ELISA-Based Method for Variant-Independent Detection of Total Microcystins and Nodularins via a Multi-immunogen Approach.

Required routine monitoring of microcystins (MCs) and nodularins (NODs) in water samples, as posed by U.S. EPA Unregulated Contaminant Monitoring Rule 4, demands cost-effective, reliable, and sensitive detection methods. To target as many MC and NOD variants as possible, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) with group-specific monoclonal antibodies for variant-independent detection of total MCs and NODs. In this ELISA method, the mice monoclonal antibodies presenting both high affinities and broad-spectrum recognition capabilities against MCs and NODs were self-produced by designing MC hapten-based multi-immunogens to minimize specificity for the particular variant. Their high affinities and variant-independent binding capabilities against MCs and NODs were validated by both wet lab and in silico methods. The developed ELISA method achieved a limit of detection of below 0.3 µg/L for 13 MC/NOD variants, well with the reported best cross-reactivities of 60-127% relative to MC-LR. As a case study, this ELISA method was used to map the variations of intracellular and extracellular total MCs/NODs in the Luoma Lake drinking water source, China, in July, 2020. Its capability to measure total MCs/NODs with high sensitivity and high throughput in a simple and affordable way would truly be a disruptive technology capable of changing our understanding of bloom/toxin dynamics and having obvious implications for monitoring efforts.

Unconventional Split Aptamers Cleaved at Functionally Essential Sites Preserve Biorecognition Capability.

Split aptamers (SPAs) are a pair of oligonucleotide fragments generated by cleaving a long parent aptamer. SPAs have many compelling advantages over the parent aptamer such as sandwich target binding, optimized concise structure, and low cost. However, only a limited number of SPAs have been developed so far because the traditional theory restricts the splitting to the functionally dispensable site that many parent aptamers do not possess. In this work, the traditional mechanism and hypothesis that SPAs can also be generated by splitting the parent aptamer at the functionally essential site while still preserving the biorecognition capability are challenged. To prove the hypothesis, three SPAs with Broken initial small-molecule binding Pockets (BPSPAs) are discovered and their binding capabilities are validated both in the wet lab and in silico. An allosteric binding mechanism of BPSPAs, in which a new binding pocket is formed upon the target binding, is revealed by all-atom microsecond-scale molecular dynamics simulations. Our work highlights the important role of MD simulations in predicting the ligand binding potency with functional nucleic acids at the molecular level. The findings will greatly promote discovery of new SPAs and their applications in molecular sensing in many fields.

An Optical Biosensor-Based Quantification of the Microcystin Synthetase A Gene: Early Warning of Toxic Cyanobacterial Blooming.

The monitoring and control of toxic cyanobacterial strains, which can produce microcystins, is critical to protect human and ecological health. We herein reported an optical-biosensor-based quantification of the microcystin synthetase A (mcyA) gene so as to discriminate microcystin-producing strains from nonproducing strains. In this assay, the mcyA-specific ssDNA probes were designed in silico with an on-line tool and then synthesized to be covalently immobilized on an optical-fiber surface. Production of fluorescently modified target DNA fragment amplicons was accomplished through the use of Cy5-tagged deoxycytidine triphosphates (dCTPs) in the polymerase chain reaction (PCR) method, which resulted in copies with internally labeled multiple sites per DNA molecule and delivered great sensitivity. With a facile surface-based hybridization process, the PCR amplicons were captured on the optical-fiber surface and were induced by an evanescent-wave field into fluorescence emission. Under the optimum conditions, the detection limit was found to be 10 pM (S/N ratio = 3) and equaled 103 gene copies/mL. The assay was triumphantly demonstrated for PCR amplicons of mcyA detection and showed satisfactory stability and reproducibility. Moreover, the sensing system exhibited excellent selectivity with quantitative spike recoveries from 87 to 102% for M. aeruginosa species in the mixed samples. There results confirmed that the method would serve as an accurate, cost-effective, and rapid technique for in-field testing of toxic Microcystis sp. in water, giving early information for water quality monitoring against microcystin-producing cyanobacteria.

Real-Time Study of Rapid Spread of Antibiotic Resistance Plasmid in Biofilm Using Microfluidics.

