Detection units and methods for detecting a target analyte
Inventors
Celedon, Alfredo A. • Russell, Joseph P.
Assignees
Publication Number
US-11371986-B2
Publication Date
2022-06-28
Expiration Date
2039-05-23
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Abstract
The present application relates to detection units and methods for detecting one or more target analytes in a sample using a complex formed by a target and first and second probes, wherein the first probe is coupled to a detectable piece, the target is coupled to the first probe and the second probe, and the second probe is coupled to a solid support. Specific binding of the detectable piece to the target analyte can be distinguished from non-specific binding of the detectable piece by measuring the number of detectable pieces that leave their initial location after exposure to a disruptor that uncouples the detectable piece from the solid support.
Core Innovation
The invention provides detection units and methods for detecting one or more target analytes in a sample by utilizing a complex formed by a target and first and second probes. The first probe is coupled to a detectable piece, the target binds both first and second probes, and the second probe is coupled to a solid support. Only in the presence of the target analyte does the detectable piece become coupled to the solid support via the complex, which includes an elongated region of at least 100 nanometers in length.
The problem addressed is the limitation of current detection systems, such as immunoassays, in detecting low concentrations of target analytes and their inability to effectively discriminate between specific and non-specific binding, leading to high background noise. Known strategies to enhance signal, such as the use of reporter markers or branched-DNA amplification, often simultaneously increase background signal from non-specific interactions and do not provide effective means to distinguish specific from non-specific binding events.
The core principle of this invention is distinguishing specific coupling of the detectable piece to the analyte from non-specific binding by exposing the complex to a disruptor that breaks the bond between the detectable piece and the solid support. The method involves measuring the number of detectable pieces that leave their initial location upon application of a disruptor. The presence of the target is revealed by detectable pieces that both (i) demonstrate displacement or Brownian motion within a predetermined range (related to the elongated region) and (ii) leave their initial location after disruption, effectively reducing background from non-specifically bound detectable pieces.
Claims Coverage
There is one independent claim in this patent that defines the key inventive features of the method for detecting a target analyte.
Detecting a target analyte with a complex comprising elongated region and disruption-based differentiation
A method of detecting a target analyte includes: - Providing at least one detectable piece coupled to a solid support via a complex formed by the target analyte and a first and second probe, such that only if the target analyte is present, the detectable piece is (directly or indirectly) coupled to the solid support at an initial location through the complex. - The complex comprises an elongated region that is at least 100 nanometers in length. - Either applying a force to the detectable piece and measuring its displacement, or measuring its Brownian motion. - Exposing the complex to a disruptor capable of uncoupling the detectable piece from the solid support. The disruptor is: - A nucleic acid that can hybridize to the target analyte in a region where the target analyte hybridizes to the first or second probe and can hybridize to a toehold region in the target analyte which does not hybridize to the probes, or - A nucleic acid that can hybridize to the first or second probe in a region where it hybridizes to the target analyte and can also hybridize to a toehold region in the probe which does not hybridize to the analyte. - Optionally applying a force to the detectable piece after disruption. - Detecting if the detectable piece has left its initial location. - Inferring the presence of the target analyte when the detectable pieces both (i) show displacement or Brownian motion within a pre-determined range and (ii) leave their initial location.
In summary, the claim covers a unique method for detecting a target analyte by forming a complex with defined elongated regions, applying physical or Brownian motion analysis, and using specific disruptor-induced uncoupling to distinguish specific from non-specific binding. The disruptor's design ensures targeted differentiation and reduction of background interference.
Stated Advantages
The invention provides capability to detect low concentrations of target analytes while distinguishing non-specific binding from specific binding in the sample.
The approach greatly reduces background signal generated by non-specifically attached detectable pieces, improving signal-to-background ratio.
The method allows for discrimination of specific analyte detection from non-specific interactions by using disruptors that preferentially uncouple specifically bound detectable pieces.
Combining displacement analysis and disruptor exposure further enhances specificity and reduces false positives from reversible or displacement-similar non-specific attachments.
Documented Applications
Detection of nucleic acids, including RNA and DNA, from biological samples such as blood, urine, and other body fluids.
Detection of short nucleic acid molecules (e.g., siRNA, miRNA, fragmented DNA).
Detection of ribosomal RNA for identification of organisms.
Use in ELISA (enzyme-linked immunosorbent assay) formats to reduce background signal.
Application in branched DNA assays for improved detection specificity.
Use in lateral flow tests (lateral flow immunochromatographic assays) to enhance signal discrimination.
Application in DNA microarray (biochip) analysis for quantifying and detecting nucleic acid targets.
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