Ratiometric fluorescent Si-FITC nanoprobe for immunoassay of SARS-CoV-2 nucleocapsid protein.

Coronavirus disease 2019 (COVID-19) highlights the importance of rapid and reliable diagnostic assays for the management of virus transmission. Here, we developed a one-pot hydrothermal method to prepare Si-FITC nanoparticles (NPs) for the fluorescent immunoassay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N protein). The synthesis of Si-FITC NPs did not need post-modification, which addressed the issue of quantum yield reduction during the coupling reaction. Si-FITC NPs showed two distinct peaks, Si fluorescence at λ em = 385 nm and FITC fluorescence at λ em = 490 nm. In the presence of KMnO4, Si fluorescence was decreased and FITC fluorescence was enhanced. Briefly, in the presence of N protein, catalase (CAT)-linked secondary antibody/reporter antibody/N protein/capture antibody immunocomplexes were formed on microplates. Subsequently, hydrogen peroxide (H2O2) and Si-FITC NPs/KMnO4 were injected into the microplate together. The decomposition of H2O2 by CAT resulted in remaining of KMnO4, which changed the fluorescence intensity ratio of Si-FITC NPs. The fluorescence intensity ratio correlated significantly with the N protein concentration ranging from 0.02 to 50.00 ng/mL, and the detection limit was 0.003 ng/mL, which was more sensitive than the commercial ELISA kit with a detection limit of 0.057 ng/mL. The N protein concentration can be accurately determined in human serum. Furthermore, the COVID-19 and non-COVID-19 patients were distinguishable by this method. Therefore, the ratiometric fluorescent immunoassay can be used for SARS-CoV-2 infection diagnosis with a high sensitivity and selectivity. Electronic Supplementary Material: Supplementary material (characterization of Si-FITC NPs (FTIR, HRXPS); stability investigation of Si-FITC NPs (photostability, pH stability, anti-interference ability); stability investigation of free FITC (pH value, KMnO4); quenching mechanism of KMnO4 (UV-vis absorption spectra, fluorescence lifetime decay curves); reaction condition optimization of biotin-CAT with H2O2 (pH value, temperature, time); detection of N protein using commercial ELISA Kit; selectivity investigation of assays for SARS-CoV-2 N protein detection; determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-5005-z.

Magnetic bead-based DNA hybridization assay with chemiluminescence and chemiluminescent imaging detection.

Simple and sensitive chemiluminescence (CL) and CL imaging methods have been developed for the magnetic bead-based DNA hybridization assay. The assay relies on the high sensitivity and long stable light signal of the CL system in which horseradish peroxidase (HRP) catalyzes the luminol-H(2)O(2) reaction with para-iodophenol (PIP) as the enhancer. In this protocol, a sandwich DNA hybridization is performed by mixing the target DNA with the magnetic bead-captured DNA and the biotinylated reporter DNA, followed through the biotin-streptavidin reaction with conjugated HRP, and then the conjugated HRP is determined by the CL system. The proposed CL protocol is suitable for for the detection of sequence-specific DNA related to the avian influenza A H1N1 virus at levels as low as 10 amol, and the CL imaging detection has a similar sensitivity. The sensitivities of the proposed methods with the HRP label are better than most of the metal nanoparticle-based methods, and are comparable with that of utilizing amplified techniques for DNA hybridization detection. In addition, the perfectly complementary DNA sequences and the single-base mismatched DNA sequences can be better distinguished by a thermally-stringent hybridization and washing steps. So, the proposed CL method can offer great promise for single-nucleotide polymorphism (SNP) analysis. Moreover, the proposed method may have significant potential for the simultaneous detection of various DNA sequences when different capture DNA sequences and reporter DNA sequences are used in a microarray.

Quantum dot-mediated detection of gamma-aminobutyric acid binding sites on the surface of living pollen protoplasts in tobacco.

gamma-Aminobutyric acid (GABA) is an inhibitory transmitter in the central nervous system of mammals. Recent investigations showed that it also plays an important role in regulating pollen tube growth and orientation in plants. To determine whether GABA receptors are also present on the membrane of pollen protoplasts, a fluorescence probe of quantum dots (QDs) was constructed and applied. The water-soluble CdSe-ZnS (core-shell) QDs were first synthesized and verified to possess good optical properties. GABA was then bioconjugated to the QDs in the presence of 1-ethyl-3-(3)-dimethylaminopropyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to make the fluorescence probe. Using the probe, GABA binding sites were detected on the protoplast membrane of both pollen and somatic cells. Both the fluorescent signals on the surface of the protoplasts and the Ca(2+) oscillation assayed via the Ca(2+) probe Fluo-3/AM inside the protoplasts provided evidence that the potential GABA(B) receptors are present on the plant protoplast membrane.

Electrochromatographic evaluation of a silica monolith capillary column for separation of basic pharmaceuticals.

A silica-based monolithic capillary column was prepared via a sol-gel process. The continuous skeleton and large through-pore structure were characterized by scanning electron microscopy (SEM). The native silica monolith has been successfully employed in the electrochromatographic separation of beta-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). Column efficiencies greater than 250 000 plates/m for capillary electrochromatography (CEC) separation of basic compounds were obtained. It was observed that retention of basic pharmaceuticals on the silica monolith was mainly contributed by a cation-exchange mechanism. Other retention mechanisms including reversed-phase and normal-phase mechanisms and electrophoresis of basic compounds also played a role in separation. A comparison of the differences between CEC and capillary zone electrophoresis (CZE) separation was also discussed.