

[0009] In the field of gene therapy, where a patient's cells are altered for therapeutic effect, viral vectors such as adenoviruses, adeno-associated viruses (AAVs), or lentiviruses are typically used. While AAVs can also be manufactured using helper viruses such as adenoviruses, herpes simplex viruses, or baculoviruses, their production process usually involves a transfection step. Once manufactured, the viral particles can be used to infect or transduce target cell types. Each of these processes—transfection, infection, and transduction—is difficult to quantify and can be another area for measurement using Raman spectroscopy and / or electrodynamics and / or optical dynamics. Several additional applications related to AAVs specifically include distinguishing between empty and full capsids, infectious and non-infectious capsids, physical titer, and infectious titer.

[0010] In another aspect of cell therapy, the application of intrinsic measurements will be useful for the research, development, production, and differentiation of stem cell products. Since various methods, including mediator supplementation and genetic modification, can be used to differentiate or dedifferentiate cells into different lineages, the ability to represent and monitor this process is crucial. Due to the heterogeneity of these populations, the exact nature of the differentiation pathway followed by each cell may differ slightly, which can make it difficult to represent cell populations using traditional antibody- or fluorescence-based methods, as it is unclear which cell types may be present. Therefore, employing a label-free method that can measure intrinsic properties (e.g., Raman spectroscopy and / or electrodynamics and / or photodynamics) can allow for unbiased and flexible characterization and monitoring of these populations and processes. Furthermore, the degree of stem cell deformability is biologically relevant and can be measured by applying photodynamics.

[0011] Measuring at least one intrinsic (or extrinsic) property of particle and cell samples using Raman spectroscopy and / or electrodynamics and / or photodynamics can provide important information for subsequent analysis and utilization of such particle and cell samples. Information about such properties can be used for a range of applications, including but not limited to: determining the viral infectivity of cell samples (the number of functionally infectious viral particles present in a particular cell population, similar to plaque assays or endpoint dilution assays) for purposes such as virus quantification, process development and monitoring, sample release assays, exogenous agent testing, clinical diagnostics, and biomarker discovery; determining cell productivity based on antibodies or proteins for process development and monitoring; determining the efficacy, quality, or activation status of cells generated by cell-based therapies or gene therapies, including CAR T and other oncology applications and stem cells; determining the effects of chemicals, bacteria, viruses, antimicrobial agents, or antiviral substances on a particular cell population; and determining the disease state or potential of research or clinical cell samples.

[0012] Therefore, what we need are devices, systems, and methods of use that can automatically sample from containers (such as vials, orifices, or perforated plates) and detect, analyze, and / or manipulate them optically and / or hydraulically and / or electrically, and coordinate the transfer of such samples to the analysis area for detection / characterization and processing. Summary of the Invention

[0013] Specific embodiments of this disclosure relate to apparatus and methods for automated nano / micro / millimeter fluid sampling and analysis of particles from fluids derived from vessels, wherein the vessels range from a single well or vial to multiple well plates. Analysis and sorting of particle samples (cells, bacteria, yeast, etc.) are accomplished using photodynamic, electrodynamic, and hydrodynamic methods and / or Raman spectroscopy to move cells within microfluidic channels for analysis, sorting or separation, and spectral sensing, as well as detection using Raman spectroscopy. More specifically, the novel apparatus and methods described herein utilize hydrodynamic, electrodynamic, and photodynamic methods to perform fluid manipulation for cell separation and analysis purposes. Additional biochemical and biological data can be obtained by using Raman spectroscopy to measure cells within microfluidic channels, wells, weirs, or other structures where cells are aggregated or concentrated for sensing purposes. In some embodiments, the use of surface-enhanced Raman (SERS) signals can be achieved by coating the channel surface with a noble metal (gold, silver, platinum, etc.) to enhance signal collection. Cells can be sensed in groups or as single cells to collect large-scale or single-cell data. Attached Figure Description

[0014] Figure 1 A schematic diagram is provided of the probe (such as a fiber optic probe) and the tube used for sampling.

[0015] Figure 2 A schematic diagram of the fiber optic probe and microfluidic sampling system is provided.

[0016] Figure 3 A schematic diagram of a microfluidic sampling system with an optical fiber probe and an integrated tip for sampling from a small aperture is provided.

[0017] Figure 4 A schematic diagram of a fiber optic probe is provided, which includes a movable / detachable reusable system tip for fiber optic probes and microfluidic sampling systems.

[0018] Figure 5(A) provides a schematic diagram illustrating a specific implementation of a microfluidic path for segmenting cells, particles, debris, or molecules. Figure 5(B) provides a schematic diagram illustrating a specific implementation of a microfluidic path for segmenting cells, particles, debris, or molecules and connecting to a fiber optic probe sampling system.

[0019] Figure 6A schematic diagram illustrating a specific implementation of a microfluidic pathway is provided for segmenting cells, particles, fragments, or molecules and for use in the analysis of photomechanical and cell interactions.

