Synthesis of two nitrogen-doped carbon quantum dots to construct fluorescence probes for sensitive Hg2+ detection with dual signal output.

The rapid and sensitive detection of heavy metal ions is of great importance in food safety and for the environment. Therefore, two novel probes, M-CQDs and P-CQDs, based on carbon quantum dots were utilized to detect Hg2+ based on fluorescence resonance energy transfer and photoinduced electron transfer mechanisms. The M-CQDs were prepared from folic acid and m-phenylenediamine (mPDA) using a hydrothermal method. Similarly, the novel P-CQDs were obtained according to the same synthetic procedure used to create M-CQDs except the mPDA was replaced with p-phenylenediamine (pPDA). Upon the addition of Hg2+ to the M-CQDs probe, the fluorescence intensity reduced significantly with a linear concentration range between 5 and 200 nM. The limit of detection (LOD) was calculated to be 2.15 nM. On the contrary, the fluorescence intensity of the P-CQDs was enhanced greatly after the addition of Hg2+. The Hg2+ detection was realized with a wide linear range from 100 to 5000 nM and the LOD was calculated to be as low as 52.5 nM. The fluorescence "quenching" and "enhancing" effect exhibited by the M-CQDs and P-CQDs, respectively, is due to the different distribution of -NH2 in the mPDA and pPDA precursors. Notably, paper-based chips modified with M/P-CQDs were established for visual Hg2+ sensing, demonstrating the possibility for real-time detection of Hg2+. Moreover, the practicality of this system was confirmed through the successful measurement of Hg2+ in tap water and river water samples.

Discrimination of Chinese green tea according to tea polyphenols using fluorescence sensor array based on Tb (III) and Eu (III) doped Zr (IV) metal-organic frameworks.

A facile and rapid fluorescence sensor array based on Tb (III) and Eu (III) doped Zr (IV) metal-organic frameworks was proposed for Chinese green tea discrimination. According to large porosity of Tb@UiO-66-(COOH)2 and Eu@UiO-66-(COOH)2, phenolic hydroxyl groups of tea polyphenols could coordinate with free carboxylic acid groups and was captured into the pores, which led to the disturbance of electronic structure of ligand and inhibited the energy transfer efficiency from ligand to Tb (III) and Eu (III) center, causing the fluorescence quenching effect. Based on Hierarchy Cluster Analysis and Linear Discrimination Analysis, the fluorescence sensor array was employed for successful tea polyphenols classification through the analysis of different fluorescence quenching effect to tea polyphenols. Green tea samples within different categories and grades were also successfully discriminated using this assay according to tea polyphenols, providing a new method for Chinese green tea identification.

The fluorescent biosensor for detecting N6 methyladenine FzD5 mRNA and MazF activity.

N6 methyladenine (m6A) modification of the FzD5 mRNA, an important post-transcriptional regulation in eukaryotes, is closely related to the occurrence and development of breast cancer. Here, we developed an ultra-sensitive biosensor based on MazF combining with cascaded strand displacement amplification (C-SDA) and CRISPR/Cas12a to detect m6A FzD5 mRNA. MazF toxin protein is a vital component of the bacterial mazEF toxin-antitoxin system that is sensitive to m6A RNA. Take advantage of it, the biosensor achieved antibody-independent and gene-specific detection for m6A RNA. Moreover, compared with traditional amplification methods, the more efficient C-SDA and the CRISPR/Cas12a system with trans-cleavage activity gave the fluorescent biosensor an excellent sensitivity with the detection limit of 0.64 fM. In addition, MazF, as a new antibacterial target, was detected by the biosensor based on C-SDA and CRISPR/Cas12a with the detection limit of 1.127 × 10-4 U mL-1. More importantly, the biosensor has good performance in complex samples. Therefore, the biosensor is a potential tool in detecting m6A FzD5 mRNA and MazF activity.

Single-nucleotide variant of PIK3CA H1047R gene assay by CRISPR/Cas12a combined with rolling circle amplification.

PIK3CA H1047R gene plays an important role in the PI3K/Akt/mTOR signaling pathway, and its mutation is closely related to the occurrence and development of breast cancer and Lipoblastoma. Therefore, it is of great value to detect the PIK3CA H1047R mutant gene. Here, an analytical method coupled CRISPR/Cas12a with rolling circle amplification (RCA) technology was constructed for ultra-sensitive and specific detection of the single-nucleotide variant (SNV) of the PIK3CA H1047R gene. With efficient amplification of RCA and CRISPR/Cas12a, the detection limit of the mutant target and mixture of the mutant with wild-type target were as low as 10 aM and 0.036%, respectively. The detection limit of the RCA-CRISPR/Cas12a method was lower than that of allelic specific PCR (AS-PCR) for detecting SNV of the PIK3CA H1047R gene. Hence, this RCA-CRISPR/Cas12a method is sensitive and specific for the detection of SNV. What's more, this strategy provides a new idea for medical diagnosis and lays a technical foundation for the research of PI3K/Akt/mTOR signaling pathways.

