Quantum Dot Reporters Designed for CRISPR-Based Detection of Viral Nucleic Acids.

Diagnostic methods based on CRISPR technology have shown great potential due to their highly specific, efficient, and sensitive detection capabilities. Although the majority of the current studies rely on fluorescent dye-quencher reporters, the limitations of fluorescent dyes, such as poor photostability and small Stokes shifts, urgently necessitate the optimization of reporters. In this study, we developed innovative quantum dot (QD) reporters for the CRISPR/Cas systems, which not only leveraged the advantages of high photoluminescence quantum yield and large Stokes shifts of QDs but were also easily synthesized through a simple one-step hydrothermal method. Based on the trans-cleavage characteristics of Cas12a and Cas13a, two types of QD reporters were designed, the short DNA strand and the hybridization-based QD reporters, achieving the detection of DNA and RNA at the pM level, respectively, and validating the performance in the analysis of clinical samples. Furthermore, based on the unique property of QDs that allowed multicolor emission under one excitation, the application potential for simultaneous detection of diseases was further investigated. Taken together, this work proposed novel QD reporters that could be applied to the various CRISPR/Cas systems, providing a new toolbox to expand the diagnosis of bioanalytical and biomedical fields.

CRISPR/Cas13a Trans-Cleavage-Triggered Catalytic Hairpin Assembly Assay for Specific and Ultrasensitive SARS-CoV-2 RNA Detection.

New coronavirus (SARS-CoV-2), which has caused the coronavirus disease 2019 (COVID-19) pandemic, has brought about a huge burden on global healthcare systems. Rapid and early detection is important to prevent the spread of the pandemic. Here, an assay based on CRISPR/Cas13a and catalytic hairpin assembly (CHA), termed as Cas-CHA, was developed for ultrasensitive and specific detection of SARS-CoV-2 RNA. Upon specific recognition of the target, the CRISPR/Cas13a collaterally cleaved a well-designed hairpin reporter and triggered the CHA reaction. Under optimized conditions, the assay detected the SARS-CoV-2 RNA with a wide range of 100 aM to 100 nM and realized a low detection limit of 84 aM. At the same time, the whole detecting process could be completed within 35 min. More importantly, the assay was able to distinguish SARS-CoV-2 RNA from common human coronaviruses and analyze in saliva samples. By the flexible design of crRNA, the assay was expanded to detect other viruses. The clinical sample analysis verified that the proposed assay held a great potential for practical testing.

Point-of-care testing (POCT) of patients with a high concentration of uric acid by using alginate hydrogel microspheres embedded with CdZnTeS QDs and urate oxidase (Alg@QDs-UOx MSs).

High concentration of uric acid is usually related to cardiovascular and cerebrovascular diseases. Developing a simple method for the rapid and efficient detection of uric acid has a great significance in clinical diagnosis. In this work, alginate hydrogel microspheres embedded with CdZnTeS QDs and urate oxidase (Alg@QDs-UOx MSs) were prepared for the first time, and further used for point-of-care testing (POCT) of patients with a high concentration of uric acid. This strategy is mainly based on visual detection of H2O2, the product of uric acid after an enzymatic reaction. The proposed sensor (Alg@QDs-UOx MSs) has several advantages. First, it can reduce the interference of the proteins to the fluorescence of QDs. Second, Alg@QDs-UOx MSs help improve the stability of the CdZnTeS QDs as well as the activity of urate oxidase during storage. Third, it is easy to use, has fast response speed, and is of low cost. Therefore, the proposed sensor shows good application prospects. Simply through the built-in camera of a smartphone, we can visualize the urine samples from patients with a high concentration of uric acid within 10 minutes, and the accuracy rates were 100%. In the range of 100.0 µM to 900.0 µM, the I/I0 values and uric acid concentrations are in a great linear relationship (R2 = 0.9973), indicating that this method can be employed for quantitative analysis of uric acid in human urine (<10 mM). The limit of detection (LOD) is 20.3 µM.

Homogeneous immunoassay for alpha-fetoprotein based on the quenching of the fluorescence of quantum dots by antibody labelled with complexed copper ion tags.

A homogeneous fluorescent immunoassay is described for the determination of alpha fetoprotein (AFP) relying on the interaction between copper ion complex and quantum dots (QDs). The copper ion complex-labelled antibody can be employed as a quencher of fluorescence of QDs and capture probe of AFP in homogeneous solution. The labelled antibody is mixed with QDs to form the immune ensemble probe. Upon the addition of AFP, the labelled antibody is stripped away from QDs by antigen-antibody combination leading to the increase in the fluorescence signal. Thus, the determination of AFP can be realized by fluorometry (best measured at excitation/emission wavelengths of 360/520 nm). The fluorescence intensity shows a good linear relationship with the AFP concentration ranging from 40 to 640 ng mL-1, and the LOD is 26 ng mL-1. The proposed method provides a new approach to incorporate metal complexes into QD-based biomolecule sensing. Graphical abstract Schematic presentation of a fluorescent probe comprised of quantum dots and antibody labelled with copper ion complex for homogeneous immunoassay of α-fetoprotein. The target antigen can break up the ground state QD/labelled antibody complex to set free the fluorescent QDs.

7-Meth-oxy-3-(4-meth-oxy-phen-yl)chroman-4-one.

The asymmetric unit of the title compound, C(17)H(16)O(4), contains two crystallographically independent mol-ecules with different absolute configurations.

Synthesis of bio-templated clickable quantum dots and a dual-emitting organic/inorganic complex for ratiometric fluorescence visual assay of blood glucose.

