J Biomed Nanotechnol.2017 Mar;13(3):337-348

Preparation and Characterization of Magnetic and Porous Metal-Ceramic Nanocomposites from a Zeolite Precursor and Their Application for DNA Separation

 

Michele Pansini1, Gianfranco Dell’Agli1, Antonello Marocco1, Paolo Antonio Netti2_ 3,

Edmondo Battista2_ 3, Vincenzo Lettera2, Paola Vergara2, Paolo Allia4, Barbara Bonelli4, Paola Tiberto5, Gabriele Barrera5, Gabriele Alberto6, Gianmario Martra6, Rossella Arletti7, and Serena Esposito1

1Department of Civil and Mechanical Engineering and INSTM Research Unit, Università degli Studi di Cassino e del Lazio Meridionale, via G. Di Biasio 43, 03043 Cassino, FR, Italy

2Center for Advanced Biomaterials for Health Care@CRIB Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53 80125 Napoli, Italy

3Interdisciplinary Research Centre on Biomaterials (CRIB) and Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale (DICMAPI), University of Naples Federico II, Piazzale Tecchio 80, 80125, Napoli, Italy

4Department of Applied Science and Technology and INSTM Unit of Torino-Politecnico, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy

5INRIM, Nanoscience and Materials Division, Strada delle Cacce 91, 10135 Torino, Italy

6Department of Chemistry and Interdepartimental Centre “Nanostructerd Interfaces and Surfaces–NIS,” University of Torino, via P. Giuria 7, 10125 Torino, Italy

7Dipartimento di Scienze della Terra, Università degli Studi di Torino, via Valperga Caluso, 35, 10125 Torino, Italy

 

Correspondence should be addressed to Michele Pansini, Department of Civil and Mechanical Engineering and INSTM Research Unit, Università degli Studi di Cassino e del Lazio Meridionale, via G. Di Biasio 43, 03043 Cassino, FR, Italy, E.mail: pansini@unicas.it

 

Abstract

In this work, metal-ceramic nanocomposite materials were produced and characterized to evaluate their uses in the separation of DNA from crude cell lysate. The production method here proposed, is based on short thermal treatments (up to 2 hours) under reducing atmosphere, applied on zeolitic Fe-exchanged precursors. The materials thus treated were subjected to xrd, TPR, N2-adsorption at 77 K and TEM analysis. Such investigations showed that they are formed by a dispersion of metallic Fe nano-sized particles in a porous ceramic matrix, prevailingly based on amorphous silica and alumina. Subsequently, these materials were magnetically characterized, and their magnetic response was critically discussed. Lastly, the nanocomposite materials were tested in the separation of Escherichia Coli DNA from crude cell lysate, using a commercial DNA purification kit as a comparison term. The results of the DNA separation experiments showed that the obtained materials could perform this type of separation.

DOI: https://doi.org/10.1166/jbn.2017.2345

 

Supplement:

In biochemical and molecular biology, is increasingly adopted the use of magnetic carriers in separation processes, thanks to the many benefits that they can guarantee. When these carriers are under the influence of a magnetic field, they become magnetic themselves and may allow the isolation or extraction of a molecule or target substance in in vitro applications.

The non-magnetic target, binds to the surface of the magnetic solid-phase support, either through specific binding interactions, or through another mechanism for example, ion exchange or hydrophobic interactions, and the resulting target-support complex is then isolated or extracted, by the application of an external magnetic field [1].

The traditional fluid phase methods of nucleic acids purification, involve the execution of elaborate series of precipitation and washing steps and are time-consuming and laborious to perform. Not least, the large number of steps required could increases the risk of degradation, loss or contamination of the sample.

In contrast, magnetic separation techniques are characterized by speeds of execution and ease in handling. The process could be performed either in manual way, or in automatic way in a large variety of automatization platforms since it requires no centrifugation or vacuum filtration procedures.

The magnetic devices most used in biological separation are particles with micro or nanometric size, formed by a nanometric iron oxide core (usually Fe3O4 magnetite or γ-Fe2O3 maghemite) coated by a, possibly, porous silica shell. The core has the magnetic properties whose permit the carrying of the particles with an external magnetic field and the porous silica shell supplies high chemical and thermal stability together with large, non toxic surfaces compatible with various chemicals and molecules for bio-conjugation.

Unfortunately, despite some of the production technics of the magnetic particles are really powerful and versatile, most of them have limits that contribute to higher running costs, as the complicated procedures and lengthy operations that must be carried out by well experienced chemists, as the expensive reactants needed, and the limited ability to supply larger scale production. Moreover, the methods used to obtain monodisperse nanoparticles with excellent magnetic properties, often, doesn’t permit the chemical modification (functionalization) of the material surface.

The process proposed by some of us, described in the International Patent WO 2015/145230 A,appears able to conjugate high performances in biological separation with moderate or low costs. The process consists of the thermally treatment at moderate temperatures (750-800 °C) of Fe2+ exchanged zeolites, under reducing atmosphere. In practice, the process can be divided in two separate phases.

The first phase consisting of cationic exchange procedures in acqueous solutions, which are useful to prepare a zeolitic precursor with the wanted cationics contents. In this work we used synthetic zeolites type 4A, that were subjected to cation exchanges by contacting them, under continuous stirring, with acqueous solutions of FeSO4∙7H2O. This operation was iterated for the proper number of times. It must be noted that the exchange of Fe2+ was performed at low temperature and under Ar bubbling to prevent Fe2+ oxidation. The cation composition of the Fe2+-exchanged zeolite was determined by atomic absorption spectrophotometry (AAS) and the same material was subjected even to TPR analysis.

