Gum arabic modified Fe3O4 nanoparticles cross linked with collagen for isolation of bacteria

Background Multifunctional magnetic nanoparticles are important class of materials in the field of nanobiotechnology, as it is an emerging area of research for material science and molecular biology researchers. One of the various methods to obtain multifunctional nanomaterials, molecular functionalization by attaching organic functional groups to nanomagnetic materials is an important technique. Recently, functionalized magnetic nanoparticles have been demonstrated to be useful in isolation/detection of dangerous pathogens (bacteria/viruses) for human life. Iron (Fe) based material especially FePt is used in the isolation of ultralow concentrations (< 102 cfu/ml) of bacteria in less time and it has been demonstrated that van-FePt may be used as an alternative fast detection technique with respect to conventional polymerase chain reaction (PCR) method. However, still further improved demonstrations are necessary with interest to biocompatibility and green chemistry. Herein, we report the synthesis of Fe3O4 nanoparticles by template medication and its application for the detection/isolation of S. aureus bacteria. Results The reduction of anhydrous Iron chloride (FeCl3) in presence of sodium borohydride and water soluble polyelectrolyte (polydiallyldimethyl ammonium chloride, PDADMAC) produces black precipitates. The X-ray diffraction (XRD), XPS and TEM analysis of the precipitates dried at 373 K demonstrated the formation of nanocrystalline Fe3O4. Moreover, scanning electron microscopy (SEM) showed isolated staphylococcous aureus (S. aureus) bacteria at ultralow concentrations using collagen coated gum arabic modified iron oxide nanoparticles (CCGAMION). Conclusion We are able to synthesize nanocrystalline Fe3O4 and CCGAMION was able to isolate S. aureus bacteria at 8-10 cfu (colony forming units)/ml within ~3 minutes.

However, the synthesis procedures for these nanomaterials are relatively complex and expensive in comparison to pure iron oxide nanoparticles [17][18][19]. Due to their unique magnetic properties, low cost synthesis [19] and low toxicity [12,16,20] Fe 3 O 4 could be widely used in numerous applications such as cellular labeling [11], magnetic separation [10], tissue repair [21], hyperthermia [21,22], magnetic resonance imaging [7], magnetically guided drug delivery [23] and molecular diagnostics [13] etc. The technique based on super paramagnetic Fe 3 O 4 nano particles, which respond to an external magnetic field, is an efficient way of separating samples linked to the magnetic particles from the liquid suspension. The particle-linked molecules can quickly agglomerate in the medium in response to a change in external magnetic field. Furthermore, the synthesis reports for iron oxide are based on co-precipitation [24], hydrothermal [15] as well as via high temperature methods [17,25]. However, the wider use of iron oxide based magnetic nanoparticles in biomedical research is still impeded due to the use of toxic chemicals [26], low yield, problems in achieving small, uniform and highly dispersed nano particles. So the desired method of synthesis needs a simpler, economical as well as a low temperature process for their enhanced applications in isolation/ detection of dangerous bacteria for humanity. Henceforth, we present a high yield, room temperature, one pot and water based new synthetic protocol that yields iron oxide nanoparticles. Moreover, we have demonstrated instant detection/isolation of pathogenic bacteria Staphylococcus aureus (S. aureus) at ultralow concentrations using CCGAMION synthesized through this new protocol, achieving a detection limit of 8 cfu/ml in 3 minutes. S. aureus is a gram-positive, perfectly spherical bacterium about 1 μm in diameter. This bacteria causes skin lesions such as boils, styes, furuncles, pneumonia, mastitis, phlebitis, meningitis and urinary tract infections etc. S. aureus shows affinity for a wide range of mammalian plasma and extracellular matrix proteins. Among the proteins, collagen was estimated to bind with receptor present on S. aureus [27]. Collagen is the main protein of connective tissues and most of the pathogenic bacteria are attached with collagen for colonization and seems to be better choice with respect to antibiotic vancomycin [4]. Further, Iron oxide particles are bio-compatible and suitable for functionalization with gum arabic (GA), a natural polymer which is known for its usage in controlled drug delivery systems and is also a surface active molecule capable of improving magnetic nano particle stability in aqueous solutions by providing steric stabilization [28]. In nutshell, this report is the first kind of demonstration via Fe 3 O 4 with GA as well as collagen.

