Automated nucleic acids isolation using paramagnetic microparticles coupled with electrochemical detection

Abstract

Easy, efficient and low demanding separation of mRNA from biological material is needed to study gene expression and to use in chip technologies. It is common knowledge that each mRNA molecule contains sequence of 25 adenines. This feature can be used for binding mRNA on the surface of the particles coated by thymine chains. The present work reports on suggesting and optimizing of mRNA separation and detection from biological material via paramagnetic microparticles coupled with electrochemical detection. Primarily we optimized cyclic and square wave voltammetric conditions to detect poly(A), which was used as standard to mimic behaviour of mRNA. Under the optimized square wave voltammetric conditions (frequency 280 Hz, accumulation time 200 s, supporting electrolyte and its temperature: acetate buffer 4.6 and 35 °C) we estimated detection limit down to 1 ng of poly(A) per ml. To enhance effectiveness and repeatability of isolation of nucleic acid automated approach for rinsing and hybridizing was proposed. We optimized the whole procedure and experimental conditions. Using automated way of isolation and under optimized conditions the yield of poly(A) (isolated concentration of poly(A)/given concentration of poly(A)*100) was approximately 75%. The suggested and optimized method for poly(A) isolation and detection was utilized for the analysis of brain tissues of patients with traumatic brain injury. The total amount of isolated mRNA varied from 40 to 760 g of mRNA per g of brain tissue. The isolation of mRNA from six samples per run was not longer than 2.5 h. Moreover, we applied the optimized procedure on fully automated pipetting instrument to isolate mRNA. The instrument was successfully tested on the analysis of extracts from roots of maize plants treated with cadmium(II) ions.

Introduction

Simple, rapid and reproducible isolation of nucleic acids with high purity is needed in a wide range of molecularly biological procedures. We are able to obtain nucleic acids with sufficient purity by the most commonly used method based on phenol–chloroform extraction; however, the whole procedure is laborious and time consuming. Due to this fact methods and approaches have been developing to shorten the time of isolation and to obtain nucleic acids with sufficient purity to be analysed by polymerase chain reaction and other molecularly biological methods [1], [2], [3], [4]. Nucleic acids isolation using paramagnetic or superparamagnetic particles (MPs) represents promising tool for this purpose [5], [6], [7]. MPs, whose size is ranging from nm to mm, respond to external magnetic field and facilitate bioactive molecules binding because of their affinity for the MPs modified surface made of biological components [8], [9], [10], [11]. The paramagnetic properties of the particles enable us to use magnetic force for transferring of the beads or for rinsing of nonbinding, otherwise commonly interfering substances. Among other advantages of MPs easy-to-use, non-laborious relatively rapid sample preparation without centrifugation and dialysis compared to conventional purification techniques belong. The time needed to get target biomolecule is also reduced due to the fact that binding of the biomolecule by MPs can protect it against physical and biological damage, e.g. denaturation [12]. Physico-chemical properties of MPs (e.g. their size, surface topography) are important to evaluate their usage in biology [13], [14], [15], [16], [17], [18], [19], [20], [21]. The mostly used MPs in biosensors applications are superparamagnetic nanoparticles composed of ferrous oxide or ferric oxide [22]. Small particles with size from 1 to 10 nm are paramagnetic, but larger particles (mm) are ferromagnetic.

Nanoparticles have a lot of physico-chemical advantages [23], [24]. Their size can be adapted to the extension and kind of a biological sample, which is a source of target biomolecules (e.g. proteins 5–50 nm, viruses 20–450 nm, cells 10–100 μm) [15], [25], [26]. Nanoparticles from ferric oxide also provide surface suitable for biomolecules binding. Methods of magnetic particles synthesis are largely discussed [9]. There are two ways to find how MPs can be modified. The first way is based on electric envelope layer for electrostatic adsorption of biomolecules. This method compared to conventional methods enables to avoid organic solvents. Matsunaga et al. modified the surface of MPs using electrically positive amino groups. Isolation was based on mutual electrostatic activities between negatively charged DNA and positively charged MPs [27]. The second way of paramagnetic particles surface modification is based on biomolecules anchored on particles. These biomolecules specifically bind target biomolecules [28], [29], [30]. On this method it is based also mRNA isolation. Oligo(deoxythymine)₂₅ anchored on the surface of MPs can be hybridized to mRNA molecule due to the presence of single chain sequence of tens adenines at the very end of all mRNA molecules [29].

MPs are commonly coupled with optical detection including fluorescent dyes. In addition, the isolated molecules can also be detected by electrochemical methods [28], [31], [32], [33], [34], [35]. The main aim of this work was to automate isolation of mRNA using paramagnetic microparticles and detect the isolated nucleic acids by cyclic and square wave voltammetry both coupled with adsorptive transfer technique.