G3-MNP

The nanoparticles were further functionalized with glutaraldehyde, washed with methanol and subsequentiy a methanohc solution of the NH2HL2 ligand was added (Fig. 8.20). After standing for 5 days at room temperature, the nanoparticles 

The IR spectrum of G3-MNP-NHHL2 showed in addition to the phenol O—H stretch at 3359 bands at 1111 cm (C—O—C) from the methyl ether arm and typical bands arising from aromatic functionalities at 818, 757 and 699 cm (Ar-H/Py-H). For its Zn(II) complex the two characteristic stretches from bridging acetate were observed at 1599 and 1414 cm [28]. It should be noted that G3-MNP that had been treated with Zn(II) acetate also showed bands typical for zinc(II) acetate. It was thus concluded that the PAMAM dendrimer took up zin-c(II) acetate which could potentially act as catalyst as well. Since this would create 

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G3-MNP (150 mg) were suspended in glutaraldehyde (25 %, 20 mL) and sonicated for one hour. After 12 h standing the nanoparticles were separated from glutaraldehyde and washed with TRIS buffer pH 8 (5 x 15 mL). G3-MNP-glutaraldehyde were used in the next step without further purification for the GpdQ immobilization and for the ligand immobilization they were washed with methanol (1 mL). 

To G3-MNP-glutaraldehyde (75 mg) in methanol (1 mL) was added NH2HL2 (50 mg) and dried potassium carbonate (75 mg) and the mixture was left standing at room temperature for live days. After this time the nanoparticles were washed by magnetic separation until the solution was clear. The nanoparticles were then resuspended in methanol (1.5 mL) and zinc acetate dihydrate (50.2 mg) was added. After stirring for 30 min the MNP were washed with methanol (5 X 1.5 mL) and dried in vacuo. 

G3-MNP-GpdQ were dried in high vacuum prior to XPS analysis. The survey spectrum shows that the elements present on the surface are mainly iron, oxygen, nitrogen and carbon (Fig. 8.21a) [34]. While the O 1 s and C 1 s peaks could be resolved into multiple species the assignment for nitrogen is less clear (Fig. 8.21c, d and e). In general the survey spectrum displays more noise as the others previously reported in this work. This is attributed to the multitude of trace elements that are bound to the enzyme and thus appear in the XPS spectrum. 

The elemental analysis of the immobilized enzyme on the nanoparticles demonstrated that both carbon and hydrogen content were elevated (4.66 %C, 1.13 %H) with respect to the G3-MNP (3.53 %C, 0.75 %H). The nitrogen content was lowered (1.22 %N) which is unsurprising since the G3-NMP have a high nitrogen content due to the PAMAM dendrimer (1.55 %N). A small amount of sulfur (0.04 %S) is found after GpdQ had been immobilized which might be due to cysteine residues and traces of sulfate bound to the enzyme. 

Fig. 8.23 Experimental setup to test activity of the GpdQ enzyme immobilized on G3-MNP...

Fig. 8.16 a Synthesis of the magnetite nanoparticles (MNP) functionalized with G3-PAMAM dendrimer. MNP suspended in water before (b) and after magnetic separation (c)... 

Infrared analysis of unsubstituted magnetite nanoparticles showed the stretches from Fe-O at 560 cm . Once amino propyl trimethyl silane (APTS) was attached to the surface, additionally C—H stretches at (2949, 2866 cm ) and Si—O stretches at 1015 cm were found. In the final G3-PAMAM MNP N-H and... 

Elemental analyses were conducted to confirm successful synthesis of the G3-PAMAM MNP and to monitor the increasing nitrogen content of the G0-G3 dendrimer (Table 8.2). Some sulfur contamination is apparent (presumably from the ferrous sulfate used in the MNP synthesis) which is, however, washed out during the repeated sonication/washing processes during subsequent PAMAM dendrimer modification. 

PAMAM dendrimer modified magnetite nanoparticles were tested as supports for GpdQ and a biomimetic complex. Previous studies have shown that a G3-PA-MAM dendrimer has a beneficial effect for protein immobilization opposed to unsubstituted MNP [1]. The dendrimer was build up stepwise by alternating additions of methanofic methyl acrylate and ethylenediamine solutions. The pendant amine functions were then further functionalized with glutaraldehyde. 

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