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Antibiotics are widely found in nitrogen-containing wastewater, which may affect the operation stability of anaerobic ammonium oxidation (anammox)-based biological treatment systems. Extracellular polymeric substances (EPSs) of anammox sludge play a pivotal role in combining with antibiotics; however, the exact role and how the structure of the leading component of EPSs (i.e., extracellular proteins) changes under antibiotic stress remain to be elucidated. Here, the interaction between sulfamethoxazole and the extracellular proteins of anammox sludge was investigated via multiple spectra and molecular simulation. Results showed that sulfamethoxazole statically quenched the fluorescent components of EPSs, and the quenching constant of the aromatic proteins was the largest, with a value of 1.73 × 10

The anaerobic ammonium oxidation (anammox) process has the advantages of high efficiency and low energy consumption, so it has broad application prospects in biological denitrification of wastewater. However, the application of anammox technology to existing wastewater treatment is still challenging. The main problems are the insufficient supply of nitrite and the susceptibility of anammox bacteria to environmental factors. In this paper, from the perspective of the diversity of anammox bacteria, the habitats and characteristics of anammox bacteria of different genera were compared. At the same time, laboratory research and engineering applications of anammox technology in treating wastewater from different sources were reviewed, and the progress of and obstacles to the practical application of anammox technology were clarified. Finally, a focus for future research was proposed to intensively study the water quality barrier factors of anammox and its regulation strategies. Meanwhile, a combined process was developed and optimized on this basis.

External stimulus such as light irradiation is able to deteriorate intracellular redox homeostasis and induce photooxidative damage to non-photogenic bacteria. Exploiting effective strategies to help bacteria resisting infaust stress is meaningful for achieving a stable operation of biological treatment system. In this work, selenium-doped carbon quantum dots (Se-CQDs) were blended into anaerobic ammonia oxidation (anammox) bacteria and an inorganic nanoparticle-microbe hybrid was successfully fabricated to evaluate its nitrogen removal performance under solar-simulated irradiation. It was found that the specific anammox activity decreased by 29.7 ± 5.2% and reactive oxygen species (ROS) content increased by 134.8 ± 4.1% under 50,000 lux light. Sludge activity could be completely recovered under the optimum dosage of 0.42 mL·(g volatile suspended solid)

The rapid development of chemical industry has induced to the large amount of phenolic wastewater production. When the promising anaerobic ammonium oxidation (anammox) was employed to treat the industrial wastewater, phenolic compounds would possibly inhibit the microbial performance. Extracellular polymeric substances (EPSs) play an essential role in protecting cells from being intoxicated by phenolic compound while the distinct mechanism remains elusive. In this work, the interaction of phenol with anammox sludge EPSs and transmembrane ammonium transport (Amt) domain was explored at molecular level by using spectral method and molecular docking simulation. It was found that phenol statically quenched the fluorescent components of EPSs and the protein component dominated the interaction between EPSs and phenol. The overall interaction was an entropy-driven process with hydrophobic interaction as the main driving force, and the CO vibration responded preferentially. As phenol continued to penetrate into the cell surface, there were hydrogen bond, hydrophobic interaction force and π-π base-stacking forces between the Amt domain and phenol. The interaction between phenol and amino acid residues of the Amt domain would interfere the NH

As one of the most promising autotrophic biological nitrogen removal technology, anaerobic ammonia oxidation (anammox) has gained intense attention for the past decades and several full-scale facilities have been implemented worldwide. However, anammox bacteria are easily affected by disturbed external environmental factors, which commonly leads to the fluctuations in reactor performance. The response of anammox sludge to external stress results in changes in components and structural characteristics of intracellular and extracellular polymer substances. Real-time and convenient spectral analysis of anammox sludge metabolites can give early warning of performance deterioration under external stresses, which is of great significance to the stable operation of bioreactor. This review summarized the research progress on characterizing the intracellular and extracellular metabolites of anammox sludge through spectroscopic techniques. The correlation between anammox sludge activity and its key metabolites was analyzed. Also, the limitations and future prospects of applying spectral analytical techniques for anammox bioreactor monitoring were discussed and outlooked. This review may provide valuable information for both scientific study and engineering application of anammox based nitrogen removal technology.

