Engineered Membranes for Residual Cell Trapping on Microfluidic Blood Plasma Separation Systems: A Comparison between Porous and Nanofibrous Membranes.

Blood-based clinical diagnostics require challenging limit-of-detection for low abundance, circulating molecules in plasma. Micro-scale blood plasma separation (BPS) has achieved remarkable results in terms of plasma yield or purity, but rarely achieving both at the same time. Here, we proposed the first use of electrospun polylactic-acid (PLA) membranes as filters to remove residual cell population from continuous hydrodynamic-BPS devices. The membranes hydrophilicity was improved by adopting a wet chemistry approach via surface aminolysis as demonstrated through Fourier Transform Infrared Spectroscopy and Water Contact Angle analysis. The usability of PLA-membranes was assessed through degradation measurements at extreme pH values. Plasma purity and hemolysis were evaluated on plasma samples with residual red blood cell content (1, 3, 5% hematocrit) corresponding to output from existing hydrodynamic BPS systems. Commercially available membranes for BPS were used as benchmark. Results highlighted that the electrospun membranes are suitable for downstream residual cell removal from blood, permitting the collection of up to 2 mL of pure and low-hemolyzed plasma. Fluorometric DNA quantification revealed that electrospun membranes did not significantly affect the concentration of circulating DNA. PLA-based electrospun membranes can be combined with hydrodynamic BPS in order to achieve high volume plasma separation at over 99% plasma purity.

Polylactic is a Sustainable, Low Absorption, Low Autofluorescence Alternative to Other Plastics for Microfluidic and Organ-on-Chip Applications.

Organ-on-chip (OOC) devices are miniaturized devices replacing animal models in drug discovery and toxicology studies. The majority of OOC devices are made from polydimethylsiloxane (PDMS), an elastomer widely used in microfluidic prototyping, but posing a number of challenges to experimentalists, including leaching of uncured oligomers and uncontrolled absorption of small compounds. Here we assess the suitability of polylactic acid (PLA) as a replacement material to PDMS for microfluidic cell culture and OOC applications. We changed the wettability of PLA substrates and demonstrated the functionalization method to be stable over a time period of at least 9 months. We successfully cultured human cells on PLA substrates and devices, without coating. We demonstrated that PLA does not absorb small molecules, is transparent (92% transparency), and has low autofluorescence. As a proof of concept of its manufacturability, biocompatibility, and transparency, we performed a cell tracking experiment of prostate cancer cells in a PLA device for advanced cell culture.

Tryptophan and Non-Tryptophan Fluorescence of the Eye Lens Proteins Provides Diagnostics of Cataract at the Molecular Level.

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of "free" Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their "free" counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.

Peptides derived from nucleoside beta-amino acids form an unusual 8-helix.

Peptides of varying length (dimers to octamers) were prepared from nucleoside beta-amino acids and conformational studies, based on NOE observations, show that the beta-peptides form an unusual 8-helix.

Investigation of novel lipid-functionalized PNA monomers as potential HIV-1 non-nucleoside reverse transcriptase and/or integrase inhibitors.

A range of novel N-terminal lipid-functionalized peptide nucleic acid (PNA) monomers have been prepared and their abilities to inhibit HIV-1 reverse transcriptase and integrase have been examined.

Foldamers derived from nucleoside beta-amino acids: PNA Or DNA? Can we have both in one place?

The synthesis of a modified thymidine (nucleoside beta-amino acid) monomer and preliminary investigations into the solid phase peptide synthesis of PNA/DNA chimeras containing a neutral, internucleoside amide linkage are described.

Single diastereomers of polyhydroxylated 9-oxa-1-azabicyclo[4.2.1]nonanes from intramolecular 1,3-dipolar cycloaddition of omega-unsaturated nitrones.

8-Benzyloxymethyl-3,4,5-tribenzoyloxy-9-oxa-1-azabicyclo[4.2.1]nonane has been prepared as the single diastereoisomer 8 from an intramolecular 1,3-dipolar cycloaddition involving 2-(benzyloxy)acetaldehyde and omega-unsaturated hydroxylamine 7 derived from methyl alpha-D-glucopyranoside. The analogous 8-methoxycarbonyl 9-oxa-1-azabicyclo[4.2.1]nonane was afforded in a similar manner, from methyl D-galactopyranoside and methyl glyoxylate, as a 3:1 mixture of diastereoisomers 15 and 16. When conducted in achiral ionic liquid 17 this ratio increased to 8:1, and in chiral ionic liquid 18, compound 15 was formed exclusively.

Synthesis of thymine derivatives of 4-hydroxyvaline.

A synthetic route to thymine derivatives of (2S,3R)- and (2S,3S)-4-hydroxyvaline has been developed starting from commercially available L-aspartic acid.

Alpha-cycloPNA: 1-aminocylopentane-1-carboxylic acid-derived peptide nucleic acid.

All four diastereoisomers of 3-thymine-1-((t)butoxycarbonyl)aminocyclopentane-1-carboxylic acid have been synthesised from (S)-dimethyl malate and thymine monomer 12 has been incorporated into an alpha-cycloPNA oligomer.

Conformationally restricted chiral peptide nucleic acids derived from azetidines.

The initial experiments towards the chemical synthesis of conformationally rigid peptide nucleic acid analogues with azetidine moieties have been described.

Estrone 3-sulfate mimics, inhibitors of estrone sulfatase activity: homology model construction and docking studies.

Steroid sulfatase (STS) is a new target for the endocrine therapy of breast cancer. To ascertain some of the requirements for inhibition of estrone sulfatase activity, a number of novel analogues of estrone 3-O-sulfate possessing sulfate surrogates were synthesized and evaluated as inhibitors of estrone sulfatase (STS) in comparison to a lead inhibitor, estrone-3-O-methylthiophosphonate (E1-3-MTP). Using a selective enzyme digestion, one of the diastereoisomers of this compound, (R(p))-E1-3-MTP, could be prepared and evaluated. From structure-activity studies, we show that chirality at the phosphorus atom, hydrophobicity, basicity, size, and charge all influence the ability of a compound to inhibit estrone sulfatase activity. Of these, hydrophobicity seems to be the most important since simple, active nonsteroidal inhibitors, based on 5,6,7,8-tetrahydronaphth-2-ol (THN), can be prepared, provided that they are lipophilic enough to partition into a nonpolar environment. Also, a negatively charged group is favorable for optimal binding, although it appears that the presence of a potentially cleavable group can compensate for lack of charge in certain cases. A homology model of STS has been constructed from the STS sequence, and molecular docking studies of inhibitors have been performed to broaden the understanding of enzyme/inhibitor interactions. This model clearly shows the positions of the key amino acid residues His136, His290, Lys134, and Lys368 in the putative catalytic region of the formylglycine at position 75, with residues Asp35, Asp36, Asp342, and Gln343 as ligands in the coordination sphere of the magnesium ion. Docking studies using the substrate and estrone-3-sulfate mimics that are active inhibitors indicate they are positioned in the area of proposed catalysis, confirming the predictive power of the model.