Rapid and Sensitive Detection of Fentanyl and Its Analogs by a Novel Chemiluminescence Immunoassay.

BACKGROUND: Abuse of fentanyl and its analogs is a major contributor to the opioid overdose epidemic in the United States, but detecting and quantifying trace amounts of such drugs remains a challenge without resorting to sophisticated mass spectrometry-based methods. METHODS: A sensitive immunoassay with a sub-picogram limit of detection for fentanyl and a wide range of fentanyl analogs has been developed, using a novel high-affinity antibody fused with NanoLuc, a small-size luciferase that can emit strong and stable luminescence. When used with human urine samples, the assay has a sub-picogram limit of detection for fentanyl, with results fully concordant with LC-MS. RESULTS: When applied to clinical samples, the novel chemiluminescence immunoassay can detect low positive fentanyl missed by routine screening immunoassays, with a limit of detection of 0.8 pg/mL in human urine. When applied to environmental samples, the assay can detect levels as low as 0.25 pg fentanyl per inch2 of environment surface. Assay turnaround time is less than 1 h, with inexpensive equipment and the potential for high-throughput automation or in-field screening. CONCLUSIONS: We have established a novel assay that may have broad applications in clinical, environmental, occupational, and forensic scenarios for detection of trace amounts of fentanyl and its analogs.

Palladium encapsulated mesoporous silica nanoparticles for the rapid detection of analytes.

We designed a simple, inexpensive, and user-friendly assay using mesoporous silica nanoparticles to detect analytes. Highly stable and uniform palladium nanoparticles covered with mesoporous silica (Pd@mSiO2) were generated and characterized extensively using physical methods. Human Serum Albumin (HSA) protein or ssDNA specific to the HIV gag region was capped onto the Pd@mSiO2 electrostatically. This "cap" prevented the Pd(0) inside the mesoporous silica nanoparticles from catalyzing the conversion of non-fluorescent molecules to fluorescent molecules. In the presence of target anti-HSA antibodies or complementary sequence (HIV gag), HSA protein or DNA cap dissociated from the surface of Pd@mSiO2-NH2 through the specific antigen-antibody reaction or DNA hybridization, allowing Pd(0) to convert the non-fluorescent molecules to fluorescent molecules. The limit and range of detection of anti-HSA antibodies were 3.8 nM and 3.8 nM to 133.3 nM, respectively. The limit and range of detection of HIV gag were 1.6 nM and 1.6 nM to 15 nM, respectively. This simple, inexpensive, "add sample and measure" diagnostic assay could potentially be incorporated into point of care diagnostics for low-resource settings.

Rapid, user-friendly, and inexpensive detection of azidothymidine.

Strict adherence to highly active antiretroviral therapy (HAART) is very important to improve the quality of life for HIV-positive patients to reduce new infections and determine treatment success. Azidothymidine (AZT) is an antiretroviral drug commonly used in HAART treatment. In this research, an "add, mix, and measure" assay was developed to detect AZT within minutes. Three different probes designed to release fluorophores when samples containing AZT are added were synthesized and characterized. The limit of detection to AZT in simulated urine samples was determined to be 4 µM in 5 min for one of the probes. This simple and rapid point-of-care test could potentially be used by clinicians and health care workers to monitor the presence of AZT in low resource settings.

Enrichment and application of nitrifying activated sludge in membrane bioreactors.

In this study, nitrifying bacteria were enriched in a membrane bioreactor (MBR, R1) and their bioaugmentation effectiveness was evaluated in another two MBRs (R2 and R3). Nitrifying activated sludge (NAS) with high nitrification activity of up to 3,000 mg-N/(L·d)-1 was successfully enriched in R1. The results showed that chemical oxygen demand concentration of 100-200 mg/L had no negative effect on NAS enrichment but reduced the ratio of bacterial nitrifiers. Moreover, the cell concentration of nitrifying bacteria in NAS, which was 3.1 × 1011 cells/L, was similar to that of the commercial bacterium agent. For the bioaugmentation test, the reactor inoculated with 14% NAS achieved a 23% higher NH4+-N removal efficiency than that of the uninoculated reactor. Along with the improvement of nitrification performance, the bacterial nitrifiers abundance and microbial richness remarkably increased after bioaugmentation. These results suggested that the MBR system could efficiently enrich nitrifying bacteria using organic carbon containing culture medium, and potentially act as a side-stream reactor to enhance the nitrification function of the wastewater treatment plant.

