Burcu
Guven
a,
İsmail Hakkı
Boyacı
*a,
Ugur
Tamer
b and
Pınar
Çalık
c
aDepartment of Food Engineering, Faculty of Engineering, Hacettepe University, Beytepe, Ankara, 06800, Turkey. E-mail: ihb@hacettepe.edu.tr; Fax: +90 312 299 21 23; Tel: +90 312 297 61 46
bDepartment of Analytical Chemistry, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey
cDepartment of Chemical Engineering, Industrial Biotechnology and Metabolic Engineering Laboratory, Middle East Technical University, 06531, Ankara, Turkey
First published on 3rd November 2011
In this study, a new method combining magnetic separation (MS) and surface-enhanced Raman scattering (SERS) was developed to detect genetically modified organisms (GMOs). An oligonucleotide probe which is specific for 35 S DNA target was immobilized onto gold coated magnetic nanospheres to form oligonucleotide-coated nanoparticles. A self assembled monolayer was formed on gold nanorods using 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and the second probe of the 35 S DNA target was immobilized on the activated nanorod surfaces. Probes on the nanoparticles were hybridized with the target oligonucleotide. Optimization parameters for hybridization were investigated by high performance liquid chromatography. Optimum hybridization parameters were determined as: 4 μM probe concentration, 20 min immobilization time, 30 min hybridization time, 55 °C hybridization temperature, 750 mM buffer salt concentration and pH: 7.4. Quantification of the target concentration was performed via SERS spectra of DTNB on the nanorods. The correlation between the target concentration and the SERS signal was found to be linear within the range of 25–100 nM. The analyses were performed with only one hybridization step in 40 min. Real sample analysis was conducted using Bt-176 maize sample. The results showed that the developed MS-SERS assay is capable of detecting GMOs in a rapid and selective manner.
Detection and identification of GMOs are gaining worldwide attention due to the expansion of the GMOs. The detection and identification of GMOs have been generally achieved with DNA- and protein-based methods. Protein-based methods are unable to detect a genetic modification if the modified gene is inactive in the cells.1 DNA-based methods for detecting GMOs are based on the complementaries of two strands of DNA double helix that hybridize in a sequence-specific manner.4
Since its discovery, SERS has received more and more attention from researchers around the world,5,6 not only because of its high sensitivity, the small volume of sample needed,7 and the possible wide applicability8,9 but also due to the stable and specific signal.10 The SERS detection of various biomolecules or species has been reported, such as proteins, enzymes, viruses, bacteria, cancer markers, and nucleotides.11 DNA can show well-distinguished SERS bands while the sugar or phosphate groups on the backbone have little interference. However, the direct detection of DNA sequence at low concentration is difficult with SERS.12 The resulting spectrum strongly depends on the sequence of the bases,13 and also on the binding modes of DNA strands,14 which results in difficulty to identify the DNA sequence. Additionally, the methodologies used for DNA recognition are usually based on the hybridization of the target nucleic acid with its complementary bases, as there is a little difference between their Raman spectra.11 Therefore, reliable and reproducible SERS active substrates are needed to label the DNA15 such as cresyl fast violet,16 Rhodamine B,17 Rhodamine 6G and others.18 Some metal nanoparticles have also been conjugated with DNA and used to enhance the Raman intensity.19,20
Nanoparticle-based Raman tags have also advantages in comparison with other nanoparticle tags. Nanoparticle-based Raman tags have been developed by some research groups.21–24 There are two kinds of nanoparticle-based Raman tags reported in the literature, one of which is prepared by the direct attachment of both a Raman reporter and biomolecule to nanoparticle; whereas the other is prepared by the dye-embedded core-shell nanoparticle-based Raman tag.25
Magnetic nanoparticles are among the most frequently used as Raman tags. Magnetic nanoparticles have become interesting tools for biological molecules and cells. They have been used for concentration, separation, purification and identification of molecules and cells26 besides immobilization of proteins and enzymes, bioseparation, assay, drug delivery and biosensors. The magnetic nanoparticles enable the isolation or extraction of a target molecule or substance by the use of an external magnetic field. The usage of magnetic nanoparticles not only possesses the advantage of superparamagnetism, but also preconcentrates the analyte in order to amplify the Raman signal efficiently.27 In this study, we have demonstrated that the magnetic nanoparticles could can be used for developing a reliable and selective SERS based homogeneous sandwich assay. The DNA probes were immobilized on spherical magnetic gold nanoparticles and rod shaped gold nanoparticles and hybridized with the target oligonucleotide. Rod shaped nanoparticles were used as SERS reporters. Then the SERS signals were obtained and the calibration curve was plotted to measure the different concentrations of target oligonucleotide. Optimum hybridization parameters were determined by using high performance liquid chromatography. The selectivity and specificity tests of the developed assay were examined with positive and negative controls. Finally, this assay was applied to the 35 S sequence of Bt-176 maize sample. The analytical performance of the SERS based sandwich assay system with respect to linear range, detection limit, response time, and selectivity is presented and discussed.