[0020] Figure 7 A schematic diagram illustrating a specific implementation of a microfluidic pathway for segmenting cells, particles, debris, or molecules for photomechanical or interaction analysis is provided, incorporating large-diameter channels or channel regions. In this specific embodiment, an example of an analytical region is provided where Raman spectroscopy can be used to obtain relevant measurements and information about the particles of interest in the sample.

[0021] Figure 8 A schematic diagram illustrating a specific implementation of a microfluidic pathway is provided for the segmentation of cells, particles, fragments, or molecules for photomechanical or Raman analysis, and for the subsequent introduction of biochemicals and / or other cells for interaction measurements.

[0022] Figure 9(A) provides a schematic diagram illustrating a specific embodiment of a microfluidic path, wherein one or more cell populations, particles, debris, or molecules bind together before entering the interrogation region. Figure 9(B) provides a schematic diagram illustrating a specific embodiment of a microfluidic path, wherein one or more cell populations, particles, debris, or molecules bind together using T-junctions before entering the interrogation region. Figure 9(C) provides a schematic diagram illustrating a specific embodiment of a microfluidic path, wherein one or more cell populations, particles, debris, or molecules bind together before entering the interrogation region, where columns within channels are used to control flow states and interactions.

[0023] Figure 10 A schematic diagram illustrating a specific implementation of the microfluidic architecture is provided, showing Raman collection pores within the microfluidic channels for sorting and accumulating cells for Raman analysis.

[0024] Figure 11 A schematic diagram illustrating a specific implementation of the microfluidic architecture is provided, showing Raman collection pores within the microfluidic channels for accumulating cells for Raman analysis, and two waste or release channels for disassembly.

[0025] Figure 12 A schematic diagram illustrating a specific implementation of the microfluidic architecture is provided, showing a Raman collection orifice within the microfluidic channel for accumulating cells for Raman analysis, and three waste or release channels for splitting the flow so that there is or no flow within the Raman collection orifice and channel.

[0026] Figures 13(A) and 13(B) provide schematic diagrams illustrating specific embodiments of a microfluidic architecture, showing collection orifices or weirs within microfluidic channels for laser-based Raman analysis or other analyses. Figure 13(A) shows a top view of a representative collection orifice or weir. Figure 13(B) shows a side view of a representative collection orifice or weir configured to capture one or more cells. Figure 13(C) shows a weir where cells are collected against a wall or other structure for analysis. Figure 13(D) shows a specific embodiment with multiple orifices, wherein the orifices are spaced apart in various configurations and optionally combined with one or more weir structures.

[0027] Figures 14(A) and 14(B) provide schematic diagrams illustrating specific embodiments demonstrating configurations for capturing cells or cell populations and analyzing them using hydrodynamic, optical, or electrodynamic forces. Figure 14(A) shows a specific embodiment demonstrating the placement of a collimating light source (630) focused into a representative collection orifice or weir. Figure 14(B) shows a top view of the representative collection orifice or weir.

[0028] Figures 15(A) and 15(B) provide schematic diagrams illustrating the integration of photodynamics, Raman detection, and a microfluidic SERS substrate for surfaces, pores, and weirs. Figure 15(A) provides a specific embodiment demonstrating a laser or collimated light source (630) providing photodynamics (1510) that can trap cells or particles (150) onto a SERS coating or substrate (1500). Figure 15(B) provides a specific embodiment demonstrating the incorporation of pores (1550) that substantially trap and separate cells (150) after they have been acted upon by photodynamics.

[0029] Figure 16 A schematic diagram is provided, illustrating the use of a device that combines optical, electrodynamic, and Raman spectroscopy for microfluidic analysis of micron or nanoparticles.

[0030] Figure 17 A schematic diagram is provided, illustrating the use of a device that combines optical, electrodynamic, and Raman spectroscopy for microfluidic analysis of micron or nanoparticles.

[0031] Figure 18 A schematic diagram is provided, illustrating the use of a device combining optical, electrodynamic, and Raman spectroscopy for microfluidic sorting of micron or nanoparticles. Detailed Implementation

[0032] The invention is described with reference to specific embodiments having various features. It will be apparent to those skilled in the art that various modifications and changes can be made in practice without departing from the scope or spirit of the invention. Those skilled in the art will recognize that these features can be used alone or in any combination based on the requirements and specifications of a given application or design. Those skilled in the art will recognize that the systems and apparatus of the specific embodiments of the invention can be used with any method of the invention, and any method of the invention can be performed using any system and apparatus of the invention. Specific embodiments including various features may also include or substantially include these various features. Other specific embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. The description of the invention provided is merely exemplary in nature, and therefore, changes without departing from the spirit of the invention are intended to be within the scope of the invention.

[0033] Before explaining at least one specific embodiment of the present invention in detail, it should be understood that the present invention is not limited to the specific constructions and component arrangements listed in the description below or illustrated in the drawings. The present invention can have other specific embodiments and can be implemented or carried out in various ways. Furthermore, it should be understood that the wording and terminology used herein are for illustrative purposes and not for limiting purposes.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood or used by one of ordinary skill in the art to which these techniques and methods are covered.

