Synthesis of Gold Nanoparticles using Schiff Base Derivative of Ceftriaxone Sodium With Isatin as A Reducing and Stabilizing Agent

Introduction

The synthesis of AuNPs via the reduction of AuCl₄^– in aqueous solution by using the antibiotic ceftriaxone(CR) has been reported earlier [1].  1H-indole-2,3-dione (isatin, Is) and  its Schiff base with β- lactam antibiotic cefotaxime  were also reported as  reducing and stabilizing agents  in the synthesis of AuNPs [2, 3].  The sizes, shapes and stability of the prepared AuNPs were found to change with type of ligand, reactant concentrations, pH and reaction temperature. The condensation of isatin with cefotaxime was found to decrease the rate of reduction of Au(III) ions compared with the free isatin molecules [2, 3] .  In  this work  the  reduction of AuCl₄^– to AuNp in aqueous solution is investigated at different conditions , using the  Schiff base ligand sodium (6S)-7-((Z)-2-(methoxyimino)-2-(2-((Z)-2-oxoindolin-3-ylideneamino)thiazol-4-yl)acetamido)-3-((2-methyl-6-oxido-5-oxo-2,5-dihydro-1,2,4-triazin-3-ylthio)methyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (ISCR) ( Fig. 1) that has been previously prepared from the condensation reaction of ceftriaxone antibiotic(CR)  with isatin [4]

Figure 1:  Structure of Schiff base Ligand ISCR.  Click here to View figure

 

Materials and Methods

Materials

Ceftriaxone sodium (C₁₈H₁₆N₈O₇S₃Na₂.3.5H₂O) (LDP), isatin (Aldrich), sodium tetrachloroaurate (III) dihydrate (NaAuCl₄.2H₂O) (BDH), were used as received from suppliers. Buffer solutions of different pH were prepared  from potassium dihydrogen orthophosphate KH₂PO₄ 99% (Fluka), dipotassium hydrogen orthophosphate, K₂HPO₄, 99%, (Fluka) and  phosphoric acid H₃PO₄ (Analar,BDH).  The synthesis and characterization of the the Schiff base ligand ISCR has been reported  earlier([4]

Instumentation

Electronic spectra of the prepared AuNPs solutions were obtained on a (200–1100 nm) SHIMADZU 1800 Double Beam UV-Visible spectrophotometer. The binding of Schiff base ligand to AuNPs was analyzed by FTIR spectra using a SHIMADZU FT-IR 8400S spectrophotometer. Size and morphology of AuNPs  were determined by Scanning electron microscope (SEM) and by  atomic force  microscopy AFM images using SEM (KYKY-EM3200)  and AFM model AA 3000 SPM 220 V-Angstrom (Advanced INC.  USA).  XRD analyses were measured by using a SHIMADZU XRD-6000 x-ray diffract ometer with a Cu-Kα (λ = 0.154060 nm) radiation source.

Synthesis of Aunps  at Different Conditions

An aqueous solution of the Schiff base  ligand ISCR (Na₂C₂₆H₁₉N₉O₈S₃.2H₂O. 1.5CH₃OH ) (1.266×10^-4 M) was prepared by dissolving 0. 1027 g of the ligand in  1000 ml deionized water (DDW). A  stock aqueous solution of AuCl ₄^– ( 2.514×10^-3 M) was prepared by dissolving  0.1 g of  NaAuCl₄.2H₂O in  100  ml  distilled  deionized water (DDW) in 100 ml volumetric flask. A standard  solution of  AuCl ₄^–(2.514×10^-4 M) was prepared by diluting 10 ml of the stock solution to 100 ml. Ten  aqueous solutions  containing a constant  concentration of NaAuCl₄.2H₂O  solution  (7.542×10^-5 M) and different concentrations of  ISCR (6.33×10^-6, 1.266×10^-5, 1.899×10^-5, 2.532×10^-5, 3.165×10^-5, 3.798×10^-5, 5.064×10^-5, 6.33×10^-5, 7.596×10^-5 and 8.862×10^-5 M) were prepared by adding 0.25, 0.5, 0.75, 1.0, 1.25,1.5, 2, 2.5, 3, 3.5 ml of  a freshly prepared solution of   ISCR   (1.266×10^-4 M) to  1.5 ml of AuCl ₄^– solution (2.514×10^-4 M) followed by dilution to 5ml. The concentration ratios of   ISCR / AuCl₄^– = 0.084, 0.168, 0.252, 0.336, 0.42, 0.504, 0.671, 0.84, 1.007  and  1.175  respectively. The uv-visible spectra of the prepared solutions were measured at room temperature to  obtain the optimum concentration ratio of  ISCR/ AuCl ₄^– . The effect of temperature on the  synthesis  rate of AuNPs  was  studied  spectrophotometrically  by heating   ten (5 ml) solutions of the selected concentration ratio of  ISCR/ AuCl₄^–,  for 5 minutes at 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80 ºC respectively and at selected temperature for different heating times (5, 10, 15, 20, 25, 30, 35, 40, 45 and 60 min) in a water bath.  The  pH  effect on AuNPs synthesis was also studied  spectrophtometrically, using  the selected ISCR/ AuCl₄^– ratio at pH  media : 2.37, 3.25, 4.15, 5.70, 6.30, 7.22, 8.38, 8.83, 10.14 and 11.16.

