Aptamer Tagged Detection Kit Inversion Strategy
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing assays for detecting and quantifying target molecules using aptamers face inefficiencies due to pre-immobilization of aptamers on solid supports, leading to lengthy reaction times, light scattering or absorption issues, and imprecision in detection, especially when using photoaptamers.
Innovation Solution
A method involving contacting a test sample with an aptamer that includes a tag, allowing an aptamer affinity complex to form, which can be converted to a covalent complex and detected using methods like mass spectrometry or Q-PCR, with the option to attach to a solid support for detection and quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If aptamers are pre-immobilized on solid support, then detection can be performed, but reaction time increases and binding efficiency decreases
Solution Approach 1:
Instead of immobilizing aptamers first and then adding sample, the invention inverts the sequence by adding sample first and then immobilizing aptamers. This allows target molecules to be present in solution during the binding phase, improving mixing efficiency and reducing reaction time, while still enabling detection through subsequent immobilization on the solid support.
Solution Approach 2:
The invention performs preliminary binding of aptamers to target molecules in solution before immobilization. This preliminary action allows the aptamer-target complexes to form under optimal mixing conditions, and then the entire complex is immobilized on the solid support, maintaining detection capability while reducing the time required for binding equilibria to be achieved.
2Stability of the object's composition
If photoaptamers are used to form covalent bonds, then binding stability improves, but light scattering or absorption by solid support interferes with the process
Solution Approach 1:
The invention performs the photoactivation and covalent bond formation step before immobilization on the solid support. By completing the photocrosslinking in solution where light penetration is optimal, the harmful light scattering and absorption effects of the solid support are avoided, while still achieving stable covalent bonding between photoaptamers and target molecules.
Solution Approach 2:
The invention inverts the conventional sequence by performing photoactivation before immobilization rather than after. This reversal allows the photoaptamer to form covalent bonds with the target molecule in solution under optimal lighting conditions, free from solid support interference, and then the stabilized complex is immobilized on the solid support for detection.
3Device complexity
If aptamers are immobilized prior to sample contact, then assay format is simplified, but detection precision decreases due to surface exposure to labeling agents
Solution Approach 1:
The invention performs preliminary binding and optional covalent crosslinking of aptamers to target molecules in solution before immobilization. This preliminary formation of aptamer-target complexes protects the target molecules from direct exposure to labeling agents on the solid support surface, reducing non-specific binding and improving detection precision, while still maintaining a simplified overall assay format.
4Ease of manufacture
If pre-immobilization step is included, then assay procedure is established, but aptamer activity or functionality is affected
Solution Approach 1:
The invention performs preliminary binding of aptamers to target molecules in solution under optimal conditions before immobilization. This preliminary action allows aptamers to exhibit their full binding activity and functionality during the solution phase, and then the formed complexes are immobilized, preserving the aptamer's functionality while still establishing a defined assay procedure.
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
This approach enhances the sensitivity and efficiency of aptamer-based assays by optimizing binding equilibria and covalent bond formation, improving the detection and quantification of target molecules while minimizing interference from the solid support.
Implementation Method 1
aptamers that include photoreactive functional groups that enable the aptamers to covalently bind, or 'photocrosslink,' their target molecules
Data Source
Figure 1A
Figure 1B
Figure 2A
AI summary
The present disclosure describes methods, devices, reagents, and kits for the detection of one or more target molecules that may be present in a test sample. In one embodiment, a test sample is contacted with an aptamer that includes a tag and has a specific affinity for a target molecule. An aptamer affinity complex that includes an aptamer bound to its target molecule is allowed to form. If the test sample contains the target molecule, an aptamer affinity complex will generally form in the test sample. The aptamer affinity complex is optionally converted to an aptamer covalent complex that includes an aptamer covalently bound to its target molecule. The aptamer affinity complex (or optional aptamer covalent complex) can then be detected and/or quantified using any of a variety of methods known to one skilled in the art, including using a solid support, using mass spectrometry, and using quantitative polymerase chain reaction (Q-PCR).