Measurement of Giardia lamblia adhesion force using an integrated microfluidic assay.

The mechanisms how Giardias attach to the intestinal epithelium remain unclear. None of the methods currently being used to measure the attachment force could provide a continuous nutrition supply and a micro-aerobic atmosphere to the Giardia. Besides, they are all labor-intensive. In the present research, a microfluidic method based on electric circuit analogy was developed. The input fluid flowed through the inlet channel with different lengths and was distributed in four assay chambers. Shear force gradients were generated in chambers, too. This allowed an easy control of fluids and the shear forces. Most importantly, the shear stress large enough to detach Giardia could be generated in laminar flow regime. Moreover, analysis could be accomplished in one single test. By applying inlet flow rates of 30, 60, and 120 µL ml-1, shear force gradients ranging from 19.47 to 60.50 Pa were generated. The adhesion forces of trophozoites were analyzed and the EC50 of the force that caused 50% trophozoites detachment was calculated as 36.60 Pa. This paper presents a novel method for measurement of Giardia adhesion force. Graphical Abstract Measurement of Giardia adhesion force. Various of flow rates were applied to generate different shear forces and Giardia trophozoites remaining attached were counted (a-c). The percentages of attachment vs shear stress were plotted and the EC50 of adhesion force was calculated (d).

Giardia duodenalis GlSir2.2, homolog of SIRT1, is a nuclear-located and NAD(+)-dependent deacethylase.

Sir2 family proteins are highly conserved and catalyze Nicotinamide Adenine Dinucleotide (NAD(+))-dependent protein deacetylation reaction that regulates multiple cellular processes. Little is known about Sir2 family proteins in Giardia. In this research, Sir2 homologs of Giardia were Phylogenetically analyzed. GL50803_10707 (GlSIR2.2) showed strong homology to SIRT1 and was the only parasite SIRT1 homolog being reported to date. Recombinant GlSIR2.2 (rGlSIR2.2) was expressed and purified. The renaturied recombinant protein showed a typical NAD-dependent protein deacetylase activity that could be inhibited by nicotinamide, with IC50 of 4.47 mM rGlSIR2.2 displayed deacetylase activity under varied NAD(+), with Km, kcat and kcat/Km values of 31.71 µM, 1.4 × 10(-3) s(-1), and 4.42 × 10(-5) µM(-1) s(-1). Similarly, the steady-state kinetic parameters with varied ZMAL, yielded Km, kcat and kcat/Km values of 96.89 µM, 4.7 × 10(-3) s(-1), and 4.85 × 10(-5) µM(-1) s(-1). Anti-rGlSIR2.2 serum was used to probe subcellular localization of GlSIR2.2 and strong staining was found predominantly in the nucleus. So we demonstrated that GlSIR2.2 was a SIRT1-like, nuclear-located, NAD(+)-dependent deacetylase. This is the first report of deacetylase activity of Sir2 family protein in Giardia.

An on-chip pollutant toxicity determination based on marine microalgal swimming inhibition.

We report the use of microalgal swimming behavior as a sensor signal integrated into microfluidics for a rapid and high-throughput determination of pollutant toxicity. There are two types of chip. A poly(dimethylsiloxane) (PDMS) 12-well chip, used for optimization of experimental conditions (i.e. light level, temperature, initial cellular density and exposure time), can perform twelve parallel tests simultaneously. In a concentration gradient generator (CGG) chip, a CGG connected with diffusible chambers enables a large number of dose-response bioassays to be performed in a simple way. Microalgal swimming was set as a microfluidic bioassay signal and was evaluated as swimming manner, motile percentage (%MOT), curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). Under optimized physical conditions, the toxicities of Cu, Pb, phenol and nonylphenol (NP) towards four mobile marine microalgae, Platymonas subcordiformis, Platymonas helgolandica var. tsingtaoensis, Isochrysis galbana and Isochrysis zhanjiangensis sp. nov, were investigated. In all cases, a toxic response (i.e. a dose-related inhibition of swimming) was detected, and a time of only 2 h was needed to predict EC50 values. The 2h-EC50s showed that I. galbana was the most tolerant and that P. subcordiformis was one of the most sensitive. Based on the relative motile percentage data, the EC50 values for Cu of I. galbana and P. subcordiformis were 6.04 and 1.67 µM, respectively, while for Pb the EC50 values were 15.30 and 3.87 µM, for phenol the EC50 values were 8.69 and 6.08 mM, and for NP the EC50 values were 29.65 and 14.47 µM, respectively. Taking into account all the swimming inhibition parameters, MOT provided more sensitive EC results. The sensitivity differences between the velocity parameters (VCL, VAP and VSL) were ascribed to differences in swimming manner of the different classes of microalgae.