Naked-eye detection of site-specific ssRNA and ssDNA using PAMmer-assisted CRISPR/Cas9 coupling with exponential amplification reaction.

Accurate and effective detection of single-stranded nucleic acids is vital in both disease diagnosis and pathological studies. Hence, we develop a PAMmer-assisted CRISPR/Cas9 system mediated G4-EXPAR (Cas-G4EX) strategy for site-specific detection of ssRNA and ssDNA. PAMmer-assisted CRISPR/Cas9 executes the site-specific cleavage of target ssRNA or ssDNA and released product fragment with the desired sequence at the 3'-terminal. This fragment serves as a primer to activate subsequent sequence-dependent exponential amplification reaction (EXPAR). The G-rich EXPAR products assembles with hemin to form a G-Quadruplex (G4/hemin). G4/hemin catalyzes ABTS-H2O2 system with the appearance of vivid green color, realizing naked-eye analysis. Cas-G4EX integrates the superiority of CRISPR/Cas9 and EXPAR, presenting outstanding site-specific recognition and high-performance amplification efficiency. Meanwhile, the programmability of CRISPR/Cas9 system makes the proposed method become a universal detection paradigm for any ssRNA or ssDNA. Cas-G4EX assay shows the linear relationship from 250 aM to 2.5 nM for ssRNA detection with the actual LOD of 250 aM, and that ranges from 100 aM to 1 nM for ssDNA detection with the actual LOD of 100 aM. Additionally, the acceptable recoveries of 101.48%-109.61% for ssRNA and 93.25%-111.98% for ssDNA in real detection of human serum are obtained for detection of single-strand nucleic acid in real samples. Cas-G4EX also exhibits the excellent discrimination for single-base mutation of single-stranded nucleic acids. Therefore, Cas-G4EX assay provides a promising platform in the applications of molecular diagnosis and pathological analysis.

Ultrasensitive detection of gene-PIK3CAH1047R mutation based on cascaded strand displacement amplification and trans-cleavage ability of CRISPR/Cas12a.

Low abundance gene-PIK3CAH1047R mutation detection is crucial for the clinical diagnosis and treatment of breast cancer. Here, a fluorescent biosensor which combines cascaded strand displacement amplification (C-SDA) and trans-cleavage ability of CRISPR/Cas12a was established to ultra-sensitively detect gene-PIK3CAH1047R mutation. The mutated gene-PIK3CAH1047R can combine with complementary sequence to form an intact recognition site for endonuclease FspI. Mediated by FspI, it breaks at the mutation site to produce DNA fragment to trigger SDA or C-SDA. Then, the fluorescent biosensors based on SDA-CRISPR/Cas12a or C-SDA-CRISPR/Cas12a were constructed. Compared with biosensor based on SDA-CRISPR/Cas12a (5 pM), the minimum detection of the biosensor based on C-SDA-CRISPR/Cas12a is reduced two orders of magnitude (50 fM). In range of 0.001%-50%, we achieved the ultrasensitive detection of gene-PIK3CAH1047R mutation low to 0.001%. Besides, the proposed biosensor works well in human serum samples, showing its application potential in low-abundance gene-PIK3CAH1047R mutation detection.

Ultra-sensitive MicroRNA-21 detection based on multiple cascaded strand displacement amplification and CRISPR/Cpf1 (MC-SDA/CRISPR/Cpf1).

MicroRNA-21 (miR-21) has been considered as a potential biomarker for cancer diagnosis and prognosis due to its high expression in tumors. Here, an analytical method which integrates the multiple cascaded strand displacement amplification and CRISPR/Cpf1 (MC-SDA/CRISPR/Cpf1) was proposed to ultra-sensitively detect it.

An ultrasensitive and point-of-care sensor for the telomerase activity detection.

Telomerase owns great application potential in diagnosis, therapy, prognosis, and drug screening of cancers. Thus, the ultrasensitive and point-of-care detection of telomerase activity meets the clinical demands extremely. Here, a sensor based on telomerase extends activators to unlock the ssDNase activity of CRISPR/Cas12a was created for the first time to detect the telomerase activity. Based on the fluorescence or CRISPR/Cas12a-based lateral flow assay, we achieve the ultrasensitive and point-of-care detection of telomerase activity in MCF-7 cells low to 57 cells·mL-1 and 5.7 × 102 cells·mL-1 in about 1 h, respectively. Besides, the detection of telomerase activity in different subtype breast cancer cells indicates that the proposed sensor possesses potential in the classification of breast cancer cell subtypes.