PCR buffer, MgCl2, dNTPs, Taq DNA polymerase and forward primer were obtained from Fermentas Inc. (Vilnius, Lithuania) and oligonucleotides were purchased from Alpha DNA (Quebec, Canada). The sequences are as shown in Table 1.
Probe 1 | 5′-NH2-AAA AAT CGG CAG AGG CAT-3′ |
Probe 2 | 5′-CGA TGG CCT TTC CAA AAA-NH2 -3′ |
Target | 5′-GGA AAG GCC ATC GTT GAA GAT GCC TCT GCC GA-3′ |
Nonsense Sequence | 5′-CAT TTT GGA CAA AGC GTC TAC GCT GCA G-3′ |
Forward Primer p 35 S-cf3 | 5′-CCACGTCTTCAAAGCAAGTGG-3′ |
Reverse Primer | 5′-NH2-AAAAATCGGCAGAGGCAT-3′ |
After and before the NH2 groups, five adenine bases of DNA probes were added as spacer parts. Bt-176 maize DNA template was obtained from Central Laboratory of Middle East Technical University (Ankara, Turkey).
PCR products were analyzed using a gel documentation system by UVP BioImaging System (UVP, Inc., Upland, CA) and pre-cast gels were stained with ethidium bromide. The quantity of products was analyzed using a micro volume spectrophotometer (NanoDrop, Wilmington, DE).
Fig. 1 Schematic illustration of MS-SERS based sandwich assay for the target oligonucleotide. |
The surfaces of the gold coated magnetic nanospheres were modified with 150 mM 11-MUA in absolute ethanol overnight to form the SAM. The nanoparticles were collected by using a permanent magnet and washed with 0.05 M MES (pH 6.5) buffer. For surface activation over the carboxyl groups, the nanoparticles were treated with 1 mL of EDC/NHS solution for 40 min. The nanoparticles were separated magnetically and were washed twice with 0.05 M MES buffer solution. After this procedure, the nanoparticle surfaces were immobilized with DNA probes by incubating for 20 min to form a covalent bond between the amino group of the probe and the carboxyl group of the nanoparticles. The nanoparticles were collected with a magnet and washed twice with MES buffer. For avoiding non-specific interaction 1% (v/v) ethanolamine was used for blocking active groups on the surface of the nanoparticles.
The surfaces of the gold nanorods were modified with 50 mM DTNB in absolute ethanol overnight to form a SAM. The nanoparticles were collected by centrifuge and washed with 0.05 M MES buffer. Similar with the magnetic nanoparticles, surface activation of the gold nanorods, probe immobilization and then blocking of the particle surfaces were performed. After each step, the washing procedure was applied twice by MES buffer.
