Core-shell of FePt@SiO2-Au magnetic nanoparticles for rapid SERS detection
© Hardiansyah et al. 2015
Received: 24 August 2015
Accepted: 8 October 2015
Published: 22 October 2015
In this study, multifunctional hybrid nanoparticles composed of iron platinum (FePt), silica (SiO2), and gold nanoparticles (AuNPs) had been developed for surface-enhanced Raman scattering (SERS) application. Core-shell structure of SiO2 and FePt nanoparticles (FePt@SiO2) was fabricated through sol-gel process and then immobilized gold nanoparticles onto the surface of FePt@SiO2, which displays huge Raman enhancement effect and magnetic separation capability. The resulting core-shell nanoparticles were subject to evaluation by transmission electron microscopy (TEM), Energy-dispersive X-ray spectroscopy (EDX), zeta potential measurement, and X-ray photoelectron spectroscopy (XPS). TEM observation revealed that the particle size of resultant nanoparticles displayed spherical structure with the size ~30 nm and further proved the successful immobilization of Au onto the surface of FePt@SiO2. Zeta potential measurement exhibited the successful reaction between FePt@SiO2 and AuNPs. The rapid SERS detection and identification of small biomolecules (adenine) and microorganisms (gram-positive bacteria, Staphylococcus aureus) was conducted through Raman spectroscopy. In summary, the novel core-shell magnetic nanoparticles could be anticipated to apply in the rapid magnetic separation under the external magnetic field due to the core of the FePt superparamagnetic nanoparticles and label-free SERS bio-sensing of biomolecules and bacteria.
Rapid, specific, accurate, and sensitive identification and detection of small molecules and pathogenic bacteria are essential toward clinical treatment of infectious diseases. Traditional approaches for the pathogen bacteria identification often requires time-consuming bacteria culture and amplification protocols. Conventional protocols for microbial detection, though reliable and gold standard, are time and cost consuming and inconvenient for field situation [1, 2]. In this respect, sensitivity of bacteria into antibiotic depends mostly on measuring the change of its proliferation in response to the drug. Thus, in order to overcome these challenges, there is a development of new protocols and its combination with nanotechnology as a new method for the higher sensitivity and low-cost strategies for fast bio-detection and identification of small molecules or bacteria [1, 3, 4].
Raman spectroscopy elaborates the molecular vibrational signal to be used to identify and detect the molecular species such as small molecules and bacteria. Surface-enhanced Raman scattering (SERS) technique has attracted a lot of attention for more than three decades [5–7] because it enhances the Raman signal of the small molecules and or bacteria by several orders of magnitude. SERS nanotechnology could observe the monolayer (single-molecule level)  and studies on the identification of biological species, i.e., three kinds species of microbes (gram-positive, gram-negative, and mycobacteria), which have been reported in our previous work [3, 5]. The enhancement is known to develop from the strong optical intensity localized between the surface of metallic nanostructure . SERS takes advantage of localized surface plasmon resonance (LSPR) in nanoscale systems based on metallic nanoparticles such as silver and gold. The enhancements ranging from a factor of 106 to 1016 have been reported in various studies [9–11]. The mechanisms for this enhancement in SERS are attributed to electromagnetic field enhancement due to localized surface plasmon resonance and also to chemical interactions of the analytic molecules with the substrate moiety. As a result of these enhancements, SERS can have the sensitivity for the identification of ultra-trace levels of analytes to single molecules and bacteria. This level of sensitivity is useful for the detection of a small number of analytic molecules normally found in a cellular compartment . Recently, the development of SERS active particles have been successfully used as labels or probes in immunoassays, bacteria, and DNA detection [3, 13–18].
The incorporation of nanoparticles through self-assembly mechanism provides an interesting characteristic in nanotechnology application. Core-shell nanoparticles have emerged as an important type of functional nanostructures with promising applications in many fields, especially in health sciences and biomaterials applications. Nanoparticles have also been assembled and bound to functionalized moiety with various mechanisms .
Various nanostructures have been synthesized in diverse formulations and functionalization whether using a single or multiple nanoparticles. For instance, iron oxide nanoparticles have been used for cellular separation and contrast agent. Silver nanoparticles have been used for bactericidal and SERS detection agent. Gold nanoparticles have been used for imaging, photothermal therapy, and LSPR detection. The most common and well investigated metallic nanoparticles, gold nanoparticles hold particularly interesting characteristics. Gold nanoparticles were applied in the significant place in medicine, material sciences, as well as diagnostics for their unique optical and physiochemical properties [1, 20–25]. Their facile synthesis and bio-conjugation procedures, along with its unique surface plasmon properties, made gold nanoparticles practicable in labs without expensive or sophisticated equipment [26–28].