Anaerobic ammonium oxidation (anammox) process exhibits advantages in treating high-ammonium livestock wastewater, while the anammox bacteria is vulnerable to heavy metal and antibiotics. Arsenic and sulfonamides extensively existed in livestock wastewater; unfortunately, their effects on the anammox process have not yet been investigated. This work comprehensively evaluated the single and joint effects of As(III) and sulfamethoxazole (SMX) on the anammox process and revealed the related inhibitory mechanism. Anammox had extraordinary resistance to As(III) stress (0–50 mg L−1), while nitrogen removal sharply deteriorated under the stress from 50 mg L−1 As(III) and 3 mg L−1 SMX. Exogenous As(III) damaged the protection of extracellular polymeric substance (EPS) and aggravated the inhibition of SMX. As(III) had direct toxic effects on anammox bacteria, while enhanced denitrifying capacity was the reason for inhibition of anammox by SMX. Moreover, the host arsenic resistance genes were changed under SMX inhibition. The findings of this study enrich our knowledge of the anammox response to various pollutants, and provide the operational guidance for anammox process to treat wastewater containing antibiotic and heavy metal.

A sensitive, rapid and homogeneous reaction measurement method for quantitation of human epididymis protein 4 (HE4) in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) was described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one biotinylated monoclonal antibody combining on the surface of streptavidin coated donor beads, and "sandwiched" by another monoclonal antibody coated on acceptor beads. The coefficient variations of the method were lower than 10%, and the recoveries were in the range of 90-110% for serum samples. A value of 0.88pmol/l was identified as the minimum detectable dose of the present method for HE4. Compared with the results from electrochemiluminescence immunoassay kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. The present assay demonstrated high sensitivity, wider effective detection range and excellent reproducibility for quantitation of HE4 can be useful for early screening and prognosis evaluation of patients with ovarian cancer.

Alpha-fetoprotein (AFP), a primary marker for many diseases including various cancers, is important in clinical tumor diagnosis and antenatal screening. Most immunoassays provide high sensitivity and accuracy for determining AFP, but they are expensive, often complex, time-consuming procedures. A simple and rapid point-of-care system that integrates Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) has been developed to determine AFP in serum with an assay time of 15 min. The approach is based on a sandwich immunoassay performed on lateral flow test strips. A fluorescence strip reader was used to measure the fluorescence peak heights of the test line (HT) and the control line (HC); the HT/HC ratio was used for quantitation. The Eu (III) chelate microparticles-based LFIA assay exhibited a wide linear range (1.0-1000 IU mL(-1)) for AFP with a low limit of detection (0.1 IU mL(-1)) based on 5ul of serum. Satisfactory specificity and accuracy were demonstrated and the intra- and inter-assay coefficients of variation (CV) for AFP were both

Sensitive, precise and rapid detection tests are needed in the quality control of rabies vaccine for rabies virus nucleoprotein. Previous studies for quantitation of rabies virus nucleoprotein focused on enzyme-linked immunosorbent assay (ELISA). A novel immunoassay for rapid determination of rabies virus nucleoprotein in rabies vaccine was first established by time-resolved fluoroimmunoassay (TRFIA). Based on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating in the wells and "sandwiched" by another monoclonal antibody labeled with europium chelates. The immunocomplex was retained after washing, and then adopted treatment with enhancement solution; fluorescence was then measured according to the number of europiumions dissociated. Levels of the rabies virus nucleoprotein were measured in a linear range (5-2500 mEU/mL) with a lower limit of quantitation (0.95 mEU/mL) under optimal conditions. The repeatability, recovery, and linearity of the immunoassay were demonstrated to be acceptable. The correlation coefficient of nucleoprotein values obtained by novel TRFIA method and ELISA method was 0.981. These results showed good correlation and confirmed that this sensitive, precise and rapid TRFIA was feasible and could be more suitable for the quality control in the process of rabies vaccine production than ELISA.