Gene transfer in biofilms is known to play an important role in antibiotic resistance dissemination. However, the process remains poorly understood. In this study, microfluidics with time-lapse imaging was used for real-time monitoring of plasmid-mediated horizontal gene transfer (HGT) in biofilms. Pseudomonas putida KT2440 harboring an antibiotic resistance plasmid RP4 was chosen as the donor while Escherichia coli and activated sludge bacteria were used as the recipient cells. Dynamic features of the transfer process, including the transfer rate, cell growth rate and kinetic changes of the transfer frequency, were determined. It was found that the routes for gene transfer strongly depend on the structure and composition of a biofilm. While intraspecies HGT is essential to initiate a transfer event, the secondary retransfer from transconjugants to the same species is more efficient and can cause cascading gene spread in single-strain biofilms. For the activated sludge biofilm, only small and scattered colonies formed and vertical gene transfer appears to be the dominant route after initial intraspecies transfer. Furthermore, more than 46% of genera in the activated sludge were permissive to plasmid RP4, many of which are associated with human pathogens. These phenomena imply early prevention and interruptions to biofilm structure could provide an effect way to inhibit rapid antibiotic resistance gene spread and reduce the likelihood of catastrophic events associated with antibiotic resistance.

Simultaneous di-oxygenation and denitrification in an internal circulation baffled bioreactor.

An internal circulation baffled bioreactor was employed to realize simultaneous di-oxygenation of phthalic acid (PA) and denitrification of nitrate, which require aerobic and anoxic conditions, respectively. Adding a small concentration of succinate as an exogenous electron donor stimulated PA di-oxygenation, which produced readily oxidizable downstream products whose oxidation also enhanced denitrification of nitrate; succinate addition also stimulated denitrification. Depending on the concentration of PA, addition of 0.17 mM succinate increased the PA removal rate by 25 and 42%, while the corresponding nitrate removal rate was increased by 73 and 51%. UV/H2O2 advanced oxidation of PA had the same effects as adding succinate, since succinate is generated by UV/H2O2; this acceleration effect was approximately equivalent to adding 0.17 mM succinate.

The importance of lag time extension in determining bacterial resistance to antibiotics.

It is widely appreciated that widespread antibiotic resistance has significantly reduced the utility of today's antibiotics. Many antibiotics now fail to cure infectious diseases, although they are classified as effective bactericidal agents based on antibiotic susceptibility tests. Here, via kinetic growth assays, we evaluated the effects of 12 commonly used antibiotics on the lag phase of a range of pure environmental isolates and of sludge bacterial communities with a high diversity. We show that an extended lag phase offers bacteria survival advantages and promotes regrowth upon the removal of antibiotics. By utilizing both lag phase extension and IC50, the killing efficiency of an antibiotic on a strain or a community can be easily revealed. Interestingly, for several strains of relevance to endemic nosocomial infections (e.g. Acinetobacter sp. and Pseudomonas aeruginosa) and the diverse sludge communities, tetracycline and quinolone antibiotics are most likely to be resisted via extended lag phase. This discovery is significant from a clinical point view since underestimation of bacteria resistance can lead to the recurrence of diseases.

Label-free fluorescence detection of melamine with a truncated aptamer.

The 2008 Chinese milk scandal caused by the adulteration of melamine encouraged the public to pay attention to melamine detection in milk products and other food stuffs. To allow simple and rapid detection of melamine, we previously isolated an 88 nt melamine aptamer (called Rd29C33) using the structure-switching SELEX. However, this 88 nt oligonucleotide is costly to synthesize, and may also complicate the rational design of biosensors for melamine detection. To overcome this obstacle, we truncated Rd29C33 at several sites, and a 34 nt Rd29C33-T7 melamine aptamer was finally found to show comparable binding affinity and better selectivity to melamine compared to the original 88 nt Rd29C33. Furthermore, a label-free bioassay method for melamine detection was designed by using Rd29C33-T7 and thiazole orange (TO). The addition of melamine to a mixture of Rd29C33-T7 and TO caused the release of TO from Rd29C33-T7, resulting in a decrease of the fluorescence intensity of the solution. A detection limit of 0.12 µM for melamine was achieved using this label-free method. Good recovery ranging from 82.6% to 97.2% for melamine detection in whole milk samples suggested the promise of this bioassay method for application in monitoring melamine in real food stuffs.

Portable Detection of Melamine in Milk Using a Personal Glucose Meter Based on an in Vitro Selected Structure-Switching Aptamer.