[Application of microfluidics in aquatic environmental pollution analysis].

Recently, a new type of chip technology, microfluidics, has received global attention for its rapid analysis speed, low reagent consumption, small size and simple operation, etc. Based on a micro-channel network and supported by a Micro-Electro-Mechanic System (MEMS), this technology integrates all the functions of a laboratory into one small piece of chip, which is called "lab on the chip". This paper presented a brief introduction about microfluidics and its representative developments. Future prospects in the aspects of instrument miniaturization, system integration, chip materials, and detection techniques, as well as the implementation of microfluidics in aquatic environmental pollutant analysis were thoroughly discussed. Some problems faced now were put forward. With the rapid progress in the microfluidics, a universal low-cost microchip capable of high speed multi-channel detection and integrated with many kinds of detection methods would be the research focus in the future.

Marine phytoplankton motility sensor integrated into a microfluidic chip for high-throughput pollutant toxicity assessment.

A microfluidic chip was designed to assess the toxicity of pollutants in a high-throughput way by using marine phytoplankton motility as a sensor signal. In this chip, multiple gradient generators (CGGs) with diffusible chambers enable large scale of dose-response bioassays to be performed in a simple way. Two mobile marine phytoplankton cells were confined on-chip and stimulated by 8 concentrations (generated by CGG) of Hg, Pb, Cu and phenol singly, as well as Cu and phenol jointly. CASA system was used to characterize motility by motile percentage (%MOT), curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). In all cases, dose-dependent inhibitions of motility were observed. In the present system, only 2h was needed to predict EC50. Thus, the developed microfluidic chip device was proved to be useful as a rapid/simple and high-throughput test method in marine pollution toxicity assessment.

Paper-based three-dimensional microfluidic device for monitoring of heavy metals with a camera cell phone.

A 3D paper-based microfluidic device has been developed for colorimetric determination of selected heavy metals in water samples by stacking layers of wax patterned paper and double-sided adhesive tape. It has the capability of wicking fluids and distributing microliter volumes of samples from single inlet into affrays of detection zones without external pumps, thus a range of metal assays can be simply and inexpensively performed. We demonstrate a prototype of four sample inlets for up to four heavy metal assays each, with detection limits as follows: Cu (II) = 0.29 ppm, Ni(II) = 0.33 ppm, Cd (II) = 0.19 ppm, and Cr (VI) = 0.35 ppm, which provided quantitative data that were in agreement with values gained from atomic absorption. It has the ability to identify these four metals in mixtures and is immune to interferences from either nontoxic metal ions such as Na(I) and K(I) or components found in reservoir or beach water. With the incorporation of a portable detector, a camera mobile phone, this 3D paper-based microfluidic device should be useful as a simple, rapid, and on-site screening approach of heavy metals in aquatic environments.

An integrated microfludic device for culturing and screening of Giardia lamblia.

In vitro culturing of trophozoites was important for research of Giardia lamblia (G. lamblia), especially in discovery of anti-Giardia agents. The current culture methods mainly suffer from lab-intension or the obstacle in standardizing the gas condition. Thus, it could benefit from a more streamlined and integrated approach. Microfluidics offers a way to accomplish this goal. Here we presented an integrated microfluidic device for culturing and screening of G. lamblia. The device consisted of a polydimethylsiloxane (PDMS) microchip with an aerobic culture system. In the microchip, the functionality of integrated concentration gradient generator (CGG) with micro-scale cell culture enables dose-response experiment to be performed in a simple and reagent-saving way. The diffusion-based culture chambers allowed growing G. lamblia at the in vivo like environment. It notable that the highly air permeable material of parallel chambers maintain uniform anaerobic environment in different chambers easily. Using this device, G. lamblia were successfully cultured and stressed on-chip. In all cases, a dose-related inhibitory response was detected. The application of this device for these purposes represents the first step in developing a completely integrated microfluidic platform for high-throughput screening and might be expanded to other assays based on in vitro culture of G. lamblia with further tests.

