Gold Nanoparticle‐Labeled CRISPR‐Cas13a Assay for the Sensitive Solid‐State Nanopore Molecular Counting

Abstract

the critical need for rapid and sensitive molecular diagnostics to combat current and future pandemics. Sensitive polymerase chain reaction (PCR) based tests are the gold standard for molecular diagnostics but rely on bulky and expensive instruments. Thus, they are not suitable for self-diagnosis or point-of-care (POC) settings. [2] High-throughput sequencing can decipher the entire genomic landscape of the pathogens but is time consuming and requires bioinformatics for data interpretation. [3] Immunoassays, such as rapid antigen tests, are simple and rapid diagnostic methods but normally lack the sensitivity to reporting the low concentration biomarkers. [4] Assays with high limits of detection and low accuracy have been acceptable out of necessity, but there are many scenarios where a rapid, sensitive, POC device would be beneficial. Therefore, developing a simple to use, portable, and sensitive diagnostic platform is one important key to addressing the current challenges for molecular diagnostics.Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are bacterial systems evolved to combat bacteriophage infections by recognizing specific nucleic acid sequences to activate nucleolytic cleavage activities. The trans-cleavage of A gold nanoparticle (AuNP)-labeled CRISPR-Cas13a nucleic acid assay is developed for sensitive solid-state nanopore sensing. Instead of directly detecting the translocation of RNA through a nanopore, the system utilizes non-covalent conjugates of AuNPs and RNA targets. Upon CRISPR activation, the AuNPs are liberated from the RNA, isolated, and passed through a nanopore sensor. Detection of the AuNPs can be observed as increasing ionic current in the chip. Each AuNP that is detected is enumerated as an event, leading to quantitative of molecular targets. Leveraging the high signal-to-noise ratio enabled by the AuNPs, a detection limit of 50 fM before front-end target amplification is achieved using SARS-CoV-2 RNA segments as a Cas13 target. Furthermore, a dynamic range of six orders of magnitude is demonstrated for quantitative RNA sensing. This simplified AuNP-based CRISPR assay is performed at the physiological temperature without relying on thermal cyclers. In addition, the nanopore reader is similar in size to a smartphone, making the assay system suitable for rapid and portable nucleic acid biomarker detection in either low-resource settings or hospitals.

Gold Nanoparticle Enabled Localized Surface Plasmon Resonance on Unique Gold Nanomushroom Structures for On‐Chip CRISPR‐Cas13a Sensing

“…The AuNP‐based CRISPR Lbu‐Cas13a reaction was carried out according to the authors’ previous work. [ 48 ] The Cas13a‐RNA reporter probe mixture contained 8 µL CRISPR complex (100 n m Lbu‐Cas13a, 110 n m crRNA, and 1× STD buffer), 4 µL of the biotin–fluorescein ssRNA reporter (10 µ m ), 8 µL RNA target (100 n m ), 16 µL 5× STD buffer, and 44 µL Nuclease Free Water. The reaction was incubated at 37 °C for 30 min.…”

Gold Nanomaterials‐Implemented CRISPR‐Cas Systems for Biosensing

“…To achieve the portable and quantitative detection of nucleic acids, Du et al developed an AuNPs-based solid-state nanopore biosensor that could produce strong ionic signal pulse (Figure 10C). [117] When SARS-CoV-2 RNA was present, the Cas13a/crRNA complex was activated to cleave the biotin-ssRNA-FAM probes, leading to the SA-coated AuNPs away from the anti-FAM-coated magnetic beads. When SARS-CoV-2 RNA was absent, the biotin-ssRNA-FAM probes bound to SA-coated AuNPs to form AuNPs-SA-biotin-ssRNA-FAM complex that was captured by the anti-FAM-coated magnetic beads.…”

“…Reproduced with permission. [117] Copyright 2022, Wiley-VCH. Using gold nanomaterials as the electrode, Zhu et al designed an electrochemical biosensor based on the CRISPR-Cas13a system and CHA for detecting miRNA-21.…”

“…C) Schematic illustration of the AuNP-Cas13-based solid-state nanopore biosensor for detecting nucleic acid. Reproduced with permission [117]. Copyright 2022, Wiley-VCH.…”

Micro- and nanosystems for the detection of hemorrhagic fever viruses

“…284,285 A typical solid-state nanopore detects biomolecules by measuring the electrical current change when the molecules pass through the nanosized hole. [286][287][288] This revolutionary technology enables real-time analysis of label-free nucleic acids and has been commercially used for disease diagnostics and precision medicine. Rozevsky et al combined transverse transcription with nanopore sensing to quantify RNA molecules.…”