Systematic process evaluation of the conjugation of proteins to gold nanoparticles.

The present work addresses some fundamental aspects in the preparation of protein-conjugated gold nanoparticles, in order to ensure an appropriate final product. Ten broadly available and/or easy to implement analytical tools were benchmarked and compared in their capacity to provide reliable and conclusive information for each step of the procedure. These techniques included transmission electron microscopy, UV/VIS spectroscopy, dynamic light scattering, zeta-potential, Fourier-transformed infrared spectroscopy, colloidal stability titration, end-point colloidal stability analysis, cyclic voltammetry, agarose gel electrophoresis and size-exclusion chromatography (SEC). Four different proteins widely used as adaptors or blocking agents were tested, together with 13 nm gold nanoparticles containing different surface chemistries. Among all tested techniques, some of the least popular among nanomaterial scientists probed to be the most informative, including colloidal stability, gel electrophoresis and SEC; the latter being also an efficient purification procedure. These three techniques provide low-cost, low time consuming, sensitive and robust ways to assess the success of the nanoparticle bioconjugation steps, especially when used in adequate combinations.

Experimental evidences support the existence of an aggregation/disaggregation step in the Turkevich synthesis of gold nanoparticles.

Turkevich method is one of the most employed techniques to synthesize gold nanoparticles. Despite its simplicity, the mechanism has been an issue of debate over the past years. The general belief is that particles are formed by a classical nucleation and growth theory, originally described by LaMer's model. In the present work, we provide new experimental evidences that supports either LaMer's theory and their detractors. In the former model, it is proposed that particles are generated by a burst nucleation form the initial 'seeds', from which their growth in a second and quasi-independent step. Instead, our experiments (DLS, UV/VIS and TEM measurements) support the idea that nanoparticles 'seeds' tend to form large intermediate clusters at the beginning of the synthesis, that afterwards disassemble to yield the final nanoparticles. However, unlike other reports, we propose that during the cluster formation the particles do not coalesce, instead they come close to each other without losing their identity. As the synthesis continues, these clusters are progressively separated into the final particles. As a consequence, a path to synthesize ultra-narrow size nanoparticles is provided, along with their stability against salt aggregation, and shelf-time. We found that these ultra-homogeneous nanoparticles are stable for several months, making them suitable for many applications in the biomedical and analytical research.

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome.

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.

Electrochemical Detection of dsDNA-Specific Antibodies.

Electrochemical biosensors have shown great promise as useful point-of-care tests since they operate on electronic circuits which can be miniaturized and whose readout process can be easily automated. Here, we describe a method for the electrochemical sensing of antibodies directed against double-stranded DNA (α-dsDNA), which are often present at higher-than-normal levels in the sera of autoimmune disease patients. The method can be easily implemented in any lab and requires little investment in equipment, namely a potentiostat. An artificial reference serum sample containing known amounts of spiked-in α-dsDNA antibodies enables reporting results in absolute scale rather than titer. Once electrodes are modified with DNA and the calibration curves are made (i.e., after the biosensor construction phase), individual measurements in test samples can be obtained in as low as 35 min.

Stable tRNA halves can be sorted into extracellular vesicles and delivered to recipient cells in a concentration-dependent manner.

Extracellular vesicles (EVs) are cell-derived nanoparticles that act as natural carriers of nucleic acids between cells. They offer advantages as delivery vehicles for therapeutic nucleic acids such as small RNAs. Loading of desired nucleic acids into EVs can be achieved by electroporation or transfection once purified. An attractive alternative is to transfect cells with the desired small RNAs and harness the cellular machinery for RNA sorting into the EVs. This possibility has been less explored because cells are believed to secrete only specific RNAs. However, we hypothesized that, even in the presence of selective secretion, concentration-driven RNA sorting to EVs would still be feasible. To show this, we transfected cells with glycine 5' tRNA halves, which we have previously shown to better resist RNases. We then measured their levels in EVs and in recipient cells and found that, in contrast to unstable RNAs of random sequence, these tRNA halves were present in vesicles and in recipient cells in amounts proportional to the concentration of RNA used for transfection. Similar efficiencies were obtained with other stable oligonucleotides of random sequence. Our results demonstrate that RNA stability is a key factor needed to maintain high intracellular concentrations, a prerequisite for efficient non-selective RNA sorting to EVs and delivery to cells. Given that glycine 5' tRNA halves belong to the group of stress-induced tRNA fragments frequently detected in extracellular space and biofluids, we propose that upregulation of extracellular tRNA fragments is consequential to cellular stress and might be involved in intercellular signalling.

An electrochemical biosensor for rapid detection of anti-dsDNA antibodies in absolute scale.

Autoimmune diseases are chronic inflammatory pathologies that are characterized by the presence of antibodies against the body's own epitopes in serum (autoantibodies). Systemic lupus erythematosus (SLE) is a common autoimmune pathology characterized by the presence of antinuclear antibodies (ANAs). These include anti-dsDNA (α-dsDNA) antibodies, which are widely used for diagnosis and disease monitoring. Their determination is carried out using traditional techniques such as Indirect Immunofluorescence (IFI) or Enzyme-Linked Immunosorbent Assay (ELISA), which are time consuming, require qualified technicians, and are not compatible with decentralized analysis outside a laboratory facility. Here, we show a sandwich-format electrochemical biosensor-based method for α-dsDNA determination in a rapid and simple manner. The total assay time is only 30 minutes, and the sensor is capable of detecting 16 ng (8 µg mL-1) of α-dsDNA antibodies. Using the current derived from the detection limit of the method as a cut-off, we could discriminate positive from negative serum samples with 90% sensitivity and 100% specificity. Using monoclonal antibodies for calibration curves, our results are presented in absolute scale (i.e., concentration instead of serum titer) which will help us to perform comparisons between methods and carry out further improvements of this protocol. In an effort to render the sensor compatible with automation, we minimized the manipulation steps without compromising the analytical performance, even in complex samples such as serum.

Electrochemical Sandwich Immunosensor for Determination of Exosomes Based on Surface Marker-Mediated Signal Amplification.

Extracellular vesicles (EVs), namely, exosomes and microvesicles, are important mediators of intercellular communication pathways. Since EVs can be detected in a variety of biofluids and contain a specific set of biomarkers which are reminiscent of their parental cells, they show great promise in clinical diagnostics as EV analysis can be performed in minimally invasive liquid biopsies. However, reliable, fast and cost-effective methods for their determination are still needed, especially if decentralized analysis is intended. In this study, we developed an electrochemical biosensor which works with 1.5 µL sample volume and can detect as low as 200 exosomes per microliter, with a linear range spanning almost 4 orders of magnitude. The sensor is specific and readily differentiates exosomes from microvesicles in samples containing 1000-fold excess of the latter. Capability of detecting exosomes in real samples (diluted serum) was shown. This was achieved by immobilizing rabbit antihuman CD9 antibodies on gold substrates and using monoclonal antibodies against CD9 for detection of captured exosomes. Signal amplification is presumably obtained from the fact that multiple detector antibodies bind to the surface of each captured vesicle. Detection is performed based on electrochemical reduction of 3,3',5,5'-tetramethyl benzidine (TMB) after addition of horseradish peroxidase (HRP)-conjugated anti-IgG antibodies. This amperometric biosensor can be easily incorporated into future miniaturized and semiautomatic devices for EV determination.