CdSe/ZnS quantum dot-encoded maleic anhydride-grafted PLA microspheres prepared through membrane emulsification for multiplexed immunoassays of tumor markers.

Early diagnosis of tumor markers is of great importance for the successful treatment of cancer. As a high-throughput and high-sensitivity detection technology, liquid suspension biochips based on quantum dot (QD) encoded microspheres have been widely used in the immunodetection of tumor markers. In this work, maleic anhydride grafted PLA (PLA-MA) microspheres based on quantum dot encoding were used as carriers for liquid phase suspension biochips for the immunoassay of tumor markers. PLA-MA fluorescent beads are prepared by embedding CdSe/ZnS quantum dots in PLA-MA using Shirasu porous glass (SPG) membrane emulsification technology, which has high fluorescence intensity, good stability, and good dispersion. Fluorescent immunoassays on dipsticks found that PLA-MA microspheres have high biological activity and good stability, which is conducive to immunoassays. Based on this, using the characteristics of CdSe/ZnS quantum dots and flow cytometry, monochromatic and two-color coding methods were developed, and 9 distinguishable coding beads were prepared. The results showed that PLA-MA fluorescent microspheres exhibited good biocompatibility, stable coding signals, low background noise, and low detection limits when performing quaternary immunoassays on tumor markers CA125, CA199, CA724, and CEA by CdSe/ZnS QD-encoded PLA-MA microsphere binding flow cytometry.

Immunoassay of SARS-CoV-2 nucleocapsid proteins using novel red emission-enhanced carbon dot-based silica spheres.

It is imminent to develop a new type of rapid COVID-19 detection method with high sensitivity. Here, we used novel red emission-enhanced carbon dot (CD)-based silica (RCS) spheres as the signals of lateral flow immunochromatography (LFI) to ultrasensitively detect novel severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins (SARS-CoV-2 NPs). The red emission of CDs can be enhanced and enriched in silica spheres by a simple way. The amino ends of the N-ß-(aminoethyl)-γ-aminopropyltrimethoxy anchor carboxyl-rich CDs and enhance the red emission, while the other end is embedded in the silica carrier. Then, the composite silica spheres werecoated with 3-(triethylsilyl) propylamine to protect the CDs, promote bioconjugation and obtain RCS spheres. The optimal emission peaks of the aqueous solution and the solid state of RCS spheres were at 634 nm and 638 nm, respectively, with quantum yields (QYs) of 48.5% and 35.7%, respectively. Their red emission has a wide excitation range (from the ultraviolet region to the red region), and the best excitation wavelength is about 580 nm. Two fluorescence detection modes of the RCS-LFI technology for the SARS-CoV-2 NP assay are available: the simple mode of observation under ultraviolet light has a sensitivity of 100 pg mL-1; the advanced mode of detection under a fluorescence microscope has a sensitivity of 10 pg mL-1. This assay also exhibits the advantages of fast detection speed, high specificity, and simple operation. In addition, the feasibility of this method in actual sample detection was verified in human serum by the standard-addition method, and the results show that the method has excellent practicability. We believe that this method will be a valuable supplement for the diagnosis of COVID-19.

Luminous silica colloids with carbon dot incorporation for sensitive immunochromatographic assay of Zika virus.

Here a novel strategy is reported of assembling silanized carbon dots (CDs) with porous silica templates to form fluorescent CD-based silica (FCS) colloids with uniformly packed CDs throughout the silica matrix. Dendritic silica spheres with highly accessible central-radial pores are adopted as a powerful absorbent host, which can form Si-O bonds with silane to directly fix the silanized CDs. The appropriate loading content of CDs on the inner surface of dendritic silica spheres is beneficial for the maximum fluorescence intensity of FCS colloids. High-quality silanized CDs endow multiple CD embedded silica spheres with excellent properties, including good fluorescence performance, excellent colloidal/optical stabilities and convenient biofunctionalization. The integration of these FCS colloids with a lateral flow strip platform provides an ultra-sensitive, specific and robust immunoassay method for the Zika NS1 protein with a visual detection limit of 10 pg mL-1, and has been successfully applied to the detection of Zika virus in clinical samples. In addition, we also prepared conventional Au NP-based lateral flow test strips and applied them to the detection of Zika NS1 protein. By comparison, the detection limit of immunofluorescent CD-based silica (iFCS)-based lateral flow test strips is 100-fold lower than that of Au NP-based lateral flow strips.

Highly-fluorescent carbon dots grown onto dendritic silica nanospheres for anthrax protective antigen detection.

Direct synthesis of carbon dots on uniform mesoporous nanospheres is an ideal way to impart fluorescence properties to the nanomaterials and retain its original uniformity. Carbon dot-based nanospheres with high quantum yield (aqueous solution, 89.3%) were synthesized by the one-step hydrothermal treatment of sodium citrate and dendritic silica spheres grafted with N-ß-(aminoethyl)-γ-aminopropyltrimethoxysilane. Its excellent chemical properties such as fluorescence, stability, homogeneity and dispersion enable it to achieve a sensitive, specific, rapid and low-cost detection of anthrax protective antigen when used as a signal for immunochromatography.

Ultrasensitive Detection of Severe Fever with Thrombocytopenia Syndrome Virus Based on Immunofluorescent Carbon Dots/SiO2 Nanosphere-Based Lateral Flow Assay.

Sensitive detection of severe fever with thrombocytopenia syndrome virus (SFTSV) by a point-of-care assay is of great significance for promoting clinical diagnosis. In this work, ultrasensitive detection of SFTSV was achieved by using fluorescent carbon dots/SiO2 nanospheres (CSNs) as reporters for a lateral flow assay. The prepared CSNs were resistant to extreme environments and had strong stability. The uniform CSNs with the size of about 200 nm were obtained by differential centrifugation. Their absolute quantum yields in the aqueous and solid phases are 56.3 and 36.6%, respectively. The excellent fluorescent properties of CSNs make the test strips more sensitive and have a longer assay lifetime. Thus, the visual detection limit of the lateral flow test strip based on immunofluorescent CSN (iCSN) was as low as 10 pg/mL SFTSV nucleoprotein. The sensitivity of this assay is 2 orders of magnitude higher than that of the colloidal gold-based lateral flow test strip. Besides, the assay owns good reproducibility and high specificity. Then, iCSN-based lateral flow test strips were evaluated in real samples of human serum of patients with satisfactory results. Furthermore, this assay has a general prospect for other fluorescent immunochromatography applications.