Multiplex Analyte Detection with Decoupled Probe Signaling
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Solution Overview
Problem
Existing methods for detecting and analyzing multiple analytes in biological samples are inflexible, expensive, complex, time-consuming, and often provide inaccurate results due to low encoding capacity and complexity.
Innovation Solution
A multiplex method using at least 20 sets of analyte-specific probes, each with unique identifier sequences, combined with decoding and signal oligonucleotides, allows for sequential signal-encoding of analytes and subgroups, enabling accurate and efficient detection through multiple hybridization cycles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If directly labeled probe sets are used for detecting analytes, then detection can be performed, but the method requires several differently tagged probe sets per transcript and needs to denature probe sets after every detection round, increasing complexity and time consumption
Solution Approach 1:
The probe set is divided into two functional segments: a binding element that specifically interacts with the analyte and an identifier element that provides a unique nucleotide sequence for decoding. This segmentation allows the same binding element to be reused across multiple detection rounds with different identifier elements, eliminating the need to denature and replace entire probe sets after each round.
Solution Approach 2:
An intermediary decoding system is introduced that uses decoding oligonucleotides to translate the identifier elements into detectable signals. This intermediary layer decouples the binding function from the signaling function, allowing probes to remain bound to analytes while enabling sequential detection through multiple decoding rounds without denaturation.
2Adaptability or versatility
If multiple fluorescent labels are used to increase encoding capacity, then more analytes can be detected, but the cost and complexity of the system increase significantly
Solution Approach 1:
The system uses periodic sequential rounds of hybridization and decoding instead of simultaneous multi-color detection. Each round uses a single fluorescent label that cycles through different analytes in a predetermined sequence, generating unique temporal codes for each analyte. This periodic action achieves high encoding capacity using only one or a few fluorescent labels.
Solution Approach 2:
The system transitions from spatial multiplexing (using different colors/spectral dimensions) to temporal multiplexing (using sequential timing). By encoding analyte identity in the time domain through sequential detection rounds rather than simultaneous spectral channels, the system achieves high encoding capacity with reduced spectral requirements.
3Measurement precision
If conventional detection methods are used, then detection can be performed, but the methods are time-consuming and reduce productivity
Solution Approach 1:
Probe sets are pre-designed with built-in unique identifier elements integrated into their structure before hybridization. This preliminary encoding allows the system to perform multiple detection rounds without redesigning or re preparing probes, significantly increasing throughput while maintaining detection precision through the predetermined decoding sequence.
4Adaptability or versatility
If existing multiplexing methods are used, then multiple analytes can be detected, but the methods are inflexible and expensive
Solution Approach 1:
The binding elements are designed to be universal and reusable across multiple detection rounds. The same binding element can hybridize to its target analyte in multiple sequential rounds with different identifier elements or decoding oligonucleotides, eliminating the need for round-specific probe redesign and reducing costs while maintaining multiplexing capability.
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 method reduces complexity, enhances flexibility, and improves accuracy by decoupling analyte-specific and signal oligonucleotides, allowing for faster, cheaper, and more reliable detection of multiple analytes and their subgroups.
Implementation Method 1
each set of analyte-specific probes interacting with a different analyte... each analyte-specific probe comprising a binding element (S) that specifically interacts with one of the different analytes
Implementation Method 2
contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence
Implementation Method 3
contacting the sample with at least a set of signal oligonucleotides, each signal oligonucleotide comprising a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c)
Data Source
AI summary
The technology provided herein relates to multiplex methods and kits for detecting different analytes and different subgroups/variations of an analyte in a sample, for example in parallel by sequential signal-encoding of said analytes, as well as in vitro methods for screening, identifying and/or testing a substance and/or drug and in vitro methods for diagnosis of a disease, and an optical multiplexing system.