Protection against Cu(II)-induced oxidative stress and toxicity to Chlorella vulgaris by 2,2'-Bipyridine-5,5'-dicarboxylic acid.

In this study, we evaluated the role of 2,2'-bipyridine-5,5'-dicarboxylic acid (Bpy-COOH) in protecting Chlorella vulgaris from the oxidative stress and toxicity induced by Cu(II). Both in vivo and in vitro tests were performed. Different addition orders of Bpy-COOH and Cu(II) were tried in the former, whereas different Bpy-COOH concentrations were used in both experiments. The in vivo experiments showed that the production of reactive oxygen species in C. pyrenoidosa treated by the addition of Bpy-COOH and Cu(II) in three orders were all significantly less than that in cases treated with only Cu(II). In vitro tests indicated that peroxidase-like complexes could be formed between Bpy-COOH and Cu(II). Based on these results, it could be concluded that the use of Bpy-COOH could significantly decrease Cu(II) toxicity to algal cells by forming peroxidase-like complexes.

Combining Bioorthogonal Chemistry with Fluorescent Silica Nanoparticles for the Ultrasensitive Detection of the HIV-1 p24 Antigen.

Early detection and viral concentration monitoring of human immunodeficiency virus in resource-poor settings are important to control disease spread and reduce mortality. Nucleic acid amplification tests are expensive for low-resource settings. Lateral flow antibody tests are not sensitive if testing is performed within 7-10 days, and these tests are not quantitative. We describe a signal enhancement technique based on fluorescent silica nanoparticles and bioorthogonal chemistries for the femtomolar detection of the HIV-1 p24 antigen. We developed a magnetic bead-based assay, wherein we used fluorescent-dye-encapsulated silica nanoparticles as reporters. The number of reporters was increased by using bioorthogonal chemistry to provide signal enhancement. The limit and range of detection of the sandwich immunoassay using alternating multiple layers for p24 in human serum were found to be 46 fg/mL (1.84 fM) and 46 fg/mL to 10 ng/mL, respectively. This simple assay was 217-fold higher in sensitivity compared to that of commercial enzyme-linked immunoassays (limit of detection of 10 pg/mL).

Point-of-Care Monitoring of Colitis Using Intestinal Alkaline Phosphatase in Inflammatory Bowel Disease.

Intestinal Alkaline Phosphatase (IAP) was investigated as a potential biomarker to monitor colitis in a mouse model of Inflammatory Bowel Disease (IBD). We developed a Point-Of-Care (POC) assay to detect IAP with a glucose meter in 15 min. We synthesized a paracetamol-bearing compound specifically cleaved by IAP to release paracetamol, which can be detected with a personal glucometer. Interleukin 10 deficient (IL 10-/-) mouse model samples were used to compare the IAP level in mice with mild or severe colitis. The results showed that fecal IAP level was significantly lower in each mouse sample with severe colitis than with mild colitis. Mice treated with anti-Tumor Necrosis Factor-alpha (anti-TNF-α) to decrease inflammation exhibited a much higher level of IAP than those without treatment (IAP levels from anti-TNF-α treated vs nontreated = 2.80 U vs 0.11 U, P < 0.0001). Taken together, IAP can be considered as a potential biomarker to monitor colitis, and a rapid, user-friendly POC glucometer-based assay can be potentially used to monitor colitis levels and inflammation flareups in IBD.

A novel technology with precise oxygen-input control: Application of the partial nitritation-anammox process.

