Metal-Organic Framework-DNA Bio-Barcodes Amplified CRISPR/Cas12a Assay for Ultrasensitive Detection of Protein Biomarkers.

CRISPR/Cas12a shows excellent potential in disease diagnostics. However, insensitive signal conversion strategies hindered its application in detecting protein biomarkers. Here, we report a metal-organic framework (MOF)-based DNA bio-barcode integrated with the CRISPR/Cas12a system for ultrasensitive detection of protein biomarkers. In this work, zirconium-based MOF nanoparticles were comodified with antibodies and bio-barcode phosphorylated DNA as an efficient signal converter, which not only recognized the protein biomarker to form the sandwich complex but also released the bio-barcode DNA activators after MOF dissociation to activate the trans-cleavage activity of Cas12a. Due to the obvious advantages, including numerous loaded oligonucleotides, a convenient release process, and the nontoxic release reagent, this MOF-DNA bio-barcode strategy could amplify the CRISPR/Cas12a system to achieve simple and highly sensitive detection of tumor protein biomarkers with detection limits of 0.03 pg/mL (PSA) and 0.1 pg/mL (CEA), respectively. Furthermore, this platform could detect PSA directly in clinical serum samples, offering a powerful tool for early disease diagnosis.

Metal Organic Framework-Based Bio-Barcode CRISPR/Cas12a Assay for Ultrasensitive Detection of MicroRNAs.

CRISPR/Cas12a has shown great potential in molecular diagnostics, but its application in sensing of microRNAs (miRNAs) was limited by sensitivity and complexity. Here, we have sensitively and conveniently detected microRNAs by reasonably integrating metal-organic frameworks (MOFs) based biobarcodes with CRISPR/Cas12a assay (designated as MBCA). In this work, DNA-functionalized Zr-MOFs were designed as the converter to convert and amplify each miRNA target into activators that can initiate the trans-cleavage activity of CRISPR/Cas12a to further amplify the signal. Such integration provides a universal strategy for sensitive detection of miRNAs. By tuning the complementary sequences modified on nanoprobes, this assay achieves subattomolar sensitivity for different miRNAs and was selective to single-based mismatches. With the proposed method, the expression of miR-21 in different cancer cells can be assessed, and breast cancer patients and healthy individuals can be differentiated by analyzing the target miRNAs extracted from serum samples, holding great potential in clinical diagnosis.

Aptazyme-induced cascade amplification integrated with a volumetric bar-chart chip for highly sensitive detection of aflatoxin B1 and adenosine triphosphate.

A simple aptazyme-induced cascade signal amplification (denoted as ACSA) was integrated with a triple-channel volumetric bar-chart chip (TV-Chip) to visually quantitate aflatoxin B1 (AFB1) and adenosine triphosphate (ATP). Bifunctional aptazymes consisting of aptamers and G-quadruplex-forming sequences were modified on magnetic silica nanoparticles (MSNPs) to construct sensing probes. The recognition of aptamers and targets leads to the formation of hemin/G-quadruplex (hGQ) DNAzymes, which can mimic the horseradish peroxidase (HRP)-accelerated signal enhancement reaction, enabling the polymerization of dopamine and subsequent deposition of polydopamine (PDA) on the MSNP probes, thereby providing abundant anchor sites to covalently immobilize numerous 4-mercaptophenylboric acid (4-MPBA)-modified platinum nanoparticles (PtNPs) (MPt). As a result, this strategy possesses high sensitivity by introducing a large number of PtNPs into the TV-Chip. The detection limits of AFB1 and ATP with 0.075 pM and 0.818 pM, respectively, were easily achieved without any additional instruments. In addition, the detection results of AFB1-spiked food samples were in good agreement with the commercial AFB1 ELISA kit, which verified the accuracy and reliability of this method. The ACSA-based TV-Chip would show great promise for on-site and real-time visual quantitation of trace targets.

Hypoxia-Responsive Platinum Supernanoparticles for Urinary Microfluidic Monitoring of Tumors.

Cancer has become a leading cause of morbidity and mortality, and there is an increasing need for versatile tools to enable sensitive, simple and early cancer monitoring. Here, we report platinum supernanoparticles as an exogenous nanosensor which can dissociate into ultrasmall platinum nanoclusters (PtNCs) under tumor-specific hypoxia conditions. The resulting PtNCs can be filtered through the kidney as urinary reporters to be quantified by a companion volumetric bar-chart chip (V-Chip) for point-of-care analysis. The V-Chip signals of triple-negative breast cancer and its lung metastasis mouse model showed a significant increase compared to healthy mice. Our nanosensor can also noninvasively monitor the course of treatment, which is significant for screening tumor recurrence and individualized evaluation of pharmacological and follow-up efficacy. Importantly, this strategy could be adapted for various diseases to form a common diagnostic platform by changing responsive linkers.