[0035] The texts and references mentioned herein are incorporated herein in their entirety, including U.S. Provisional Patent Application Serial No. 62 / 654335, filed April 7, 2018, and U.S. Patent Application Serial No. 16 / 378067, filed April 8, 2019.

[0036] This paper provides novel devices for automated sample analysis, wherein the sample is contained in vials, dishes, wells, multi-well plates, etc.; and also provides methods for using such devices. In this paper, these devices may be referred to as microfluidic devices, and these devices can be used to analyze particulate samples, wherein the samples include a variety of particles, cells, biological and / or chemical entities. Microfluidic devices deliver samples in suitable media / preparations / dishes for analysis of liquids, particles, or cells using fluid-based instruments or systems. Suitable fluid-based instruments for analysis may utilize laser-force cytology (LFC) or other methods. In some specific embodiments, a series of novel methods combining hydrodynamics, optical force, and electrodynamics with Raman spectroscopy are used to characterize particles / cells. The applications can be subdivided into several categories, including but not limited to: (1) offline Raman spectroscopy analysis using probes in holes or tubes, (2) on-chip microfluidic Raman detection or cell separation for subsequent Raman analysis, (3) cell electrodynamic and / or optical sorting and / or Raman spectroscopy based on intrinsic parameters without the use of labels, (4) cell separation using holes or weirs with optical, hydrodynamic or electrodynamic and Raman spectroscopy to measure the biochemical properties of cells, and (5) the use of metal coatings to enhance Raman signals, and weirs, holes or channels in microfluidic devices using hydrodynamic or optical forces.

[0037] In one specific embodiment, the microfluidic device described herein may further include dual fiber optic probes. For example, as... Figure 1 As shown, a dual-fiber probe and a fluid tube (120) for sampling particles containing fluid from a vessel (130) can be incorporated into the instrument for spectral measurements at the sampling point. In some embodiments, the probe is made of optical fibers (100, 110) for transmitting excitation light and collecting Raman signals. The probe may also be made of other materials suitable for its purpose. The integrated sampling tube allows cells or particles (150) to be introduced into the instrument. The probe, well plate, or both can be converted into one to three sizes for automated sampling. In some embodiments, the base of the vessel may be SERS-active to enhance or enable Raman signal capability.

[0038] In such Figure 2 In the specific embodiment shown, a dual-fiber probe and a microfluidic sampling tube with a shell (240) are provided to allow cells or particles (150) to interact with SERS or other Raman-active surfaces to enhance the Raman signal (260). This is used for Raman measurements on a per-sample basis while simultaneously introducing samples into another device. If necessary, a flushing liquid can be applied in front of the Raman or other sensor fibers to clean between samples and prevent sample cross-contamination.

[0039] Figure 3A fiber optic probe and a microfluidic sampling system with an integrated tip (160) for sampling from a small orifice or tube are illustrated. If necessary, a liquid can be flushed in front of the Raman or other sensor fibers to clean between samples and prevent cross-contamination. SERS or other Raman-active surfaces may optionally be included to enhance the Raman signal (260). By using the sampling tip, the device enables access to higher-density well plates with smaller orifices. The tip can be made of any suitable material, including but not limited to polymers, plastics, glass, metals, or composites. The size and dimensions of the sampling tip can be customized according to the application and can be produced by methods known to those skilled in the art, such as by 3D printing.

[0040] Figure 4 Additional specific embodiments of the invention are provided, wherein, for Figure 3 The fiber optic probe and microfluidic sampling system features a removable / detachable reusable system tip (170). A glass (or other material) tip is attached to a metal Raman probe housing (180), which may be metal, plastic, or other material, and includes a strap or clip (310) that holds a seal or gasket (300) between them for sealing in the fluid. In an alternative embodiment, the tip (170) is attached to the housing (180) via a magnet or electromagnet. The probe tip (175) is replaceable, and a laser sensor can be selectively used to check the integrity of the probe. In an alternative embodiment, the bottom (190) is optically opaque or dark to prevent fluorescence from the bottom of the well plate.

[0041] Figure 5(A) provides a specific embodiment in which one or more paths of a microfluidic device (e.g., an autosampler) can be designed to segment cells, particles, fragments, or molecules by having branches in a microfluidic channel network. This network can be fluidly placed... Figures 1-4 This can be a downstream device of any probe system described herein, or a stand-alone device. The orientation of particles or cells (150) can be controlled by guiding them to the waste channel (510) or the analysis channel (520) by changing the flow rate or pressure between the two channels, or by using some other means, such as optical, electrodynamic, or acoustic forces. Depending on their characteristics or type, cells can be guided to the analysis area. For example, Figure 5(A) shows a second cell type (155) in the population that is preferentially guided to the analysis area. Figure 5(B) shows a fiber optic probe sampling system (such as...) Figures 1-4 One specific embodiment of the connection between the system (described in Figure 5(A)) and the bifurcated channel. In this specific embodiment, the sample is made from a mixed population of cells (150) and (155). Depending on their characteristics, some cells from each population may be directed to the analysis area.

