Holographic Optical Tweezers and Boosting Upconversion Luminescent Resonance Energy Transfer Combined Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a Biosensors

Abstract

Taking advantage of outstanding precision in target recognition and trans-cleavage ability, the recently discovered CRISPR/Cas12a system provides an alternative opportunity for designing fluorescence biosensors. To fully exploit the analytical potential, we introduce here some meaningful concepts. First, the collateral cleavage of CRISPR/Cas12a is efficiently activated in a functional DNA regulation manner and the bottleneck which largely applicable to nucleic acids detection is broken. After selection of a representative aptamer and DNAzyme as the transduction pathways, the sensing coverage is extended to a small organic compound (ATP) and a metal ion (Na + ). The assay sensitivity is significantly improved by utilizing a beadsupported enrichment strategy wherein emerging holographic optical tweezers are used to enhance imaging stability and simultaneously achieve multiflux analysis. Last, a sandwich-structured energy-concentrating upconversion nanoparticle triggered boosting luminescent resonance energy transfer mode is comined to face with complicated biological samples by skillfully confining the emitters into a very limited inner shell. Following the above attempts, the developed CRISPR/Cas12a biosensors not only present an ultrasensitive assay behavior toward these model non-nucleic acid analytes but also can serve as a formidable toolbox for determining real samples including single cell lysates and human plasma, proving a good practical application capacity.

Detection of Amyloid β Oligomers by a Fluorescence Ratio Strategy Based on Optically Trapped Highly Doped Upconversion Nanoparticles-SiO2@Metal–Organic Framework Microspheres

“…The OT instrument was constructed on the basis of our previous works. − In Scheme B, a 980 nm continuous wave laser (power ≤ 800 mW, TEM 00 Gaussian beam) as the UCL excitation and optical trapping source is first collimated to a 3.3 mm beam through an optical fiber collimator. The light beam is set parallel to the optical platform and the light spot is expanded to about 8.2 mm, which is larger than the pupil of the objective ( d = 7.5 mm), through a 2.5-fold beam expander system ( L 1 = 20 mm, L 2 = 50 mm).…”

Advances in Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-Based Diagnostic Assays Assisted by Micro/Nanotechnologies

“…Clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based biosensors were employed for detection of non-nucleic acid analytes by bead-supported holographic optical tweezers analysis . Adapted from ref . Copyright 2021 American Chemical Society.…”

Challenges and Opportunities for Clustered Regularly Interspaced Short Palindromic Repeats Based Molecular Biosensing

“…Multiplexing and amplification-free strategies have not been extensively explored. To make the CRISPR biosensing more robust and versatile, especially for massive screening in pandemics, it is possible to revolutionize the CRISPR-Cas systems by incorporating protein engineering, , advanced materials, , microfluidic techniques, , and/or artificial intelligence. , In addition to the proposed improvements, possible future work will be discussed in the following paragraphs.…”