Steric-Hindrance Hybridization Assay for Whole Blood Protein Detection
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Solution Overview
Problem
Current methods for detecting proteins or biomarkers, such as antibodies, in point-of-care settings are often multi-step, costly, and require specialized technicians, while existing DNA-based electrochemical sensors face challenges like signal drift and low nano-amp electrochemical signals when used in whole blood, limiting their real-world application.
Innovation Solution
The use of a Steric-Hindrance Hybridization Assay (eSHHA) system that employs densely-packed surface-bound anchoring oligonucleotides and complementary signaling oligonucleotides with a reporter moiety, which generates a signal by modulating electrochemical currents based on the presence of macromolecular targets, allowing for detection and quantification in complex samples like whole blood without the need for extensive sample processing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA-based electrochemical sensors are used for protein detection in whole blood, then detection capability is achieved, but signal drift and low electrochemical signal output occur
Solution Approach 1:
The sensor system is divided into distinct functional segments: surface-bound anchoring oligonucleotides, free signaling oligonucleotides, and reporter moieties. This segmentation allows each component to perform its specific function optimally, with the anchoring oligonucleotides providing stable surface attachment and the signaling oligonucleotides providing detection capability, thereby resolving the contradiction between signal stability and output
Solution Approach 2:
Signaling oligonucleotides act as intermediaries between the surface-bound anchoring oligonucleotides and the reporter moieties. These intermediaries facilitate electron transfer and signal generation while maintaining signal stability, effectively mediating between the stable surface attachment and the electrochemical signal output requirements
2Adaptability or versatility
If unimolecular probes are used for target detection, then conformational modification upon binding is achieved, but detection is limited to targets capable of binding two distinct target-binding moieties
Solution Approach 1:
The detection system is segmented into separate anchoring and signaling oligonucleotides, allowing the signaling oligonucleotide to bind to a single target-binding moiety without requiring simultaneous binding to two distinct moieties. This segmentation enables broader target detection capability while maintaining reliable signal generation
Solution Approach 2:
Instead of requiring the target to induce conformational modification in a unimolecular probe, the system inverts the approach by using surface-bound anchoring oligonucleotides that remain stable while free signaling oligonucleotides provide the detection function. This inversion removes the restrictive requirement for targets to bind two distinct moieties
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
eSHHA provides a rapid, cost-effective, and multiplexable method for detecting and quantifying proteins in whole blood with enhanced signal stability and sensitivity, enabling precise measurements and improved penetration into real-world diagnostics.
Implementation Method 1
Each of the signaling oligonucleotide has a core nucleic acid sequence which is substantially complementary to a region of each of the anchoring oligonucleotides and is capable of hybridizing with the anchoring oligonucleotide
Implementation Method 2
The signaling oligonucleotide is configured such that, upon hybridizing with the anchoring oligonucleotide, the second end of the signaling oligonucleotide is located in the vicinity of the first end of the anchoring oligonucleotide
Implementation Method 3
the density of the plurality of anchoring oligonucleotides on the first substrate prevents or limits the formation of a complex between at least one anchoring oligonucleotide, at least one signaling oligonucleotide and the macromolecular entity at two adjacent locations on the first substrate
Data Source
Figure 1A~1B
Figure 2A~2F
Figure 3~5B
AI summary
The present disclosure provides target detection and quantification systems as well as related methods based on the use of steric hindrance (either created by the target itself or a macromolecular entity used to bind to the target) to prevent or limit the hybridizing between an anchoring oligonucleotide (associated to a substrate) and a signaling oligonucleotide or a combination of signaling oligonucleotides (capable of specifically binding the target or the macromolecular entity).