Carlos D. M. Filipe

Characterization of the denitrifying fraction of phosphate accumulating organisms in biological phosphate removal

Results of experimental investigations are presented that strongly support the hypothesis that PAO from activated sludge systems consist of two groups: a) denitrifying PAO (DNPAO) capable of using oxygen and nitrate and b) non-denitrifying PAO (non-DNPAO) only able to use oxygen. Batch experiments were performed in which activated sludge obtained from a pilot scale BiodeniphoTM was submitted to a sequence of anaerobic/anoxic/aerobic, anaerobic/aerobic or anaerobic/anoxic conditions while monitoring the course of NOx-N, NH4-N, PO4-P, PHB and PHV. Several methods for the determination of the two fractions of PAO are performed and compared. This study extends on previously reported results (Kern-Jespersen and Henze, 1993) in that the pH was controlled to around pH 7 to assure that phosphate precipitation was minimal, and in the measurement of PHB and PHV. With regards to the latter, the paper also examines the influence of the size of the internal PHA pool on the anoxic phosphorus uptake rate. Simulations implementing existing models for the growth of non-DNPAO and DNPAO are used to confirm the experimental results and to gain a better understanding of some of the observations.

Microgel-based inks for paper-supported biosensing applications.

As a first step for the development of biosensing inks for inexpensive paper-based biodetection, we prepared paper strips printed with carboxylic poly( N-isopropylacrylamide) microgels that were modified either with an antibody or with a DNA aptamer. We found that the antibody and the DNA aptamer retained their recognition capabilities when coupled to microgel. The printed microgel remains stationary during chromatographic elution while the microgel-supported molecular recognition elements are accessible to their intended targets present in the elution solution. Our work indicates that microgels, large enough to isolate the biosensors from the paper surface, are sufficiently hydrophilic to be wetted during chromatographic elution, exposing the gel-supported affinity probes to their targets.

Detection of DNA using bioactive paper strips.

Paper strips containing DNA-conjugated microgels (MG) are used to achieve sensitive DNA detection in three steps: target DNA promoted ligation of a DNA primer to the MG-bound DNA, rolling circle amplification (RCA) between the primer and a circle DNA, and hybridization of the RCA products and a fluorescent DNA probe.

Tools for water quality monitoring and mapping using paper-based sensors and cell phones

In this paper we describe a combination of paper-based sensors and a novel smart-phone application for on-site quantification of colorimetric readouts as an ultra-low cost solution to monitoring water quality. The system utilizes a paper-based analytical device (μPAD) that produces a colorimetric signal that is dependent on the concentration of a specific target; a cell phone equipped with a camera for capturing images of two μPADs - one tested with a water sample and the other tested with clean water that is used as a control; and an on-site image processing app that uses a novel algorithm for quantifying color intensity and relating this to contaminant concentration. The cell phone app utilizes a pixel counting algorithm that performs with less bias and user subjectivity than the typically used lab-based software, ImageJ. The use of a test and control strip reduces bias from variations in ambient lighting, making it possible to acquire and process images on-site. The cell phone is also able to GPS tag the location of the test, and transmit results to a newly developed website, WaterMap.ca, that displays the quantitative results from the water samples on a map. We demonstrate our approach using a previously developed μPAD that detects the presence of organophosphate pesticides based on the inhibition of immobilized acetylcholinesterase by these contaminants. The objective of this paper is to highlight the importance and potential of developing and integrated monitoring system consisting of μPADs, cell-phones and a centralized web portal for low-cost monitoring environmental contaminants at a large-scale.

Target-induced and Equipment-free DNA Amplification with a Simple Paper Device.

We report on a paper device capable of carrying out target-induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA-enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps.

Pullulan Encapsulation of Labile Biomolecules to Give Stable Bioassay Tablets

A simple and inexpensive method is reported for the long-term stabilization of enzymes and other unstable reagents in premeasured quantities in water-soluble tablets (cast, not compressed) made with pullulan, a nonionic polysaccharide that forms an oxygen impermeable solid upon drying. The pullulan tablets dissolve in aqueous solutions in seconds, thereby facilitating the easy execution of bioassays at remote sites with no need for special reagent handling and liquid pipetting. This approach is modular in nature, thus allowing the creation of individual tablets for enzymes and their substrates. Proof-of-principle demonstrations include a Taq polymerase tablet for DNA amplification through PCR and a pesticide assay kit consisting of separate tablets for acetylcholinesterase and its chromogenic substrate, indoxyl acetate, both of which are highly unstable. The encapsulated reagents remain stable at room temperature for months, thus enabling the room-temperature shipping and storage of bioassay components.

