ABSTRACT
An aerosol jet printing-enabled dual-function biosensor for the sensitive detection of pathogens using SARS-CoV-2 RNA as an example has been developed. A CRISPR-Cas13:guide-RNA complex is activated in the presence of a target RNA, leading to the collateral trans-cleavage of ssRNA probes that contain a horseradish peroxidase (HRP) tag. This, in turn, catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by HRP, resulting in a color change and electrochemical signal change. The colorimetric and electrochemical sensing protocol does not require complicated target amplification and probe immobilization and exhibits a detection sensitivity in the femtomolar range. Additionally, our biosensor demonstrates a wide dynamic range of 5 orders of magnitude. This low-cost aerosol inkjet printing technique allows for an amplification-free and integrated dual-function biosensor platform, which operates at physiological temperature and is designed for simple, rapid, and accurate point-of-care (POC) diagnostics in either low-resource settings or hospitals.
Subject(s)
Biosensing Techniques , COVID-19 , Humans , SARS-CoV-2 , Limit of Detection , Colorimetry/methods , RNA, Viral , COVID-19/diagnosis , Respiratory Aerosols and Droplets , Horseradish Peroxidase , Biosensing Techniques/methodsABSTRACT
An aerosol jet printing enabled dual-function biosensor for the sensitive detection of pathogens using SARS-CoV-2 RNA as an example has been developed. A CRISPR-Cas13: guide-RNA complex is activated in the presence of a target RNA, leading to the collateral trans-cleavage of ssRNA probes that contain a horseradish peroxidase (HRP) tag. This, in turn, catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by HRP, resulting in a color change and electrochemical signal change. The colorimetric and electrochemical sensing protocol does not require complicated target amplification and probe immobilization and exhibits a detection sensitivity in the femtomolar range. Additionally, our biosensor demonstrates a wide dynamic range of 5 orders of magnitude. This low-cost aerosol inkjet printing technique allows for an amplification-free and integrated dual-function biosensor platform, which operates at physiological temperature and is designed for simple, rapid, and accurate point-of-care (POC) diagnostics in either low-resource settings or hospitals.
ABSTRACT
Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR Associated (Cas) proteins to target and cleave foreign genetic elements in an RNA-guided manner [1-3]. Type VI CRISPR-Cas systems contain a single effector ribonuclease, Cas13, that binds and processes a CRISPR-RNA (crRNA; also known as a guide-RNA), forming an RNA-guided RNA-targeting effector complex [4,5]. Previous studies have shown that Cas13 can be engineered to target and modulate RNA processes in human cells, illustrating the versatility and specificity of Cas13 as an RNA knockdown (KD), splicing, editing, or imaging tool [6-8]. While Cas13 has been successfully used by several groups, our lab has observed significant variability in Cas13 KD ability depending which protocol is being followed [9-12]. To further understand this variability and generate a robust Cas13 KD protocol we thoroughly tested which Cas13 ortholog to use, the duration of KD experiments, the amount of plasmid DNA transfected, methods for analyzing KD efficiency, and report an optimized method for carrying out and analyzing Cas13 mediated RNA KD experiments. The method outlined in this paper illustrates a faster and more reliable protocol to iteratively test gRNA performance and target gene KD.
