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Understanding the origins of lipid membrane bilayer rearrangement in response to external stimuli is an essential component of cell biology and the bottom-up design of liposomes for biomedical applications. The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorus–oxygen bonds of phosphate esters in phosphatidylcholine (PC) lipids. The atomic position of this hydrolysis reaction has huge implications for the stability of PC-containing self-assembled structures, such as the cell wall and lipid-based vesicle drug delivery vectors. While PLC converts PC to diacylglycerol (DAG), the interaction of PC with PLD produces phosphatidic acid (PA). Here we present a combination of small-angle scattering data and all-atom molecular dynamics simulations, providing insights into the effects of atomic-scale reorganization on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated incorporation of DAG or PA. We observed that PC liposomes completely disintegrate in the presence of PLC, as conversion of PC to DAG progresses. At lower concentrations, DAG molecules within fluid PC bilayers form hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules and burrow into the hydrophobic region. This leads initially to membrane thinning followed by a swelling of the lamellar phase with increased DAG. At higher DAG concentrations, localized membrane tension causes a change in lipid phase from lamellar to the hexagonal and micellar cubic phases. Molecular dynamics simulations show that this destabilization is also caused in part by the decreased ability of DAG-containing PC membranes to coordinate sodium ions. Conversely, PLD-treated PC liposomes remain stable up to extremely high conversions to PA. Here, the negatively charged PA headgroup attracts significant amounts of sodium ions from the bulk solution to the membrane surface, leading to a swelling of the coordinated water layer. These findings are a vital step toward a fundamental understanding of the degradation behavior of PC lipid membranes in the presence of these clinically relevant enzymes, and toward the rational design of diagnostic and drug delivery technologies for phospholipase-dysregulation-based diseases., Phospholipase C activity destabilizes PC liposomes, ultimately leading to nonlamellar phases. Phospholipase D activity attracts cations to PC liposome membranes but does not affect their morphology.

Recognition of chemical modifications in canonical nucleobases of nucleic acids is of key importance since such modified variants act as different genetic encoders, introducing variability in the biological information contained in DNA. Herein, we demonstrate the feasibility of direct SERS in combination with chemometrics and microfluidics for the identification and relative quantification of 4 different cytosine modifications in both single- and double-stranded DNA. The minute amount of DNA required per measurement, in the sub-nanogram regime, removes the necessity of pre-amplification or enrichment steps (which are also potential sources of artificial DNA damages). These findings show great potentials for the development of fast, low-cost and high-throughput screening analytical devices capable of detecting known and unknown modifications in nucleic acids (DNA and RNA) opening new windows of activity in several fields such as biology, medicine and forensic sciences.

Recently, plasmonic-based biosensing has experienced an unprecedented level of attention, with a particular focus on the nucleic acid detection, offering efficient solutions to engineer simple, fast, highly sensitive sensing platforms while overcoming important limitations of PCR and microarray techniques. In the broad field of plasmonics, surface-enhanced Raman scattering (SERS) spectroscopy has arisen as a powerful analytical tool for detection and structural characterization of biomolecules. Today applications of SERS to nucleic acid analysis largely rely on indirect strategies, which have been demonstrated very effective for pure sensing purposes but completely dismiss the exquisite structural information provided by the direct acquisition of the biomolecular vibrational fingerprint. Contrarily, direct label-free SERS of nucleic acid shows an outstanding potential in terms of chemical-specific information which, however, remained largely unexpressed mainly because of the inherent poor spectral reproducibility and/or limited sensitivity. To address these limitations, we developed a fast and affordable high-throughput screening direct SERS method for gaining detailed genomic information on nucleic acids (DNA and RNA) and for the characterization and quantitative recognition of DNA interactions with exogenous agents. The simple strategy relies on the electrostatic adhesion of DNA/RNA onto positively-charged silver colloids that promotes the nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at picogram level (i.e. the analysis can be performed without the necessity of amplification steps thus providing realistic direct information of the nucleic acid in its native state). We anticipate this method to gain a vast impact and set of applications in different fields, including medical diagnostics, genomic screening, drug discovery, forensic science and even molecular electronics.

The standard protocols for DNA analysis largely involve polymerase chain reaction (PCR). However, DNA structures bound to chemical agents cannot be PCR-amplified, and therefore any sequence changes induced by external agents may be neglected. Thus, the development of analytical tools capable of characterizing the biochemical mechanisms associated with chemically induced DNA damage is demanded for the rational design of more effective chemotherapy drugs, understanding the mode of actions of carcinogenic chemicals, and monitoring the genotypic toxicology of environments. Here we report a fast, high-throughput, low-cost method for the characterization and quantitative recognition of DNA interactions with exogenous agents based on surface-enhanced Raman scattering spectroscopy. As representative chemical agents, we selected a chemotherapeutic drug (cisplatin) which forms covalent adducts with DNA, a duplex intercalating agent (methylene blue), and a cytotoxic metal ion (Hg(II)) which inserts into T:T mismatches. Rich structural information on the DNA complex architecture and properties is provided by the unique changes of their SERS spectra, which also offer an efficient analytical tool to quantify the extent of such binding.

Rapid advances in nanofabrication techniques of reproducibly manufacturing plasmonic substrates with well-defined nanometric scale features and very large electromagnetic enhancements paved the way for the final translation of the analytical potential of surface-enhanced Raman scattering (SERS) to real applications. A vast number of different SERS substrates have been reported in the literature. Among others, discrete particles consisting of an inorganic micrometric or sub-micrometric core homogeneously coated with plasmonic nanoparticles stand out for their ease of fabrication, excellent SERS enhancing properties, long-term optical stability and remarkable experimental flexibility (manipulation, storage etc). In this article, we performed a systematic experimental study of the correlation between the size of quasi-spherical gold and silver nanoparticle and the final optical property of their corresponding assembles onto micrometric polystyrene (PS) beads. The size and composition of nanoparticles play a key role in tuning the SERS efficiency of the hybrid material at a given excitation wavelength. This study provides valuable information for the selection and optimization of the appropriate PS@NPs substrates for the desired applications.