Fluorescent Nanoclusters for Portable Nucleic Acid Detection
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Solution Overview
Problem
Existing nucleic acid detection systems, particularly those using CRISPR-Cas assays, require expensive equipment like fluorometers and metals, making them inconvenient for first responders to use for rapid on-site detection of pathogens.
Innovation Solution
A portable nucleic acid detection system combining CRISPR technology with photoluminescence, utilizing fluorescent (nano)clusters that exhibit high fluorescence yield and photo-stability, allowing for visible emission under UV light, which does not require expensive metals or high-end equipment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If FRET-based reporters with fluorometers are used for nucleic acid detection, then measurement precision is improved, but device complexity and cost increase
Solution Approach 1:
The invention extracts the detection function from complex fluorometer equipment and transfers it to simple visual observation. By using naked-eye visible fluorescent nanoclusters instead of FRET reporters requiring fluorometers, the system removes the need for expensive measurement equipment while maintaining detection capability through direct visual observation of fluorescence color changes
Solution Approach 2:
The invention replaces expensive, complex fluorometer equipment with simple, disposable visual detection methods. The fluorescent nanoclusters serve as temporary, single-use detection agents that provide sufficient measurement precision through direct observation without requiring sophisticated instrumentation
2Measurement precision
If FRET-based reporters are used for nucleic acid detection, then measurement precision is improved, but loss of substance increases due to filter discards
Solution Approach 1:
The invention extracts the useful fluorescent signal from the complex FRET system and isolates it as direct visible emission from nanoclusters. This eliminates the need for spectral filtering that discards portions of the emission spectrum, allowing full utilization of the fluorescent signal without information loss
Solution Approach 2:
The invention changes the detection parameter from filtered spectral measurement to direct visual observation of visible fluorescence. By shifting from measuring specific wavelength ranges through filters to observing overall visible emission, the system captures complete spectral information without discarding any portion of the fluorescent signal
3Difficulty of detecting and measuring
If FRET fluorescence measurement in biological samples is performed, then detection capability is improved, but object-affected harmful factors increase due to interference
Solution Approach 1:
The invention uses composite fluorescent nanoclusters with unique optical properties that differ fundamentally from traditional FRET fluorophores. These nanoclusters exhibit distinct fluorescence characteristics that are less susceptible to biological interference, allowing detection in complex biological samples with reduced background noise and autofluorescence
Solution Approach 2:
The invention changes the fluorescent detection parameter from FRET-based energy transfer measurement to direct visible emission detection. This parameter change reduces sensitivity to biological interference sources such as autofluorescence and light scattering, while maintaining the ability to detect nucleic acid presence through colorimetric fluorescence observation
4Measurement precision
If expensive metals are used in nucleic acid detection systems, then measurement precision is improved, but cost increases
Solution Approach 1:
The invention replaces expensive metal-based detection reagents with cost-effective fluorescent nanoclusters. These nanoclusters provide equivalent or superior measurement precision without requiring precious metals, significantly reducing the quantity of expensive substances needed while maintaining detection accuracy
Solution Approach 2:
The invention changes the detection reagent from metal-based compounds to carbon-based fluorescent nanoclusters. This material substitution maintains measurement precision through optical detection while eliminating dependence on expensive metals, reducing both cost and quantity of precious substances required
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, easy, and portable detection of nucleic acids, including pathogenic DNA, with improved sensitivity and reduced interference from biological samples, suitable for first responders to use near the patient or on-site.
Implementation Method 1
combining CRISPR technology and photoluminescence, in particular fluorescence. Fluorescent (nano)clusters exhibit great potential for fluorescent bioassays thanks to the advantages of high fluorescence yield, good photo-stability and a visible emission (orange/red) when excited with ultraviolet light
Implementation Method 2
the effector protein exhibiting target-activated nucleic acid cleavage activity capable of cleaving nucleic acid reporter molecules to generate nucleic acid fragments
Implementation Method 3
a polymerase exhibiting catalytic activity capable of transferring nucleotides to the fragments to form polynucleotide tails
Implementation Method 4
synthetic RNA/DNA oligonucleotides that have a reporter fluorophore (donor) on one end and a quencher (acceptor) in proximity on the other end. Intact, these fluorescence resonance energy transfer (FRET) based reporters show no fluorescence
Data Source
AI summary
The invention relates to a nucleic acid detection system, a diagnostic device, use of the nucleic acid detection system as a diagnostic agent, a kit-of-parts for detecting nucleic acids, a method for detecting nucleic acids, and a method for diagnosing a disease state of a subject. The nucleic acid detection system comprises a CRISPR-Cas system which comprises an effector protein and one or more guide RNAs having a guide sequence, the guide sequence being capable of targeting the effector protein to a target sequence of a target, and the effector protein exhibiting target-activated nucleic acid cleavage activity capable of cleaving nucleic acid reporter molecules to generate nucleic acid fragments; and a polymerase exhibiting catalytic activity capable of transferring nucleotides to the fragments to form polynucleotide tails, wherein preferably the detection system is a nucleic acid fluorescence detection system.