Melamine detection in milk and other foods has attracted much attention since the discovery that melamine-adulterated food causes severe kidney damage. Although many methods have been developed to detect melamine, few methods can provide quantitative results using an affordable and portable device that is suitable for home use or field application. To achieve this goal, we herein report the first in vitro selection of a melamine responsive aptamer using a structure-switching method. A personal glucose meter (PGM) based melamine sensor was designed and subsequently tested using the newly isolated aptamer. Conversion of melamine concentration to glucose amount was achieved by including an invertase-conjugated DNA that is complementary to part of the aptamer. Melamine binding triggers the release of the invertase-DNA conjugate, which hydrolyzes sucrose into glucose. The glucose produced is then measured directly using an off-the-shelf PGM. The described sensor shows high selectivity for melamine against several closely related melamine analogues, such as cyanuric acid, ammeline, and ammelide, and has low detection limits of 0.33 µM (or 41.1 ppb) in buffer and 0.53 µM (or 67.5 ppb) in 80% whole milk without any pretreatment. The detection limits meet the threshold of 2.5 ppm for non-infant-formula products and 1 ppm for melamine in infant milk products as defined by the U.S. Food and Drug Administration (FDA). In addition to the PGM sensor demonstrated here, the same aptamer can be converted into other types of sensors with different signal outputs, allowing portable detection of melamine under a variety of conditions.

Distribution and population structure characteristics of microorganisms in urban sewage system.

The sewage system functions as an important public infrastructure. The survived microbial population inside the sewage system plays an important role in the biochemical process during wastewater transportation within the system. The study aims to investigate the microbial communities spatial distribution inside manholes and sewage pipes by using the massive parallel 454 pyrosequencing combined with denaturing gradient gel electrophoresis of V1-V3 regions of 16S rRNA. The microbial structure, distribution characteristic, taxonomic composition analysis, and compositional overlaps of the microbial community both were conducted. The result indicated that the changes in microbial diversity exhibited a consistent trend with average dehydrogenase activity. Proteobacteria, Firmicutes, and Anaerolineae were the dominant bacteria in the sewage system. The microbial community exhibited distinguishing characteristics in comparison with fecal, surface water, and wastewater treatment process. Parachlamydia acanthamoebae, Zymophilus paucivorans, and uncultured Epsilon proteobacterium were mainly found at the upper position of the manhole, while Microbacterium sp. was mainly found at the lower position. Longilinea, Georgenia, and Desulforhabdus were mainly observed in the sewage pipe. The microbial bacteria that survived in the anaerobic environment (i.e., sulfate reduction bacteria groups) exhibited a significant positive relationship with anaerobic crucial environmental factors in the redundancy analysis.

Portable and reusable optofluidics-based biosensing platform for ultrasensitive detection of sulfadimidine in dairy products.

Sulfadimidine (SM2) is a highly toxic and ubiquitous pollutant which requires rapid, sensitive and portable detection method for environmental and food monitoring. Herein, the use for the detection of SM2 of a portable optofluidics-based biosensing platform, which was used for the accurate detection of bisphenol A, atrazine and melamine, is reported for the first time. The proposed compact biosensing system combines the advantages of an evanescent wave immunosensor and microfluidic technology. Through the indirect competitive immunoassay, the detection limit of the proposed optofluidics-based biosensing platform for SM2 reaches 0.05 µg·L(-1) at the concentration of Cy5.5-labeled antibody of 0.1 µg·mL(-1). Linearity is obtained over a dynamic range from 0.17 µg·L(-1) to 10.73 µg·L(-1). The surface of the fiber probe can be regenerated more than 300 times by means of 0.5% sodium dodecyl sulfate solution (pH = 1.9) washes without losing sensitivity. This method, featuring high sensitivity, portability and acceptable reproducibility shows potential in the detection of SM2 in real milk and other dairy products.

Effect of variation of liquid condition on transformation of sulfur and carbon in the sediment of sanitary sewer.

This study aims to estimate the influence of the typical variation in liquid conditions on the biochemical reaction characteristics of sulfur and carbon in the sediment of gravity sanitary sewers. Thus, a series of experimental tests were conducted with real wastewater and sewage sediment to investigate the potential biochemical process of carbon and sulfur in sediment. Results indicated that the sulfur and carbon biochemical process in sediment with neutral pH is significant in the gravity sewage system. The changes in concentration and the ratios of wastewater component substrates are the key factors in chemical oxygen demand and sulfate reduction rates. Furthermore, the condition of dissolved oxygen in liquid significantly affected the biochemical reaction processes of sulfur and carbon. Finally, the frequent alternation of anaerobic and anoxic with low dissolved oxygen effectively inhibits sulfide accumulation and simultaneously reduces carbon loss in the sewage system.

Sulfide elimination by intermittent nitrate dosing in sewer sediments.