Moreover, gold nanoparticles have also been combined with various nanostructures in order to develop SERS substrate [27, 29]. In this work, the core-shell nanoparticles were developed by functionalized amino-silane around FePt nanoparticles (FePt@SiO2) nanoparticles and then grated the gold nanoparticles onto the surfaces of FePt@SiO2 nanoparticles. Systematic characterizations have been conducted in order to elaborate the morphology and chemical properties of the hybrid nanoparticles. The coverage of the gold nanoparticles and clusters on the surfaces of the silica nanoparticles was evaluated using transmission electron microscopy (TEM). Zeta potential measurement was conducted to evaluate the surface charges of nanoparticles. Furthermore, SERS detection was conducted toward adenine and Staphylococcus aureus to elaborate the Raman enhancement signals using the novel core-shell nanoparticles.
Iron (II) chloride tetrahydrate (FeCl2.4H2O) (>99 %), platinum (II) acetylacetonate, (Pt(acac)2) (>99.9 %), oleylamine (cis-1-amino-9-octadecene (≥98 %), oleic acid (cis-9-octadecenoic acid (>99 %), benzyl ether, cyclohexane (99.5 %)), IGEPAL® CA-520 (octylphenoxy poly(ethyleneoxy) ethanol, branched), tetraethyl orthosilicate (TEOS) (99.99 %), ammonia (≥99 %), N-[3-(trimethoxysilyl) propyl] ethylenediamine (energy-dispersive X-ray spectroscopy (EDS)) (97 %), acetic acid (CH3COOH, ≥99 %), trisodium citrate dihydrate (Na3C6H5O7 · 2H2O, ≥99.5 %), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4 · 3H2O, 99 %), and adenine (C5H5N5, ≥99 %) were purchased from Sigma Aldrich, St. Louis, MO, USA. Ethyl alcohol (ethanol, analytical standard) was purchased from Fluka Analytical, USA. Agar Bacteriological was purchased from Oxoid. Luria-Bertani (LB Broth) was purchased from DifcoTM. High-purity water purified by a Milli Q Plus water purifier system (Milipore, USA), with a resistivity of 18.3 M Ω cm, was used in all experiments. All the chemicals were used without further purification.
Synthesis of Iron Platinum Nanoparticles
Preparation of FePt@SiO2
FePt nanoparticles were encapsulated into the silica nanoparticles through sol-gel processes. Briefly, 10 mL of FePt nanoparticles (26.5 mg/mL) and 8 ml of IGEPAL® CA-520 were mixed with 170 mL cyclohexane under vigorous stirring. Afterwards, 1.3 mL ammonia (30 %) was added slowly to the mixture followed by adding 1 mL triethoxysilane (TEOS), and the reaction was mixed under vigorously stirring for 72 h. After 72 h, the encapsulated particles were mixed with ethanol and centrifuged 4500 rpm for 30 min to remove the excess silica formed during the hydrolysis-condensation processes. The resultant of nanoparticles was separated using a strong neodymium magnet and then dissolve in ethanol. The product termed as FePt@SiO2.
Surface Modification of FePt@SiO2 Using EDS Reaction
The N-[3-(trimethoxysilyl) propyl] ethylenediamine (EDS) was used for surface modification of FePt@SiO2. Briefly, an equal volume of FePt@SiO2 (183 mg/mL) and acetic acid were mixed with EDS under vigorously stirring for 1 h at room temperature which will resulted in amine-functionalized FePt@SiO2 (FePt@SiO2-N). The solution was subject to centrifugation at 4500 rpm for 30 min to remove any traces. The resulting nanoparticles were collected using a strong neodymium magnet and then dissolve in deionized water. FePt@SiO2-N with different concentration of EDS in the range of 0–0.5 M was prepared.
Preparation of Gold Nanoparticles
Gold nanoparticles were prepared through well-established citrate reduction techniques as method described previously with minor modification . In a typical experiment, 103 mL of HAuCl4 0.33 mM was place on a three-neck round-bottom flask on a stirring hot plate. To the rapidly stirred boiling solution, 3.5 mL of a 1 % solution of trisodium citrate dihydrate (Na3C6H5O7.2H2O) was quickly added. The gold sol gradually forms as the citrate reduces the gold (III). It was remove from heat when the solution has turned deep red. The product was termed as gold nanoparticles (AuNPs).
Preparation of Au-FePt@SiO2-N
Gold-surface modified FePt@SiO2 was prepared through the simple mixing of gold solution and FePt@SiO2-N. Briefly, FePt@SiO2-N and Au NPs were mixed under vigorously stirring for 1 h at room temperature. The resulting nanoparticles were then centrifuged at 4500 rpm for 30 min to remove any traces and impurities. The resultant termed as gold nanoparticles-modified FePt@SiO2-N (Au-FePt@SiO2-N). Au-FePt@SiO2-N with different Au concentration in the range of 0–238 μM was prepared.
