CRISPR Graphene Biosensor for Digital Nucleic Acid Detection

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Solution Overview

Problem

Conventional nucleic acid-based molecular diagnostic tests require complex instrumentation and trained personnel, limiting their use in resource-constrained settings for point-of-care (POC) applications, especially for detecting DNA mutations in diseases like tuberculosis, HIV, and ZIKA.

Innovation Solution

A CRISPR-based graphene biosensor that utilizes a substrate with electrodes and a ribonucleoprotein (RNP) complexed with guide RNA to detect target nucleic acids without the need for PCR amplification, providing a portable and integrated solution for digital DNA mutation detection.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional PCR-based molecular diagnostic methods are used, then detection sensitivity and reliability are improved, but device complexity and requirement for trained personnel increase

Engineering Contradiction:

Improvedetection reliabilityVSAvoidinstrumentation complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent extracts and eliminates the PCR amplification step from the diagnostic workflow. By using CRISPR-Cas9 system with engineered Cas9 variants that have reduced non-specific DNA binding, the system achieves direct detection of target nucleic acids without requiring PCR amplification, thereby simplifying the instrumentation while maintaining detection reliability

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent introduces engineered Cas9 protein variants as intermediaries that mediate between the target DNA and the detection system. These Cas9 variants with reduced non-specific binding act as selective mediators that enhance target recognition accuracy, allowing reliable detection without complex PCR instrumentation

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If PCR amplification is used to detect target nucleic acids, then detection sensitivity is improved, but the time required for diagnosis and loss of time increase

Engineering Contradiction:

Improvedetection sensitivityVSAvoiddiagnosis time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent applies preliminary action by pre-engineering Cas9 protein variants with optimized binding characteristics before the diagnostic test. These pre-engineered proteins have enhanced target specificity and reduced non-specific binding, allowing direct detection of target nucleic acids without time-consuming PCR amplification steps

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent changes the biochemical parameters of the Cas9 protein through engineering modifications. By altering the protein's DNA binding properties to reduce non-specific interactions, the system achieves high detection sensitivity directly from genomic samples, eliminating the need for PCR amplification and significantly reducing diagnosis time

Inventive Principle:

Principle #35Parameter changes

3Device complexity

If CRISPR system is used for direct detection without PCR, then device complexity is reduced, but detection precision and reliability may worsen due to non-specific binding

Engineering Contradiction:

Improveinstrumentation simplicityVSAvoiddetection precision

Core Design Contradiction:

Device complexityVSMeasurement precision

Solution Approach 1:

The patent applies local quality by engineering specific local regions of the Cas9 protein to have reduced non-specific DNA binding affinity. By modifying particular amino acid residues in the Cas9 protein structure, the system maintains high target specificity while enabling direct detection without PCR, thus preserving detection precision with simplified instrumentation

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent changes the biochemical parameters of Cas9 protein through targeted engineering modifications. By adjusting the protein's DNA binding parameters to reduce non-specific interactions, the system achieves reliable direct detection of target nucleic acids, maintaining high detection precision while eliminating complex PCR instrumentation

Inventive Principle:

Principle #35Parameter changes

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

Enables rapid, sensitive, and specific detection of target nucleic acids directly from genomic samples within minutes, reducing the need for amplification and complex equipment, making it suitable for POC diagnostics.

Implementation Method 1

a guide ribonucleic acid (gRNA) comprising a first sequence capable of binding to the RNP and a second sequence capable of binding to the target nucleic acid

Methodology Applied

Scientific EffectNucleic acid hybridization: Absorption (physical)

Implementation Method 2

a ribonucleoprotein (RNP) conjugated to a second moiety of the linker molecule

Methodology Applied

Scientific EffectMolecular recognition: Absorption (physical)

Data Source

PatentUS11905552B2Immobilized RNPs for sequence-specific nucleic acid capture and digital detection

Publication Date: 2024.02.20 RGT UNIV OF CALIFORNIA

AI summary

A digital biosensor for assaying a target nucleic acid and methods of using the biosensor for digitally detecting the target nucleic acid are disclosed wherein the biosensor includes a substrate having a substrate surface and at least two electrodes, a linker molecule having a first moiety conjugated to the substrate surface, a ribonucleoprotein (RNP) conjugated to a second moiety of the linker molecule, and a guide ribonucleic acid (gRNA) having a first sequence capable of binding to the inactive RNP and a second sequence capable of binding to the target nucleic acid.