The fluorescent aptasensor based on CRISPR-Cas12a combined with TdT for highly sensitive detection of cocaine.

Ultrasensitive and specific detection of cocaine is of great significance for monitoring cocaine abuse. Herein, a fluorescent aptasensor via coupling CRISPR-Cas12a, with magnetic nanoparticles (MNPs), split-aptamer, and terminal deoxynucleotidyl transferase (TdT), was developed for the detection of cocaine. In short, the complete cocaine aptamer is split into two parts, one is modified on magnetic nanoparticles (MNPs) and the other is free. The presence of cocaine will mediate the binding of these two segments. Then TdT will mediate the extension to form an ultra-long sequence that can bind with multiple CRISPR-Cas12a resulting in the trans-cleavage activity of CRISPR-Cas12a being triggered. Thence, the DNA reporter which is bi-labeled with fluorophore and quencher is cleaved resulting in the generation of a fluorescence signal. The developed fluorescent aptasensor realizes the detection of cocaine with excellent sensitivity and specificity. The detection limit is low down to 33 pM, and the linear range is from 330 to 1.65 × 105 pM. Most importantly, this fluorescent aptasensor can be successfully applied to the determination of cocaine in human plasma samples.

Correction: Ultrasensitive fluorescent aptasensor for CRP detection based on the RNase H assisted DNA recycling signal amplification strategy.

[This corrects the article DOI: 10.1039/C9RA01352K.].

A highly sensitive electrochemical aptasensor for vascular endothelial growth factor detection based on toehold-mediated strand displacement reaction.

An electrochemical aptasensor with high sensitivity, specificity, and good intra-day reproducibility is reported to meet the detection needs of vascular endothelial growth factor (VEGF). The toehold-mediated strand displacement recycling amplification and VEGF aptamer are integrated in the biosensor. The probe A is hybridized with the VEGF aptamer to form the probe A-aptamer complex. When VEGF is introduced, the aptamer specifically binds with VEGF, and probe A can be liberated. Then, the free probe A captures the toehold region of the Hp1, leading the exposure of the toehold region on the other end of Hp1. Similarly, Hp2 and Hp3 are also immobilized on the surface of the electrode; thus, the methylene blue labelled on Hp2 and Hp3 causes the current response. With the signal transduction mechanism, the expression level of VEGF can be detected quantitatively. With a series of optimizations of sensor parameters, high sensitivity and specificity of the VEGF detection sensor can be achieved with a detection limit as low as 10 pg mL-1. This significant performance has good intra-day reproducibility, and it can be applied to human biological samples such as serum, urine, and saliva to detect the VEGF content.

[Extraction of exosome by gel electrophoresis microfluidic chip and determination of miRNA-21 in exosome of human plasma].

We developed a high-efficiency microfluidic chip for extracting exosomes from human plasma. We collected peripheral blood from normal human, designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes. The extracted exosomes were characterized by transmission electron microscopy, nanosight and Western blotting, the morphology, concentration and particle size of exosomes were identified and analyzed. Meanwhile, we used ultracentrifugation and microfluidic chip to isolate exosomes separately. The particle size and concentration of the exosomes extracted by two methods were compared and analyzed, and their respective extraction efficiency was discussed. Finally, the expression level of miRNA-21 in exosomes was analyzed by RT-PCR. The microfluidic chip isolated (in 1 hour) high-purity exosomes with size ranging from 30-200 nm directly from human plasma, allowing downstream exosomal miRNA analysis. By comparing with ultracentrifugation, the isolation yield of microfluidic chip was 3.80 times higher than ultracentrifugation when the volume of plasma sample less than 100 µL. The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip were: voltage: 100 V; concentration of agarose gel: 1.0%; flow rate of injection pump: 0.1 mL/h. The gel electrophoresis microfluidic chips could rapidly and efficiently isolate the exosomes, showing great potential in the research of exosomes and cancer biomarkers.

Ultrasensitive fluorescent aptasensor for CRP detection based on the RNase H assisted DNA recycling signal amplification strategy.

An aptamer-based method for the ultrasensitive fluorescence detection of C-reactive protein (CRP) was developed using the ribonuclease H (RNase H) assisted DNA recycling signal amplification strategy. In this assay, CRP can specifically bind to the aptamer of CRP and the DNA chain of P1 is released from the aptamer/P1 (Ap/P1) complexes. After the addition of the fluorescence labeled (5-FAM) RNA, P1 hybridizes with fluorescence labeled RNA to form a P1/RNA double strand. When RNase H is added, the RNA with fluorescence labeling in the double strand is specifically cut into nucleotide fragments, which cannot be adsorbed on the surface of the GO, so as to generate a fluorescence signal. In the absence of CRP, fluorescence labeled RNA cannot hybridize with P1 to form double strands, which is able to directly adsorb on the surface of GO, resulting in no fluorescence signal. The detection limit is as low as 0.01 ng mL-1, with a linear dynamic range from 50 pg mL-1 to 100 ng mL-1. This sensor is able to detect CRP in spiked human serum, urine and saliva. Thus, it shows a great application prospect in disease diagnosis and prognosis.