Aptamer-Based Multiplexed Detection via Solution-Phase Complex Formation
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Solution Overview
Problem
Existing aptamer-based assays face challenges such as inefficient mixing, lengthy reaction times, light scattering or absorption by the solid support, and imprecision in detection due to immobilization of aptamers before sample contact, which affects sensitivity and specificity in detecting target molecules.
Innovation Solution
The development of methods and kits that enable the purification of aptamer affinity complexes, allowing for the separation of aptamers from their complexes, using slow off-rate aptamers and photocrosslinking, and employing kinetic challenges to enhance binding efficiency and specificity, enabling improved detection and quantification of target molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If aptamers are immobilized on a solid support prior to contact with the sample, then the assay enables determination of target molecules through binding, but the mixing of aptamers with target molecules on the surface is inefficient, leading to lengthy reaction times and extended incubation periods
Solution Approach 1:
Instead of immobilizing aptamers first and then adding sample, the patent inverts the sequence by first contacting aptamers with target molecules in solution to form complexes, and then immobilizing these pre-formed complexes on the solid support. This inversion resolves the contradiction by enabling efficient mixing in solution phase while maintaining the reliability of surface-based detection.
Solution Approach 2:
The patent applies preliminary action by pre-forming aptamer-target complexes in solution before immobilization. This preliminary binding step occurs under optimal mixing conditions, avoiding the inefficiency of surface-based mixing, and the pre-formed complexes are then transferred to the solid support for detection.
2Reliability
If photoaptamers are used to covalently bind target molecules, then harsh wash conditions can be used to remove non-specifically bound molecules, but the solid support may scatter or absorb the light used to effect covalent bond formation
Solution Approach 1:
The patent extracts the photocrosslinking step from the solid support environment and performs it in solution phase. By removing photoaptamers from the solid support before light activation, the harmful light scattering and absorption by the solid support are eliminated, while still enabling covalent bond formation through subsequent photocrosslinking in a clear solution medium.
Solution Approach 2:
The patent introduces solution phase as an intermediary medium between the photoaptamers and the solid support. This intermediary allows light to penetrate effectively for photocrosslinking without the interference of solid support materials, while still achieving the desired covalent binding and subsequent solid support immobilization.
3Reliability
If aptamers are immobilized on the solid support, then the assay can proceed with binding and detection, but the surface of the solid support may be exposed to and affected by labeling agents, subjecting detection to imprecision
Solution Approach 1:
The patent inverts the sequence by performing labeling in solution phase before solid support immobilization. This inversion protects the detection system by ensuring that labeling agents do not interact with the solid support surface, eliminating a source of measurement imprecision while maintaining reliable detection through subsequent immobilization of labeled complexes.
4Ease of manufacture
If pre-immobilization of aptamers is performed, then the assay can be structured with a solid support, but it results in inefficient mixing and may affect the activity or functionality of the aptamers
Solution Approach 1:
The patent inverts the traditional sequence by forming aptamer-target complexes in solution first, then immobilizing them on the solid support. This inversion preserves aptamer activity and functionality by avoiding premature immobilization that could affect aptamer conformation or binding capability, while still achieving the structural benefits of a solid support-based assay.
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
These methods provide high sensitivity and specificity for detecting and quantifying target molecules by optimizing aptamer activity, binding efficiency, and covalent bond formation, while reducing noise and improving multiplexed analysis capabilities.
Implementation Method 1
A variation of this assay employs aptamers that include photoreactive functional groups that enable the aptamers to covalently bind or 'photocrosslink' their target molecules
Implementation Method 2
the solid support may tend to scatter or absorb the light used to effect the formation of covalent bonds between the photoaptamers and their target molecules
Implementation Method 3
the solid support may tend to scatter or absorb the light used to effect the formation of covalent bonds between the photoaptamers and their target molecules
Data Source
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. The described methods, devices, kits, and reagents facilitate the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., an aptamer). The methods described create a nucleic acid surrogate for a non nucleic acid target, thus allowing the wide variety of nucleic acid technologies, including amplification, to be applied to a broader range of desired targets, especially protein targets. The disclosure further describes aptamer constructs that facilitate the use of aptamers in a variety of analytical detection applications.