Quasi-spherical silver nanoparticles for human prolactin detection by surface-enhanced Raman spectroscopy.

Prolactin is a polypeptide hormone made of 199 amino acids; 50% of the amino acid chain forms helices, and the rest forms loops. This hormone is typically related to initiating and maintaining lactation, although it is also elevated in various pathological conditions. Serum prolactin levels of 2 to 18 ng ml-1 in men, up to 30 ng ml-1 in women, and 10 to 210 ng ml-1 in pregnant women are considered normal. Immunoassay techniques used for detection are susceptible to error in different clinical conditions. Surface-enhanced Raman spectroscopy (SERS) is a technique that allows for obtaining the protein spectrum in a simple, fast, and reproducible manner. Nonetheless, proper characterization of human prolactin's Raman/SERS spectrum at different concentrations has so far not been deeply discussed. This study aims to characterize the Raman spectrum of human prolactin at physiological concentrations using silver nanoparticles (AgNPs) as the SERS substrate. The Raman spectrum of prolactin at 20 ng ul-1 was acquired. Quasi-spherical AgNPs were obtained using chemical synthesis. For SERS characterization, decreasing dilutions of the protein were made by adding deionized water and then a 1 : 1 volume of the AgNPs colloid. For each mixture, the Raman spectrum was determined. The spectrum of prolactin by SERS was obtained with a concentration of up to 0.1 ng ml-1. It showed characteristic bands corresponding to the side chains of aromatic amino acids in the protein's primary structure and the alpha helices of the secondary structure of prolactin. In conclusion, using quasi-spherical silver nanoparticles as the SERS substrate, the Raman spectrum of human prolactin at physiological concentration was determined.

Double genetically modified symbiotic system for improved Cu phytostabilization in legume roots.

Excess copper (Cu) in soils has deleterious effects on plant growth and can pose a risk to human health. In the last decade, legume-rhizobium symbioses became attractive biotechnological tools for metal phytostabilization. For this technique being useful, metal-tolerant symbionts are required, which can be generated through genetic manipulation.In this work, a double symbiotic system was engineered for Cu phytostabilization: On the one hand, composite Medicago truncatula plants expressing the metallothionein gene mt4a from Arabidopsis thaliana in roots were obtained to improve plant Cu tolerance. On the other hand, a genetically modified Ensifer medicae strain, expressing copper resistance genes copAB from Pseudomonas fluorescens driven by a nodulation promoter, nifHp, was used for plant inoculation. Our results indicated that expression of mt4a in composite plants ameliorated plant growth and nodulation and enhanced Cu tolerance. Lower levels of ROS-scavenging enzymes and of thiobarbituric acid reactive substances (TBARS), such as malondialdehyde (a marker of lipid peroxidation), suggested reduced oxidative stress. Furthermore, inoculation with the genetically modified Ensifer further improved root Cu accumulation without altering metal loading to shoots, leading to diminished values of metal translocation from roots to shoots. The double modified partnership is proposed as a suitable tool for Cu rhizo-phytostabilization.

Improving legume nodulation and Cu rhizostabilization using a genetically modified rhizobia.

The rhizobia-legume interaction has been proposed as an interesting and appropriate tool for rhizostabilization of soils contaminated with heavy metals. One of the main requirements to use this symbiosis is the availability of tolerant and symbiotically effective rhizobia. The aim of this work was to improve the symbiotic properties of the arsenic-resistant wild-type strain Ensifer medicae MA11 in Cu-contaminated substrates. The copAB genes from a Cu-resistant Pseudomonas fluorescens strain were expressed in E. medicae MA11 under the control of the nifH promoter. The resulting strain E. medicae MA11-copAB was able to alleviate the toxic effect of Cu in Medicago truncatula. At 300 µM Cu, root and shoot dry matter production, nitrogen content, number of nodules and photosynthetic rate were significantly reduced in plants inoculated with the wild-type strain. However, these parameters were not altered in plants inoculated with the genetically modified strain. Moreover, nodules elicited by this strain were able to accumulate twofold the Cu measured in nodules formed by the wild-type strain. In addition, the engineered E. medicae strain increased Cu accumulation in roots and decreased the content in shoots. Thus, E. medicae MA11-copAB increased the capacity of M. truncatula to rhizostabilize Cu, decreasing the translocation factor and avoiding metal entry into the food chain. The plasmid containing the nifH promoter-copAB construct could be a useful biotool for Cu rhizostabilization using legumes, since it can be transferred to different rhizobia microsymbionts of authoctonous legumes growing in Cu-contaminated soils.