Probe Array Hybridization Using Amplified Probes for Low-Abundance Detection
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Solution Overview
Problem
Current hybridization assays for detecting nucleic acid sequences face challenges with low concentration of target nucleic acid molecules, requiring time-consuming amplification steps and special equipment, which prolongs the assay response time and may not efficiently utilize the signal-to-noise ratio.
Innovation Solution
A method and device where probes are amplified to create multiple copies, localized on a specific location, allowing target nucleic acid molecules to hybridize without prior amplification, using a probe array on a solid surface with an accompanying sensor array, where the probes are not covalently attached to the surface.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If target nucleic acid amplification is performed to detect low concentration targets, then detection sensitivity is improved, but assay time increases and special equipment is required
Solution Approach 1:
Instead of amplifying the target nucleic acid as in conventional methods, this invention inverts the approach by amplifying the probe molecules. Multiple copies of the probe are generated and localized to specific locations on a solid surface, enabling detection of low-concentration targets without target amplification. This inversion eliminates the need for special amplification equipment and reduces assay time while maintaining detection sensitivity.
2Measurement precision
If target nucleic acid amplification is performed, then detection sensitivity is improved, but device complexity increases due to special equipment requirements
Solution Approach 1:
The invention simplifies the device by inverting the amplification strategy - instead of requiring complex target amplification equipment, it uses probe amplification followed by localized delivery to the solid surface. This approach can be performed with standard molecular biology equipment and eliminates the need for specialized real-time amplification systems, reducing overall device complexity while achieving the same detection sensitivity.
Solution Approach 2:
The probe amplification and localization steps are performed in advance before the actual hybridization assay. By preparing multiple copies of the probe and positioning them at specific locations on the solid surface beforehand, the system eliminates the need for complex real-time amplification equipment during the assay itself, thereby reducing device complexity.
3Productivity
If probe density is increased to enhance hybridization probability, then detection efficiency is improved, but probe attachment stability may be compromised
Solution Approach 1:
The invention replaces covalent chemical bonding with physical adsorption for probe attachment to the solid surface. Multiple copies of the probe are localized to specific locations through adsorption onto the solid support, which provides sufficient stability for hybridization while allowing easier modification and replacement of probes. This substitution of attachment mechanism enables high probe density without compromising reliability.
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
Enhances the chances of hybridization by increasing probe density, enabling efficient detection of low-concentration target nucleic acids with improved signal-to-noise ratio and reduced assay time.
Implementation Method 1
hybridization between a target nucleic acid and a plurality of copies of a probe may be detected in a single sample chamber using a probe array on a solid surface
Implementation Method 2
nucleic acid hybridization may be a molecular biology technique, in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA sequences
Data Source
AI summary
The present disclosure provides methods, device, and system for a hybridization assay to detect nucleic acid targets. The assay may use a probe array format with addressable microwells. Each microwell may retain a plurality of copies of one probe which may be complementary to a portion of a specific nucleic acid target. The plurality of copies of probes may be produced from a pooled library of probe sequences by amplification techniques, including, rolling circle amplification and emulsion-PCR. Detection of hybridization may rely on methods using ion-sensitive field effect transistor (ISFET) and optical detectors to detect signals indicating the presence of the nucleic acid targets.