[Effects of typical organic pollutants on marine dioxide carbon system in simulated experiments].

The effects of typical organic pollutants (ethanol, acetone, ON, OP) on pH and marine carbon dioxide system were investigated in laboratory simulated experiments. The results indicated that the growth of biological carbon fixed system was promoted by ethanol and acetone of low concentration (< 0.5 mmol x L(-1)) (t = 7 d). So pH increased while DIC, HCO3(-) and p(CO2) significantly decreased compared to the controls (p <0.01). The concentrations of ethanol and acetone which resulted in the maximum decrease of carbon dioxide parameters were 0.25 mmol x L(-1) and 0.1 mmol x L(-1), respectively. When the infusion of organic pollutants were higher than the "critical concentration", the carbon dioxide parameters increased and pH decreased compared to the controls with the decrease of carbon fixation strength of alga. As for ON and OP groups, when they were in the range of experimental concentration, decreasing trends of inorganic carbon in sea water were always found. The concentration changes of DIC (DeltaDIC) under the press of the four typical organic pollutants were significantly negatively correlated with the changes of dry weight of alga (p < 0.01 or p < 0.05) and the coefficient parameters were -0.902, -0.945, -0.898 and -0.918, respectively (t = 7 d). The main controlling factor to the variation of carbon dioxided system was the adaption of biological carbon fixed system to different concentration and kind of organic pollutants.

[Effects of excessive inorganic nitrogen on inorganic carbon system in seawater simulated experiments].

The influence of excessive inorganic nitrogen (NO3-, NH4+, NO2-) on the pH, inorganic carbon system and air-sea CO2 flux was studied in laboratory seawater simulation experiments. The result indicates that the excessive inorganic nitrogen significantly has an important influence on the inorganic carbon system in all conditions. At the NO3- addition groups, HCO3- and p (CO2) increase while pH and CO3(2-) decrease when the concentration of NO3- < or = 37.60 micromol x L(-1). But high concentrations > or = 188 micromol x L(-1) is just the reverse. The seawater changes from carbon source to carbon sink of atmospheric CO2 with NO3- addition increased. At the NH4+ addition groups, HCO3- and p (CO2) decrease while pH and CO3(2-) increase when the concentration of NH4+ < or = 25.20 micromol x L(-1). However, when the concentration of NH4+ > or = 126 micromol x L(-1), the concentrations of HCO3- and p (CO2) increase with NH4+ addition and the seawater changes from carbon sink to carbon source of atmospheric CO2 and the intensity of carbon source has a positive relationship with the concentration of NH4+. At the NO2- addition groups, the concentrations of HCO3- and p (CO2) decrease while pH and CO3(2-) increase with time scale and the seawater acts as a strong carbon sink with NO2- addition of 7.90 micromol x L(-1). While at the other NO2- concentration levels, the carbon sink is weak in seawater. The Chl-a concentration of nutrient addition groups compared with control (delta Chl-a) shows a significant negative correlation with delta p (CO2) (r = -0.87, p < 0.000 1, n = 16). The main controlling factor to the variation of inorganic carbon system is the adaptation of phytoplankton to different form and concentration of nitrogen.

[Variation characteristics and controlling factors of heavy metals in the South Yellow Sea surface seawaters].

Based on the 8 cruises data of surface seawater heavy metals and other related environmental factors from 1997 to 2004 of the South Yellow Sea (SYS), distribution patterns, mechanisms controlling the distributions and pollution levels of heavy metals (As, Cd, Cu, Hg, Pb, Zn) were studied with the data of 2003-10, and 8-year-fluctuation trends of heavy metals were also discussed. The average concentrations of heavy metals in surface seawater were 2.33, 0.078, 1.41, 0.003 6, 0.37, 6.21 microg/L respectively. The average concentrations showed a relatively stable trend in 8 years, except Zn's distinct upward tendency. The distribution patterns corresponded to the distance away from the coastline,that was, the content of heavy metals (except Pb) was low in central area while high inshore. Those were responses of human activity in the marginal sea, however, in some local areas, Pb was controlled by atmospheric deposition, Cd was relative to pH and salinity, Hg was related to organic carbon, Cu and Zn were influenced by runoff and drainage, and the resuspending of sediments played important roles on the content and distribution of As. Compared to the Marine Water Quality Standard of China, heavy metals indicated that SYS was a first class sea, and Ecological Risk Index analysis showed that SYS was a low ecological risk sea. In conclusion, seawater quality of heavy metals in SYS surface seawater is relatively good in general.

