Label free molecular detection methods, systems and devices
Inventors
Lo, Yu-hwa • Qiao, Wen • Song, Junlan • Chen, Longchuan
Assignees
US Department of Veterans Affairs • University of California San Diego UCSD
Publication Number
US-9862987-B2
Publication Date
2018-01-09
Expiration Date
2034-01-16
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Abstract
Methods, systems, and devices are disclosed for capturing, concentrating, isolating, and detecting molecules. In one aspect, a molecular probe device includes a molecular probe having a complimentary base pair region initially zipped and structured to include a binding agent to chemically attach the molecular probe to an outer surface of a magnetic bead, and a binding molecule to chemically attach the molecular probe to a substrate of a microfluidic device, in which the complimentary base pair region is configured to hybridize to a complementary nucleic acid sequence of a DNA or RNA molecule.
Core Innovation
The invention relates to methods, systems, and devices for capturing, concentrating, isolating, and detecting molecules, specifically nucleic acids such as DNA or RNA, including microRNAs (miRNAs), in a label-free manner using molecular probes integrated into microfluidic devices. These molecular probes have a complementary base pair region initially zipped in a hair-pinned structure and chemically attached to a magnetic bead and a substrate. Application of a magnetic field causes the molecular probe to unzip, exposing sequences that hybridize to target nucleic acids, enabling sensitive and specific molecular detection on-chip.
The problem solved is the technical challenge of detecting circulating nucleic acids present at very low concentrations within biological fluids, such as miRNAs at femtomolar levels in blood or other biofluids. Existing microfluidic devices lack sufficient flow rate and collection efficiency to extract and concentrate these molecules rapidly and at low cost for clinical diagnostics. Furthermore, current miRNA assays face challenges due to the need for amplification steps, time-consuming processes, and inability to provide point-of-care solutions with high specificity and sensitivity. The disclosed invention addresses these issues by providing a lab-on-a-chip platform capable of capturing, enriching, and optically detecting nucleic acids in a label-free, high-specificity and high-sensitivity manner.
The disclosed technology enables molecular probes tethered to transparent iron oxide magnetic beads to convert the binding events of target nucleic acids into optical signals without the need for labels or amplification. The magnetic field controls the conformation of the hair-pinned probe, facilitating selective hybridization, and the change in probe length upon binding can be detected optically via changes in scattering intensity. The invention also includes microfluidic device architectures with integrated electrodes for electrophoretic capture and concentration of target molecules, a recapture region for enrichment, and optical detection regions comprising arrays of molecular probes designed for multiplexed detection of nucleic acid targets in parallel.
Claims Coverage
The patent includes two independent claims covering a molecular probe device and a device for capture, enrichment, and detection of biomolecules from a fluid. The main inventive features relate to the molecular probe structure, its interaction with magnetic beads and magnetic fields for controlled hybridization, and integrated microfluidic system components for capture and detection.
Molecular probe device with magnetic bead and hair-pinned nucleic acid sequence
The device comprises a magnetic bead bound to a molecular probe that includes a single-stranded nucleic acid sequence with a base pair region complementary to a target miRNA. This region is zipped in a hair-pinned structure without a magnetic field and unzipped to an extended configuration when a magnetic field is applied, enabling hybridization with the target miRNA.
Binding chemistry for molecular probe attachment
The molecular probe includes a binding agent chemically binding it to the magnetic bead's surface and a binding molecule chemically attaching it to a substrate surface, such as digoxigenin and anti-digoxigenin antibody coating on the substrate, and biotin-streptavidin interaction with the magnetic bead.
Magnetic field-controlled conformational change and hybridization
The molecular probe is operable to interact with an applied magnetic field via the magnetic bead, which moves the bead and unzips the complementary base pair region to expose the binding sequence. Upon hybridizing with the target nucleic acid, the probe resists re-zipping when the magnetic field is reduced, but unbound probes can reform the hairpin structure under reduced magnetic field.
Device including microfluidic channel, electrode array, and microchamber with molecular probes for capture and detection
The device comprises an electrically insulating substrate and microfluidic channel containing biofluids, an array of electrodes producing electric fields to immobilize miRNA in a capture region, a smaller-volume chamber connected to the channel to collect released miRNA at higher concentration, and molecular probes (as described) attached to the substrate in the chamber capable of hybridizing to target miRNA with magnetic field-induced conformational changes.
The independent claims collectively cover a molecular probe device that uses magnetic beads and hair-pinned nucleic acid probes whose conformation is controlled by magnetic fields to hybridize with target miRNAs, as well as a microfluidic device integrating electrophoretic capture, enrichment, and molecular probe-based detection to provide sensitive, label-free molecular detection.
Stated Advantages
Label-free, high-specificity, and high-sensitivity detection of nucleic acids without the need for amplification steps.
Capability to detect low concentration circulating nucleic acids such as miRNAs in femtomolar ranges suitable for point-of-care clinical diagnostics.
Minimally invasive detection methods suited for early disease diagnosis, including cancers such as non-small-cell lung cancer.
Integration in a low-cost, lab-on-a-chip platform enabling rapid capture, enrichment, and optical detection of target molecules from biological fluids.
Multiplexed detection capability for multiple nucleic acid targets in parallel on the same device.
Documented Applications
Early detection and diagnosis of cancer, particularly non-small-cell lung cancer, through detection of circulating miRNAs serving as biomarkers.
Point-of-care miRNA-based diagnostic devices and research tools for rapid and sensitive detection of nucleic acid biomarkers from biofluids such as blood, saliva, sputum, urine, or vitreous fluid.
Use as the back-end detection component of lab-on-a-chip devices integrating capture, enrichment, and optical molecular detection using the disclosed molecular probes.
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