Characterization of Fe 3 O 4 nano particles
The X-ray diffraction data ( Figure 1) corresponds to the formation of magnetite (Fe 3 O 4 ) nanocrystals [18]. All the peaks of Figure 1 can be indexed to Fe 3 O 4 structure (JCPDS-88-0315). One can easily observe the broadening of peaks of Fe 3 O 4 due to crystallite size reduction. The crystallite size calculated using Debye Scherer formula [29] is found to be~11 nm. The inset (upper one) of Figure 1 shows obvious black appearance of assynthesized powder. The other inset (lower one) of Figure 1 shows that the nanoparticles dispersed in water can be drawn by applying an external magnetic field. Also, due to XRD pattern similarities of Fe 3 O 4 and Fe 2 O 3 , the survey XPS spectrum together with spectra of Fe (2p 3/2 , 1/2 ) (inset of Figure 2) and O(1s) (inset of Figure 2) is collected and shown in Figure 2. The measured peaks are Fe (1s 2 , 2s 2 , 2p 6 , 3s 2 , 3p 6 , 3d 6 , and 4s 2 ), oxygen (1s), nitrogen (1s), carbon (1s) and chlorine (2 s, 2p). The peak at~715 eV corresponds to Fe2P 3/2 of Fe 3+ and a small peak at~723 eV corresponds to Fe2P 1/2 confirming formation of magnetite [30,31]. In addition, the peaks corresponding to sodium, nitrogen, chlorine and carbon originate from PDADMAC indicating the existence of PDADMAC on the surface of iron oxide. Moreover, morphology as well as size of thusobtained magnetic nanoparticles was investigated through transmission electron microscopy and represented in Figure 3. As it's obvious from Figure 3 the particle size is~20 nm with non spherical morphology, which is again supported by our observation of crystallite size through XRD peak broadening ( Figure 1).

Bacterial Isolation
A representative image of the captured bacteria with the help of CCGAMION at various cfu/ml is shown in Figure 4. This figure clearly shows that CCGAMION can capture only S. aureus from 8-40 cfu/ml ( Figure 4c and Figure 4f) and it is not able to capture other bacteria such as S. albus and E. coli (Figure 4i and Figure  4l) which may be due to less affinity of S. albus/E. coli towards collagen binding sub segment CNA [27]. It is obvious that with the increase of concentration of bacterial solution, we can easily isolate bacteria from the solution. Herein, mixing of gum arabic modified Fe 3 O 4 with collagen to bacterial solution ( Figure 5a) results in sufficient number of magnetic nanoparticles binding onto S. aureus due to its affinity towards collagen. A small magnet placed near to these simply attracted ( Figure 5a) bacteria-nanoparticle composites for the analysis and scanning electron microscopy (SEM) easily detected the bacteria from aggregates due to its micron size and shape ( Figure 4c and Figure 4f).

Discussion
The reduction of iron salt (FeCl 3 ) in the presence of structure directing agent PDADMAC, may have nucleated iron (Fe) nanoparticles in the channel created by electrostatic adsorption of BH -4 on the surface of PDADMAC, followed by subsequent oxidation of iron (Fe) to Fe 3 O 4 on heating at 100°C. However, the role of surfactants in the formation of directed morphologies is a matter of thorough investigations of nucleation, growth and also of interaction energies of surfactants with that of the embryos [32]. Furthermore, the CCGA-MION was separated along with the bacteria using the interactions of magnetic nanoparticle-aggregate with an external magnetic field ( Figure 5a) and ligand-receptor with that of the bacteria (Figure 5b).
Herein, it is very important to discuss about the interaction of GA with Fe 3 O 4 and also with collagen. GA attracts Fe 3 O 4 via electrostatic attraction between carboxylic group of GA and surface hydroxyl group of Fe 3 O 4 which is due to the glycoprotein present in gum arabic [33]. Furthermore, it is also well known that the adsorption of GA on to the surface of Fe 3 O 4 follows the Langmuir isotherm indicating that absorption is likely independent of molecular weight of gum Arabic (GA) [28]. As we know that GA is a mixture of branched polysaccharides and glycoprotein containing numerous functional groups which may be responsible for the cross-linking with collagen via covalent bonding [34]. Even naked magnetic nano particles can be absorbed by some bacteria like E. coli and Salmonella randomly at much higher concentrations (10 5 cfu/ml) [35]. On the other hand, our motive is to make Fe 3 O 4 nano particles to adhere on the surface of S. aureus and this can be done only by coating GA modified nanoparticles with collagen. Furthermore, the collagen is a well known mammalian fibrous proteins found in the connective tissues of mammals for providing structural support for tissues, bones, tendons and skin. S. aureus has been shown to bind with specific affinity towards collagen due to microbial surface component recognizing adhesive matrix molecules (MSCRAMM) as they are found on the cell surface of the S aureus. The collagen binding MSCRAMM on S. aureus is called collagen adhesion (CNA) [27,36] and plays an important role in pathogenesis. CNA has structural characteristic of cell wall anchored proteins on gram-positive bacteria and it consists of an N-terminal signal peptide, a non-repetitive region, one to four repeated units, followed by a cell-wall anchor region, a transmembrane segment and a short positively charged cytoplasm tail. The non-repetitive region of CNA is found to be fully responsible for the collagen-binding activity of CNA. Here, it is worth mentioning the presence (Figure 2) of some of PDADMAC Furthermore, high sensitivity, selectivity and affinity of bio-functional Fe 3 O 4 particles for the detection/isolation of bacteria, also depends on the size of magnetic nanoparticles. The size of Fe 3 O 4 nanoparticles should be such that it can allow the presence of sufficient number of ligands to achieve a multiple interaction, simultaneously it should also be able to yield high surface to volume ratio, stability as well as high binding rates. Due to the significant size differences between Fe 3 O 4 (~20 nm, Figure 3) nanoparticles and bacteria (micron size, Figure 4), a scanning electron microscope (SEM) easily distinguishes S. aureus from the aggregates. Moreover, the smaller size as well as high surface/volume ratio of Fe 3 O 4 nanoparticles increases its biding efficiency with bacteria. Besides, the above stated size requirements; Fe 3 O 4 nano particles have biocompatibility and biodegradability for in vivo applications. However, for our in vitro application good chemical stability of Fe 3 O 4 is adequate.

