RESUMEN

The application of analytical chemistry to the exploration of the World Cultural Heritage represents a major challenge in that most protocols and strategies are invasive and require micro-sampling. We report a novel methodology for harvesting material deposited on the surface of ancient documents while avoiding their damage or contamination. The technology here described relates to the capture of metals on these specimens. It is based on the use of plastic films (ethylene vinyl acetate, EVA) impregnated with different metal chelators (sodium 2,3-dimercapto-1-propanesulfonate, DMPS, meso-2,3-dimercaptosuccinic acid, DMSA and ethylene diamino tetra acetic acid, EDTA, as calcium salt), for harvesting from surfaces of different supports potential traces of metals therein deposited. The EVA film technology has been used to explore the pages of a manuscript written by Kepler concerning the movements of the moon and catalogued under the title "Hipparchus" at the Archives of the Russian Academy of Sciences (St. Petersburg branch). The EVA-based chelating diskettes were able to capture very significant amounts of different metals, namely: Au, Ag, Hg, As, Pb, suggesting that Kepler, well known as astronomer, astrologist, mathematician and Lutheran theologian, might have started practicing alchemy, a pseudo-chemical science he had learned from his colleague Tycho Brahe in Prague.

RESUMEN

A miniaturized chemical sensor is here described for the analysis of environmental pollutants (VOC: volatile organic chemicals). It is used for remote detection of formaldehyde (FA) fumes in the atmosphere, and is based on the redox reaction between FA and silver nitrate. The sensor is worn as a bracelet and the data acquired are transferred via a Bluetooth channel to a smartphone. A dedicated software transforms the signal from a grey to a color scale. The signal response has been assessed over low (20 to 120 ppb) as well as higher (1-15 ppm range) levels. The sensor has been applied to monitor potential FA fumes of some artwork in the Summer Palace in Beijing and the modifications induced by FA treatment on a precious Stradivarius violin. The performance of this novel sensor is compared with a commercial apparatus widely adopted, namely the Honeywell MultiRAE Lite wireless portable multi-gas monitor (pumped model).

Asunto(s)

RESUMEN

Ten pages, selected from a total of 127, of the last manuscript of Master i Margarita, written by Bulgakov in the last four years of his life, have been analysed in order to harvest and identify any trace proteome left on the margin by the novelist, in the hope of finding biomarkers of his fatal nephrotic syndrome. To that aim, we prepared a special ethyl-vinyl acetate film as binder of ground AG 501 Bio-Rad mix-bed strong cation/strong anion exchange resins for adsorbing any protein left on the margins of the pages via saliva and/or sweat. After eluting, digesting and interrogating the peptides by LC-MS/MS, we could identify three proteins, periostin, N-acetyl-ß-glucosaminidase and nephrin, reported as biomarkers of renal pathologies. Additionally a further 29 unique gene products, of saliva and skin origin, have been identified, together with two bacterial proteins. The novel method here reported could be safely applied to any other research on manuscripts stored in public libraries and repositories of the World Cultural Heritage. SIGNIFICANCE: The present manuscript aims at finding proteomics traces in a 75-year old manuscript in order to confirm the health state of the author. In the case of Bulgakov it was known that he died of renal disease, possibly leaving traces and/or biomarkers of this pathology on the margins of the pages analysed. Three proteins, stated to be biomarkers of nephrotic syndrome, could be identified. In order not to contaminate the manuscript pages with resin particles, we have devised a novel harvesting film, by which strong cation and anion exchangers are embedded in ethyl-vinyl acetate foils. It is felt that this technology could be safely applied to other specimens belonging to the Word Heritage.

Asunto(s)

RESUMEN

The manuscript pages of the final draft of Master i Margarita, the masterpiece by Mikhail Bulgakov, written in the last four years of his life (1936-1940), have been treated with a mixture of chromatographic beads, namely a strong cation exchanger and a C8 resin. Potential substances captured by the beads, after harvesting them, were eluted with a mixture of isopropyl alcohol, dichloromethane and ammonium hydroxide and the eluate subjected to GC-MS analysis in order to detect the presence, if any, of drugs, due to the fact that the writer suffered intense pains caused by an inherited nephrotic syndrome. Indeed all the pages under investigation (a total of ten, taken at random among 127 foils) contained traces of morphine, from as little as 5 up to 100ng/cm(2). In addition to the intact drug, we could detect one of its metabolites, namely 6-O-acetyl morphine. The significance of these findings in terms of a possible improvement of the novel and in terms of drug use (or abuse) in the modern world is discussed and evaluated. BIOLOGICAL SIGNIFICANCE: The extraction of metabolites/proteins from the surface of the original manuscript pages of Bulgakov masterpiece Master i Margarita has permitted to monitor his health state and intake of medicaments over the last four years of his life. We have ascertained that: (1) he was assuming large doses of morphine as pain killers; (2) he was affected by a nephrotic syndrome, since we could identify three proteins known as biomarkers of this pathology. The double extraction procedure here reported could open up a novel field of investigation of (relatively) ancient manuscripts for metabolome/proteome analysis on the health status of the writer/artist.