Objectives Diabetes mellitus is a chronic disease affecting millions of people globally and resulting in significant death rates each year. A fast, inexpensive alternative to traditional testing and monitoring techniques is desirable, since secretion of insulin and C-peptide is impaired in diabetes mellitus. Design and methods A highly sensitive immunoassay was developed for the simultaneous measurement of C-peptide and insulin levels in human serum, utilizing dual-label time-resolved fluoroimmunoassay (TRFIA) and magnetic particle technologies. This assay was characteristic for a single-step sandwich-type immunoassay, wherein antibody-coated magnetic particles were used as the solid phase and Eu 3 + and Sm 3 + chelate labels were used for detection. Results Antibody-coated magnetic particles in a TRFIA format performed well in addressing a number of quantitative needs. Conclusions The results of this assay correlated well with commercial chemiluminescence assays and provided a number a advantages, including reduced sample volume, reduced reagent and personnel costs and reduced assay time, while maintaining the required clinical sensitivity.

A highly sensitive, rapid and novel simultaneous measurement method for cytokeratin 19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA) in human serum by magnetic nanoparticle-based dual-label time-resolved fluoroimmunoassay was developed. On the basis of a sandwich-type immunoassay format, analytes in the samples were captured by antibodies coated onto the surface of the magnetic beads and sandwiched by other antibodies labeled with europium and samarium chelates. The lower limit of quantitation of the present method for CYFRA 21-1 was 0.77 ng mL−1 and CEA was 0.85 ng mL−1. The coefficient variations of the method were less than 7%, and the recoveries were in the range of 90–110% for serum samples. The concentrations of CYFRA 21-1 and CEA serum samples determined by the present method were compared with those obtained by the chemiluminescence immunoassay. A good correlation was obtained with the correlation coefficients of 0.961 for CYFRA 21-1 and 0.938 for CEA. This novel method demonstrated high sensitivity, wide effective detection range and excellent reproducibility for the simultaneous determination of CYFRA 21-1 and CEA, which can be useful for the early screening and prognosis evaluation of patients with lung cancer.

Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02-700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7-8.7% and 3.8-7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X - 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus.

Background Cytokeratin 19 fragment antigen (CYFRA 21–1) is used to diagnose and monitor neoplasms. However, the main disadvantages of the currently available CYFRA 21–1 assays include heterogenous technology, being time-consuming, and having low through-put with low insensitivity. This study investigated the use of amplified luminescent proximity homogeneous immunoassay (AlphaLISA) for the quantization of CYFRA 21–1 in human serum. Methods The AlphaLISA kit was developed based on AlphaScreen detection technology with two different anti-CYFRA 21–1 monoclonal antibodies. One was coated on AlphaLISA acceptor beads and the other was biotinylated. Donor beads were coated with streptavidin. The test conditions were optimized and analytical performance was studied. Results The measurement range of AlphaLISA CYFRA 21–1 kit was 0.08–500 ng/ml. Assay detection limit was 0.08 ng/ml. The intra- and interassay coefficients of variation were 3.00–9.00% and 4.00–10.00%, respectively. There was no cross-reaction to alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 19–9 (CA19–9), cytokeratins 8 (CK8), and cytokeratins 18 (CK18). The correlation coefficient of blood samples involved was 0.974 between CYFRA 21–1-AlphaLISA assay and a commercial electrochemiluminescence immunoassay (ECLIA) CYFRA 21–1 kit (Roche). Conclusions The AlphaLISA CYFRA 21–1 kit developed in this study had favorable performance characteristics for clinical application with acceptable analytical sensitivity, specificity, and accuracy.

In order to early screen and detect suspected biomarkers from pathogens and the human body itself, tracers or reaction strategies that can act as signal enhancers have been proposed forth at purpose. In this paper, we discussed the applicability of magnetic microparticles-assisted time-resolved fluoroimmunoassay (MMPs-TRFIA) for sensitive determination of potential analytes. Hepatitis B e antigen, antibody to hepatitis B surface antigen and free triiodothyronine were used as biomarker models to explore the reliability of the method. By coupling with bioprobes, MMPs were used as immunoassay carriers to capture target molecules. Under optimal condition, assay performance, including accuracy, precision and specificity, was outstanding and demonstrated satisfactory. To further evaluate the performance of the MMPs-TRFIA in patients, a total of 728 serum samples from hospital were analyzed for three biomarkers in parallel with the proposed method and chemiluminescence immunoassay kit commercially available. Fairly good agreements are obtained between the two methods via data analysis. Not only that but the reliability of MMPs-TRFIA has also been illustrated by three different reaction models. It is confirmed that the novel method modified with MMPs has been established and showed great potential applications in both biological detection and clinical diagnosis, including big molecule protein and low molecular weight haptens.