Results and Discussion

Uv-Vis Spectrophotometry

Concentration Effect

Fig. 2  shows the uv-visible spectra of Schiff base ligand, ISCR(a),  AuCl₄^– (b) and AuNPs solution prepared from mixing 1.5 mL of  ISCR (1.266×10^-4 M) with 0.75 mL of AuCl₄^– (2.514×10^-4 M) diluted to 5 mL(c).  The spectrum of ISCR displayed two bands at λ 230 and 297 nm attributed to π→π* transition as was reported earlier[4], while the spectrum of AuCl₄^– solution corresponds to  square planar tetrachoroaurate complex [5,6].  After 24h of preparation, the solution mixture developed a pink color with absorption maxima at 533 nm assigned to the  surface plasmon band SPB of spherical AuNPs[7-13]. This indicates that AuCl₄^– has been reduced by Schiff base ligand to form AuNPs.

Figure 2: The uv-visible spectra of a- ISCR (1.266×10-4 M), b- AuCl4– (2.514×10-4 M) and c-synthesized AuNPs  in aqueous solutions after 24 h. Click here to View figure

 

Fig. 3  shows the variation in the absorption spectra of AuNPs solutions prepared from a constant concentration of AuCl₄^– (3.771×10^{-5 M)} and different concentrations of  ISCR  (6.33×10^-6, 1.266×10^-5, 1.899×10^-5, 2.532×10^-5, 3.165×10^-5, 3.798×10^-5, 5.064×10^-5,    6.33×10^-5, 7.596×10^-5 and 8.862×10^{-5 M)}.  (1-10 respectively). No color change  and no SPB were detected in all solutions until 24 h when the  spectra gave  increased absorption of  single bands at λ=(538, 538, 534, 549, 541, 546, 541, 541, 538 and 535 nm respectively assigned  to  SPR of spherical AuNPs [ 7-12].  After 48h the spectra of all solutions showed increased absorptions with the bands of solutions 1, 4, 6-10 being shifted to lower wavelengths and appeared at λ= (531, 542, 541, 538, 535, 537 and 533 nm respectively). Absorption bands of solutions 2, 3 and 5 were shifted to higher wavelengths and appeared at   λ= 541, 545, 543 nm respectively, referring to larger size or aggregation of AuNPs.  After one week, solution 2 exhibited two high absorption bands appeared at 538 and 776 nm which may be attributed to the formation of non-spherical AuNPs[5, 14-17] .  After three weeks, band absorptions increased and their positions were shifted to higher wavelengths and appeared at λ= 560, (540 and 830), 540, 542, 540, 538, 543, 543, 540 and 542 nm respectively. After four weeks the intensity of SPBs decreased. The best performance was recorded by solutions 9 and 10 (ISCR/AuCl₄^– 1.007 and 1.175 respectively) the spectra of which exhibited higher SPB absorbance and higher stability with time compared with the other solutions. This result is quite different from those  reported on using  ceftriaxone (CR) and  isatin (Is) separately  when  optimum concentration ratios of  CR/ AuCl₄^–  and Is/ AuCl₄^–  were (0.172) [1] and (9.52) [2] respectively  which reflects the effect of different ligand structures  on  gold nanoparticle synthesis.