The formation of hydrogen sulfide in biofilms and sediments in sewer systems can cause severe pipe corrosions and health hazards, and requires expensive programs for its prevention. The aim of this study is to propose a new control strategy and the optimal condition for sulfide elimination by intermittent nitrate dosing in sewer sediments. The study was carried out based on lab-scale experiments and batch tests using real sewer sediments. The intermittent nitrate dosing mode and the optimal control condition were investigated. The results indicated that the sulfide-intermittent-elimination strategy by nitrate dosing is advantageous for controlling sulfide accumulation in sewer sediment. The oxidation-reduction potential is a sensitive indicator parameter that can reflect the control effect and the minimum N/S (nitrate/sulfide) ratio with slight excess nitrate is necessary for optimal conditions of efficient sulfide control with lower carbon source loss. The optimal control condition is feasible for the sulfide elimination in sewer systems.

[Sensing of Cu²âº Based on Fenton Reaction and Unmodified Gold Nanoparticles].

Heavy metal pollution has received great attentions in recent years. The traditional methods for heavy metal detection rely on the expensive laboratory instruments and need time-consuming preparation steps; therefore, it is urgent to develop quick and highly sensitive new technologies for heavy metal detection. The colorimetric method based on the gold nanoparticles (AuNPs) features with simple operation, high sensitivity and low cost, therefore, enabling it widely concerned and used in the environmental monitoring, food safety and chemical and biological sensing fields. This work developed a simple, rapid and highly sensitive strategy based on the Fenton reaction and unmodified AuNPs for the detection of Cu²âº in water samples. The hydroxyl radical ( · OH) generated by the Fenton reaction between the Cu²âº and sodium ascorbate (SA) oxidized the single stranded DNA (ssDNA) attached on the AuNPs surface into variable sequence fragments. The cleavage of ssDNA induced the aggregation of AuNPs in a certain salt solution, therefore, resulting in the changes on the absorbance of solution. The assay conditions were optimized to be pH value of 7.9, 11 mg · L⁻¹ ssDNA, 8 mmol · L⁻¹ SA and 70 mmol · L⁻¹ NaCl. Results showed that the absorbance ratio values at the wavelengths of 700 and 525 nm (A700/A525) were linearly correlated with the Cu²âº concentrations. The linear detection range was 0.1-10.0 µmol · L⁻¹ with a detection limit of 24 nmol · L⁻¹ (3σ). Spiked recoveries ranged from 87%-120% in three sorts of water, including drinking water, tap water and lake water, which confirmed that the potentials of the proposed assay for Cu²âº detection in reality.

Hapten-grafted graphene as a transducer for homogeneous competitive immunoassay of small molecules.

A hapten-grafted graphene-based biosensor by integrating both the graphene nanosheets and immunoassay sensing technologies was developed for ultrasensitive homogeneous competitive immunoassay of small molecules. The structure of hapten-grafted graphene avoids the activity loss of biomolecules immobilized onto the graphene surface and is beneficial to preserve the binding affinity between small molecule and its specific antibody. The sandwich structure formed between hapten-grafted graphene nanosheets and fluorescence-labeled antibody increases the quenching efficiency of the organic dye, thereby resulting in high signal-to-background ratios and improved sensitivity for Bisphenol A (BPA) detection. On the basis of fluorescence resonance energy transfer (FRET) and homogeneous competitive immunoassay mechanism, high BPA concentrations in the sample reduce the amount of fluorescence-labeled anti-BPA antibody bound to graphene-BPA nanosheets, thus resulting in remarkable fluorescence signals. The linear quantification of BPA over concentration ranges from 0.5 to 50 nM with a detection limit determined as 0.12 nM. These findings show that the proposed method provides a powerful tool for the rapid and sensitive detection of small molecules in biological and environmental samples.

Gradient microfluidics enables rapid bacterial growth inhibition testing.

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask).

Single cell growth rate and morphological dynamics revealing an "opportunistic" persistence.

Bacteria persistence is a well-known phenomenon, where a small fraction of cells in an isogenic population are able to survive high doses of antibiotic treatment. Since the persistence is often associated with single cell behaviour, the ability to study the dynamic response of individual cells to antibiotics is critical. In this work, we developed a gradient microfluidic system that enables long-term tracking of single cell morphology under a wide range of inhibitor concentrations. From time-lapse images, we calculated bacterial growth rates based on the variations in cell mass and in cell number. Using E. coli and Comamonas denitrificans to amoxicillin inhibition as model systems, we found the IC50 determined via both methods are in a good agreement. Importantly, the growth rates together with morphological dynamics of individual cells has led to the discovery of a new form of persistence to amoxicillin. Normal cells that are sensitive to amoxicillin gain persistence or recover from the killing process, if they have had an opportunity to utilise the cytoplasm released from lysed cells close-by. We term this acquired persistence in normal growing cells "opportunistic persistence". This finding might shed new insights into biofilm resistance and the effect of antibiotics on environmental microbes.

