Fabrication and Characterization of Gold Nano Particles for DNA Biosensor Applications
*Ahmed Mishaal Mohammed 1, 2
1 Chemistry Department, College of Science, Anbar University Ramadi, Iraq.
2 Institutes of Nano Electronic Engineering, University Malaysia Perlis, 01000 Kangar, Perlis, Malaysia
Corresponding author: Ahmed Mishaal Mohammed
E-mail address: [email protected]
ARTICLE INFO ABSTRACT
Received in revised form
Available online This research is concerned with the preparation of biosensing using silicon oxide in biomedicine application, effectively used for the detection of target DNA hybridization. An electrochemical DNA biosensor was successfully fabricated by using (3-Aminopropyl) tri ethoxysilane (APTES) as a linker molecule combined with the gold nanoparticles (GNPs) on thermally oxidized SiO2 thin film. The GNPs size was calculated by utilizing the UV-Vis data and the average calculated particle size was within the range of 30±5 nm, characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The GNPs modified SiO2 thin films were electrically characterized through the measurement of capacitance, permittivity and conductivity, using a low-cost dielectric analyzer. The capacitance, permittivity and conductivity profiles of the biosensor clearly DNA immobilization and hybridization.
Nanotechnology has become increasingly popular because of their unique physical, chemical, optical and catalytic properties compared to their bulk counterparts. Nanotechnology revolutionizes many technology and industry sectors and medical instrumentation, homeland security, and many others [1-3]. Currently, the detection of DNA is an area of great interest as it is the key feature in the research for specific nucleotide sequences of DNA detection. This technique plays an important role in biodiagnostics , determination of genetic diversity , criminal investigation in forensics and immigration , food analysis [7,8] and environmental monitoring . Currently, a variety of high selectivity and sensitivity of DNA detection such as polymerase chain reaction (PCR) , terminal-restriction fragment length polymorphis (T-RFLP) , chromatography in tandem with mass spectrometry  and surface plasmon resonance (SPR)  are widely studied. However, these techniques have their limitation including long assay time, labour complexity and high cost. Therefore, a variety of approaches for the detection of DNA which overcome those weakness have been actively studied, such as electrochemical sensing , fluorescence , bio-field effect transistor (Bio-FET)  and oligonucleotide microarray and DNA . Among these techniques, electrochemical DNA biosensor with its simplicity, good sensitivity, low cost, and possible miniaturization has attracted significant research interest.
Gold nanoparticles (GNPs) have gained considerable care in recent years for potential applications in nanomedicine due to their interesting size dependent electronic, chemical and optical properties. Also, gold nanoparticles shows make a promise in enhancing the effectiveness of various aimed cancer treatments such as photo thermal therapy and radiotherapy . Nanoparticles have been synthesized by a wide variety of techniques such as pulsed laser deposition , chemical reduction , flame metal combustion , electrolysis , electrochemical reduction , solvothermal , photo-reduction , sono-electrochemical , microwave-induced , green method , aerosol flow reactor , spray pyrolysis , chemical fluid deposition , photochemical reduction  , and spark discharge .
Gold nanoparticles offer some advantages such as high surface-volume ratio, optical properties, chemical stability and robustness. It is successfully used in the modification of semiconductor nanostructure for biological diagnostic . GNPs are also widely applied in bio-labeling, self-assembled monolayer (SAM), electron-transfer theories and immunoassays [33,34] and applied in various kind of bimolecular immobilization, such as DNA , proteins  and enzymes .
