DOI: 10.1007/s00339-008-4420-7 Appl. Phys. A 91, 241–246 (2008) Materials Science & Processing Applied Physics A r. herrera-becerra 1 c. zorrilla 1 j.l. rius 1 j.a. ascencio 2, ✉ Electron microscopy characterization of biosynthesized iron oxide nanoparticles 1 Instituto de F´ ısica, Universidad Nacional Aut´ onoma de M´ exico, A.P. 20-364, Distrito Federal, C.P. 01000, M´ exico 2 Instituto de Ciencias F´ ısicas, Universidad Nacional Aut´ onoma de M´ exico, A.P. 48-3, Cuernavaca, Morelos, C.P. 62210, M´ exico Received: 18 December 2007/Accepted: 3 January 2008 Published online: 23 February 2008 • © Springer-Verlag 2008 ABSTRACT We biosynthesized iron oxide nanoparticles with four different pH in the solution to see its influence in the oxides obtained. This method allowed for generating aggre- gates of 1–10 nm, and under optimal conditions (pH = 10) we could control the size in the range of 1–4 nm. With the purpose to analyze the biosynthesized iron oxide clusters we employed electron transmission microscopy techniques. Be- cause the biosynthetic method with alfalfa has been used, the presence of the biomass, which is dense and within which are contained the nanoparticles, makes their observation difficult. Using the HAADF ( Z contrast) technique it is possible to locate the nanoparticles, which are then characterized using EDS and HRTEM. PACS 61.46.-w; 68.37.Lp; 81.07.-b; 81.16.Be 1 Introduction Magnetite nanoparticles show remarkable new phenomena such as superparamagnetism. These phenomena arise from the finite size and surface effects that dominate the magnetic behavior of individual nanoparticles [1]. In this way, magnetite nanoparticles have attracted great attention for many important technological and biomedical applications such as magnetic separation, drug delivery, cancer hyperther- mia and magnetic resonance imaging (MRI) enhancement, due to their non-toxicity property and high chemical stability. To date, various techniques, but most often chemical synthesis methods for preparing magnetite nanoparticles, already have been reported, such as coprecipitation [2] (Massart’s method; coprecipitation of Fe +2 and Fe +3 salts in saturated base solu- tions [3, 4]), microemulsions [5], solvothermal processing [6] and high-temperature organic phase decomposition [7, 8]. For biomedical applications the nanomagnetic particles must be in a medium after their synthesis that prevents the for- mation of large aggregates, changes from their original struc- ture and that is biocompatible when exposed to biological systems. In particular, magnetic nanoparticles are required to be water-soluble, monodisperse, superparamagnetic at room ✉ Fax: +52 777 3291775, E-mail: ascencio@fis.unam.mx temperature (no remanence along with rapidly changing mag- netic state) [9] and easy to produce on a large scale. However, as has been mentioned the physical and chemical properties of magnetic nanoparticles greatly depend upon the synthesis route [10], and the synthesis of nanomagnetite particles that can meet all the above requirements remains a challenge. As different groups experiment with the development of new chemical or physical methods to produce nanoparticles, the concern for a negative impact on the environment is also pointed out: some of the chemical procedures involved in the synthesis of nanoparticles use toxic solvents that could poten- tially generate hazardous byproducts, and often involve high energy consumption. This is leading to a growing awareness of the need to develop clean, nontoxic and environmentally friendly procedures for synthesis and assembly of nanopar- ticles. New methods for growing nanoparticles are exploring the use of biological systems. Jose-Yacaman et al. firstly re- ported the formation of gold and silver nanoparticles by living plants [10, 11]. New reports have increased knowledge of the bioreduction by plants also for other elements that can be found in polluted water [13, 14]. Therefore, the utilization of biomass has emerged as a novel method for the synthesis of nanoparticles. For ex- ample, dead biomasses of oat and wheat [15] have shown the ability to create different types of gold nanoparticles when exposed to Au +3 -rich aqueous solutions. Furthermore, alfalfa biomass has proven to bind Au +3 and form nanopar- ticles when exposed to gold solutions [16]. There are also reports of synthesis of Eu–Au, Yb, Sm, Zn nanoparticles from alfalfa biomass [17–21], including the production of one dimensional nanostructures [22, 23]. Sastry et al. at- tained the biosynthesis of metal nanoparticles by plant leaf extracts [24, 25]. Huang et al. reported a bioreduction method using sun-dried biomass of cinnamomum camphora leaf (camphora tree) [26]. Only recently the production of iron oxide nanoparticles by a biosynthesis method using alfalfa has been reported [27]. It has been reported by several authors that the major chemical structures responsible for metal ion bind- ing are found within the biomass. Biosorption by inactivated biological mass occurs through the coordination of metal ions to different functional groups. In almost all applications the preparation method of the nanomaterials represents one of the most important chal- lenges that will determine the particle size and shape, the size