The second phase of thermal treatment is functional to the ceramization of the material jointly with the reduction of the Fe2+ cations to zero number of oxidation. The single thermal treatment applied on the Fe2+exchanged zeolite was performed at temperature of 750°C (sample 1) and 800°C (sample 2) with short time of treatment (respectively 120 minutes and 0 minutes of dwell time at max temperature) in a Al2O3 tubular furnace in wich the reducing atmosphere was created by a continuous flow of a H2-Ar gaseous mixture, bearing 3% vol. H2.

The product of these operations is a composite formed by metallic Fe nanoparticles (few nanometers or tenths of nanometers) evenly dispersed in a prevailingly amorphous silica and alumina matrix, which keeps at least a part of the original porosity typical of the zeolite precursor. This achievement is in agreement with the X-ray diffraction, N2-adsorption at 77 K and TEM analysis performed. To complete the microstructural characterization of the two samples presented in this work, we also report the data of the surface area, the distribution of the pores size and the distribution of the size of the iron particles. Quantitative phase analyses of the amount metallic Fe dispersed in the composite materials were performed on the two thermally treated samples by using synchrotron radiation powder diffraction. The synchrotron XRPD experiments were carried out on the high-resolution beamline ID22 at ESRF (Grenoble), with a fixed wavelength of 0.41067 Ã.

The nanostructured composite materials produced with this technics belongs to the category of the magnetic solid carrier equipped with magnetic component evenly dispersed in a matrix material to which the nucleic acid molecules bind [1]. The magnetic characterization was performed on both the metal-ceramic nanocomposite samples tehermally treated at 750°C and 800°C and on pure zeolite A at room temperature using a Lakeshore 720 vibrating sample magnetometer under a magnetic field ranging from −17 to 17 kOe. The room-temperature hysteresis loops of both metal-ceramic nanocomposites are reported after careful subtraction of the diamagnetic signal from the zeolite A. Anyhow, both the samples exhibits a magnetic response sufficient to easily separate the solid adsorbent from the supernatant liquid through the exposition to an external magnet.

To verify the usability of these materials as magnetic carriers in biological separation processes, we have chosen to use them for the separation of the DNA of Escherichia coli from a crude cell lysate. After the proper operations of cellular cultivation and lysis, the DNA was extracted from the lysates both using a nucleic acid binding column deriving from a commercial kit, and the magnetic samples 1 and 2, produced within this work.

A crude cell lysates, containing a certain amount of bacteria culture, was added with a proper volume of a water suspension containing 20 mg/L of magnetic sample 1 or 2, and with a binding buffer containing a chaotropic agent (5.5 M guanidine thiocyanate in 20 mM TrisHCl, pH 6.6). Each suspension containing either sample1 or sample 2, and DNA, were well dispersed by vortex for 30 s and then continuously shaken for 12 h at room temperature. Subseguently, the magnetic powders dispersed in the suspensions were immobilazed by applying an external magnet (Magnetic Rack, Millipore) to allow the removal of the supernatant liquid and then they have been washed with 70wt. % ethanol twice and dryied completely. The following step was the resuspension in a volume of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) that allowed the magnetic particle bound nucleic acids to be eluted by incubation at room temperature for 20 min with continuous agitation.

Finally, the identity and purity of the nucleic acids adsorbed on the materials surfaces were investigated by UV spectrophotometry. The ratio A260 nm/A280 nm was used as a test of nucleic acid purity [2] and in both extractions performed using the magnetic sample 1 or 2, the values ranged between 1.7 and 1.8 that means remarkably high purity, and low protein impurities of the obtained nucleic acids extracts. The yield of the purified nucleic acids using magnetic sample 1 or 2 resulted in 1.6 and 3 times, respectively, higher than the one achieved from the commercial kit, with a similar separation purity. The fluorescence confocal scanning laser microscopy was used to observe the distribution of the nucleic acids on the magnetic samples 1 and 2. The results showed that the DNA was not only located on the outer surface, but even on the interior of the particles; the nucleic acids were able to diffuse within the pores of the magnetic samples 1 and 2.

In order to determine the maximum nucleic acid adsorption capacity of such samples, standard DNA adsorption isotherms were produced.The adsorption characteristics were studied using the common Langmuir model [3] assuming a monolayer adsorption onto homogeneous surfaces. The maximum adsorbed capacity of these materials was determined to be 14.2 and 31.1 μg DNA/mg for sample 1 (as said a material obtained by a thermal treatment at 750°C for 120 minutes applyed on the zeolitic precursor) and sample 2 (obtained by a thermal treatment at 800°C-0 min), respectively. Concluding with an overview, this work shows that:

1) The materials produced by the method described have the necessary characteristics to be profitably used as magnetic separators of nucleic acids in in vitro applications

2) The whole process can be reasonable considerated cheap since it consists of simple cation exchange operations and short thermal treatments under reducing atmosphere, at moderate temperatures. Moreover, the raw material from which we started (the type A zeolites), is very cheap as its process of production was already optimized on account of its many technological applications.

 

 

Figure 1. Symbolic representation of the whole process

 

Acknowledgments: The authors would like to thank ALFED S. P. A. for the kind financial contribution. We are particularly grateful to Avv. Massimo Napolitano for believing in this research even more than the authors themselves and to Professor E. Garrone for the useful discussions.

 

REFERENCES

1. Bruce, I.J., J. Taylor, M. Todd, M.J. Davies, E. Borioni, C. Sangregorio, T. Sen, “Synthesis, characterization and application of silica-magnetite nanocomposite,” Journal of Magnetism and Magnetic Materials,” vol. 84, pp. 145-160, 2004.
2. Sinden, R.R., “DNA Structure and Function,” Academic Press, San Diego, p. 34, 1994.
3. Langmuir, I., “The adsorption of gases on plane surfaces of glass, mica and platinum,” Journal of the American Chemical Society,” vol. 40, pp. 1361-1403, 1918.