Nucleic acid sample preparation
Inventors
Krishnan, Rajaram • Charlot, David J. • Tu, Eugene • MCCANNA, James • Kumosa, Lucas • Swanson, Paul D. • Turner, Robert • Yang, Kai • DOBROVOLSKAYA, Irina • Liu, David • HINESTROSA SALAZAR, Juan Pablo • MENEZES, Juscilene
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10006083-B2
Publication Date
2018-06-26
Expiration Date
Abstract
The present invention includes methods, devices and systems for isolating a nucleic acid from a fluid comprising cells. In various aspects, the methods, devices and systems may allow for a rapid procedure that requires a minimal amount of material and/or results in high purity nucleic acid isolated from complex fluids such as blood or environmental samples.
Core Innovation
The invention provides a method for detecting a cell free biomarker using a device that comprises an array of electrodes capable of establishing an AC electrokinetic field region and at least two chambers. The sample is applied to a first chamber and known quantities of a cell-free biomarker standard are applied to a second chamber, with the sample applied at a conductivity of greater than 100 mS/m to enable electrokinetic field-based manipulation and signal capture.
In the first chamber, the method establishes a first AC electrokinetic high field region configured to isolate larger nanoparticulate molecular targets and a second AC electrokinetic low field to concentrate cells or micron-sized entities that may be present in the sample. In the second chamber, the method establishes a third AC electrokinetic high field to isolate the molecular nucleic acid standard applied to that chamber.
After field-based isolation and concentration, the method flushes cells and micron-sized entities from the first chamber. The method detects bound cell-free biomarker signal on the array in the first chamber and the second chamber, and quantifies the bound cell-free biomarker by comparing the detected signal from the first chamber to the detected signal from the second chamber.
Claims Coverage
The document includes one independent claim, which recites eight principal method features for detecting and quantifying a cell-free biomarker using an AC electrokinetic multi-chamber device and a cell-free biomarker standard. The remaining dependent claims refine the independent claim by adding specific quantitative constraints and device/material features, and by adding optional additional operations.
AC electrokinetic multi-chamber biomarker detection with conductivity threshold
Applying a sample having a conductivity of greater than 100 mS/m to a device with an array of electrodes establishing an AC electrokinetic field region and at least two chambers, applying the sample to a first chamber, and applying known quantities of a cell-free biomarker standard to a second chamber.
High-field isolation of larger nanoparticulate targets
Establishing a first AC electrokinetic high field region in the first chamber, where the first AC electrokinetic high field isolates larger nanoparticulate molecular targets.
Low-field concentration of cells or micron-sized entities
Establishing a second AC electrokinetic low field in the first chamber, where the second AC electrokinetic low field concentrates cells or micron-sized entities that may be present in the sample.
High-field isolation of nucleic-acid standard in second chamber
Establishing a third AC electrokinetic high field to the second chamber, where the third AC electrokinetic high field isolates the molecular nucleic acid standard applied to the second chamber.
Flushing non-target entities from the sample chamber
Flushing cells and micron-sized entities that may be present in the sample from the first chamber.
Dual-chamber detection and comparative quantification
Detecting bound cell-free biomarker signal on the array in the first chamber and second chamber, and quantifying the bound cell-free biomarker by comparing the detected signal from the first chamber to detected signal from the second chamber.
Defined AC electrokinetic operating parameters
Producing an AC electrokinetic field using an alternating current with a voltage of 1 volt to 40 volts peak-peak, a frequency of 5 Hz to 5,000,000 Hz, and a duty cycle of 5% to 50%.
Tightened conductivity condition for the fluid
Using a fluid having a conductivity greater than 500 mS/m.
Hydrogel layer over the electrode array
Forming a hydrogel layer on the array of electrodes with a thickness between about 0.1 microns and about 1 micron.
Wavy repeating dot electrode geometry with tapering linker
Using an electrode array with a wavy repeating pair-of-dots shape connected by a tapering linker, with dot diameters widest at a repeating unit and equidistant or roughly equidistant edge-to-edge spacing between parallel repeating units.
Elution for further characterization
Eluting the bound cell-free biomarker from the first chamber for further characterization.
Across the independent claim and its refinements, the core inventive concept is AC electrokinetic processing in a multi-chamber device using a cell-free biomarker standard, with chamber-specific high-field isolation for larger nanoparticulate targets and nucleic-acid standard isolation, low-field concentration of cells or micron-sized entities, flushing to remove non-target entities, and comparative detection and quantification of bound biomarker signal between chambers. Dependent claims further specify AC field operating ranges, conductivity thresholds, hydrogel coating thickness, and a wavy repeating dot electrode geometry, and add optional elution for further characterization.
Stated Advantages
Enables quantifying bound cell-free biomarker by comparing detected signal from the first chamber to detected signal from the second chamber.
Isolates larger nanoparticulate molecular targets using an AC electrokinetic high field region.
Concentrates cells or micron-sized entities using an AC electrokinetic low field region.
Isolates a molecular nucleic acid standard using an AC electrokinetic high field region in the second chamber.
Flushes cells and micron-sized entities from the first chamber.
Supports further characterization by eluting the bound cell-free biomarker from the first chamber.
Documented Applications
Detecting a cell free biomarker using a device that uses AC electrokinetic field regions and multiple chambers, including comparison to a cell-free biomarker standard for quantification.
Further characterization of a bound cell-free biomarker by eluting the bound cell-free biomarker from the first chamber.
Interested in licensing this patent?