[Environmental biogeochemical significance of nitrogen in Jiaozhou Bay sediments].

The geochemical characteristics of nitrogen forms in different grain size sediments of Jiaozhou Bay were investigated employing the sequential extraction technique. Results showed that the transferable nitrogen in Jiaozhou Bay sediments can be divided into four forms, ion exchangeable form (IEF-N), weak acid extractable form (WAEF-N), strong alkaline extractable form (SAEF-N) and strong oxidant extractable form (SOEF-N). The percentages of nitrogen forms for transferable nitrogen in different grain size sediments were different. Generally, for all grain size sediments, the SOEF-N was the primary form accounted for 35.38%, 44.38% and 58.69% of transferable nitrogen for fine, medium and coarse sediments. And for medium and coarse grain size sediment, SAEF-N was the dominant inorganic nitrogen accounted for 26.31% and 25.85%, respectively. And for fine-grained sediments, IEF-N was the primary inorganic nitrogen accounted for 27.67% of transferable nitrogen. The correlation analysis showed that generally, nitrogen was positive correlated with fine and medium grain size but negative correlated with coarse sediments. In addition, nitrogen in different grain size sediments had also positive correlations with phytoplankton abundance, chlorophyll-a and NO3- concentrations of overlying water. The results showed that sediments had great influences on element concentrations of overlying water, and on the other side, it also illustrated that sedimentary nitrogen was an excellent indicator of hypereutrophication and had essential environmental significance.

[Phosphorus and its environmental marker function in Jiaozhou Bay sediments].

Phosphorus is a key biogenic element and plays an essential role in global biogeochemical cycles. The geochemical characteristics of phosphorus as well as the controlling factors and the environmental marker function in Jiaozhou Bay sediments were presented. The different forms of sedimentary phosphorus were studied in three sediment cores, which are forms of inorganic phosphorus, exchangeable phosphorus, phosphorus bound to Al-Fe, and calcium, occluded phosphorus as well as total phosphorus and organic phosphorus, respectively. The vertical profiles of various forms of phosphorus in response to 210Pb chronological studies of sediment cores were investigated in order to reveal sedimentary environment changes. The results showed that inorganic phosphorus was the dominant form of total phosphorus and organic phosphorus was only the minor part. The detrital carbonate-bound phosphorus was the largest part of total phosphorus. The concentrations and burial fluxes of phosphorus were controlled by source and environmental factors including pH, salinity, temperature and grain size of sediments etc. Moreover, the analysis of OC/OP ratios suggested that the terrestrial inputs were the dominant source of Jiaozhou Bay sediments. The sedimentation fluxes of phosphorus increased in the past two decades, as a result, the pollution was still serious from the 1980 to 2000. But the state has greatly improved since 2000. The Fe-P, Al-P and Oc-P have close relationships with the degree of pollution and may act as good indicators for environment pollution in Jiaozhou Bay.

[Effects of heavy metal (copper and cadmium) coupled with Ulca pertusa on marine inorganic carbon system in simulated experiments].

Simulated experiments coupled with ocean biota dynamics were performed in laboratory. In these experiments, effects of heavy metal (copper and cadmium) coupled with Ulca pertusa on marine inorganic carbon system and CO2 fluxes were investigated. The results indicated that concentration changes (delta) of components in carbon dioxide system with time scale were correlated with the concentrations and kinds of heavy metal. In copper groups and cadmium groups (0.1 micromol x L(-1) and 1 micromol x L(-1)), DIC HCO3- and PCO2 significantly decreased comparing to the control experiment data( p = 0.01). However, when the heavy metal infusions were higher than the "critical concentration", the above mentioned parameters increased with time scale and their increments followed the uptrend with increasing heavy metal concentrations. The "critical concentration" in copper groups was much lower than that in cadmium groups, which attributed to the tolerance diversity of Ulca pertusa to copper and cadmium. Furthermore, CO2 fluxes under the influences of heavy metal were also regularly changed with time. Sea waters with low infusions of heavy metal represented as sinks to the atmosphere CO2. These sinks would probably convert into CO2 sources after a period of time. Sea waters with comparatively high amount of heavy metal were always to be CO2 sources, and their release fluxes of CO2 augmented along with the increasing infusions of heavy metal.