Asunto(s)

RESUMEN

A novel method for separation of RNA fragments is reported here, based on migrating the polyanionic RNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations are typically performed in a 0-10 mM, pK 10.3 Immobiline gradient under denaturing conditions (6 M urea). In the 100-1000 bp length, it is shown that separations of RNA are optimal and very sharp bands can be obtained, in comparison with conventional electrophoresis, due to the "focusing" effect originated by the charge balancing between the positively charged gel matrix and the negatively charged RNA species. Excellent separations are also obtained from micro-RNAs, single-stranded RNA molecules of 21-23 nucleotides in length, which appear to regulate gene expression in animal and plant tissues. As a third example, 2-D runs in control and polycationic gels are shown. Under native conditions, RNAs are not aligned in a diagonal, suggesting that molecular shape has a strong influence on the interaction between RNA and the charged gel matrix. Thus, 2-D runs in cationic matrices might be exploited for structural studies of RNA molecules.

Asunto(s)

RESUMEN

A novel method for separation of low-molecular-mass heparins is reported here, on the basis of migrating the polyanionic heparins in a polycationic polyacrylamide gel, made by incorporating a gradient of positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated either in linear or nonlinear gradients of positive charges, thus modulating at whim the separation power. This allows the polydisperse heparins to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization. It is shown that the separations obtained are a complex function of both size and charge distribution along the oligosaccharide chains. This novel methodology represents a marked improvement over existing techniques and appears to hold promise for applications in screening of commercial lots of heparins, also in view of possible presence of contaminants, such as those recently detected in imported heparins.

Asunto(s)

RESUMEN

A novel method for separation of DNA fragments is here reported, based on migrating the polyanionic DNA fragments in a polycationic polyacrylamide gel, made by incorporating positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated under two working conditions: either against a gradient of positive charges, to allow the various DNA fragments to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization, or in a plateau gel (i.e., in a gel containing a constant level of positive charges from anode to cathode). In this last case, separation is still obtained due to differential charge modulation of the various DNA fragments. In the 100-1000-bp length, it is shown that separation can be obtained even for fragments differing in length by <0.5%, as shown in the splitting of a 656- and 659-bp doublet, that could not be resolved by conventional polyacrylamide gels. In the 10-100-bp range, it is shown that the present method can resolve single nucleotide polymorphisms, i.e. fragments of identical number of nucleotides but differing by one base substitution. In this last case, separations are obtained only in gradient gels containing a much steeper gradient of charges (0-20 mM Immobiline pK 10.3 and pK 12, as opposed to gradients of only 2-4 mM positive charges for larger size fragments). This novel methodology represents a marked improvement over existing techniques and appears to hold promises for applications in diverse fields, such as molecular biology, forensic medicine, and genetic screening.

Asunto(s)

RESUMEN

A miniature electrophoretic device is developed on the basis of a new isoelectric focusing (IEF) method, namely parallel isoelectric focusing. We report here the theory and the results of operation of a new parallel isoelectric device (PID). The main advantages and limitations of the method are discussed for miniaturization purposes. It is shown that the method guarantees the fast and complete separation of any complex protein mixtures under acceptable conditions, such as voltage source, temperature, size of the device, and separation process duration. It is shown that the main problem of PID miniaturization is the buffer design, and the relation between Immobiline buffer capacity and solution buffer capacity. The main experimental limitation of PID resolution is protein sensitivity to pH changes.

Asunto(s)

RESUMEN

Investigation of isoelectric focusing (IEF) kinetics has been performed to provide the theoretical basis for miniaturization of classical IEF in immobilized pH-gradients. Standard IEF demands colinearity of the electric field and pH-gradient directions (serial devices). It is shown that the IEF separation process based on a continuous, serial pH gradient is incompatible with miniaturization of separation devices. The new realization of the IEF device by a parallel IEF chip is suggested and analyzed. The main separation tool of the device is a dielectric membrane (chip) with conducting channels that are filled by Immobiline gels of varying pH. The membrane is held perpendicular to the applied electric field and proteins are collected (trapped) in the channels whose pH are equal to the pI of the proteins. The pH value of the surrounded aqueous solution is not equal to any channel's pH. The fast particle transport between different channels takes place due to convection in the aqueous solution. The new device geometry introduces two new spatial scales to be considered: the scale of transition region from a solution to the gel in a channel and a typical channel size. The corresponding time scales defining the IEF process kinetics are analyzed and scaling laws are obtained. It is shown both theoretically and experimentally that parallel IEF accelerates the fractionation of proteins by their pI down to several minutes and enables possible efficient sample collection and purification.

Asunto(s)

RESUMEN

A new universal method for the generation of nonlinear electrophoretic mobility of a packet of any particles is suggested. The method is based on the investigation of particle packet dynamics under the influence of an external force. The system under consideration is a homogeneous and isotropic medium with traps for these particles. Packet dynamics is described by a linear diffusion equation. The measured packet parameters are the position and the velocity of a packet maximum. It is shown that these parameters are nonlinear in the external field under definite limitations on the trap properties. This statement is proved both theoretically and experimentally for the simple model of diffusive substrate, the so-called comb structure. The prospects of designing new supporting substrates (microfluidic systems) with a nonlinear response are discussed.

Asunto(s)