Tecnología Nanopore para la secuenciación de DNA

Nanopore-based sequencer will open the path to the fourth-generation DNA sequencing technology. The main differences between this technique and the previous ones are: DNA molecule that will be sequenced does not need a previous amplification step, is not necessary any type of specific label both molecular adaptors, and it has been abolished enzymatic process in the nucleotide sequence identification event. These differences have as result a more economic method since don’t spend the necessary reagents for the previous techniques, furthermore it lets to sequence samples with a low DNA concentration. This technique is based in the use of a membrane with a biologic nanopore inserted in it whereby the molecule to analyze (analyte) it made to pass, this membrane is placed between two reservoirs containing ions, when an external volatage is applied in both sides this lead to an ion current through the nanopore. When an analyte cross the nanopore the ion current is modified, that modification in the amplitude and duration of ion current determine the physical and chemical properties of that analyte. By means of subsequent statistical analyzes it can be determined to what sequence own this ion current blockade patterns. More used nanopores are the biologic ones, although they are working to develop synthetic nanopores. The main biologic nanopores are: α-Hemolysin from Staphylococcus aureus (α-HL), Mycobacterium smegmatis porin A (MspA) and bacteriophage phi29 pore (phi29). Α-HL and MspA have in their narrowest point a diameter similar to nucleotide size, they are functional at high temperature both wide range of pH (2-12) but MspA is able to read four nucleotide at the same time while α- HL just can read one by one. Finally, phi29 present a bigger diameter what let to get information about DNA spatial conformation and their interaction with proteins (Feng et al., 2015). Nowaday Oxford Nanopore Technologies (ONT) is the only company which has developed Nanopore technology; they have two devices available to sequencing (PromethION and MinION). The MinION is a single-use DNA sequencing device with the size of a USB memory with a total of 3000 nanopores that can sequence until 200kb. The PrometheION is big size sequencer that own 48 different cells, what let to sequence different samples at the same time, with a total of 144.000 nanopores and reading of several megabases (https://www.nanoporetech.com/). The high processivity and low cost become this technique in a great option to massive- sequencing.

Cold vapour generation electrothermal atomic absorption spectrometry and solid phase extraction based on a new nanosorbent for sensitive HG determination in environmental samples (sea water and river water)

Mercury is not an essential element for plant or animal life and it is a potential environmental toxic because of its tendency to form covalent bonds with organic molecules and the high stability of the Hg-C bond. Reports estimate a total mercury concentration in natural waters ranging from 0.2 to 100 ng L-1. Due to this fact, highly sensitive methods are required for direct determination of such extremely low levels. In this work, a rapid and simple method was developed for separation and preconcentration of mercury by flow injection solid phase extraction coupled with on-line chemical vapour generation electrothermal atomic absorption spectrometry. The system is based on chelating retention of the analyte onto the mini column filled with a mesoporous silica functionalized with 1,5 bis (di-2-pyridyl) methylene thiocarbohydrazide. The main aim of this work was to develop a precise and accurate method for the determination of the Hg. Under the optima conditions and 120 s preconcentration time, the detection limit obtained was 0.009 μg L-1, with RSDs 3.7 % for 0.2 μg L-1, 4.8 % for 1 μg L-1 and enrichment factor 4, Furthermore, the method proposed has permitted the determination of Hg with a reduction in the analysis time, the sample throughput was about 18 h-1, low consumption of reagents and sample volume. The method was applied to the determination of Hg in sea water and river water. For the quality control of the analytical performance and the validation of the newly developed method, the analysis of two certified samples, TMDA 54.4 Fortified Lake, and LGC6187 River sediment was addressed. The results showed good agreement with the certified values.