With the prevalence of diabetes, rapid and simple blood glucose monitoring has become more and more important. Here, we report the synthesis of bio-templated N3-CdZnTeS quantum dots (QDs), which are great fluorescent biological labels and were used for the fabrication of dual-emissive dye@protein-QD conjugates via copper-free click chemistry, such as the 5(6)-carboxyfluorescein@glucose oxidase-quantum dot (FAM@GOx-QDs) complex. When adding glucose, the red fluorescence of the CdZnTeS QDs sharply decreased, while the green fluorescence of FAM was invariable. A good linear relationship ranging from 0.3 to 30 µM was obtained for glucose detection, with the limit of detection as low as 0.035 µM. Notably, the DNA-bridging FAM@GOx-QDs complex exhibited enhanced enzyme activity and stability, and was applied for the differentiation of diabetic and healthy people by the naked eye.

4-alkoxy-3-arylfuran-2(5H)-ones as inhibitors of tyrosyl-tRNA synthetase: synthesis, molecular docking and antibacterial evaluation.

A series of novel 4-alkoxy-3-arylfuran-2(5H)-ones as tyrosyl-tRNA synthetase inhibitors were synthesized. Of these compounds, 3-(4-hydroxyphenyl)-4-(2-morpholinoethoxy)furan-2(5H)-one (27) was the most potent. The binding model and structure-activity relationship indicate that replacement of morpholine-ring in the side chain of 27 with a substituent containing more hydrophilic groups would be more suitable for further modification. Antibacterial assay revealed that the synthetic compounds are effective against growth of Gram-positive organisms, and 27 is the most potent agent against Staphylococcus aureus ATCC 25923 with MIC(50) value of 0.23 µg/mL.

3-(4-Hy-droxy-phen-yl)-7-meth-oxy-chroman-4-one monohydrate.

In the title compound, C(16)H(14)O(4)·H(2)O, the dihedral angle betwen the benzene rings is 71.4 (6)°. The pyran ring is in a sofa conformation. In the crystal, O-H⋯O hydrogen bonds connect the components into a two-dimensional network parallel to (010), incorporating C(2) (2)(4) and C(2) (2)(11) chains. In addition, weak C-H⋯O, C-H⋯π and π-π stacking inter-actions [centroid-centroid distance = 3.768 (2) Å] are present.

Investigating the effect of 6-mercaptohexanol on the performance of a biosensor based on nanosurface energy transfer between gold nanoparticles and quantum dots.

Nanosurface energy transfer (NSET)-based sensors have been widely developed using various pairs of nanomaterials including gold nanoparticles (AuNPs) and quantum dots (QDs). However, a low signal to background ratio is one of the most important problems that researchers are continually trying to solve. Herein, we present a 6-mercaptohexanol (MCH) modified MCH/DNA-Au-QD sensor for the detection of nucleic acids and MUC1. Interestingly, an unexpected effect of MCH was found in enhancing the fluorescence recovery ratio, therefore yielding a higher signal to background ratio. Through further investigation, we perceive the enhancement as a result of lowering of the NSET efficiency between free DNA-AuNPs and free DNA-QDs, which arises from the stretching of adsorbed DNA on the surface of AuNPs. The employment of MCH endowed the sensor with a wider linear range from 5 nM to 120 nM and a relatively lower LOD of 1.19 nM in nucleic acid detection, outperforming the original DNA-Au-QD sensor. Furthermore, the application of the sensor can be further extended to MUC1 detection. This study offers a better understanding of the NSET process between QDs and AuNPs and also initiates a new approach for the performance optimization of analogous NSET-based sensors.

DNA-templated quantum dots and their applications in biosensors, bioimaging, and therapy.

Over the past 10 years, DNA functionalized quantum dots (QDs) have attracted considerable attention in sensing and imaging of disease-relevant biological targets, as well as cancer therapy. Considerable efforts have been devoted to obtaining DNA functionalized QDs with enhanced stability and quantum yield. Here, we focus on a one-pot method, in which phosphorothioate-modified DNA is used as the co-ligand on the basis of the strong binding of sulfur and Cd2+. After a short summary of the preparation of DNA-templated QDs, versatile bioapplications based on the constructed ratiometric fluorescent probes, nanobeacons and multiple bottom-up assemblies will be discussed. A substantial part of the review will focus on these applications, ranging from small molecule, biological macromolecule, cancer cell and pathogen sensing to in vitro and in vivo imaging. Besides, drug or siRNA delivery based on DNA-templated QD assemblies will also be briefly discussed here.

Facile synthesis of stable CdTe/CdS QDs using dithiol as surface ligand for alkaline phosphatase detection based on inner filter effect.

Alkaline phosphatase (ALP) is a universal and important hydrolase that has been proved to be associated with several diseases. Herein, a simple and effective method was proposed for ALP detection based on the inner filter effect of p-nitrophenol (pNP) on the fluorescence of CdTe/CdS quantum dots (QDs). For the preparation of CdTe/CdS QDs, Na2TeO3 was used as the Te source, and dithiol as the S source and surface ligand. The as-prepared CdTe/CdS QDs show good fluorescence properties, such as high quantum yield (∼80%), and good chemical/photo-stability. pNP is a hydrolysate of p-nitrophenol phosphate disodium salt under the catalysis of ALP, which could effectively quench the fluorescence of QDs due to the absorption spectra of pNP overlaps well with the excitation spectra of the CdTe/CdS QDs. Therefore, the prepared CdTe/CdS QDs could be applied for ALP detection. A good linear relationship ranging from 2.2 to 220 U/L was obtained with the limit of detection as low as 0.34 U/L. In addition, this method was successfully applied for the assay of ALP in human serum with the satisfactory results.