A microfluidic Braille valve platform for on-demand production, combinatorial screening and sorting of chemically distinct droplets.

Droplet microfluidics is a powerful tool for a variety of biological applications including single-cell genetics, antibody discovery and directed evolution. All these applications make use of genetic libraries, illustrating the difficulty of generating chemically distinct droplets for screening applications. This protocol describes our Braille Display valving platform for on-demand generation of droplets with different chemical contents (16 different reagents and combinations thereof), as well as sorting droplets with different chemical properties, on the basis of fluorescence signals. The Braille Display platform is compact, versatile and cost efficient (only ~US$1,000 on top of a standard droplet microfluidics setup). The procedure includes manufacturing of microfluidic chips, assembly of custom hardware, co-encapsulation of cells and drugs into droplets, fluorescence detection of readout signals and data analysis using shared, freely available LabVIEW and Python packages. As a first application, we demonstrate the complete workflow for screening cancer cell drug sensitivities toward 74 conditions. Furthermore, we describe here an assay enabling the normalization of the observed drug sensitivity to the number of cancer cells per droplet, which additionally increases the robustness of the system. As a second application, we also demonstrate the sorting of droplets according to enzymatic activity. The drug screening application can be completed within 2 d; droplet sorting takes ~1 d; and all preparatory steps for manufacturing molds, chips and setting up the Braille controller can be accomplished within 1 week.

Directed and Modular Protein Immobilization on Gold and Silver Nanoparticles.

Conjugation of proteins to gold nanoparticles (AuNP), silver nanoparticles (AgNP), or other metal nanoparticles (NPs) can often be achieved using passive adsorption. Although such an approach is simple and effective, there is usually no control over the orientation of the protein and denaturation due to close contact with the metal surface. The method described here makes use of adapter proteins which have the ability to adsorb to the NP surface in an oriented and stable way and at the same time enable straightforward attachment to other proteins of interest.

[Partial Nitrification and Denitrification of Low C/N Ratio Sewage Based on Zoning Oxygen and Dissolved Oxygen Control].

Poor nitrogen removal from municipal sewage is mainly due to insufficient carbon source and low C/N ratio. The A2/O pilot plant was established to investigate the accumulation rate of nitrous nitrogen and the removal of nitrogen pollutants by adjusting the ratio of anoxic/aerobic zoning and dissolved oxygen levels in the aerobic zone. The results showed that when DO is 2.0-2.5 mg·L-1, changing the ratio of anoxic to aerobic zoning had little effect on the reaction system, and it was difficult to realize partial nitrification. When DO is 0.5-0.8 mg·L-1, VAnoxic:VAerobic=1:1, this is the best working condition of the system. The accumulation rate of nitrous nitrogen at the end of aerobic zone is stable at more than 62%, and the total nitrogen of effluent is reduced to 9.0 mg·L-1, which can achieve the goal of deep denitrification. Analyzing the apparent activity of nitrifying bacteria, it was found that the SAOR and SNOR (according to N/VSS calculation) were 0.14 g·(g·d)-1 and 0.04 g·(g·d)-1, respectively, under the optimum conditions. The difference between them was more obvious than that in other stages of the experiment, that is, the higher inhibition of NOB activity was the direct reason for the increase of nitrite accumulation rate. Illumina MiSeq sequencing showed that the number of NOB in this stage was significantly lower than that in other stages. Intermittent OUR method was used to analyze the composition of carbon sources at the inlet and outlet of the anoxic zone. The results showed that short-cut nitrification and denitrification could save 27.3% of the carbon sources under the optimal operating conditions. The biodegradable COD consumption in the anoxic zone was 63.6%, which was much higher than that in other stages.

Modular assembly of proteins on nanoparticles.

Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum, for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold-sulfur bonds (Au-S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes, which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond.

Rapid on-site detection of Acidovorax avenae subsp. citrulli by gold-labeled DNA strip sensor.

Acidovorax avenae subsp. citrulli (AAC) is one of the most harmful diseases in cucurbit production. A rapid and sensitive DNA strip sensor was constructed based on gold nanoparticle-labeled oligonucleotide probes for the detection of AAC. Both the qualitative and semi-quantitative detections of target DNA were successfully achieved using the developed DNA strip sensor. The qualitative limit of detection (LOD) of the strip sensor was determined as 4 nM. The LOD for the semi-quantitative detection was calculated to be 0.48 nM in the range of 0-10 nM. The genomic DNA was detected directly using the DNA strip sensor without any further treatment. This DNA strip sensor is a potentially useful tool for rapid on-site DNA screening.

Fluorescent strip sensor for rapid determination of toxins.

Here, we report a simple fluorescent strip sensor based on aptamer-quantum dots technology that can meet toxin monitoring demands using ochratoxin A (OTA) as a model toxin. The limit of the detection (LOD) for the fluorescent strip was 1.9 ng mL(-1), while the time needed for the detection is only 10 min; this conforms to the standards of World Health Organization (WHO) or better. Overall functional parameters are also better than the analogous characteristics of gold nanoparticle strips. High selectivity was maintained as well, making them suitable for the samples with complex solution composition.

Wash-free magnetic oligonucleotide probes-based NMR sensor for detecting the Hg ion.

An easily applied and sensitive sensor for the detection of heavy metal ion residues based entirely on magnetic nanoparticle and oligonucleotide was developed. The tool is established on the relaxation of magnetic nanoparticles with different dispersion states. The target analyte, Hg ions, induce the aggregation of the MNP oligonucleotide probes. Accordingly, the light produced by the magnetic relaxation image and the transverse relaxation time (T(2)) all change due to the effect of the aggregation. The limit of qualitative detection of the sensor is 0.15 ppt. The recoveries from test samples range between 97.1-101.8%. Using the nuclear resonance instrument, the method is a high throughput and sensitive sensor.

An aptamer-based chromatographic strip assay for sensitive toxin semi-quantitative detection.

An aptamer-based chromatographic strip assay method for rapid toxin detection was developed. The aptamer-based strip assay was based on the competition for the aptamer between ochratoxin A and DNA probes. The sensing results indicated that the sensitivity of the aptamer-based strip was better than that of conventional antibody-based strips. The visual limit of detection of the strip for qualitative detection was 1 ng/mL while the LOD for semi-quantitative detection could down to 0.18 ng/mL by using scanning reader. The recoveries of test samples were from 96% to 110%. All detections could be achieved in less than 10 min, indicating that the aptamer-based strip could be a potential useful tool for rapid on-site detections.