Single-Step, Salt-Aging-Free, and Thiol-Free Freezing Construction of AuNP-Based Bioprobes for Advancing CRISPR-Based Diagnostics

Abstract

The recently reported freezing-based labeling method for constructing DNA-AuNP probes is rapid but still requires thiol modification. Here, we evaluated a poly(A)-tagged DNA sequence using the freezing-based labeling method, and the results demonstrated that approximately 10 A bases at the sequence ends are essential. More detailed observations revealed that some DNA sequences tend to form secondary structures and thus shield exposed A bases, resulting in inefficient or failed labeling. However, successful labeling was restored by simply increasing the poly(A)-base number. Building on these discoveries, we developed three kinds of AuNP-based bioprobes, DNA-AuNP, RNA-AuNP, and DNA-enzyme-AuNP, using the freezing-based labeling method. This method was completed in a single mixing step with no need for thiol modification, representing one of the most convenient and lowest cost AuNP bioprobe labeling techniques ever reported. In addition, the resulting AuNP bioprobes were further used to advance CRISPR-based diagnostics through the development of user-friendly colorimetric, fluorescence, and lateral flow detection strategies.

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…In their work they showed simple and fast freezing-based dual labeling of AuNPs with both DNA and HRP proteins. The HRP protein can catalyzed a color reaction, resulting in test lines being better visual even at 1–10 nM concentrations of target sequence ( Hu et al, 2020 ).…”

CRISPR-Cas based virus detection: Recent advances and perspectives

“…Combining the trans -cleavage activity of Cas12a and DNA functionalized AuNPs, Li et al generated a plasmonic CRISPR-Cas12a assay for grapevine red blotch virus (GRBV) detection by naked eye ( Li et al, 2019d ). Hu et al developed a magnetic pull-down assisted colorimetric diagnosis based on the CRISPR-Cas12a system, termed as M-CDC ( Hu et al, 2020 ). African swine fever virus (ASFV) was successfully detected by M-CDC method with 100% sensitivity and 100% specificity.…”

“…Broughton et al designed a CRISPR-Cas12a coupled lateral flow assay for RNA of SARS-CoV-2 sensitive detection extracted from respiratory swabs ( Broughton et al, 2020 ). To simplify the operation, some studies reported the detection of viral nucleic acid directly from the raw sample matrix without nucleic acid extraction ( Barnes et al, 2020 ; Hu et al, 2020 ; Myhrvold et al, 2018 ; Schermer et al, 2020 ). Myhrvold et al developed HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases).…”

Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay

“…[1][2][3] Recent advances in microbial clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) enzymes offer exciting opportunities for developing novel isothermal nucleic acid amplication assays that are both sensitive and specic. [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] In particular, the combination of recombinase polymerase amplication (RPA) with the unique target-binding induced indiscriminative singlestranded DNase (ssDNase) activity of Cas12 and Cas13 has led to the development of a series of isothermal assays, such as specic high-sensitivity enzymatic reporter unlocking (SHERLOCK) [4][5][6] and DNA endonuclease-targeted CRISPR trans reporter (DETECTR). 7 We have also introduced an isothermal plasmonic CRISPR assay by integrating Cas12a with loopmediated isothermal amplication (LAMP) and plasmonic nanoparticles.…”