Conclusions
In summary, we have succeeded in synthesizing Fe 3 O 4 nanoparticles through a wet chemical route using PDADMAC as capping agent. Reactive Fe nanoparticles initially formed due to redox reaction using PDADMAC as a soft template which subsequently turns into Fe 3 O 4 nanoparticles on heating at 100°C through oxidation process. Using collagen coated gum arabic modified Fe 3 O 4 nano particles, we are able to capture, collagen binding bacteria S. aureus at various concentration ranging from 8 cfu/ml to 40 cfu/ml. However, we need a deeper study of surface chemistry for attaching bioactive molecules onto a magnetic nanoparticles as well as more precise control of numbers and orientations of molecules. Moreover, the CCGAMION may provide a new technology platform for isolation/detection of S. aureus and we can expect a major role of nanosized Fe 3 O 4 materials in diagnostics and clinical applications in near future.

Materials
The fish collagen (Biofill) was purchased from Eucare pharmaceuticals (origin fish), Chennai (India). The anhydrous ferric chloride was purchased from Central Drug House (India), New Delhi. Gum arabic (GA), nutrient broth and agar-agar were purchased from Himedia, Mumbai (India). PDADMAC from Sigma Aldrich chemicals. Staphylococcus aureus, staphylococcus albus, Escherichia coli was isolated from patients sample at Bose Clinical Laboratory, Madurai (India).

Synthesis of Fe 3 O 4
The anhydrous ferric chloride salt (10 mM) was dissolved in 50 ml of ethanol and allowed to react with a solution containing a mixture of 10 ml PDADMAC, 50 ml of water and NaBH 4 (100 mM) in ethanol for about one hr with constant magnetic stirring. The final reaction mixture is allowed to stand for one hour for the precipitation. The mixture was centrifuged and precipitate was removed. Further stoving was done at 100°C for 12 hours for late ripening of the powders and used for various characterizations. The X-ray powder diffraction (XRD) was carried out on an X'Pert PRO PANalytical instrument with Cu K α radiation at a scanning rate of 2°per min. The morphological pictures of nanoparticles were taken with the help of Technai G 2 type of transmission electron microscope (TEM). Preparation of collagen solutioñ 0.5 g of fish collagen particles were dissolved in 0.5 M acetic acid and sonicated for 10 min. Finally, the mixture is filtered through 5 micron filter paper to get a clear collagen solution.

Preparation of bacterial solution
Bacterial cells were suspended in peptone water and serial dilutions were made until the desired concentration of~8 to~40 cfu/ml was established.

Procedure
In trial, 30 mg of GA modified Fe 3 O 4 was dissolved to 5 ml vial containing a mixture of 200 μl PBS, 200 μl water and 160 μl fish collagen with a constant stirring up to 5 minutes. Then immediately after adding 500 μl (10 cfu/ml) of bacterial solution, magnetization was done using a bar magnet of 100 gauss up to 3 minutes, taken as the capture time of bacteria. After 10 min the aqueous solution was carefully removed using a micro pipette, 0.5 ml from each sample is poured on nutrient agar plates and incubated overnight at 37°C.
The aggregates were analyzed with the help of Hitachi model S-3000 H scanning electron microscope. All the materials except bacterial solutions were sterilized with UV-rays before use. Further, similar experiments were repeated with S. albus (~30-40 cfu/ml) and E. coli (~30-40 cfu/ml) strains. The concentration of bacteria left over in the vial in terms of cfu/ml for all the samples analyzed and tabulated in Table 1.

Acknowledgements
The corresponding author Dr. Jai Prakash Tiwari (JPT) would like to thank our previous Director (Professor A. K. Shukla, presently at Solid State and Structural Chemistry Unit, Indian Institute of Sciences, Bangalore India) for his encouragement and love during his stay at Central Electrochemical Research Institute, Karaikudi, Tamilnadu-630006, India. This paper is dedicated to mine uncle (Shiv Sahai Tiwari) as we lost him in January-2010.