At the hybridization step, the probe coated magnetic nanospheres and probe coated nanorods were mixed, the mixed solution was centrifuged to separate the liquid phase and then the target oligonucleotide solution was added to the mixed nanoparticle-probe complex. The hybridization step was performed at 55 °C in PBS solution for 30 min to form a probe-target conjugation. Then, the washing procedure was performed with PBS and PBST buffers. In all washing steps, nanoparticles were dispersed in liquid media using an ultrasonic bath.
The immobilization and hybridization parameters such as probe concentration, immobilization time, hybridization time, hybridization temperature, buffer salt concentration and pH were optimized by using HPLC. The probe oligonucleotide was immobilized on the gold coated magnetic nanospheres. The effects of the probe concentration and probe-nanoparticle immobilization time were determined. The gold coated magnetic nanospheres were mixed with the probe solutions at concentrations between 2 and 10 μM. Nanoparticles were mixed with the probe solutions at immobilization times between 1 and 45 min. The target-probe complexes were hybridized between 10 and 60 min. Furthermore, the target-probe complexes were hybridized between 25 and 95 °C. The buffer salt concentration and pH parameters were also examined in the range of 150–1500 mM and 6.5–8.2, respectively. In order to eliminate non-specific interaction, the washing procedure was modified after hybridization. The modified washing procedure was performed 3 times with PBS, 3 times with PBST, and 3 times with PBS, respectively.
The specificity of the developed assay was evaluated by negative and positive control assays. The negative control was performed by interaction of probe 1 and probe 2 without target oligonucleotide sequence. Two positive controls were done with the target oligonucleotide sequence. There were interactions of probe 1 and target (without probe 2) and probe 2 and target (without probe 1). The selectivity of the assay was examined with a nonsense sequence. The accuracy of the developed method was investigated with the Bt-176 maize genome which was isolated by the Gene Amp 2400 method.31 The forward primer p 35 S-cf3 and the reverse primer probe 1 of the 35 S regions were used. PCR amplification was carried out in 50 μL 1x PCR buffer (pH 8.3). The contents of the PCR buffer were MgCl2 (2.5 mM), dNTPs (0.2 mM), forward and reverse primers (0.5 μM), Taq DNA polymerase (0.025 U/μL) and Bt-176 maize DNA template (2 μL % 0.5). Amplification was achieved with an initial denaturation step at 95 °C for 4 min followed by a 40 cycle process that includes a denaturation step for 1 min at 95 °C, an annealing step for 1 min at 62 °C, an extension step for 1 min at 72 °C, and a final extension at 72 °C for 10 min. Dilution of the PCR product was prepared. The amount of diluted PCR product was analyzed using the developed MS-SERS assay. The PCR product was denatured at 94–96 °C to obtain a single strand in the presence of the probe immobilized gold coated magnetic nanospheres and gold nanorods. Hybridization was performed at 55 °C for 30 min, then the washing procedure was applied and the SERS measurement was performed. The target sequence concentration was calculated using the SERS intensity and calibration graph. They were also quantified using a micro volume spectrophotometer and the results of the methods were compared.
Experiments were performed in triplicate, average and standard deviations were calculated, and data analysis was performed using Microsoft Excel®.