[0042] Figure 6 This illustrates how selective analysis of certain cells or particles (150) or (155) can be achieved by guiding cells into a dedicated analysis channel (520) and a discard channel (510) within a microfluidic device. In one embodiment, as a cell enters the channel structure, the flow is altered to displace the cell into the analysis channel by fluid pressure controlled by a feedback loop based on cell optical imaging. Other embodiments may use other forces, such as optical, magnetic, electrodynamic, or acoustic forces, to guide or trap cells in the analysis channel for interrogation. Optical-based methods may include using optical tweezers to hold the cell, dual laser beams, or cells trapped between a flowing and a lightly focused laser beam (laser-force cytology). In one embodiment, a collimated light source (630) focused into the analysis channel (520) is shown. Depending on the analytical requirements, a significant distance or offset (620) may exist between the entrance of the bifurcation (610) and the analysis channel (520). This is to prevent the laser or other force sources on the cells from affecting cells that have not entered the analysis channel.

[0043] Figure 7 A microfluidic pathway for segmenting cells, particles, debris, or molecules for photomechanical or interaction analysis is depicted in a specific embodiment incorporating a larger diameter channel or channel region. This larger region (710) reduces the fluid rate to facilitate capture and increase residence time for analysis. The analysis may include, for example, Raman spectroscopy, fluorescence, or other measurements.

[0044] The addition of molecules, viruses, exogenous substances, cells, or other materials can be achieved through a separate channel (810) for introduction. Figure 8 This can be a single analytical channel with a single input, or multiple analytical channels with one or more inputs for introducing other materials. Photodynamic and hydrodynamically captured cells can undergo injection of chemicals, biochemicals, bacteria, viruses, exogenous bodies, cells, or other materials in a controlled manner (820).

[0045] Another method for creating and measuring cell interactions is to mix samples before analysis. Figure 9A~9C). In one embodiment, samples flowing from vessel 1 (910) and samples flowing from vessel 2 (920) can be combined. Before analysis, the samples will travel through an interaction region (930). The samples may or may not interact or combine chemically or biologically. The travel distance of the mixed population before analysis can be customized to achieve precise analysis time. This can also be achieved by adjusting the flow rate over a fixed distance. Furthermore, the residence time of cell or particle interactions before analysis can be controlled by fluid, optical, electrodynamic, or magnetic forces. The channel structure can be oriented horizontally, vertically, or at an angle relative to gravity. Interactions can be controlled by incorporating T or Y nodes before the analysis region. Figure 9(B) shows one embodiment depicting a T node. Shapes or other features can also be employed to facilitate or improve interactions between two different samples or populations. Figure 9(C) shows one embodiment in which a column array (930) exists within the channel to control interactions between any cells or particles introduced into the channel.

[0046] Figure 10 Specific embodiments of a microfluidic architecture having collection or analytical regions (1030) within a microfluidic channel are provided for sorting and accumulating cells or particles (150) for Raman or other spectroscopic analyses. Cells may be guided to a waste channel (1010) or collected within the analytical region (1030) using fluid, optical, magnetic, electrodynamic, or acoustic forces (1015). After analysis, cells or particles may be guided out of the device via a channel (1040).

[0047] Figure 11 Provided Figure 10 The microfluidic architecture shown in the figure illustrates a collection pore or region (1030) within a microfluidic channel for accumulating cells for Raman or other analyses, and additional waste or release channels (1020) for disassembly prior to analysis.

[0048] Figure 12 Provided Figure 11 The microfluidic architecture shown illustrates collection or regions within microfluidic channels for accumulating cells for Raman analysis, and includes two additional discard or release channels (1020 and 1050) for splitting the flow so that there is or is no flow within the Raman collection orifice (1030) and channel (1040).

[0049] Figures 13(A) through 13(D) illustrate microfluidic architectures, showing sample collection orifices or weirs within microfluidic channels for laser-based Raman or other spectroscopic analyses. Figure 13(A) shows a top view of one specific embodiment of the orifice, although its shape can be circular (as shown), rectangular, elliptical, or polygonal. Several specific embodiments are shown. In Figure 13(B), the orifice is used to capture one or more cells. The depth (1310) and width (1315) of the orifice can be adjusted to fine-tune its behavior, including the number and type of cells (150) collected. The bottom of the orifice may include an electrode or SERS material (1320) to facilitate Raman or other spectroscopic analyses. Figure 13(C) shows a weir where cells are collected against a wall or other structure for analysis. The height of the weir (1330) can be adjusted, and the weir area may also contain electrodes or SERS material (1320) similar to the orifice. Figure 13(D) shows a specific embodiment with multiple orifices. These orifices can be spaced apart in various configurations and can be combined with one or more weir structures. Multiple weir structures can also be used individually to create multiple collection or analysis zones. In each specific implementation, fluid, optical, magnetic, electrodynamic, or acoustic forces can be used to assist in the collection or release of cells trapped in the structure.