An integrated photoluminescence sensing platform using a single-multi-mode fiber coupler-based probe.

We demonstrate an integrated fiber optic photoluminescence sensing platform using a novel single-multi-mode fiber coupler (SMFC)-based probe with high collection efficiency for fluorescence signals. The SMFC, prepared using fused biconical taper technology, not only transmits excitation light, but also collects and transmits fluorescence. The entire system does not use complex optical components and rarely requires optical alignment. The simple structure of the SMFC considerably improves the light transmission efficiency, signal-to-noise ratio, and sensitivity of the system. Theoretical and experimental results show that the proposed probe increases the collection efficiency by more than eight-fold compared with a bifurcated fiber probe. The performance of the proposed probe was experimentally evaluated by measuring the fluorescence spectra of well-known targets and a fresh Tall Fescue leaf.

Microsensor determination of multiple microbial processes in an oxygen-based membrane aerated biofilm.

Microsensor techniques were used to investigate in situ the simultaneous occurrence of sulfate reduction and nitrogen removal in a membrane aerated biofilm reactor. H2S, O2, pH, ORP, NH4(+) and NO3(-) microsensors were fabricated and used to measure the profiles inside the membrane aerated biofilm. Production and consumption rates of H2S, O2, NH4(+) and NO3(-) were estimated using corresponding concentration profiles. The results showed that in anoxic zone, located from the interface between biofilm and bulk liquid to about 550 µm below the interface, both sulfate reduction and denitrification occurred. Highest H2S production rates (around 0.27 mg L(-1)s(-1)) were found about 400 to 450 µm below the interface. Below the anoxic zone, an aerobic zone was present. High H2S oxidation activity occurred at around 550-700 µm below the interface. High oxygen consumption rates (0.34 mg L(-1)s(-1)) occurred at around 750-900 µm below the interface. Nitrification activity occurred at about 500-650 µm below the interface. Along the entire biofilm depth, pH changed slightly (within 0.2 unit). Near the interface of the aerobic and anoxic zone, there was a drastic redox potential change. These results demonstrated simultaneous sulfate reduction and nitrogen removal in a piece of membrane aerated biofilm.

Mesophilic and thermophilic fermentation of activated sludge for volatile fatty acids production: focusing on anaerobic degradation of carbohydrate and protein.

The volatile fatty acids (VFAs) productions, as well as particulate organics decomposition, soluble chemical oxygen demand (SCOD) yield, and the VFAs production pathways from mesophilic and thermophilic anaerobic fermentation in waste activated sludge were investigated. Batch experiments showed that the decomposition rate of volatile suspended solids (VSS), particulate carbohydrate (P-C) and particulate protein (P-P) followed the first-order kinetic model at different temperatures. However, the intermediates, accumulated in the process of protein or carbohydrate digestion had a more significant inhibitory effect on the production of VFAs during the mesophilic anaerobic acidification process. The production of VFAs by thermophilic anaerobic fermentation is 2086.05 mg COD/L, which is about twice the production under mesophilic conditions. Among them, the concentration and proportion of high molecular weight organic acids such as isobutyric acid (320.29 mgCOD/L) and isovaleric acid (745.75 mgCOD/L) are relatively high. Then 13C stable isotope labelling experiment demonstrated that, the decomposition of carbohydrates yields 77% acetic acid and 86% butyric acid, while protein breakdown produces 85% propionic acid and 99% valeric acid. This confirms that carbohydrates are more favourable for the formation of even-carbon organic acids, while proteins tend to yield odd-carbon organic acids. Additionally, this helps refine the pathway for valeric acid formation during anaerobic acidogenesis.

Preconcentration and detection of SARS-CoV-2 in wastewater: A comprehensive review.

Severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) affected the health of human beings and the global economy. The patients with SARS-CoV-2 infection had viral RNA or live infectious viruses in feces. Thus, the possible transmission of SARS-CoV-2 through wastewater received great attentions. Moreover, SARS-CoV-2 in wastewater can serve as an early indicator of the infection within communities. We summarized the preconcentration and detection technology of SARS-CoV-2 in wastewater aiming at the complex matrices of wastewater and low virus concentration and compared their performance characteristics. We described the emerging tests that would be possible to realize the rapid detection of SARS-CoV-2 in fields and encourage academics to advance their technologies beyond conception. We concluded with a brief discussion on the outlook for integrating preconcentration and the detection of SARS-CoV-2 with emerging technologies.