Fabrication of Gold Nanoparticles/Graphene-PDDA Nanohybrids for Bio-detection by SERS Nanotechnology
© Mevold et al. 2015
Received: 22 August 2015
Accepted: 29 September 2015
Published: 12 October 2015
In this research, graphene nanosheets were functionalized with cationic poly (diallyldimethylammonium chloride) (PDDA) and citrate-capped gold nanoparticles (AuNPs) for surface-enhanced Raman scattering (SERS) bio-detection application. AuNPs were synthesized by the traditional citrate thermal reduction method and then adsorbed onto graphene-PDDA nanohybrid sheets with electrostatic interaction. The nanohybrids were subject to characterization including X-ray diffraction (XRD), transmission electron microscopy (TEM), zeta potential, and X-ray photoelectron spectroscopy (XPS). The results showed that the diameter of AuNPs is about 15–20 nm immobilized on the graphene-PDDA sheets, and the zeta potential of various AuNPs/graphene-PDDA ratio is 7.7–38.4 mV. Furthermore, the resulting nanohybrids of AuNPs/graphene-PDDA were used for SERS detection of small molecules (adenine) and microorganisms (Staphylococcus aureus), by varying the ratios between AuNPs and graphene-PDDA. AuNPs/graphene-PDDA in the ratio of AuNPs/graphene-PDDA = 4:1 exhibited the strongest SERS signal in SERS detection of adenine and S. aureus. Thus, it is promising in the application of rapid and label-free bio-detection of bacteria or tumor cells.
Raman scattering was discovered by CV Raman in 1928, and further, the use of surface-enhanced Raman scattering (SERS) technology was developed by Fleischman and others in 1974. SERS is used widely in various applications such as label-free sensing of bacteria Escherichia coli (E. coli) and various molecules. This is possible because of the enhancement of the Raman signal. Gold and silver nanoparticles are widely used for SERS enhancement [1–3] via their localized surface plasma resonance (LSPR). LSPR can increase the intensity of the Raman signal by at least 109, thereby easily detecting the presence of various bacteria or molecules. Once metal (gold and silver) become nanoparticles with a unique size and morphology, their optical, electrical, and magnetic properties also change. Recent research on SERS technology emphasize on controlling the size and morphology of the nanoparticles. When the gap of the metal nanoparticles is within 10 nm, it will produce “hot spot” effect, which will further enhance the intensity of the SERS signal. Therefore, it is important to develop the SERS bio-detection by controlling the gap and the particle size of the metals nanoparticles.
Graphene  is an allotrope of carbon in the form of a two-dimensional hexagonal lattice, with its sp2 hybridization and very thin atomic thickness (of 0.345 Nm). It is formed from a single layer of graphite structure. Graphene was successfully isolated in 2004 by physicist Novoselov and Geim from graphite . What make graphene so unique are its remarkable strength, electricity, and heat conduction, as well as many others. Graphene has many very specific physical properties, such as (1) high mechanical strength, Young’s modulus can reach 1000 GPa ; (2) thermal conductivity that can reach 5300 W/mK, which is higher than metals or diamond ; (3) electron mobility that can exceed 200,000 cm2/Vs with a resistance value (10–6 Ω·cm) even lower than silver or copper , which is the least resistance value of any currently known materials. Since graphene has excellent electrical and thermal conductivity and physical properties, it can be widely applied in different fields and has become a very popular research topic in recent years [9–12].
In this experiment, Au/graphene-PDDA nanocomposite was fabricated by adsorbed gold nanoparticles (AuNPs) onto the graphene-PDDA nanosheets as shown in Fig. 1. Graphite was a chemical exfoliated into graphene, PDDA was adsorbed onto graphene by reduction method, and AuNPs (negative charge) was then bonded onto the resulting graphene-PDDA nanosheets (positive charge) by ionic binding. Graphene-PDDA nanosheets are the supporting substrate to uniformly embed the AuNPs for creating more homogenous “hot spots” and controlling the interparticle gap of AuNPs. The positive charge of AuNPs/graphene-PDDA nanosheets is used to easily capture the negative charge of Staphylococcus aureus for SERS rapid detection. Various ratios of AuNPs/graphene-PDDA were evaluated in order to create an optimum surface-enhanced Raman scattering (SERS) signal for SERS bio-detection [17–20] of small molecules (adenine) and microorganisms (S. aureus).
The materials used in this study were as follows: graphite powder, <20 μm, synthetic, Aldrich; sulfuric acid, H2SO4, 96.5 %, Baker; fuming nitric acid, HNO3, ≧99.5 %, Sigma-Aldrich; potassium permanganate, KMnO4, Baker; PDDA, 35 % (average M w < 100,000), Aldrich; hydrogen peroxide, H2O2(aq), 35 %, Acros; hydrochloric acid, HCl(aq), 37 %, Scharlau; sodium citrate dehydrate, Na3Ct·2H2O, ≧99.5, Sigma-Aldrich; hydrogen tetrachloroaurate(III) trihydrate, HAuCl4·3H2O, 99 %, Sigma-Aldrich; nitric acid, HNO3(aq), 69 %, Panreac; silicon Oil, Choneye Pure Chemical; Luria-Bertani (LB broth), Difco™ (Agar Bacteriological), Oxoid; and adenine, C5H5N5, ≧99 %, Sigma.
