A versatile CRISPR/Cas12a-based sensitivity amplifier suitable for commercial HRP-based ELISA kits
TL;DR: The CRISPR/Cas12a-based ELISA Sensitivity Amplifier (CES-Amplifier) approach is based on a hybrid single strand DNA oligo and antibody conjugate targeting the HRP enzyme, which provides a versatile and affordable approach for expanding CRISpr/Cas-based biosensing to a wide range of non-nucleic acid analytes.
Abstract: The collateral cleavage function is only a corollary of programmable nuclease activity of certain Cas effectors, such as Cas12 and Cas13, but it can be utilised to amplify fluorescence signals in various CRISPR/Cas-based biosensing systems. In this work, this special signal amplification capability of CRISPR/Cas12a ribonucleoproteins has been employed to increase the sensitivity of a broad class of commercial ELISA systems with undisclosed chemistry except for the use of horseradish peroxidase (HRP), a common signal reporting molecule. We demonstrated that such ELISA systems with HRP on the detection antibody, can be amplified by 2 orders of magnitude using an example commercial IFN-γ ELISA kit where the detection limit was decreased from 312.5 pg/mL to 1.2 pg/mL. The detection range was simultaneously increased from 2 orders to 3 orders of magnitude. Our CRISPR/Cas12a-based ELISA Sensitivity Amplifier (CES-Amplifier) approach is based on a hybrid single strand DNA oligo and antibody conjugate targeting the HRP enzyme. The CES-Amplifier can be directly integrated into commercial ELISA kits to replace their original last step, without any additional changes of the ELISA kit reagents or setup. In this way, our CES-Amplifier provides a versatile and affordable approach for expanding CRISPR/Cas-based biosensing to a wide range of non-nucleic acid analytes.
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01 Apr 2018
TL;DR: Progress is presented in the application of immobilization strategies including the classical adsorption process, affinity attachment, random cross-linking and specific covalent linking.
Abstract: Recent advances in the development of immunosensors using polymeric nanomaterials and nanoparticles have enabled a wide range of new functions and applications in diagnostic and prognostic research. One fundamental challenge that all immunosensors must overcome is to provide the specificity of target molecular recognition by immobilizing antibodies, antibody fragments, and/or other peptides or oligonucleotide molecules that are capable of antigen recognition on a compact device surface. This review presents progress in the application of immobilization strategies including the classical adsorption process, affinity attachment, random cross-linking and specific covalent linking. The choice of immobilization methods and its impact on biosensor performance in terms of capture molecule loading, orientation, stability and capture efficiency are also discussed in this review.
37 citations
TL;DR: In this article , a CRISPR/Cas12a assisted on-fibre immunosensor (CAFI) was developed based on an antibody-analyte-aptamer sandwich structure.
11 citations
TL;DR: In this paper , a facile fluorescent CRISPR-Cas12a-based sensing strategy is presented for prostate specific antigen (PSA), as a prostate cancer biomarker, with the assistance of a cruciform DNA nanostructure and PicoGreen (PG) as a fluorochrome.
3 citations
TL;DR: In this paper , the interaction of molecularly imprinted polymers with the target molecule lidocaine, which is an amino amide pharmaceutical, was analyzed using gas chromatography-mass spectrometry.
Abstract: Molecularly imprinted polymers are materials that can capture target analytes using molecular recognition. These polymers can efficiently detect trace target chemical species in biological samples, such as urine and blood, containing various matrix components. We aimed to analyze the interaction of molecularly imprinted polymers with the target molecule lidocaine, which is an amino amide pharmaceutical. Lidocaine-templated molecularly imprinted polymers were synthesized by bulk polymerization. Gas chromatography-mass spectrometry showed there was a strong interaction between lidocaine and the recognition site in the molecularly imprinted polymers. Non-selective adsorption of lidocaine on the molecularly imprinted polymers was confirmed and characterized by Scatchard analysis. The washing solution was optimized for the separation of lidocaine from the structurally similar anesthetics procaine and tetracaine by taking advantage of differences in binding strength. The molecularly imprinted polymers could adsorb lidocaine in urine samples and were not affected by interfering compounds. Consequently, they could be applied to the analysis of biological materials. These results suggest that the molecularly imprinted polymers can be used for separation and detection with high selectivity and could be applied in forensic and clinical analysis.
2 citations
TL;DR: This work provides an alternative strategy to expand the application of the CRISPR/Cas system for the detection of non-nucleic acid targets by using the DNA activator of Cas to label the secondary antibody of traditional ELISA.
Abstract: Using the DNA activator of Cas to label the secondary antibody of traditional ELISA, the CRISPR/Cas12a-based fluorescence immunoassay was presented. The sensing strategy ingeniously combines the efficient signal generation of the CRISPR/Cas12a system with antigen-antibody-specific recognition. This work provides an alternative strategy to expand the application of the CRISPR/Cas system for the detection of non-nucleic acid targets.
2 citations
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TL;DR: It is shown that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA cleavage activity by Cas12a that completely degrades ssDNA molecules, which is also a property of other type V CRISPR-Cas12 enzymes.
Abstract: CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics
1,989 citations
TL;DR: A Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), is used to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA.
Abstract: Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.
1,946 citations
TL;DR: ShERLOCK as discussed by the authors is a platform that combines isothermal preamplification with Cas13 to detect single molecules of RNA or DNA, which can detect Dengue or Zika virus single-stranded RNA and mutations in patient liquid biopsy samples via lateral flow.
Abstract: Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) that combines isothermal preamplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCK version 2 (SHERLOCKv2) (i) four-channel single-reaction multiplexing with orthogonal CRISPR enzymes; (ii) quantitative measurement of input as low as 2 attomolar; (iii) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxiliary CRISPR-associated enzyme; and (iv) lateral-flow readout. SHERLOCKv2 can detect Dengue or Zika virus single-stranded RNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.
1,356 citations
TL;DR: This brief note addresses the historical background of the invention of the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA).
Abstract: This brief note addresses the historical background of the invention of the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA). These assays were developed independently and simultaneously by the research group of Peter Perlmann and Eva Engvall at Stockholm University in Sweden and by the research group of Anton Schuurs and Bauke van Weemen in The Netherlands. Today, fully automated instruments in medical laboratories around the world use the immunoassay principle with an enzyme as the reporter label for routine measurements of innumerable analytes in patient samples. The impact of EIA/ELISA is reflected in the overwhelmingly large number of times it has appeared as a keyword in the literature since the 1970s. Clinicians and their patients, medical laboratories, in vitro diagnostics manufacturers, and worldwide healthcare systems owe much to these four inventors.
1,339 citations
01 Apr 2018
TL;DR: SHERLOCKv2 can detect Dengue or Zika virus single-stranded RNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.
Abstract: Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) that combines isothermal preamplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCK version 2 (SHERLOCKv2) (i) four-channel single-reaction multiplexing with orthogonal CRISPR enzymes; (ii) quantitative measurement of input as low as 2 attomolar; (iii) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxiliary CRISPR-associated enzyme; and (iv) lateral-flow readout. SHERLOCKv2 can detect Dengue or Zika virus single-stranded RNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.
988 citations