Figure 3: Absorption spectra of ISCR- synthesized AuNPs at different concentration ratios of ISCR / AuCl4– (0.084, 0.168, 0.252, 0.336, 0.42, 0.504, 0.671, 0.84, 1.007 and 1.175 (1-10 respectively). Click here to View figure

 

Temperature Effect

Fig. 4 shows the spectra of   ten solutions of ISCR / AuCl₄^– (1.007) after being heated at 35-80°C for 5 minutes.   All solutions exhibited weak absorption bands appeared at λ = 549, 550,

Figure 4: Absorption spectra with time for AuNPs synthesized from a solution of ISCR / AuCl4– 1.007 heated at 35° -80°C. Click here to View figure

 

540,536, 539, 535, 529, 534, 531 and 525 nm respectively, assigned to the SPB of spherical AuNPs with estimated size range about 10-60 nm [7, 9,10,13]. The reaction time to form AuNPs  was reduced with increasing temperatures and all solutions exhibited continuous increase  in absorbance of SPBs for more than 48h. As  in the case of  CR[1], the highest rate of GNPs synthesis was recorded at  80 °C which remained stable for more than  two weeks . Fig. 5 shows the spectra of  AuNPs in ten solutions of the concentration ratio ISCR / AuCl₄^– (1.007) heated  at 80°C  for 5 min to 60 min.  The spectra of all solutions exhibited single absorption bands in the visible region appeared at λ= (524, 529, 538, 530, 528, 541, 534,

Figure 5: Absorption spectra of AuNPs prepared from a solution of ISCR/ AuCl4– (1.007) heated at 80°C for different time (5-60 min). Click here to View figure

 

534, 530  and 533 nm respectively.  After 24h the bands of solutions heated at 5, 25, 40, 45 and 60 min. were shifted to higher wavelengths and appeared at (530, 531, 538, 533 and 534 nm respectively), while those of the solutions heated for 15,30 and 35 min were shifted to lower wavelength and appeared at λ=(531, 537 and 519 nm respectively). The spectra of all solutions showed increased absorption of SPB with time for one week, followed by decreased absorption after two weeks. The best heating times were 5 and 30 minutes. However, the reduction process by ISCR was of lower rate compared with that of free CR and isatin[1,2] which may be attributed to the removal of free amino group of CR moiety and  the change redox behavior of isatin moiety .

PH Effect

Fig. 6 shows the absorption spectra of ISCR- synthesized AuNPs   solutions at different pH media (2.37, 3.25, 4.15, 5.70, 6.30, 7.22, 8.38, 8.83, 10.14 and 11.16, 1-10 respectively) using ISCR / AuCl₄^– concentration ratio (1.007).

Figure 6: Absorption spectra of AuNPs synthesized at different pH value. Click here to View figure

 

After 1h of preparation no color change and no SPB were detected in all solutions. After 24h only the solutions (2, 3 and 4) of  pH (3.25-5.70) gave pink colors and their spectra exhibited a single weak absorption band  at λ 533, 540 and 537 nm respectively. The best performance was exhibited at pH=5.7 and to less extent at pH= 4.15   as the two solutions remained stable   for two weeks.  Despite the difference in ligand / AuCl₄^– ratio, the performances of ISCR were almost similar to that of CR and Is at nearly the same pH values [1, 2]. The low rate of reduction of  Au(III)  in highly acidic solutions may be attributed to the protonation of functional groups responsible for electron donation while neutral and basic solutions may result in the formation of  stable Au(III) hydroxyl anion complexes such as [AuCl₃(OH)]^–, [AuCl₂(OH)₂]^–,  [AuCl(OH)₃]^– or [Au(OH)₄]^– [18,19].  The reduction process by ISCR was also of  lower rate compared with that of CR and isatin[1,2].