[0050] Figures 14(A) and 14(B) provide specific embodiments for capturing particles, cells, or cell populations in a Raman analysis well (1030) using fluidic, optical, magnetic, electrodynamic, or acoustic forces for Raman or other spectroscopic analyses. Figure 14(A) illustrates one embodiment in which a collimated light source (630) is used to apply optical force to capture cells or particles (150) within the Raman analysis well (1030). Figure 14(B) illustrates another embodiment in which electrodynamic forces are used to capture cells or particles within the Raman analysis well (1030).

[0051] Figures 15(A) through 15(B) provide schematic diagrams illustrating the integration of photodynamics, Raman detection, and a microfluidic SERS substrate for the surfaces, pores, and weirs of Figures 13 and 14. Photodynamics can be used to capture cells or particles onto the SERS coating or substrate for analysis. Figure 15(A) shows a specific embodiment where a laser or collimated light source (630) provides photodynamics (1510) that captures cells or particles (150) onto the SERS coating or substrate (1500), which are then analyzed using Raman spectroscopy. Fluid flows from an inlet (1530) of a channel or channel network to an outlet (1520), e.g., as described in Figures 13 and 14. Figure 15(B) shows a specific embodiment with pores (1550) that substantially capture and separate cells (150) after they are acted upon by photodynamics (1510).

[0052] Figure 16A device combining photodynamic, electrodynamic, and / or Raman spectroscopy is illustrated for microfluidic analysis of cells, micrometers, or nanoparticles. Electrodynamic forces drive nanoparticles from a cathode (1600) to an anode (1610), and they accumulate in a potential well (1410) where Raman signals can be measured. Activity analysis is then performed using the laser force of multiple lasers (1400) located at each node or by scanning a single laser through the node. The device may contain one or more nodes. These lasers apply photodynamic forces to drive the particles against fluid flow to measure the properties of the nanoparticles. In one embodiment, cells or particles with different properties (1620, 1622, 1624) accumulate at each node according to their electrical properties. In an alternative embodiment, particles move according to their electrodynamic forces and encounter the laser beam, automatically propelling themselves different distances by the photodynamic forces generated by their respective biochemical and biophysical properties.

[0053] Figure 17 A device combining optical, electrodynamic, and / or Raman spectroscopy is illustrated for microfluidic analysis of cells, micrometers, or nanoparticles. Electrodynamic forces drive the nanoparticles toward the anode (1610), and at each node 1505, 1515, 1525, a laser beam (1550) drives the particles through channels. The device may contain one or more nodes. The degree to which the electrophoretic force / flow and the laser optical force drive the particles will determine their trajectory. Particles with higher refractive indices (1626) will be driven more along the laser beam than particles with lower refractive indices; similarly, particles with greater electrophoretic forces will be driven away from the laser beam and toward a cascade of other analytical regions. The competition between electrophoretic and optical forces provides the ability to analyze particles with different compositions. Other specific implementations may include various combinations of magnetic, acoustic, electrodynamic, or optical forces, as well as Raman spectroscopy and / or pressure-based pumping. For example, a device combining optical and Raman spectroscopy in which the fluid is driven by pressure-based flow.

[0054] Figure 18 A device combining optical, electrodynamic, and / or Raman spectroscopy is illustrated for microfluidic sorting of micron or nanoparticles. Electrodynamic forces drive the nanoparticles toward the anode (1610), and at each node 1505, 1515, 1525, separation in the flow is introduced, while a laser beam drives the particles to orifices or outlets 1510, 1520, and 1530 via photopressure. The device may contain one or more nodes. The degree to which the electrophoretic force / flow and the laser optical force drive the particles determines their separation path. Particles with higher refractive indices (1626) are driven more towards the separation channel / orifice than particles with lower refractive indices; similarly, particles with greater electrophoretic forces are driven away from the separation channel or orifice and toward a cascade of other separation regions. The competition between electrophoretic and optical forces provides the ability to separate particles with different compositions.

[0055] In one specific embodiment, this document provides an apparatus for the automated analysis of one or more samples, wherein the automated analysis process includes automated flow, wherein the sample comprises liquid or particles in a sample vessel, and wherein the apparatus includes component assemblies capable of processing the sample (or multiple samples) for analytical evaluation using fluid- and / or particle-based instruments. The apparatus may include microfluidic components for analyzing samples containing particles / cells. In some specific embodiments, the microfluidic component may include an autosampler. In some specific embodiments, the apparatus may include Radiance® (LumaCyte, Virginia, USA). Methods of using such apparatus are also provided. In some specific embodiments, the apparatus of the present invention includes additional capabilities for particle analysis and characterization of cells using a series of novel methods combining hydrodynamics, optical dynamics, and electrodynamics with Raman spectroscopy.

