Impact of Processing Temperature and Composition on Foaming of Biodegradable Poly(hydroxyalkanoate) Blends

Poly­(3-hydroxybutyrate-<i>co</i>-3-hydroxyhexanoate) (PHBHHx) was melt-foamed using a single screw extrusion process and found to be susceptible to cell coalescence at the higher processing temperatures used for achieving low bulk density. The addition of poly­(3-hydroxybutyrate-<i>co</i>-3-hydroxyvalerate) (PHBV) allowed for the generation of a range of crystallization temperatures much broader than can be achieved through small molecular crystal nucleation agents. This broader range can induce solidification to occur earlier during cooling and so can be used to minimize cell coalescence. Several PHBHHx/PHBV blend compositions were produced and characterized; initial understanding of the crystallinity and impact of blending miscible crystallizable polymers was gained through thermal analysis. PHBHHx and PHBV appeared to be fully miscible across all compositions used in this study. The impacts of blend composition and extruder temperature profile on foam properties were investigated. It was found that just 2% PHBV consistently led to 30% improvement in cell density over pure PHBHHx when the highest extruder zone temperature was 170 °C. As a result, PHBHHx/PHBV was able to achieve greater than twofold expansion with reduced cell coalescence, which has not been previously achievable by PHBV with the same blowing agent

A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

<div><p>Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. <i>Rhodobacter sphaeroides</i> cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to <i>R. sphaeroides</i>. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.</p></div

Effects of cell loading rate, visualized before and after PCR.

<p>Microscope images of 100 μm alginate microspheres containing: (A) no cells, (B) one cell per microsphere, and (C) multiple cells per microsphere. After PCR or whole genome amplification (2-step method), the DNA is stained by GelGreen and visualized by fluorescence microscopy. Images show: (D) no amplification, (E) amplification that is characteristic for one cell per microsphere, and (F) amplification of many cells, showing multiple foci of amplification and “comet tails” from cells that are trapped in the border region of the microsphere.</p

Strategy for WGA in two steps.

<p>This diagram outlines the whole genome amplification strategy in 2 steps, plus an additional step for adding the full length sequencing adapters. In Step 1, primers with 2 different tag sequences are added in a 50:50 mix. The primers anneal to the template, and a thermophilic strand-displacing enzyme (Vent exo- polymerase) is used to generate a population of fragments. For Step 2, the tag sequences are used as primers to further amplify the population of fragments. Half of the fragments are expected to have two different primer sequences on each end. After isolation of a microsphere containing fluorescent products, the sample is processed in a third reaction for addition of the full length sequencing adapters, or tails.</p

Results from Illumina MiSeq paired-end sequencing run, 2 × 150 bp.

<p>Results from Illumina MiSeq paired-end sequencing run, 2 × 150 bp.</p

Sequence coverage of the <i>Rhodobacter sphaeroides</i> genome after whole genome amplification.

<p>Sequence coverage of the <i>Rhodobacter sphaeroides</i> genome after whole genome amplification.</p

Illustration of the process workflow.

<p>1.) Cells are diluted in alginate polymer to a concentration of approximately 10⁵ cells per microliter, resulting in a 10% occupancy rate in 100 μm microspheres. 2.) Cells are lysed using heat, and the bulk microsphere solids are mixed with reagents for a 2-step whole genome amplification reaction. 3.) After amplification, microspheres are diluted to extinction in a 384 well plate, and scanned for presence of single microspheres that fluoresce with PicoGreen DNA stain. 4.) An isolated microsphere is transferred to a fresh tube and the DNA products are recovered by dissolving the alginate matrix. These amplified products are submitted to further rounds of amplification to add sequencing adapters. 5.) The products are prepared for high throughput sequencing using the Illumina MiSeq platform. Intermediate steps include fluorescence microscopy and quantitative PCR for quality control.</p

Primers for 2-step PCR whole genome amplification, tailing, and qPCR.

<p>Primers for 2-step PCR whole genome amplification, tailing, and qPCR.</p

Langmuir–Blodgett Deposition of Graphene OxideIdentifying Marangoni Flow as a Process that Fundamentally Limits Deposition Control

Langmuir–Blodgett deposition is a popular route to produce thin films of graphene oxide for applications such as transparent conductors and biosensors. Unfortunately, film morphologies vary from sample to sample, often with undesirable characteristics such as folded sheets and patchwise depositions. In conventional Langmuir–Blodgett deposition of graphene oxide, alcohol (typically methanol) is used to spread the graphene oxide sheets onto an air–water interface before deposition onto substrates. Here we show that methanol gives rise to Marangoni flow, which fundamentally limits control over Langmuir–Blodgett depositions of graphene oxide. We directly identified the presence of Marangoni flow by using photography, and we evaluated depositions with atomic force microscopy and scanning electron microscopy. The disruptive effect of Marangoni flow was demonstrated by comparing conventional Langmuir–Blodgett depositions to depositions where Marangoni flow was suppressed by a surfactant. Because methanol is the standard spreading solvent for conventional Langmuir–Blodgett deposition of graphene oxide, Marangoni flow is a general problem and may partly explain the wide variety of undesirable film morphologies reported in the literature

Langmuir–Blodgett Deposition of Graphene OxideIdentifying Marangoni Flow as a Process that Fundamentally Limits Deposition Control

Langmuir–Blodgett deposition is a popular route to produce thin films of graphene oxide for applications such as transparent conductors and biosensors. Unfortunately, film morphologies vary from sample to sample, often with undesirable characteristics such as folded sheets and patchwise depositions. In conventional Langmuir–Blodgett deposition of graphene oxide, alcohol (typically methanol) is used to spread the graphene oxide sheets onto an air–water interface before deposition onto substrates. Here we show that methanol gives rise to Marangoni flow, which fundamentally limits control over Langmuir–Blodgett depositions of graphene oxide. We directly identified the presence of Marangoni flow by using photography, and we evaluated depositions with atomic force microscopy and scanning electron microscopy. The disruptive effect of Marangoni flow was demonstrated by comparing conventional Langmuir–Blodgett depositions to depositions where Marangoni flow was suppressed by a surfactant. Because methanol is the standard spreading solvent for conventional Langmuir–Blodgett deposition of graphene oxide, Marangoni flow is a general problem and may partly explain the wide variety of undesirable film morphologies reported in the literature