A sensitive, rapid and novel measurement method for cytokeratin 19 fragment (CYFRA 21–1) in human serum by magnetic particle-based time-resolved fluoroimmunoassay (TRFIA) is described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating onto the surface of magnetic beads and “sandwiched” by another monoclonal antibody labeled with europium chelates. The coefficient variations of the method were lower than 7 %, and the recoveries were in the range of 90–110 % for serum samples. The lower limit of quantitation of the present method for CYFRA 21–1 was 0.78 ng/ml. The correlation coefficient of CYFRA 21–1 values obtained by our novel TRFIA and CLIA was 0.980. The present novel TRFIA demonstrated high sensitivity, wider effective detection range and excellent reproducibility for determination of CYFRA 21–1 can be useful for early screening and prognosis evaluation of patients with non-small cell lung cancer.

Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02-700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7-8.7% and 3.8-7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X - 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus.

Quantum dots are not widely used in clinical diagnosis. However, the homogeneous time-resolved fluorescence assay possesses many advantages over current methods for the detection of carcinoembryonic antigen (CEA), a primary marker for many cancers and diseases. Therefore, a novel luminescent terbium chelates- (LTCs) and quantum dots-based homogeneous time-resolved fluorescence assay was developed to detect CEA. Glutathione-capped quantum dots (QDs) were prepared from oil-soluble QDs with a 565 nm emission peak. Conjugates (QDs-6 F11) were prepared with QDs and anti-CEA monoclonal antibody. LTCs were prepared and conjugates (LTCs-S001) were prepared with another anti-CEA monoclonal antibody. The fluorescence lifetime of QDs was optimized for sequential analysis. The Forster distance (R0) was calculated as 61.9 A based on the overlap of the spectra of QDs-6 F11 and LTCs-S001. Using a double-antibody sandwich approach, the above antibody conjugates were used as energy acceptor and donor, respectively. The signals from QDs were collected in time-resolved mode and analyzed for the detection of CEA. The results show that the QDs were suitable for time-resolved fluoroassays. The spatial distance of the donor-acceptor pair was calculated to be 61.9 A. The signals from QDs were proportional to CEA concentration. The standard curve was LogY = 2.75566 + 0.94457 LogX (R = 0.998) using the fluorescence counts (Y) of QDs and the concentrations of CEA (X). The calculated sensitivity was 0.4 ng/mL. The results indicate that water-soluble QDs are suitable for the homogenous immunoassay. This work has expanded future applications of QDs in homogeneous clinical bioassays. Furthermore, a QDs-based homogeneous multiplex immunoassay will be investigated as a biomarker for infectious diseases in future research.

In this paper, a novel time-resolved fluoroimmunoassay (TRFIA) protocol using magnetic particles for the simultaneous determination of α-fetoprotein (AFP) and the free β-subunit of human chorionic gonadotropin (free β-hCG) in human serum is described. The new approach uses magnetic particles as an immobilization matrix and means of separation, while the luminescent europium and samarium chelates are used as probes. The proposed method was evaluated via a single-step, sandwich-type TRFIA immunoassay of AFP and free β-hCG as model analytes in serum. With the advantages of magnetic particles, the TRFIA immunoassay exhibited a wide dynamic range for AFP of 0.1-750 ng mL(-1), with a lower detection limit of 0.05 ng mL(-1). The dynamic range for free β-hCG was 0.16-450 ng mL(-1), with a lower detection limit of 0.08 ng mL(-1). Satisfactory specificity, reproducibility, and recovery of the immunoassay were demonstrated. Good correlations were obtained in the analysis of 446 human serum samples between the proposed method and a commercial TRFIA kit. These results demonstrate the feasibility and potential of the new method as a rapid and highly sensitive immunoassay that could be developed into a platform for multi-analyte determinations in clinical practice.