The morphology of silver nanoparticles prepared by enzyme-induced reduction

Abstract

Silver nanoparticles were synthesized by an enzyme-induced growth process on solid substrates. In order to customize the enzymatically grown nanoparticles (EGNP) for analytical applications in biomolecular research, a detailed study was carried out concerning the time evolution of the formation of the silver nanoparticles, their morphology, and their chemical composition. Therefore, silver-nanoparticle films of different densities were investigated by using scanning as well as transmission electron microscopy to examine their structure. Cross sections of silver nanoparticles, prepared for analysis by transmission electron microscopy were additionally studied by energy-dispersive X-ray spectroscopy in order to probe their chemical composition. The surface coverage of substrates with silver nanoparticles and the maximum particle height were determined by Rutherford backscattering spectroscopy. Variations in the silver-nanoparticle films depending on the conditions during synthesis were observed. After an initial growth state the silver nanoparticles exhibit the so-called desert-rose or nanoflower-like structure. This complex nanoparticle structure is in clear contrast to the auto-catalytically grown spherical particles, which maintain their overall geometrical appearance while increasing their diameter. It is shown, that the desert-rose-like silver nanoparticles consist of single-crystalline plates of pure silver. The surface-enhanced Raman spectroscopic (SERS) activity of the EGNP structures is promising due to the exceptionally rough surface structure of the silver nanoparticles. SERS measurements of the vitamin riboflavin incubated on the silver nanoparticles are shown as an exemplary application for quantitative analysis.

Keywords: EGNP; SERS; enzymatically grown silver nanoparticles; enzyme-induced deposition; nanoflower.

Figure 1

Schematic sketch displaying the growth mechanism of the silver EGNP: (a) the substrates are immobilized with single-strand DNA as linker molecules; (b) this is followed by binding of HRP at the target DNA; (c) finally the growth of the nanoflower-like silver nanoparticles is induced by the enzyme.

Figure 2

SEM images of silver EGNPs grown from different starting DNA concentrations of 0.16, 0.625, 1.25, 2.5, and 10 µM (as labelled above the columns). The reaction time was 10 s, 1 min, 5 min, or 30 min, respectively. The scale bar of 1 µm, as indicated, is valid for all parts of the figure. The images were taken at normal incidence.

Figure 3

SEM cross-section images of silver EGNP grown from a starting DNA concentration of 25 µM. The reaction times were 2, 3, and 5 min, respectively. The scale bar of 500 nm as indicated is valid for all three images.

Figure 4

Fraction of the substrate surface covered by silver nanoparticles obtained by grey-tone analysis of SEM images (analysed field size: 12 × 9 µm²) in dependence on (a) the initial concentration of DNA with the time of synthesis as a parameter and (b) time of particle growth with the DNA concentration as a parameter. Hidden error bars are too small to be displayed on the chosen axis scale. The curves are only a guide for the eye. Please note the differently scaled ordinates.

Figure 5

High-resolution TEM image of a silver nanoparticle grown by enzymatic synthesis. The right part shows a detail at higher magnification with clearly observable crystallographic planes of silver.

Figure 6

TEM cross section image of a silver nanoparticle on a glass substrate (a), an EDX spectrum recorded across the cross section (b), and mappings of the elements silicon (c), silver (d), or carbon (e), respectively.

Figure 7

Experimentally recorded RBS spectrum for silver nanoparticles synthesized on a silicon substrate (red squares) together with a RBS simulation of a homogeneous silver film (blue triangles). The left shoulder below 750 keV corresponds with the silicon substrate and the right peaks correspond to the silver thin film or the silver nanoparticles, as appropriate.

Figure 8

Surface coverage of silver (blue triangles) and absolute number of silver atoms (green circles), both belonging to the left ordinate, as well as the maximum height of the silver nanoparticles assuming the silver bulk density for the nanoparticles (red squares; belonging to right ordinate) of EGNP films on silicon in dependence on the DNA concentration, determined by means of RBS measurements. The reaction time was always 5 min. The lines are only a guide for the eye.

Figure 9

(a) A typical SERS spectrum of riboflavin (5 µM) on enzymatically generated silver nanoparticles measured for an excitation wavelength of 532 nm and (b) the dependence of the integrated SERS intensity at 1087 cm⁻¹ (flagged with the arrow in part (a)) on the riboflavin concentration. The inset shows the strongly increasing SERS intensity for low riboflavin concentrations on a linear scale.