[0056] In one specific embodiment, cell and particle analysis is performed in an offline Raman setup. For example, the analysis may occur within wells (i.e., similar to an autosampler). In this embodiment, sorted or unsorted cells can be directed into a reservoir capable of Raman measurement using a fiber optic probe. Such analyses can be performed in a variety of well plates, including but not limited to 6, 12, 24, 48, 96, 192, 288, 384, or 1536-well plates, and furthermore, analyses can be performed in wells or reservoirs of variable or fixed capacity. Probes with cavities and mixing capabilities to prevent background noise from the well plates on Raman measurements, such as fiber optic probes, can be used. During the sampling step, as fluid is drawn out (using a hydrodynamic flow from a syringe pump or air pressure over a fluid-driven flow, or electroosmotic flow), it fills the cavity for typical measurements. In some embodiments, a suitably sized sampling tube can be attached to the cavity portion of the probe to draw fluid into the autosampler while Raman measurements are performed. This type of configuration allows large fiber optic probes to be designed with filters and still access small samples. In some specific embodiments, the analysis can be performed in a well plate with a SERS-active bottom, wherein cells accumulate; such a well plate may have a glass bottom, and Raman measurements can be performed from below or above.

[0057] In alternative embodiments, the apparatus and methods of the present invention may include particle or cell analysis occurring on a chip in a separate region of an autosampler dedicated to Raman analysis. Various configurations of the apparatus may be suitable depending on the purpose and function of the task. For example, in some embodiments, the microfluidic channel may be split, i.e., bifurcated or trifurcated (or more) to guide a subset of cells to the region for Raman measurement; particles may be clamped using a laser beam, DEP, or a combination of laser-DEP with a specific geometry; optical force may clamp cells against a wall with a SERS-active surface. In some configurations, individual regions may exist between samples that are flushed away during a cleaning cycle (including a release channel with an electronic pressure controller to handle flow after sample loading. Analysis can switch back to the main waste channel to leave cells loaded in the Raman chamber on the chip). In some embodiments, a disposable chip with a region for collecting and measuring 96 samples may be used. In some other embodiments, a user-replaceable node may be used, wherein a chip holder and tubing connection or integrated connection to the tubing is used (preferably). Other features include sample interaction channels, where target cells are clamped using laser / hydrodynamic or laser / electrodynamic methods and then biochemicals or other cells or materials are introduced; electrodynamics in zigzag or sawtooth regions for capturing and measuring Raman signals; laser separation of nanoparticles from the electrodynamically captured region; and real-time on-chip Raman measurements using LFC lasers (a combination of lasers to introduce optical force and simultaneously generate Raman signals).

[0058] In one specific embodiment, the samples analyzed by the microfluidic device described herein may include, but are not limited to, polymers, metals, glass or alloy-based particles, biological cells, plant cells (algal cells or others), prokaryotic cells (bacteria), eukaryotic cells, yeast, fungi, mold cells, erythrocytes, neurons, oocytes (eggs), sperm, leukocytes, basophils, neutrophils, eosinophils, monocytes, lymphocytes, macrophages, platelets, vesicles, exogenous bodies, stromal cells, multicellular structures (such as spheroids), mesenchymal stem cells, and induced pluripotent stem cells (iPSCs), tumor cells, primary cancer cells, T cells, B cells, monocytes, macrophages, other leukocytes, erythrocytes, genetically modified T cells or any other modified cells or gene therapy modified products, and subcellular components, including cell nuclei, mitochondria, or chloroplasts. Samples may be synthetically manufactured or obtained from natural sources. Samples can be obtained from bodily fluids or substances, including but not limited to tears, saliva, sputum, blood, plasma, lymph, urine, sweat, pus, nasal mucus, or semen.

[0059] In one specific implementation, the analysis and evaluation performed by fluid- and / or particle-based instruments includes, but is not limited to, measurement of photodynamics, laser-based cytology, Raman spectroscopy, automated microscopy, capillary electrophoresis, single-cell droplet microfluidics, single-cell genomics, sequencing devices, mass spectrometry, and nucleic acid or protein analysis, amplification, or modification.

[0060] In some embodiments, the fluid handling components of the apparatus include an outer tube housed within a sample vessel, one or more discrete inner tubes within the diameter of the outer tube, optical fibers for spectral sensing (including, but not limited to, Raman spectroscopy), and connections to one or more destination vessels to which the fluid is connected, and one or more separate systems for controllably moving fluid into or out of the sample vessel. In some embodiments, valves may be used to preferentially drive fluid into one or more inner tubes or prevent fluid from entering one or more inner tubes, and the systems for moving the fluid may include vacuum systems, pressure-based systems, or pumps (e.g., peristaltic pumps, diaphragm pumps, syringes, or others). In some embodiments, the fluid handling apparatus is located within a manifold that creates an hermetically sealed seal for the sample vessel or its compartment. The outer tube may be made of metal, plastic, ceramic, composite material, glass / capillary, or other materials; the inner tube may be made of metal, plastic, ceramic, composite material, glass / capillary, or other materials. In some embodiments, the inner tube is reversibly connected to the outer tube using connectors (e.g., fittings, sleeves, ferrules, or other housings), or permanently connected using glue, epoxy resin, adhesives, or other bonding agents. In some embodiments, the outer and inner tubes are fabricated as single pieces of one or more material types using additive manufacturing techniques, including 3D printing such as stereolithography, digital light processing, fused deposition modeling, selective laser sintering, selective laser melting, electron beam melting, layered solid fabrication, spray molding, material spraying, or other techniques. Furthermore, the outer and inner tubes can be fabricated from glass using laser patterning and hydrofluoric acid (HF) or potassium hydroxide (KOH) etching and bonding methods. In other embodiments, the inner tube of the device is connected to one or more fluid reservoirs that can be transferred to sampling vessels or other vessels within the system. Liquid can be transferred to separate adherent cells grown in the sample device. Sample containers may include vials or well plates with 6, 12, 24, 48, 96, 192, 288, 384, 1536 or any custom number of wells.