Characterization of AuNPs

FT-IR Spectra

The infrared band assignment of the FTIR spectrum of ISCR shown in Fig. 7 a   was  reported earlier [4].  The spectrum of  ISCR- synthesized AuNPs (Fig. 7b) shows that the positions of the bands attributed to azomethine (-HC=N-)(1625.88 cm^-1) , υ C=O of  lactam(1733.89 cm^-1),    υ C=O overlapped amide and ester(1660.10 cm ^-1), ν_asy(COO^–) and ν_sy(COO^–) (1640.25 and 1430.15 cm^-1 respectively) of the free ligand were  shifted and  appeared at  1604, 1680, 1641.31 and (1646 and 1434) cm^-1 respectively. The two bands observed at 3425.34 and 3236.33 cm^-1 in Fig. 7b may be assigned to N-H stretching vibration of NH₂ group attached to the benzene ring of isatin moiety. This band  may be resulted from the oxidation of the indole  ring leading to ring opening [2]. These data refer to the binding of AuNPs with functional groups of the Schiff base ligand.

Figure 7: FT-IR spectrum of a- ISCR and  b- ISCR – synthesized AuNPs Click here to View figure

 

X-Ray Diffraction (XRD)

Fig. 8 shows the diffraction pattern of AuNPs prepared from aqueous solution of ISCR / AuCl₄^– = (1.007).  Four diffraction peaks were observed at 2θ = (38.2522, 44.4910, 64.6450 and 77.919) degrees  corresponding to the planes (111), (200), (220) and (311) of  face centered cubic lattice structure of AuNPs [9, 20] which are  in agreement with the XRD pattern data available in  the  JCPDS file no.(04-0784). The crystalline sizes of the nanoparticle were estimated using unmodified Scherrer’s equation [21, 22].  The average size was found 38.5 nm corresponding to the planes (111) and (200), and 80 nm according to the planes (111), (200), (220) and (311).

Figure 8: XRD pattern of ISCR-conjugated AuNPs Click here to View figure

 

Scanning Electron Microscopy (SEM

The SEM image of AuNPs  at room temperature and  at heating temperature range  35-80°C  for 5 min. showed  particles of regular spherical shapes with  a wide size  distribution ranging between 44-97 nm and average size of 73 nm  (Fig. 9).  However, heating the aqueous solution at 80°C for 30 minutes gave rise to different shapes and sizes of AuNPs which appeared as spherical, nanorods and irregular shapes as is shown in Fig.10. The average diameter of nanosphers was 43.39 nm, while average diameter and length of nanorods were 43 and 178 nm respectively. This result shows that the heating period at 80°C affects the size and morphology of AuNPs in this study.

Figure 9a: SEM b-particle size distribution of Schiff base ligand -synthesized AuNPs heated at 80°C  for 5min Click here to View figure

Figure 10:  a -SEM and particle size distribution of  spherical (b)  and  rod like(c)  ISCR-synthesized AuNPs heated at 80°C for 30 min. Click here to View figure

 

Atomic Force Microscopy (AFM)

Fig. 11 shows the  AFM micrographs of the  ISCR- synthesized  AuNPs  at heating temperature 80°C for 5 minutes which  have spherical shapes with average diameter of  82.28 nm.  The AFM image of the same solution heated at 80°C for 30 minutes( Fig.12)  showed the presence of different sizes and shapes of AuNPs  with average particle sizes 85.52 nm.

Figure 11: AFM pictures and size distribution of ISCR-synthesized AuNPs heated at 80°C for 5 min . Average diameter 82.28 nm. Click here to View figure

 

Figure 12: AFM pictures and size distribution of ISCR-synthesized AuNPs heated at 80°C for 30 min. Average diameter 85.52 nm. Click here to View figure

 

Antibacterial Activity

The antibacterial activity of ISCR and ISCR-capped Au NPs have been tested against the pathogenic bacteria gram negative Escherichia coli, and gram positive Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumonia compared with the activity of CR and CR-capped AuNPs that has been reported earlier[1], using drop diffusion method. The diameter of inhibition zones caused by each test sample was measured in (mm) and the results are described in Table 1. The ISCR-capped AuNPs showed the highest activity against all bacterial cultures compared with CR, ISCR and CR- capped AuNPs. These results indicate that AuNPs enhanced the activity of Schiff base ligand especially against Pseudomonas Aeruginosa and Staphylococcus aurous. The Escherichia coli was more sensitive to the four tested solutions compared with the other three cultures.

Table  1: Inhibition  zones (mm) exhibited by CR, ISCR and their conjugated AuNPs against some pathogenic bacteria.

---

This work is licensed under a Creative Commons Attribution 4.0 International License.