Electrode Oligonucleotide Assay for High-Throughput Nucleic Acid Detection
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Solution Overview
Problem
Current methods for detecting nucleic acid analytes, such as microRNAs, suffer from low throughput, poor reproducibility, high cost, and long, complicated protocols, limiting their effectiveness in identifying specific isomiRs and other nucleic acid biomarkers.
Innovation Solution
An electrochemical luminescence (ECL) method involving the hybridization of detection oligonucleotides to capture oligonucleotides and nucleic acid analytes, followed by ligation and washing to stabilize the hybridization complex, allowing for efficient detection through an ECL reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If qPCR-based detection, microarrays, sequencing, mass spectrometry, or hybridization methods are used, then nucleic acid analytes can be detected, but the throughput is low and the protocol is long and complicated
Solution Approach 1:
The patent combines multiple detection functions into a single integrated assay. The detection oligonucleotide simultaneously binds to the capture oligonucleotide and the nucleic acid analyte in a single hybridization step, eliminating the need for separate detection steps required by traditional methods. This merging of functions achieves high throughput while maintaining sensitivity.
Solution Approach 2:
The detection oligonucleotide serves multiple functions: it acts as both a capture probe and a detection probe, binds to both the immobilized capture oligonucleotide and the target analyte, and enables signal generation through ECL. This multi-functionality reduces the number of reagents and steps required, thereby shortening the protocol and increasing throughput.
2Reliability
If current detection methods are used, then nucleic acid analytes can be detected, but the performance is limited by poor reproducibility and sensitivity
Solution Approach 1:
The detection oligonucleotide acts as an intermediary that bridges the capture oligonucleotide and the target analyte. By requiring the detection oligonucleotide to hybridize to both components, the system creates a stable three-way complex that enhances signal specificity and reduces background noise, thereby improving both reproducibility and sensitivity.
Solution Approach 2:
The patent replaces traditional mechanical or chemical detection mechanisms with electrochemical luminescence (ECL) signaling. The ECL detection reagent produces a luminescent signal when activated by the detection oligonucleotide, providing a highly sensitive and reproducible measurement approach that overcomes the limitations of fluorescence-based or colorimetric methods.
3Measurement precision
If current detection methods are used, then nucleic acid analytes can be detected, but the cost is high
Solution Approach 1:
The assay uses inexpensive oligonucleotide probes and standard ECL reagents that can be easily replaced. The detection system relies on consumable items like the detection oligonucleotide and ECL reagent, which are cheaper than the specialized reagents required for sequencing or mass spectrometry, thereby reducing the overall cost while maintaining detection capability.
Solution Approach 2:
The patent extracts the essential detection function from complex, expensive systems like sequencing or mass spectrometry and implements it through a simplified oligonucleotide-based ECL assay. By removing unnecessary complexity and using only the minimal required components (capture oligo, detection oligo, ECL reagent), the system achieves cost-effectiveness without sacrificing measurement precision.
4Measurement precision
If current detection methods are used, then nucleic acid analytes can be detected, but the protocol is long and complicated
Solution Approach 1:
The patent segments the detection process into three clear functional steps: (1) hybridization of the detection oligonucleotide to the capture oligonucleotide and analyte, (2) ligation of the detection oligonucleotide to the analyte, and (3) ECL signal generation. This segmentation simplifies the protocol by making each step distinct and manageable, reducing overall complexity while maintaining accuracy.
Solution Approach 2:
The capture oligonucleotide is pre-immobilized on the electrode surface before the assay begins. This preliminary action eliminates the need for complex sample preparation and preprocessing steps, as the detection oligonucleotide can directly hybridize to the immobilized capture oligonucleotide and target analyte in a single step, thereby simplifying the overall protocol.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method enhances the sensitivity and specificity of nucleic acid detection, enabling differentiation between miRNA and siRNA variants and detecting 3′ modifications, with improved throughput and reduced complexity.
Implementation Method 1
permitting at least one of the plurality of detection oligonucleotides to hybridize to at least one of the plurality of capture oligonucleotides and at least one of the plurality of nucleic acid analytes
Implementation Method 2
incubating the analyte hybridization complex from b) under ligation conditions to ligate the nucleic acid analyte to the capture oligonucleotide
Implementation Method 3
performing an ECL reaction by inducing a charge at the electrode surface in the presence of an ECL detection reagent complexed with the detection oligonucleotide and an ECL co-reactant, and measuring any luminescence signal emitted
Data Source
AI summary
The present disclosure relates to a method of detecting a nucleic acid analyte, the method comprising contacting an electrode surface comprising a plurality of capture oligonucleotides immobilized on the electrode surface with a solution comprising: a plurality of nucleic acid analytes; and a plurality of detection oligonucleotides, wherein each of the plurality of detection oligonucleotides comprises a first portion and a second portion adjacent to each other, wherein the first portion comprises a sequence that is complementary to a sequence in the capture oligonucleotide and the second portion comprises a sequence that is complementary to a sequence in the nucleic acid analyte.