Synthesis of Gold Nanoparticles
Citrate thermal reduction method was used to prepare gold nanoparticles. It is an oxidation-reduction reaction, which uses sodium citrate (Na3Ct·2H2O) as a reducing agent to reduce Au3+ of HAuCl4·3H2O. The experimental procedure is as follows. (1) Ninety-six milliliters of 0.307 mM tetrachloroauric acid solution was prepared. (2) The solution was heated, and after boiling for 10 min, 4 mL of 1 % sodium citrate solution was added and the color changed from yellow to dark red.
Synthesis of Graphene Oxide
Graphene oxide (GO) was prepared by using the modified Hummers method. Potassium permanganate was added to the graphite, and the surface of the graphite will have many oxidized functional groups. The experimental procedure is as follows. (1) Thirty-six milliliters of concentrated sulfuric acid was added to 1.0 g of graphite powder and stirred for 1 h. (2) The solution was stirred continuously in ice bath, 12 mL of fuming nitric acid was added dropwise, and then, 5 g of potassium permanganate was slowly added. (3) The solution was stirred for 120 h under room temperature. (4) One hundred twenty milliliters of deionized water was added slowly and stirred for 2 h under room temperature. (5) Six milliliters of hydrogen peroxide solution was added and stirred for 2 h and left at room temperature for 24 h. (6) The top layer of the clear solution was removed, and 200 mL of deionized water, 1 mL of hydrogen peroxide solution, and 1 mL of hydrochloric acid were added. The solution was mixed for 2 h and centrifuged. (7) Step 6 was repeated three times. (8) The top layer was washed with deionized water until the solid pH is close to 7. (9) After the solid was collected, it was placed in a vacuum oven and dried at 40 °C for 48 h. The final product was graphene oxide powder.
Synthesis of Graphene-PDDA
The experimental procedure is as follows. (1) Sixty milligrams of graphene oxide powder was mixed with 20 mL of deionized water. (2) The solution was sonicated for 10 min. (3) Eight hundred microliters of PDDA was added and stirred for 10 min. (4) The solution was heated to 90 °C under reflux for 12 h. (5) The solution was centrifuged, and the upper layer solution was removed. The step was repeated for several times. (6) Deionized water was added to the final product.
Synthesis of Au/Graphene-PDDA
Various ratios of AuNPs to graphene-PDDA
Graphene-PDDA (3 mg/mL) μL
HAuCl4 (0.1 mg/mL) mL
DI water μL
SERS Measurements by AuNPs/Graphene-PDDA Nanohybrids
A Raman microscope (HR800, Horiba, Japan) with He-Ne laser (632.8 nm) was used to detect the presence of S. aureus (ATCC 6538P). The experimental procedure is as follows. (1) Fifty microliters of the varied AuNPs/graphene-PDDA and 50 μL of S. aureus solutions (1 × 105 CFU/mL grown for 18 h at 37 °C) or adenine (concentration of adenine is 10−4 M) were placed in 1.5 mL micro-centrifuge tubes and mixed well. (2) Five microliters of each sample was dropped on the aluminum sheet. Raman spectra in the range of 400 to 1800 cm−1 were evaluated for the samples. The intensity of the Raman signal at 733 cm−1 (SERS signal from the cell wall of S. aureus) was investigated also for the samples.
Characterization Analysis of AuNPs/Graphene-PDDA Nanohybrids
The interaction between AuNPs and graphene-PDDA were analyzed by X-ray photoelectron spectroscope (XPS, VG ESCA Scientific, Theta Probe), and surface electric properties of AuNPs/graphene-PDDA samples were analyzed by zeta potential analyzer (Nano S90, Malvern Instruments) as described below.
Results and Discussion
Characteristics of Au/Graphene-PDDA
SERS Application of Au/Graphene-PDDA
Au/graphene-PDDA and their SERS intensity integral of S. aureus (integrated range of SERS intensity, 700~770 cm−1)
SERS intensity*10−3 (integral)
43.89 ± 4.64
48.30 ± 6.33
114.26 ± 14.95
64.39 ± 4.11
47.39 ± 1.90
29.75 ± 3.60
Au/graphene-PDDA and their SERS intensity integral of adenine (integrated range of SERS intensity, 700~770 cm−1)
174.76 ± 5.84
322.91 ± 6.87
593.43 ± 10.29
488.96 ± 13.79
410.37 ± 38.32
177.00 ± 2.04
This paper demonstrates in detail the synthesis of Au/graphene-PDDA nanocomposites and its application in SERS detection of S. aureus and adenine. Graphite was chemically exfoliated, and PDDA was π-π stacked onto the surface of graphene nanosheets, and later, gold nanoparticles were synthesized and attached onto the surface of graphene-PDDA by surface charge interaction. The resulting Au/graphene-PDDA nanocomposites greatly enhanced the Raman signal of S. aureus and adenine. Various ratios of AuNPs to graphene-PDDA were tested to make optimum SERS enhancement effects. AuNPs/graphene-PDDA in the ratio of AuNPs/graphene-PDDA = 4:1 exhibited the strongest SERS signal in the bio-detection of small biomolecules (adenine) and microorganisms (S. aureus). AuNPs/graphene-PDDA was shown to have enhanced Raman signal capability and unique ability to adsorb onto the microorganisms. Thus, it can be further applied in the rapid and label-free bio-sensing of biomolecules and microorganisms.
This work was financially supported by the Ministry of Science and Technology of Taiwan (MOST 104-2221-E-131-010 and MOST 104-2628-M-001-009) and partially supported by Academia Sinica.
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