[0061] In one specific embodiment, this document provides a microfluidic device, system, and method of use thereof, comprising one or more fiber optic probes and one or more fluidic tubes, wherein the one or more fiber optic probes are designed to perform spectroscopic measurements, and wherein the one or more fluidic tubes include a sampling tube that allows a sample containing particles to be introduced into the instrument, and wherein the sampling tube enables the particles to be moved relative to the fiber optic probes. The probes may include optical fibers for transmitting excitation light and collecting Raman signals, the fluidic tubes are capable of introducing cells or particles into a sampling area, and the device may further include a shell capable of enabling the particles to interact with SERS or other Raman-active surfaces. The probes may be constructed of polymers, plastics, glass, fused silica, liquid-core waveguides, metals, composite materials, or any other suitable material.

[0062] In some embodiments, the apparatus of this disclosure may include an integrated sampling tip; wherein the integrated sampling tip has custom dimensions to enable access to higher-density well plates using smaller orifices. The integrated sampling tip may be manufactured by 3D printing or any other method known to those skilled in the art. In some embodiments, the sampling tip may be removable or detachable, or reusable. A removable or detachable tip may be secured to a probe housing using a strap or clip seal, wherein the probe housing may further include Raman features. To ensure probe integrity, a laser sensor may be employed.

[0063] In some specific embodiments, the tube used for sample loading may include a capillary and may act as a liquid core waveguide to distribute excitation light throughout the capacity and collect emitted or Raman signals throughout the same capacity to enhance sensitivity.

[0064] This document provides an apparatus and method for the automated analysis of one or more samples, wherein the automated analysis process includes automated flow, wherein the sample comprises liquid or particles in a sample vessel, and wherein the apparatus includes component assemblies capable of processing the sample for analytical evaluation of fluid- and / or particle-based instruments. In some specific embodiments, one or more paths of the microfluidic device may be designed to segment cells, particles, debris, or molecules by having one or more channels in a microfluidic channel network. The device may further include one or more fiber optic probes and one or more fluid tubes, wherein the one or more fiber optic probes are designed to perform spectroscopic measurements, and wherein the one or more fluid tubes include sampling tubes that allow the introduction of a sample containing particles into the instrument, and wherein the sampling tubes enable particle movement relative to the fiber optic probes.

[0065] Analytical evaluations using fluid- and / or particle-based instruments include the use of Raman spectroscopy, optical mechanics, hydrodynamics, and / or electrodynamics.

[0066] In some embodiments, the apparatus of this disclosure further includes one or more features capable of varying flow rates or pressures between one or more channels in a microfluidic channel network. The flow rates or pressures between one or more channels in the microfluidic channel network are adjusted by optical, electrodynamic, or acoustic forces. As contemplated herein, optical forces may include optical tweezers, dual laser beams, or laser-force cytology and equivalents.

[0067] The device may further include additional features, such as one or more channels in the microfluidic channel network including channels dedicated to analysis and channels for disposal. The dedicated analysis and disposal channels can be adjusted, the diameter of the channels in the microfluidic channel network can be adjusted, and the device may further include one or more additional channels for introducing materials, wherein the one or more additional channels include a single analysis channel with a single input, or multiple analysis channels with one or more inputs. The additional materials include, but are not limited to, molecules, exogenous substances, cells, biochemicals, bacteria, or viruses.

[0068] In some embodiments, the apparatus of this disclosure may further include one or more regions that facilitate sample mixing prior to analysis. The channels of the microfluidic channel network can be customized for any desired configuration, including incorporating T or Y nodes, adjusting the length, diameter, shape, or other dimensions of the channels to adjust flow rates and achieve precise analysis. The channel network may further include one or more collection wells or analytical regions within the microfluidic channel network for sorting and accumulating cells for Raman or other spectroscopic analyses. Some embodiments may include additional waste or release channels for splitting the sample. Some embodiments may include one or more collection wells or regions within the microfluidic channels for collecting cells for Raman analysis, and may also include one or more waste or release channels for splitting the sample flow to allow for flow or no flow in the Raman collection wells (and channels). In some embodiments, the apparatus herein may further include one or more collection wells or weirs within the microfluidic channels for laser-based Raman or other spectroscopic analyses, and the depth and width of the wells may be adjustable for fine-tuning behavior. Optionally, the bottom of the wells or weirs may include electrodes or SERS material to facilitate Raman or other spectroscopic analyses. In some embodiments, cells, particles, fragments, or molecules of a sample are collected against a wall or other structure for analysis, and fluid, light, magnetic, electrodynamic, or acoustic forces are used to assist in the collection or release of cells retained in a collection hole or weir.

