Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.

Riverine CO2 variations in permafrost catchments of the Yangtze River source region: Hot spots and hot moments.

Rivers and streams are pivotal modulators in regional and global carbon cycles, but riverine CO2 flux is still uncertain for permafrost watersheds. Here we present the seasonal CO2 partial pressure (pCO2) and CO2 emission flux (FCO2) of 8 rivers and streams in the Yangtze River source region (YRSR), which have high permafrost coverage and seasonally thawed active layer. The YRSR rivers and streams are generally supersaturated with CO2, although there are a few sites with CO2 undersaturation during spring. The small headwater streams are CO2 hot spots that show significantly higher pCO2 (52 % higher) and FCO2 (792 % higher) than larger rivers. Both pCO2 and FCO2 show distinct seasonality across the study sites. pCO2 and FCO2 peak in summer and exhibit much lower levels in autumn and spring, indicating that hot moments of riverine CO2 occur in summer. Seasonal pCO2 and FCO2 variations are jointly controlled by hydrology, active layer dynamics and associated processes. The warm summer causes active layer thaw and highly active soil respiration, which release a large quantity of soil carbon and increase the CO2 sources via strengthened hydrologic connectivity. The high rainfall and more thaw-released water in summer bring high discharge, which can increase the water velocity and gas exchange rate and thus CO2 emission flux. Most of the variances of seasonal FCO2 (95 %) can be explained by hydrology and active layer thaw depth. Nevertheless, the hydrological process and seasonally thawed active layer over Qinghai-Tibet Plateau (QTP) play crucial roles in riverine carbon export due to the summer monsoon-dominated climate in QTP. Our results suggest that full seasonal coverage of CO2 dynamics is essential to quantify the annual CO2 flux accurately. Changing climate and warming permafrost may alter the annual CO2 emission due to deeper flow paths, hydrology changes, and longer emission windows throughout the year.

Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has necessitated rapid, easy-to-use, and accurate diagnostic methods to monitor the virus infection. Herein, a ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed using Si-fluorescein isothiocyanate nanoparticles (FITC NPs) for detecting SARS-CoV-2 nucleocapsid (N) protein. Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane (APTES)-FITC as the Si source. This method did not need post-modification and avoided the reduction in quantum yield and stability. The p-nitrophenyl (pNP) produced by the alkaline phosphatase (ALP)-mediated hydrolysis of p-nitrophenyl phosphate (pNPP) could quench Si fluorescence in Si-FITC NPs via the inner filter effect. In ELISA, an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody. ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs. The change in fluorescence intensity ratio could be used for detecting N protein, with a wide linearity range (0.01-10.0 and 50-300 ng/mL) and low detection limit (0.002 ng/mL). The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum. Moreover, this proposed method can accurately distinguish coronavirus disease 2019 (COVID-19) and non-COVID-19 patient samples. Therefore, this simple, sensitive, and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection. Electronic Supplementary Material: Supplementary material (characterization of Si-FITC NPs (FTIR spectrum, XRD spectra, and synchronous fluorescence spectra); condition optimization of ALP response (fluorescence intensity ratio change); mechanism investigation of ALP response (fluorescence lifetime decay curves and UV-vis absorption spectra); detection of N protein using commercial ELISA Kit; analytical performance of assays for ALP detection or SARS-CoV-2 N protein detection; and determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-4740-5.

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.

Fluorescence recovery based on synergetic effect for ALP detection.

Alkaline phosphatase (ALP) is an important biomarker associated with diabetes, liver dysfunction, bone diseases, and breast cancer. Here we developed a method based on synergetic fluorescence recovery for the sensitive detection of ALP. Cadmium-zinc-selenium (CdZnSe) quantum dots (QDs) were prepared by one-pot water bath method without any complicated and rigorous conditions. CdZnSe QDs displayed high luminous efficiency, good stability, and good biocompatibility. KMnO4 and ascorbic acid phosphate (AAP) can dynamically quench the fluorescence of CdZnSe QDs. Ascorbic acid, produced by ALP-catalyzed hydrolysis of AAP, reacted with KMnO4, causing the synergetic fluorescence recovery of CdZnSe QDs. The synergetic recovery efficiency correlates well with the logarithmic ALP concentration in the range of 2.5-250 U/L with a detection limit of 0.21 U/L. In addition, good recoveries were obtained in the detection of ALP in human serum. This method provided a new research idea to improve the detection sensitivity and selectivity of ALP detection.

Construction of ratiometric Si-Mn:ZnSe nanoparticles for the immunoassay of SARS-CoV-2 spike protein.

The continuing global spread of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, has led to an unprecedented global health crisis. Effective and affordable methods are needed to diagnose SARS-CoV-2 infection. In this work, a ratiometric fluorescence probe, Si-Mn:ZnSe nanoparticles, was constructed through the electrostatic interaction between Si dots and Mn:ZnSe QDs, and the fluorescence of Mn:ZnSe QDs has a specifical response to H2O2. An immunocomplex was formed by the recognition of capture antibody/spike (S) protein/spike neutralizing antibody/biotinylated second antibody/streptavidin/biotinylated catalase (CAT). In the presence of S protein, CAT effectively catalyzed the decomposition of H2O2 in the system, and the fluorescence of Mn:ZnSe QDs was not specifically quenched. Based on this principle, a ratiometric immunoassay of SARS-CoV-2 S protein was established. The sensitivity of the proposed ELISA method was comparable to that of the commercial kit. In addition, this method can effectively distinguish the pseudo-SARS-CoV-2 virus and other pseudovirus. Therefore, this method provided a reliable and potential direction for diagnosing SARS-CoV-2 infection.