The detection relies on the immobilization of single stranded DNA (ssDNA) probes that are complementary and specific for a DNA sequence of the pathogenic target, on gold nanoparticles (GNPs) (Fig. 2). GNPs are used to report the immobilization and hybridization. GNPs are used here because of their ease of production and functionalization [37, 38]. The nanoparticles conjugated with ssDNA probes specific for the pathogenic target of interest are then hybridized with the DNA test sample, isolated using magnetic separation, and detected through electrochemical analysis [39, 40-42]. While biosensors using this detection strategy that has been shown to detect specific DNA fragments from various pathogens [43-45], many of these biosensors have only been tested for the detection of purified and gold nanoparticles amplified DNA targets. Similar to some of the limitations of commercially available detection strategies, GNPs is often criticized for its complex, expensive, time-consuming, and labor-intensive procedure requirements.
consequently, the need of GNPs for biosensor detection of pathogenic DNA is greatly restrictive for both field-based and resource limited settings, resulting in the increased need for GNPs independent biosensor detection methods. The scope of this work, we have developed, an easy to fabricate and low cost using GNPs/APTES/SiO2/Si/Ti which were acted as the electrode. Immobilization and hybridization of DNA were performed using electrochemical detection with potassium hexacyanoferrate for sensitivity of the DNA detection.
2.1 Preparation of GNPs solution
Gold nanoparticles (GNPs) were used in this project for the immobilization and hybridization of the DNA on the SiO2 thin films. Firstly, a HAuCl4 solution with the concentration of 0.49 mol/L was prepared by dissolving 500 mg of HAuCl4 into 3 ml of 10% HCl. Then, a diluted 0.2 mM of HAuCl4 solution was made by adding 40 µL (19.6 µmol) of HAuCl4 solution into 100 ml of deionized water as to produce solution A. Secondly, 558.79 mg of Trisodium Citrate was added into 50 ml of deionized water to make solution B. The concentration of the solution was controlled at 38.8 mmol/L. Solution A was brought to a rolling boil at 150°C with stirring vigorously as to get a homogenous size of the GNPs solution. 10 mL of 38.8 mM of sodium citrate was added rapidly into the vortex of the solution. The solution resulted in a color change from pale yellow to red. Boiling and stirring was continued for another 10 min. The heating was then removed, and stirring was continued for an additional 15 min. When the solution cooled down to room temperature, it was filtered through a 0.8 µm membrane filter paper. The prepared solution was kept in the refrigerator with the temperature 4ºC and measured by using UV-Vis with the wavelength 400 nm to 800 nm, TEM and AFM.
2.2 Modification of SiO2 with GNPs
A p-type silicon (100) wafer (1 cm × 1 cm) was cleaned by using acetone and isopropanol in ultrasonic for about 15 minutes and was immersed into the buffered oxide etch (BOE) solution and washed with deionized water followed by oxidation process for 30 minutes. After oxidation, the silicon oxide (SiO2) layer of thickness ~50 nm, the titanium was deposited on the backside of the Si using thermal evaporator. The selectivity of the DNA biosensor was studied using the GNPs/APTES/SiO2/Si/Ti electrode. The SiO2 surface was functionalized with APTES solution which was prepared by mixing of 2% APTES with 93% of ethanol and 5% of deionized water. The silanyl group (-SH3) presented in APTES was used for the process of silanization, which was chemically attached with the hydroxyl-rich SiO2 . Besides that, amino group (NH2-) presented in APTES was served as a glue layer to attached the GNPs which linked to probe DNA. Attachment between APTES and GNPs is shown in (Fig.1). For the surface modification of SiO2 with APTES, 10 µl of prepared APTES solution on the SiO2 surface and incubated for 2 hours. Then, the surface washed for 3 times in blow dried the surface and drop 10 µl of GNPs on the surface at 150°C for 20 min on hot plate. This step was repeated 3 times to obtain enough GNPs on the SiO2 surface and electrode to be ready for electrical characterization.
2.3 Probe DNA immobilization on modified GNPs
The oligonucleotides used in this project were purchased from 1st BASE Pte Ltd (Malaysia) and are shown in (Table 1). Probe DNA was dropped onto the GNPs modified SiO2 electrode for immobilization incubated at room temperature for 2 hours. After period times, the electrode was carefully washed by using deionized water to remove any un-bonded DNA probe and dried at room temperature. The probe modified devices were denoted as DNA/GNPs/APTES/SiO2/Si/Ti, then ready for electrochemical measurements.