[0069] In one specific embodiment of this disclosure, a method for microfluidic analysis of a sample comprising cells, particles, debris, or molecules includes using an apparatus for automated analysis of one or more samples, wherein the automated analysis process includes automated flow, wherein the sample comprises liquid or particles in a sample vessel, wherein the apparatus includes component assemblies capable of processing the sample for analytical evaluation by fluid- and / or particle-based instrumentation, wherein one or more paths of the microfluidic apparatus are designed to segment cells, particles, debris, or molecules by having one or more channels in the microfluidic channel network, wherein one or more channels in the microfluidic channel network are connected at one or more nodes, wherein the apparatus further includes one or more collection orifices or weirs within the microfluidic channels for laser-based Raman or other spectral analysis, wherein the sample is loaded into the apparatus and the flow rate and direction of the sample are controlled by a combination of optical and electrodynamic forces, wherein the cells, particles, debris, or molecules of the sample accumulate in one or more collection orifices or weirs in which Raman signals can be measured. In some embodiments, the device further includes one or more fiber optic probes and one or more fluidic tubes, wherein the fiber optic probes are designed to perform spectroscopic measurements, and wherein the fluidic tubes include sampling tubes that allow the introduction of a particle-containing sample into the instrument, and wherein the sampling tubes enable particle movement relative to the fiber optic probes. In some embodiments, further analysis is performed using the laser force of multiple lasers optionally located at each node or scanned to a single laser passing through the node. Electrodynamic forces can drive cells, particles, debris, or molecules of the sample toward the anode, and laser beams can be used to drive particles through a network of microfluidic channels.

[0070] In one specific embodiment, this disclosure includes a method for microfluidic analysis of a sample comprising cells, particles, debris, or molecules, comprising using an apparatus for automated analysis of one or more samples, wherein the automated analysis process includes automated flow, wherein the sample comprises liquid or particles in a sample vessel, wherein the apparatus includes component assemblies capable of processing the sample for analytical evaluation by fluid- and / or particle-based instrumentation, wherein one or more paths of the microfluidic apparatus are designed to segment cells, particles, debris, or molecules by having one or more channels in the microfluidic channel network, wherein one or more channels in the microfluidic channel network are connected at one or more nodes, wherein the apparatus further includes one or more collection orifices or weirs within the microfluidic channels for laser-based Raman or other spectral analysis, wherein the apparatus further... The device includes one or more fiber optic probes and one or more fluid tubes, wherein the fiber optic probes are designed to perform spectral measurements, and wherein the fluid tubes include sampling tubes that allow the introduction of particles containing a sample into the instrument, and wherein the sampling tubes allow the particles to move relative to the fiber optic probes, wherein the sample is loaded into the device, and the flow rate and direction of the sample are controlled by a combination of optical and electrodynamic forces, wherein cells, particles, debris, or molecules of the sample accumulate in one or more collection orifices or weirs capable of measuring Raman signals, wherein electrodynamic forces drive the cells, particles, debris, or molecules of the sample toward the anode, wherein flow splitting is introduced at one or more nodes, and a laser beam drives the cells, particles, debris, or molecules of the sample into the orifices by photopressure, and wherein the competition between electrophoretic and optical forces provides the ability to separate particles according to their different compositions.

Claims

1. A microfluidic device comprising one or more fiber optic probes and one or more fluid tubes, wherein, The one or more fiber optic probes are designed to perform spectral measurements. Furthermore, the one or more fluid tubes include a sampling tube that allows a sample containing particles to be introduced into the instrument, and wherein the sampling tube enables the particles to move relative to the fiber optic probe.

2. The apparatus according to claim 1, wherein, The probe includes optical fibers for transmitting excitation light and collecting Raman signals.

3. The apparatus according to claim 1, wherein, The fluid tube is capable of introducing cells or particles into the sampling area.

4. The apparatus of claim 1, further comprising a shell capable of enabling the particles to interact with surface-enhanced Raman (SERS) or other Raman-active surfaces.

5. The apparatus of claim 1, further comprising an integrated sampling tip.

6. The apparatus according to claim 5, wherein, The integrated sampling tip has a custom size to allow access to higher density well plates using smaller orifices.

7. The apparatus according to claim 5, wherein, The integrated sampling tip is made by 3D printing.

8. The apparatus according to claim 1, wherein, The probe is made of polymer, plastic, glass, metal or composite material.

9. The device according to claim 1, further comprising a movable / detachable tip.

10. The apparatus according to claim 9, wherein, The movable / detachable tip is reusable.

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