Gradient elution isotachophoretic apparatus for separating, purifying, concentrating, quantifying, and/or extracting charged analytes and methods thereof
Inventors
Henry, Alyssa • Konek, Christopher • Ross, David • Strychalski, Elizabeth
Assignees
Applied Research Associates Inc • National Institute of Standards and Technology NIST • United States Department of Commerce
Publication Number
US-10067088-B2
Publication Date
2018-09-04
Expiration Date
2035-06-22
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Abstract
Gradient elution isotachophoretic apparatus, and systems for performing gradient elution isotachophoresis to separate, purify, concentrate, quantify, and/or extract charged analytes from a sample. The isotachophoretic apparatus include an electrophoretic assembly, a sampling assembly connected to the electrophoretic assembly, and/or a support structure connected to the electrophoretic assembly and/or to the sampling assembly. The system includes an isotachophoretic apparatus, and a controller communicatively coupled to the isotachophoretic apparatus. The controller includes a storage medium and a processor for executing computer readable and executable instructions.
Core Innovation
Embodiments of the invention disclose a gradient elution isotachophoretic (GEITP) apparatus and systems designed to separate, purify, concentrate, quantify, and/or extract charged analytes from a sample. The apparatus includes a moveable electrophoretic assembly comprising a separation unit and a detection unit, with the detection unit accommodating a conductivity detection device, a light source, and a light source detection device. The electrophoretic assembly is configured for movement in horizontal and vertical directions via moveable support structures. The sampling assembly is operably connected to the electrophoretic assembly, and a support structure connects components of the apparatus. The system further includes a controller with a processor and storage medium for executing instructions to automate GEITP processes.
The apparatus’s separation unit includes at least one separation channel oriented vertically, a leading electrolyte reservoir in open fluidic communication with the channel, a voltage supply device communicatively coupled to the channel, and a pressure control device connected to the reservoir for producing pressure-driven counterflow. The detection unit’s support structure aligns the conductivity detection device, light source, light source detection device, and separation channel for operation, where fluorescence can be excited and detected in biomolecules contacted with sensor molecules. The sampling assembly includes trailing electrolyte and delivery reservoirs to hold samples and receive extracted analytes respectively.
Methods disclosed include optional pre-treatment of the separation channel, insertion of leading electrolyte fluid and fluorescence sensor molecules into the channel, contacting the channel with the sample and trailing electrolyte fluid, and separating charged analytes via GEITP by producing pressure-driven counterflow, applying voltage to induce electrophoretic migration, and varying counterflow pressure to control analyte focusing. Detection of charged analytes is achieved via conductivity and/or fluorescence detection, followed by delivery to a clean reservoir. The apparatus and method operate to isolate and quantify charged analytes such as DNA, including from crude or complex samples, improving throughput and potentially automating sample preparation and analysis.
Claims Coverage
The patent includes multiple independent claims describing an apparatus and system for performing gradient elution isotachophoresis with specific structural and functional features.
Moveable electrophoretic assembly with detection unit
An electrophoretic assembly that is moveable, comprising a separation unit and a detection unit that includes a support structure accommodating a conductivity detection device, a light source, and a light source detection device, with at least one moveable electrophoretic assembly support structure connected to the separation or detection unit, communicatively coupled to a motor for movement in Z or X directions.
Separation unit comprising vertical separation channels and associated reservoirs and devices
The separation unit includes at least one separation channel oriented vertically, a leading electrolyte reservoir in open fluidic communication with the separation channel, a voltage supply device communicatively coupled to the channel, and a pressure control device connected to the leading electrolyte reservoir.
Detection unit support structure with alignment for fluorescence excitation and detection
The detection unit support structure defines cavities to accommodate the separation channel, the light source at a fluorescence excitation angle (30° to 150°), and the light source detection device at a fluorescence detection angle (30° to 150°), providing an interface such that fluorescence from charged analytes contacted with sensor molecules in the channel can be excited and detected.
Moveable support structures with motors for three-axis translation
The apparatus includes first and second moveable support structures for the electrophoretic assembly that provide movement in Z and X directions respectively, each communicatively coupled to a motor, and a third moveable support structure for the sampling assembly providing movement in the Y direction, also communicatively coupled to a motor.
Automated control of GEITP process via controller executing instructions
A controller communicatively coupled to the electrophoretic assembly and sampling assembly, executing instructions to (1) optionally pre-treat the separation channel, (2) insert leading electrolyte fluid and fluorescence sensor molecules into the separation channel, (3) contact the separation channel with sample and trailing electrolyte fluid, (4) separate charged analytes by producing pressure-driven counterflow, applying voltage, and varying pressure to focus analytes, (5) direct light to excite fluorescence, (6) detect charged analytes via conductivity and/or fluorescence detection, and (7) deliver charged analytes to a delivery reservoir.
The independent claims collectively cover a moveable isotachophoretic apparatus with vertical separation channels and integrated detection accommodating fluorescence and conductivity measurements, controlled movement in three orthogonal axes, and an automated system controller for performing gradient elution isotachophoresis to separate, detect, and deliver charged analytes from samples.
Stated Advantages
Enables separation, purification, concentration, quantification, and delivery of charged analytes rapidly, e.g., DNA in less than 5 minutes.
Requires minimal sample preparation, e.g., no filtration, centrifugation, or precipitation needed prior to analysis.
Allows automated, controlled positioning and operation to improve throughput and reduce labor intensity.
Improves exclusion of contaminants and inhibitors, enhancing purity of analytes suitable for downstream analysis like STR analysis for human identification.
Delivers analytes in volumes compatible with downstream processes and maintains analyte concentration effectively.
Documented Applications
Separating, purifying, concentrating, quantifying, and extracting charged analytes, including biomolecules such as DNA and RNA, from crude samples including environmental contaminants.
Preparation of DNA samples for forensic applications such as short tandem repeat (STR) analysis for human identification.
On-line DNA quantification during analysis to potentially replace traditional qPCR quantification steps.
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