Temperature and magnetic dual responsive microparticles for DNA separation

Abstract

The use of solid support in DNA separation from biological mixtures for diagnostics offers great potential for developing versatile separating tools. Although different polymer materials have been developed and studied for DNA separation, the application of such non-magnetic particles for DNA separation has remained limited. In this work, we describe the adsorption and desorption behavior of DNA on the temperature sensitive poly(N-isopropylacrylamide-co-aminoethylmethacrylate) (PNIPAM-co-AEMH) coated magnetic particles. Submicron sized temperature responsive magnetic/PDVB/P (NIPAM/AEMH/MBA) hybrid particles were prepared by a precipitation polymerization of NIPAM in the presence of previously prepared divinylbenzene (DVB) cross-linked magnetic particles as seed. Wherein, N,N-methylenebisacrylamide (MBA), aminoethylmethacrylate hydrochloride (AEMH) and 2,2′-azobis (2-methylpropionamidine) dihydrochloride (V-50) were used as a crosslinker, functional monomer and initiator respectively. The amino (−NH₂) functional groups derived from AEMH and V-50 are able to be cationic charged ($-{\text{NH}}_3^{+}$) by being protonated and can interact electrostatically with negatively charged DNA. The adsorption and desorption behavior of Herring Sperm DNA onto these functional magnetic particles were investigated as a function of pH, temperature, time and ionic strength. Interestingly, it was found that there is a significant effect of temperature on the DNA adsorption onto these magnetic particles. The results obtained indicate that the maximum amount of DNA adsorption was found at lower salt concentration, acidic pH condition and below the volume phase transition temperature (VPTT) of the PNIPAM shell. The highest desorption was found at higher salt concentration in basic pH and above the VPTT (40 °C) temperature. The temperature responsive magnetic particles and procedure described here can be used in microsystem separation technology for nucleic acid extraction, purification and concentration.

Introduction

As an emerging area, magnetic particles have gained much interest because of their promising applications in different fields such as biocatalysis [1], target drug delivery [2], cell labeling [3], detection [4], bioseparation [5], metal removing [6], and water purification [7].

To elaborate magnetic particles capable of being utilized for carrying biomolecules, surface modification or functionalization with polymer is required. Among different polymers, poly(N-isopropylacrylamide) (PNIPAM) hydrogel is a well known temperature responsive polymer that makes a coil-to-globule transition and undergoes dehydration behavior at its lower critical solution temperature (LCST) [8], [9], eventually showing volume phase transition at 32 °C. PNIPAM based magnetic polymeric particles have been studied for different applications such as biocatalysts [10], protein purification [11] and bioprocess [12]. However, there are very few reported works on the use of core–shell particles having both of temperature responsive and magnetic property for nucleic acid separation.

The use of magnetic particles for the separation of nucleic acids has many advantages over other methods. The magnetic particles can be used for nucleic acid isolation directly from crude sample mixture and due to the magnetic properties of the particles; they can be removed easily and quickly even from viscous samples. Magnetic particles can save time and money, even in the techniques based on both manual and automated low- to high-throughput processes. The time consuming traditional long time centrifugation, precipitation and filtration steps can be avoided by using magnetic particles [13]. Different magnetic particles have been used as a solid support for DNA separation [14], [15] and RNA [16], [17] due to the ease of handling, rapid processing and less chemical requirements. Despite ongoing research progress in magnetic particle synthesis for nucleic acid separation and purification, better understanding about the interaction of DNA with magnetic polymer particles in the case of adsorption and desorption is still required. In this process, the role of external parameters such as temperature, pH, ionic strength, etc., as driving forces should be well explained to develop an automated separating system that can be guided by external magnetic field. Melzak et al. [18] and Balladur et al. [19] reported some explanation about such driving forces for DNA adsorption on silica particles in different conditions. The contribution of phosphate ion and chaotropic agents for DNA adsorption and desorption on amine modified magnetic silica particles has been reported in another literatures [14], [20].

Recently, we have successfully prepared temperature responsive core shell magnetic/PDVB/P (NIPAM/AEMH/MBA) polymer particles by seed polymerization using oil in water magnetic emulsion as the seed [21]. Now, we report here the feasibility of using previously prepared magnetic particles for DNA separation and the different aspects of the procedure for DNA adsorption and desorption onto temperature responsive magnetic/PDVB/P (NIPAM/AEMH/MBA) particles. The effect of pH, temperature and ionic strength on the adsorption and desorption of DNA were studied. It was found that the adsorption and desorption principally depend on the electrostatic interaction between cationic magnetic particles and negatively charged DNA molecules. Most importantly, the highlight of this report is a significant effect of temperature as a driving force with pH effect on the adsorption of DNA. The results suggest that this magnetic particle preparation and application processes can offer a wide applicable technique for nucleic acid separation in the biomedical field.