Ecological Engineering of Bioreactors for Wastewater Treatment

Biological nutrient removal (BNR) systems remove carbon, nitrogen, and phosphorus from wastewaters through biodegradation of organic compounds, oxidation of ammonia-N to nitrate-N, reduction of nitrate-N to N2 gas, and sequestration of phosphorus as polyphosphate. The microbial community in such systems is complex because it must contain heterotrophic bacteria capable of aerobic respiration, anaerobic respiration, and fermentation; specialized heterotrophic bacteria that can store polyphosphate; and autotrophic nitrifying bacteria that can withstand long periods without oxygen. Although the basic design principles for BNR systems are reasonably well established, it is becoming apparent that a greater understanding of the microbial interactions involved is required to increase system reliability. For example, although the environment established for the selection of phosphorus accumulating organisms was thought to give them a strong competitive advantage over other heterotrophic bacteria, this has turned out not to be the case. Rather, glycogen accumulating organisms can compete quite effectively in the same environment. Furthermore, BNR systems have suffered from problems with sludge settleability, even though many of the system characteristics are considered to be conducive to the suppression of filamentous bacteria. Finally, the use of molecular techniques has revealed that the autotrophic nitrifying bacteria are different from those that had been considered to be present. This paper reviews the microbial ecology of BNR systems, establishes how molecular biology techniques are changing our understanding of that ecology, and suggests ways in which engineering control can be exerted over community structure, thereby increasing the reliability of BNR systems.

Printed paper sensors for serum lactate dehydrogenase using pullulan-based inks to immobilize reagents.

In this study, a paper-based point-of-care (POC) colorimetric biosensor was developed for the detection of lactate dehydrogenase in serum using a nonporous, oxygen impermeable reversibly gelling polysaccharide material based on pullulan. The pullulan could be printed onto paper surfaces along with all required assay reagents, providing a means for high-stability immobilization of all reagents on paper. Serum containing lactate dehydrogenase (LDH) was directly spotted on to the pullulan-coated bioactive paper and provided quantitative colorimetric data that was comparable to that obtained with a conventional plate-reader method. The paper strip was found to be highly stable and could be stored at 4 °C for at least 10 weeks with no loss in performance, as compared to a complete loss in performance within 1 day when the reagents were printed without the stabilizing polysaccharide. The ease of fabrication coupled with the high stability of the printed reagents provides a facile platform for easily manufactured POC sensors.

In Vivo Formation of Protein Based Aqueous Microcompartments

In this paper, we report the formation of protein based liquid droplets resulting in the formation of in vivo microcompartments in E. coli or tobacco cells. These microcompartments were generated by expressing elastin-like polypeptides (ELP), which have the ability to undergo a reversible phase transition, resulting in the formation of an aqueous two-phase system (ATPS) in the cytoplasm of the cell. We prove that these microcompartments are liquid by expressing a fusion protein consisting of ELP and GFP and by performing fluorescence recovery after photobleaching (FRAP) experiments at different stages of cell cultivation. In the initial phases of cell growth, the fusion protein concentration is low and is not sufficient to drive the formation of a second aqueous phase. As the intracellular fusion protein concentration increases with longer cultivation time, droplets start forming, and as protein expression continues, the droplets coalesce at the poles of the E. coli cells. FRAP experiments with cells at different growth stages reveals that the protein in these ELP based droplets is comprised of aqueous and not solid aggregates, as seen in typical inclusion bodies. Staining of the ribosomes and coimaging of the ELP-GFP fusion protein showed that these compartments exclude the protein making machinery of the cell, acting as depots for newly formed protein. It is also shown, in vitro, that ELP based droplets result in the exclusion of proteases, protecting proteins from degradation. Additional studies are still required to test this possibility in vivo. To the best of our knowledge, this is the first report characterizing the formation of an engineered extra aqueous phase in a living organism.

Effects of temperature and relative humidity on the stability of paper-immobilized antibodies.

The stability of a paper-immobilized antibody was investigated over a range of temperatures (40-140 °C) and relative humidities (RH, 30-90%) using both unmodified filter paper and the same paper impregnated with polyamide-epichlorohydrin (PAE) as supports. Antibody stability decreased with increasing temperature, as expected, but also decreased with increasing RH. At 40 °C, the half-life was more than 10 days, with little dependence on RH. However, at 80 °C, the half-life varied from ~3 days at low RH to less than half an hour at 90% RH, demonstrating that hydration of the antibody promotes unfolding. Antibody stability was not influenced by the PAE paper surface treatment. This work shows that antibodies are good candidates for development of bioactive paper as they have sufficient stability at high temperature to withstand printing and other roll-to-roll processing steps, and sufficient low temperature stability to allow long-term storage of bioactive paper materials.