2.4 Target DNA hybridization detection
Hybridization of DNA used in this project was purchased from 1st BASE Pte Ltd (Malaysia) and are shown in (Table 1). Hybridize DNA, 10 µl of 10 µM complementary DNA was dropped onto GNPs electrode and incubated for 2 hours. After that, the GNPs electrode was washed by using deionized water to remove any non-hybridized DNA and dried at room temperature. 10 µl of 0.5 µM methylene blue then dropped onto the GNPs electrode and incubated again for 3 minutes. Finally, the GNPs electrode will once again wash with deionized water to remove any excess of methylene blue and the GNPs electrode is ready to be electrical measured once it has dried.
2.5 Electrochemical characterization by cyclic voltammetry
Electrochemical measurement was performed by using dielectric analyser. The tests were conducted by using Ag/AgCl as the reference electrode and GNPs modified electrode as a working electrode. The Ti act as a back gate. The responses of the DNA immobilization and hybridization were investigated in 10 µM Potassium Hexacyanoferrate III, K3Fe(CN)6 aqueous solution containing 0.1 M KCl as electrolyte. A schematic view of testing measurement for DNA detection is shown in (Fig.2).
The morphology of the GNPs was characterized using a transmission electron microscopy (TEM, Libra 120-Carl Zeiss). Ultraviolet-visible (UV-Vis-NIR, Perkin Elmer lambda 950) spectroscopy was then used to study the optical properties of the GNPs at room temperature and atomic force microscopy (AFM) was used to study the structural properties of the GNPs. DNA immobilization and hybridization was tested using a dielectric analyzer (Alpha-A High Performance Frequency Analyzer, Novocontrol Technologies, and Germany).
3 Results and discussion
3.1 Measurement of UV-Vis spectroscopy
The characterization of prepared GNPs solution was examined using an UV-Vis spectroscopy. The measurements were carried out within the wavelength range of 400-800 nm under ambient conditions and the result is shown in (Fig. 3). The absorbance maximum was found at 530 nm, which was indicative of GNPs of diameter 30 ± 5 nm . The particle-size was further confirmed using the method described by Haiss et al.  shown in equation (1).
Where d = particle diameter; λspr = peak position of Au-nanoparticles, λο = 512, L1 = 6.53, L2 = 0.0216. From equation (1), the obtained nanoparticles size was around 28 nm in diameter.
3.2 Characterization of surface morphology
Morphology and microstructure of the GNPs were investigated by transmission electron microscopy (TEM). (Fig. 4) shows typical surface morphologies of GNPs as obtained from TEM. The GNPs can be clearly recognized without showing any preferred direction. The GNPs have structure which is a typical feature of relatively 20-30 nm in diameter.
3.3 Atomic force microscopy (AFM)
AFM demonstrated the particle size and structure of GNPs. (Fig. 5) shows AFM images which present a two-dimensional (2-D) and three-dimensional (3-D) view of the surface structure of the GNPs. The images confirmed that the GNPs have a roughness surface of 228.89 nm and small particles size distribution.