Reliable and accurate oxygen-input control, which is critical to maintaining efficient nitrogen removal performance for partial nitritation-anammox (PN-A) process, remains one of the main operational difficulties. In this study, a novel, yet simple system (a simple process for autotrophic nitrogen-removal, SPAN) with precise oxygen-input control was developed to treat ammonium-rich wastewater via PN-A process. SPAN brings oxygen to biomass by circulating water and creating water spray (shower) at the water-air interface, and effectively balances the activities of core functional microorganisms through precise oxygen-input control. The oxygen-input rate is decided by the water circulation rate and shower rate and is measurable and predictable. Therefore, the required amount of oxygen for ammonium oxidation can be precisely delivered to the biomass by adjusting the circulation rate and shower rate. The results of two parallel SPAN reactors demonstrated that during long-term operation, the required oxygen input was precisely and reliably controlled. More than 99% of NH4+-N and 81% - 85% of total nitrogen were stably removed, with anammox bacteria contributing to more than 96% of total nitrogen removal. Anammox bacteria were efficiently enriched to the highest level among the key nitrogen-converting microbial groups, both in terms of abundance (8.17%) and nitrogen-conversion capacity, while ammonium oxidizing bacteria were well controlled to provide sufficient ammonium-oxidizing capacity. Nitrite oxidizing bacteria were maintained stable (relative abundance of 1.08%-1.88%) and their activity was effectively suppressed. This study provided a novel technology, SPAN, to precisely control oxygen input in PN-A system, and proved that SPAN was effective and reliable in achieving long-term high-efficiency nitrogen removal.

[Nitrification and Bioaugmentation of Biological Treatment System of Sewage Treatment Plant at High Temperature in Summer].

The influence of temperature (30-45℃) and ammonia-nitrogen volume load on the nitrification function and microbial community of activated sludge in an aerobic tank of a sewage treatment plant were investigated under simulated high-temperature stress in the summer. Meanwhile, the bioaugmentation effectiveness of the middle-temperature-enriched nitrifying sludge (with or without acclimation) was evaluated in two biological treatment systems under high-temperature shock. The results showed that the ammonium-nitrogen (NH4+-N) removal efficiency and the nitrifying bacteria content of the aerobic activated sludge at 30-40℃ were above 90% and up to 4.55% and decreased to 40% and 1.97% at 45℃, respectively. To quickly recover the nitrification function of the biological system under high-temperature shock in the summer, the middle-temperature-enriched nitrifying sludge was acclimated at 40℃ for 61 d and achieved (60±5) mg·(L·h)-1 nitrification activity. Then, its bioaugmentation efficiency was compared with that of the middle-temperature-enriched nitrifying sludge. In the bioaugmentation test, 10% of NH4+-N was removed in the reactor inoculated with 5% (volume fraction) of the acclimated nitrifying sludge, while the reactor needed inoculate with 10% (volume fraction) of the middle-temperature-enriched sludge to achieve the same removal efficiency. The results suggested that middle-temperature-enriched nitrifying sludge, after acclimating at 40℃, has a better enhancement effect under a high-temperature shocking load.

Start up of partial nitritation-anammox process using intermittently aerated sequencing batch reactor: Performance and microbial community dynamics.

This study investigated the performance and microbial community dynamics of a start-up method for the partial nitritation-anammox (PN-A) process: start-up from return sludge in an intermittently aerated sequencing batch reactor (IASBR). The robustness of this PN-A IASBR system in achieving long-term efficient nitrogen removal was also investigated. Stable partial nitritation with nitrite accumulation ratio of about 80% was firstly achieved in the IASBR. Then, PN-A process with total nitrogen removal of up to 81.5% was established due to the thriving of anammox bacteria Candidatus Kuenenia resulting from the reduction of the aeration rate. Molecular analysis showed that both bacterial and archaeal communities shifted greatly throughout the start-up stage and the PN-A stage. Besides bacterial genus Nitrosomonas, ammonium-oxidizing archaea (AOA) Candidatus Nitrososphaera with a high abundance of 3.44% also contributed to partial nitritation. Nitrospira was effectively restrained (abundance <1.6%) while methanogens co-existed with the aerobic and anaerobic nitrogen-conversion microorganisms. This study showed that IASBR configuration was efficient in starting up the PN-A process from return sludge, maintaining long-term efficient nitrogen removal and triggering the thrive of AOA.

