Stable Nanoreporter Probes for Sensitive Biomolecule Detection
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Solution Overview
Problem
Current methods for detecting and quantifying gene expression in biological samples are inefficient, requiring large sample amounts and lacking sensitivity, especially in cases of limited sample supply, and do not allow for individual-level detection of target molecules.
Innovation Solution
Development of uniquely labeled nanoreporters with designed single-stranded nucleic acid backbones and complementary polynucleotide sequences, enabling accurate and sensitive detection and quantification of target molecules by forming molecular complexes and generating distinct detectable signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA microarrays are used for detecting gene expression, then multiple genes can be detected simultaneously, but significant amounts of biological sample are required
Solution Approach 1:
The invention segments the detection process into two distinct phases: (1) hybridization of target molecules to nanoreporter probes in solution phase, and (2) immobilization of formed complexes on a surface for detection. This segmentation allows the hybridization to occur in a small volume where kinetics are efficient, while the surface serves only for stable positioning and signal detection, thereby reducing the total sample volume required while maintaining high detection throughput
Solution Approach 2:
The nanoreporter probes act as intermediaries that first bind to target molecules in solution to form stable complexes, and then these complexes are immobilized on the surface. This intermediary approach allows the detection to proceed with minimal sample volume because the nanoreporters concentrate and stabilize the target molecules before surface attachment, improving both sample efficiency and detection capability
2Device complexity
If DNA microarrays are used for detecting target molecules, then hybridization can occur on a miniaturized surface, but hybridization kinetics are less efficient compared to solution phase
Solution Approach 1:
The invention separates the hybridization function from the immobilization function. Hybridization occurs in solution phase where molecules have free movement and optimal kinetics, while immobilization occurs separately on the surface after complex formation. This temporal and spatial segmentation resolves the contradiction by allowing fast solution-phase hybridization while still achieving miniaturized surface-based detection
Solution Approach 2:
The nanoreporter probes perform preliminary binding to target molecules in solution before the complexes are immobilized on the surface. This preliminary action in the solution phase allows hybridization kinetics to proceed optimally without the constraints of surface attachment, and only after binding is complete are the complexes transferred to the miniaturized surface for detection
3Device complexity
If DNA microarrays are used for detecting target molecules, then detection can be performed on a miniaturized surface, but detection of individual target molecules and direct quantification are not achieved
Solution Approach 1:
The invention uses nanoreporter probes that are essentially copies or replicas of the target sequence, designed to bind specifically to individual target molecules. Each nanoreporter probe acts as a molecular copy that can be individually tracked and detected. This copying approach, combined with the solution-phase hybridization followed by surface immobilization, enables the detection and quantification of individual target molecules while maintaining miniaturization
Solution Approach 2:
The invention replaces the traditional microarray mechanical system where targets are immobilized and probes are added, with a system where nanoreporter-probe complexes form in solution and then attach to the surface. This substitution of the detection mechanism allows for individual molecule resolution and direct quantification because the solution-phase formation preserves molecular integrity and the subsequent immobilization maintains spatial information for precise measurement
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 nanoreporters provide a significant increase in detection efficiency, allowing for the identification and quantification of multiple target molecules with higher accuracy and sensitivity compared to existing technologies, particularly in limited sample scenarios.
Implementation Method 1
each label attachment region is hybridized to a complementary polynucleotide sequence
Data Source
AI summary
The present invention relates to compositions and methods for detection and quantification of individual target molecules in biomolecular samples. In particular, the invention relates to improved, stable nanoreporter probes that are capable of binding to and identifying target molecules based on the probes' uniquely detectable signal. Methods for identifying target-specific sequences for inclusion in the probes are also provided, as are methods of making and using such probes. Polynucleotide sequences of certain nanoreporter components are also provided. The probes can be used in diagnostic, prognostic, quality control and screening applications.