Sensitive CRISPR-Cas12a-Assisted Immunoassay for Small Molecule Detection in Homogeneous Solution

Zhu, Fengxi; Zhao, Qiang

doi:10.1021/acs.analchem.3c00218

Cited by 12 publications

(3 citation statements)

References 51 publications

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“…These results show that the CRISPR/Cas12a-powered immunosorbent assay can be applied to detect different targets by using the corresponding small molecule labeled acDNA probe and antibody. Compared with the homogeneous immunoassays, , this CRISPR/Cas12a powered competitive immunosorbent assay has more robustness for complex sample matrices as separation steps are involved. In addition, preconcentration of targets may be achieved in the immunosorbent assays by affinity capture with the immobilized antibody to further lower the detection limits.…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules

Zhu,

Zhao

2023

Anal. Chem.

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CRISPR/Cas systems are powerful tools for sensitive nucleic acid molecular diagnosis due to their specific nucleic acid recognition and high transcleavage activity and have also allowed for quantification of non-nucleic acid targets, relying on a strategy to convert the target detection to analysis of nucleic acids. Here, we describe a CRISPR/Cas12a-powered immunosorbent assay for sensitive smallmolecule detection by using the antibody coated on the microplate to recognize the target and the small molecule-labeled active DNA (acDNA) to trigger the activity of CRISPR/Cas12a. In the absence of small-molecule targets, acDNA probes are captured by the antibody on the microplate and then activate Cas12a in catalytic trans-cleavage of fluorescent DNA reporters, generating strong fluorescence. The presence of smallmolecule targets displaces the acDNA probes from the antibody, causing a decrease of acDNA probes on the microplate and reduction of activated Cas12a, so the fluorescence signal decreases, and small molecules can be detected by monitoring the fluorescence change. After systematically optimizing experimental conditions (e.g., Cas12a reaction), the proposed method achieved the detection of three model small molecules, biotin, digoxin, and folic acid, with low detection limits, and a flexible detection concentration range was obtained by simply changing the amount of acDNA probes and immobilized antibodies. The assay showed high selectivity and good applicability in complex media. The integration of the CRISPR/Cas12a system improves the analytical performance of immunoassay, broadening and facilitating its applications in rapid, simple, and sensitive small molecule analysis.

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Section: Resultsmentioning

confidence: 99%

CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules

Zhu,

Zhao

2023

Anal. Chem.

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“…In a recent paper, biotin (among other analytes) was measured in blood plasma of healthy volunteers living in a low-altitude area who had been transported to high altitude using ultrahigh-performance liquid chromatography—tandem mass spectrometry (UHPLC-MS/MS) [ 133 ]. New analytical methods have also been described in the recent literature, including an artificial intelligence-aided, competitive massive parallel spectroscopy (MPS) bioaffinity assay [ 134 ], a chemiluminescent binding assay based on avidin and biotinylated quinone [ 135 ], an impedimetric immunosensor assay [ 136 ], a competitive immunoassay based on magnetic beads and gold nanoparticle probes [ 137 ], a method based on column-switching liquid chromatography–tandem mass spectrometry [ 138 ], as well as two types of CRISPR/Cas12a-powered competitive immunoassays [ 139 , 140 ].…”

Section: Biotin Levels And/or Biotin Supplementation Proposed For Spe...mentioning

confidence: 99%

Biotin Homeostasis and Human Disorders: Recent Findings and Perspectives

Karachaliou,

Livaniou

2024

IJMS

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Biotin (vitamin B7, or vitamin H) is a water-soluble B-vitamin that functions as a cofactor for carboxylases, i.e., enzymes involved in the cellular metabolism of fatty acids and amino acids and in gluconeogenesis; moreover, as reported, biotin may be involved in gene regulation. Biotin is not synthesized by human cells, but it is found in food and is also produced by intestinal bacteria. Biotin status/homeostasis in human individuals depends on several factors, including efficiency/deficiency of the enzymes involved in biotin recycling within the human organism (biotinidase, holocarboxylase synthetase), and/or effectiveness of intestinal uptake, which is mainly accomplished through the sodium-dependent multivitamin transporter. In the last years, administration of biotin at high/“pharmacological” doses has been proposed to treat specific defects/deficiencies and human disorders, exhibiting mainly neurological and/or dermatological symptoms and including biotinidase deficiency, holocarboxylase synthetase deficiency, and biotin–thiamine-responsive basal ganglia disease. On the other hand, according to warnings of the Food and Drug Administration, USA, high biotin levels can affect clinical biotin-(strept)avidin assays and thus lead to false results during quantification of critical biomarkers. In this review article, recent findings/advancements that may offer new insight in the abovementioned research fields concerning biotin will be presented and briefly discussed.

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“…[22][23][24] Among them, the CRISPR/Cas12a system for nucleic acid detection has been rapidly adopted due to its inherent excellent performance aspects such as programmability, specificity, sensitivity, and easy operation. [25][26][27][28][29] After the pairing of guide RNAwith target DNA, the activity of the CRISPR/Cas12a system is activated to cleave DNA indiscriminately. [30] This combination with DNA-based bio-barcodes and CRISPR/Cas12a assays is expected to provide a novel strategy for f/t-PSA ratio identification and analysis.…”

Section: Introductionmentioning

confidence: 99%

Smartphone‐Based Free‐to‐Total Prostate Specific Antigen Ratio Detection System Using a Colorimetric Reaction Integrated with Proximity‐Induced Bio‐Barcode and CRISPR/Cas12a Assay

Luo,

Zhou,

Zhan

et al. 2024

Small

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The free‐to‐total prostate‐specific antigen (f/t‐PSA) ratio is of great significance in the accurate diagnosis of prostate cancer. Herein, a smartphone‐based detection system is reported using a colorimetric reaction integrated with proximity‐induced bio‐barcode and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a assay for f/t‐PSA ratio detection. DNA/antibody recognition probes are designed to bind f‐PSA or t‐PSA and induce the release of the DNA bio‐barcode. The CRISPR/Cas12a system is activated by the DNA bio‐barcode to release Ag+ from the C‐Ag+‐C structure of the hairpin DNA. The released Ag+ is used to affect the tetramethylbenzidine (TMB)‐H2O2‐based colorimetric reaction catalyzed by Pt nanoparticles (NPs), as the peroxidase‐like activity of the Pt NPs can be efficiently inhibited by Ag+. A smartphone with a self‐developed app is used as an image reader and analyzer to analyze the colorimetric reaction and provide the results. A limit of detection of 0.06 and 0.04 ng mL−1 is achieved for t‐PSA and f‐PSA, respectively. The smartphone‐based method showed a linear response between 0.1 and 100 ng mL−1 of t‐PSA or f‐PSA. In tests with clinical samples, the smartphone‐based method successfully diagnosed prostate cancer patients from benign prostatic hyperplasia patients and healthy cases with high sensitivity and specificity.

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Sensitive CRISPR-Cas12a-Assisted Immunoassay for Small Molecule Detection in Homogeneous Solution

Cited by 12 publications

References 51 publications

CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules

CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules

Biotin Homeostasis and Human Disorders: Recent Findings and Perspectives

Smartphone‐Based Free‐to‐Total Prostate Specific Antigen Ratio Detection System Using a Colorimetric Reaction Integrated with Proximity‐Induced Bio‐Barcode and CRISPR/Cas12a Assay

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