Silver nanoparticles or free silver ions work? An enantioselective phytotoxicity study with a chiral tool.

Nowadays, silver nanoparticles (AgNP) have been widely used and there are raising concerns about their potential adverse effects on organism. As for the exact toxicity mechanism of AgNP, opinions still vary and whether the released silver ions (Ag+) or AgNP themselves are responsible for the toxicity remains debatable. In the present study, we have designed two exposure systems where Ag+ and AgNP coexisted but differed in quantification by using photo-reduced method with cysteine enantiomers, and their toxicities to freshwater microalgae Scenedesmus obliquus and model plant Arabidopsis thaliana were determined. In the results, Ag+ was in suit photo-reduced by cysteine enantiomers, and the UV-Vis and circular dichroism spectrum evidence confirmed the quantification difference between Ag-l-cysteine (Ag-l-Cys) and Ag-d-cysteine (Ag-d-Cys), where there was more AgNP and less Ag+ in Ag-l-Cys. Furthermore, the toxicity assay data revealed that Ag-d-Cys was more toxic to S. obliquus but A. thaliana was more susceptible to Ag-l-Cys. The metal element distribution in Arabidopsis leaves was also influenced in an enantioselective manner, which was related to the oxidative stress. Considering the quantification difference between Ag-l-Cys and Ag-d-Cys, it can be concluded that AgNP exhibited their toxicity to S. obliquus by the action of Ag+, but toxicity brought to A. thaliana was attributed to AgNP themselves rather than Ag+. The results of the present study help to better clarify the role of Ag+ in AgNP toxicity and offer a chiral tool and a new sight to investigate the toxicity mechanism of AgNP.

[Pilot-scale Experiment on Enrichment of Nitrifying Activated Sludge and Its Application in Enhancing a Wastewater Biological Treatment System Against Ammonia Shocking Loads].

Nitrifying activated sludge (NAS) was enriched in a membrane bioreactor (MBR) with pre-treated municipal wastewater and additional ammonium sulfate as the culture medium. The influences of temperature, dissolved oxygen (DO), ammonia nitrogen volumetric load, free ammonia (FA), and free nitrite (FNA) on the enrichment of NAS were investigated, the cost of the process was evaluated, and then NAS's application in enhancing a wastewater biological treatment system against ammonia shocking loads was attempted. The results showed that after 182 days of cultivation in an MBR, NAS had a nitrification activity of 98.41 mg·(L·h)-1, which was 30-times higher than that of the seeding sludge. The yield of NAS was 14.96 mg·(L·d)-1, costing 3.52 Yuan for 1 kg. Temperature was found to be a key factor affecting the sludge nitrification activity. The sludge nitrification activity was decreased to 1/3 of the maximum value at temperatures below 15.0℃, while lowering the ammonium volumetric load retarded the decrease in the sludge nitrification activity to some extent. In addition, dissolved oxygen deficiency resulted in nitrite accumulation, and thereby slowed down the NAS enrichment rate. The enriched NAS was then applied to a wastewater biological treatment pilot equipment, which had just been exposed to an ammonium shocking load. The removal rate of ammonia nitrogen in the biological system increased from 29.4% to 88.4% after 2.0% of NAS was inoculated. The enhanced biological system retained ammonia removal rates of as high as 99.0%, even as the temperature dropped to 13.3℃±1.6℃ afterwards. The above pilot-experiment results suggested that enriched nitrifying sludge is suitable for quickly increasing the start-up or recovery rates of the nitrifying function in a biological system.

Co-exposure of silver nanoparticles and chiral herbicide imazethapyr to Arabidopsis thaliana: Enantioselective effects.