Structure and morphology of the resultant nanoparticles were characterized by transmission electron microscopy using TEM-8100, Hitachi, at an acceleration voltage of 200 kV. Prior to the TEM observation, an aliquot of suspension of samples was diluted with water until optically clear. Briefly, an aliquot of suspension were placed on a carbon-coated Formvar copper grid (300 mesh, Electron Microscopy Sciences) to form a thin film specimen. The excess of the sample was blotted using a filter paper then air-dried overnight in a dust-free area before loading into the specimen chamber. Elemental analysis was performed using EDS analysis system attached to the same instrument. Zeta (ζ) potential was determined through electrophoretic mobility measurement and calculated using Helmholtz-Smoluchowski’s equation. The zeta (ζ) potential of the samples were determined at 25 °C by using dynamic light scattering (DLS) spectrophotometer, Horiba Instrument, Horiba, Kyoto, Japan. All characterization measurements were repeated three times. The resultant of nanoparticles were further investigated using X-ray photoelectron spectroscopy (XPS) (ESCALAB 250, Thermo VG Scientific, West Sussex, UK) equipped with Mg Kα at 1253.6 eV at the anode to evaluate their chemical binding energy characteristics. Briefly, an aliquot of solution was put on silicon slice and dried overnight. Furthermore, the sample was then placed to XPS chamber and excited with X-rays with a monochromatic Al Kα1,2 radiation. Magnetization as a function of the field was evaluated using a vibrating sample magnetometer (VSM) Lakeshore model 7400 at room temperature.
S. aureus (ATCC 6538P) was used as the model of pathogenic bacteria. Briefly, frozen preserved stock was thawed at room temperature, and then 0.1 mL were pipetted and streaked into a quadrant on a nutrient agar plate. Nutrient agar was composed of 6 g Agar Bacteriological (Agar No. 1) LP0011 powder mixed with 10 g Difco™ LB Broth, Miller (Luria-Bertani) powder and diluted with 400 mL deionized (DI) water and cultured at 37 °C for 18–24 h to allow the formation of colonies. Afterward, a single colony was scraped with a loop and swabbed onto a 15°-slant medium (nutrient agar) and then incubated at 37 °C for 18–24 h.
Surface-enhanced Raman Spectroscopy
Raman spectroscopy was conducted using Raman microscope instrument (HORIBA Jobin Yvon S.A.S.) equipped with laser (λ = 633 nm). The instrument was calibrated against a Si crystal. Prior to the experiment, an aliquot of Au-FePt@SiO2-N solution was mixed equally with the concentrations of adenine (5 × 10−5 M) or S. aureus (5 × 106 CFU/mL) and then homogenized using vortex (VTX-3000 L Mixer Uzusio, LMS Co., Tokyo, Japan). Afterwards, the mixed suspension was dropped wisely into the alumina chip followed by drying in free dust-air atmosphere.
Results and Discussion
Structure and Morphology of FePt Nanoparticles
Physicochemical Properties of FePt@SiO2, FePt@SiO2-N, and Gold Nanoparticles
Structure and Morphology of Au-FePt@SiO2-N
Figure 4b shows the zeta potentials of Au-FePt@SiO2-N with various gold concentrations. It revealed that the surface charged of the core-shell nanoparticles becomes negatively charged after gold immobilization by zeta potential measurement. This might be that the Au-trisodium citrate dihydrate nanoparticles coverage of FePt@SiO2-N develops the net negatively charged nanoparticles, which further prove the successful immobilization of Au nanoparticles onto the surface of FePt@SiO2-N. Figure 4c displays the region of the XPS spectra corresponding to Au-4f. Pristine gold nanoparticles exhibited binding energy of Au 4f7/2 and Au 4f5/2 at 83.4 and 87.1 eV, respectively. These peaks were shifted to 83.1 (Au-4f7/2) and 86.7 eV (Au-4f5/2) in the Au-FePt@SiO2-N. The shifted peaks further confirmed the reaction between gold (Au) nanoparticles and FePt@SiO2-N nanoparticles through electrostatic interaction.
SERS Detection of Adenine
SERS Detection of Bacteria (S. aureus)
We fabricated multifunctional core-shell nanostructures of gold nanoparticles-decorated silica encapsulated FePt (Au-FePt@SiO2-N), which could be developed in the SERS detection, such as small biomolecules (adenine) and pathogenic bacteria (S. aureus). SERS spectra of adenine and S. aureus obtained from the Au-FePt@SiO2-N core-shell nanoparticles showed finger-print peaks with significant enhancement on the Raman signal. Furthermore, the magnetic core of FePt nanoparticles could be applied in the rapid magnetic separation of the biomolecules and bacteria and achieve the condensed effect. Therefore, the novel SERS platform of core-shell structure of Au-FePt@SiO2 nanoparticles (Fig. 8) would provide the potential to apply in the magnetic separation and bio-sensing at the same time, which could save the diagnosis time significantly.
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 the Academia Sinica. Technical support of VSM from Nano group Public Laboratory, Institute of Physics, at Academia Sinica in Taiwan, is acknowledged.
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