3.4 Capacitance measurement
Previous studies documented that the dielectric properties of ssDNA is different from those of dsDNA, especially in low frequency region . Therefore, we proceeded to detect target DNA hybridization through the measurement of dielectric properties of the Au-modified SiO2 thin films. The change in capacitance before and after hybridization of the DNA at different frequencies was measured using a dielectric analyzer. The measurements were carried out at frequency range of 1Hz to 1MHz. Before the detection of DNA targets, dielectric properties of Au-doped SiO2 thin films were measured to draw the base line for the further detection of DNA targets. The capacitance measurement was also performed when the ssDNA-probe was immobilized onto the Au-doped SiO2 thin films. For hybridization, complementary was tested on the same device. The results are demonstrated in (Fig. 6). It was clearly observed that the capacitance values of the bare, GNPs-modified surface and immobilization and target DNA hybridization were 47 × 10-12 F, 41 × 10-8 F, 21 µF and 14 µF, respectively at 1 Hz. The capacitance value for GNPs-modified surface is higher than bare device. The immobilization and hybridization of DNA was successfully detected by showing the highest capacitance values of 21 µF and 14 µF respectively onto the modified GNPs electrode. The capacitance value between GNPs-modified surface and DNA immobilization and hybridization had presented the successful of using GNPs on thermally oxidized SiO2 thin film for DNA detection. Vetrone et al.  investigated the ability of a gold nanoparticle-DNA (AuNP-DNA) biosensor to detect non-PCR amplified genomic Salmonella enterica serovar Enteritidis (S. enteritidis) DNA, from pure or mixed bacterial culture and spiked liquid matrices. Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry. Reyes et al.  report a ZnO-nanostructure-based quartz crystal microbalance (nano-QCM) device for biosensing applications. ZnO nano tips are directly grown on the sensing area of a conventional QCM by metalorganic chemical vapor deposition (MOCVD). The selective immobilization and hybridization of DNA oligonucleotide molecules are confirmed by fluorescence microscopy of the nano-QCM sensing areas.
3.5 Permittivity measurement
The permittivity measurements were also performance on the same device as shown in (Fig. 7). These measurements have the same direction with the capacitance measurement whereby it gives the largest changes in permittivity with complementary targets and probe DNA immobilization. However, it clearly demonstrated that permittivity measurement starts to increase dramatically from 343×101 to 193×103 and 155×103 at the frequency range of ~200 Hz to 1 Hz for bare and DNA immobilization and hybridization respectively whereas the capacitance measurements profile started to significantly increase from a frequency of ~1 Hz and tend as the frequency increases. The result revealed that permittivity measurement giving more sensitivity at lower frequency during hybridization. This work demonstrated changes in capacitance and permittivity value of the GNPs-modified electrode during probe DNA immobilization and hybridization ensures the presence of the DNA during the measurement using GNPs electrode.
3.6 Conductivity measurement
Conductivity measurements were also carried out to investigate the effect of probe DNA immobilization and target DNA hybridization on the GNPs-modified SiO2 thin films. The measured conductivity values for bare, the GNPs-modified SiO2, immobilized and hybridization device were (3.9×10-13, 2.1×10-9, 1.2×10-7 and 1.1 ×10-7) S-cm-1, respectively. It can be observed from (Fig. 8) that probe DNA immobilization and target DNA hybridization on the GNPs-modified SiO2 thin films, conductivity was increased on the bare electrode; therefore the resistivity of the device was decreased. This might be due to a strong interaction occurred between potassium hexacyanoferrate III, K3Fe(CN)6 and the unpaired guanine base in the probe DNA . Furthermore, the differences value of conductivities between GNPs-modified surface and probe DNA immobilization and target DNA hybridization had shown the electron transfer occurred during the immobilization of DNA and target DNA hybridization. Therefore, the conductivity measurement had confirmed the reaction of the DNA immobilization and target DNA hybridization onto GNPs-modified surface were realized in the electrolyte solution.
A DNA biosensor was successfully fabricated using gold-nanoparticles (GNPs) modified SiO2 thin films with IDE electrodes. The gold-nanoparticles were synthesized through a low-cost and easily performable technique and GNPs modification on SiO2 thin films was performed using a low-cost doping process. The fabricated biosensor successfully differentiated the detection of DNA immobilization and hybridization through the measurement of dielectric and conductivity properties in a label free approach, suggesting its applications in low-cost bio diagnostics, forensic testing, food analysis and environmental monitoring.
The work was supported by INEE at (UniMAP), through the Nano Technology project therefore, the authors thanked and wished to acknowledge the Institute of Nano Electronic Engineering (INEE) at University Malaysia Perlis (UniMAP) for supporting this work.
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