In this study, we investigated the possible combined exposure effects of silver nanoparticles (Ag-NPs) and chiral herbicide imazethapyr (IM) on Arabidopsis thaliana. Herein, we show that co-exposure of Ag-NPs and chiral herbicide IM to A. thaliana can amplify the enantioselective ecotoxicity. It was found that after co-exposure of the herbicidally active 0.2 µM (R)-IM and 100 µM Ag-NPs, the silver concentration in roots was 1.40-fold higher than the co-exposure of Ag-NPs and (S)-enantiomer, as well as occurring in shoots that Ag-NPs combined with (R)-IM increased the Ag(+) concentration 77.78% than that with (S)-IM, suggesting an (R)-enantiomer preferential silver uptake. Increase of Ag(+) release under co-exposure of Ag-NPs and (R)-enantiomer was also observed. Our experiments indicated that under co-exposure of Ag-NPs and (R)-enantiomers, more accumulated amino acids can form more adducts with Ag(+), resulting in more Ag(+) release from Ag-NPs and higher ecotoxicity.

[Enhanced Pollutants Removal in a Municipal Wastewater Treatment Plant with Multistage A/O Process].

Removal of conventional pollutants as well as genotoxicity was studied along a multistage A/O process, which was based on the monitoring data in a Municipal Wastewater Treatment Plant (MWWTP) of Yixing City. The results showed that the multistage A/O process removed (67.3±7.0)% of COD, (93.7±1.5)% of NH4+-N, (65.3±7.9)% of TN and (60.0±18.7)% of TP, respectively, which played a dominant role in the removal performance of the whole wastewater treatment process. The multistage A/O process showed significant ability to reduce alkanes, halogenated hydrocarbons and alcohols in the municipal wastewater, while it failed to remove the aromatic proteins which were the main fluorescent substances of this wastewater. Furthermore, the process removed 82.8% genotoxicity from its influent. Low organic load, single-phase influent and undesirable carbon source feeding pattern, which caused the downstream A/O stages being not fully utilized, were considered as the predominant reasons for the relatively low performance of the multistage A/O process. Multi-phase feeding and adjusting carbon source feeding pattern were thereby proposed. The results were considered to be helpful for improving the operational performance of the MWWTP and useful for performance evaluation of MWWTPs with similar process.

New insights into the effects of the herbicide imazethapyr on Cu(II) ecotoxicity to the aquatic unicellular alga Scenedesmus obliquus.

Interactions between heavy metals and organic contaminants can result in speciation changes of heavy metals. Therefore, it is unknown whether organic contaminants in aquatic environments act as selective agents to mediate the toxicity of heavy metals. To elucidate these interactions, the effects of the herbicide imazethapyr (IM) on Cu(II) ecotoxicity to the aquatic unicellular alga Scenedesmus obliquus were investigated. It was found that the toxicity of Cu could be mediated by IM. To explore the mechanisms involved, complex formation, the catalytic activity of the complex, the Cu species and the distribution of Cu and Fe in the algal cell were characterized. The results showed that Cu(II) and IM formed an octahedral complex with an IM:Cu molar ratio of 2:1. These complexes also catalyzed the disproportionation of hydrogen peroxide. Analysis of the K-edge of Cu using XAFS spectroscopy indicated that when treated with Cu, the Cu was bound to polygalacturonic acid (on the cell wall), and once inside the cell, Cu may complex with the reduced glutathione (GSH) (in the cell). When the cell is treated with IM and Cu simultaneously, IM-Cu may be the primary complex formed. Once Cu combines with IM, it is difficult for it to interact with the cell wall. In addition, using scanning transmission soft X-ray microscopy, it was found that Cu could induce changes in the distribution of the essential trace element Fe, whereas IM-Cu cannot. This finding demonstrates the importance of interactions between heavy metals and organic contaminants, which are able to mediate the toxicity of heavy metals and should be considered in future risk assessments.