Non-spherical gold nanorod particles act as electromagnetic hot spots for SERS owing to the facile tunability of the plasmonic properties and presence of sharp corners and edges. Such anisotropic nanoparticles display strong polarization dependence in their plasmonic properties, exhibiting significantly higher SERS intensity with certain orientations. In addition, the degree of surface plasmon coupling increases at the gaps or junctions of nanoparticles with the creation of hot spots. Based on these considerations, creating hot spots using magnetic nanoparticle-nanorod interactions results in high sensitivity and selectivity of the developed systems.35
Fig. 2 Optimization parameters (a) immobilization concentration, (b) immobilization time, (c) hybridization time, (d) hybridization temperature, (e) buffer salt concentration, (f) buffer salt pH. |
As it can be seen from the published studies, the immobilization times change between 15 min36 to 30 min.37 The immobilization time of the developed method was shown to be one of the shortest methods. The hybridization time for the nanoparticle based electrochemical DNA analysis method, absorbance based, SERS based and fluorescence based methods were found as 20 min,38 40 min,39 60 min25 and 120 min,40 respectively. The hybridization temperature was chosen as 55 °C in most studies.39,41 Tsuruoka and Karube42 showed that high salt concentrations have accelerated the hybridization process. The optimum buffer salt concentration was found as 750 mM in our study. Hybridization buffer salt concentrations were given as 600 mM25 and 1000 mM43 in published studies. Also the hybridization buffer pH was chosen as 725,44 and 7.4.43
Fig. 3 Symmetric NO2 stretching bands of DTNB range from 1000 nM to 0 nM target concentration obtained with rod shaped nanoparticles (a) 1000 nM, (b) 500 nM, (c) 100 nM, (d) 75 nM, (e) 50 nM, (f) 25 nM (g) 0 nM target concentration. |
The spectra contain features which are attributable to the Raman label (reporter molecule) and are dominated by bands representative of the DTNB-based adlayer (e.g., the symmetric nitro stretch (Vs (NO2)) at 1370 cm−1. The detection is based on the characteristic feature of the Raman tag and then quantified by its intensity. The calibration curve was plotted with the changes of the band intensities of DTNB vs. the different concentrations of target oligonucleotide (0–1000 nM). As shown in Fig. 4, the intensities of the Raman spectra were obtained in the presence of different concentrations of target oligonucleotide. A good linear correlation (R2 = 0.998) was obtained within the range of 25–100 nM target oligonucleotide concentration. The sensitivity of the developed assay was investigated and the LOD and LOQ of the proposed method for the target were found to be 11 nM and 34 nM, respectively.
Fig. 4 Calibration curve for the target oligonucleotide in the range of 0–1000 nM. |
The specificity of the developed assay was evaluated and the band intensities of the negative and positive controls were obtained. The selectivity test was performed using the nonsense sequence. Intensities obtained from the specificity and selectivity tests were compared with the intensities of the LOD and LOQ concentrations (Fig. 5). Shown in Fig. 5, the band intensities of the specificity and selectivity test data were lower than the intensity of the LOD value. It means that the developed MS-SERS assay is reliable and selective to only the target oligonucleotide. When the band intensities of the target oligonucleotide (Fig. 3) were compared with the specificity and selectivity data (Fig. 5), the intensities that belong to the target oligonucleotide were found to be much greater.
Fig. 5 Evaluation of the specificity and selectivity of the developed method (a) without probe 1, (b) without target, (c) without probe 2, (d) nonsense sequence, (e) intensity at LOD concentration, (f) intensity at LOQ concentration. |
The amplification of the Bt-176 maize sample was performed with PCR. In order to observe the variation of intensity, different concentrations of PCR product were obtained and analyzed with the developed assay and also with NanoDrop. The concentrations were found to be 27.0 ± 0.21 nM and 81.0 ± 0.43 nM by NanoDrop. The concentrations measured by using the developed MS-SERS assay are 28.2 ± 4.3 nM and 91.8 ± 5.4 nM. As a comparison, similar results were obtained with NanoDrop and the proposed assay system. As a consequence, it is said that the proposed method could be applied in real samples.
The working range of the developed assay is between 25 and 100 nM with a detection limit of 11 nM and the assay time is less than 40 min including the hybridization procedure (30 min) and SERS measurement (less than 10 min). The detection limit in SERS based methods varies between 10 nM,45 100 nM43 and 10 pM,46 20 fM.18
As a comparison, it is shown that the hybridization time of the developed assay is quite short. The hybridization procedure was performed with different reaction times such as 2–3 h at room temperature47 40 min,48 60 min,49 1 h at 35 °C50 and microarray technology requires a long hybridization time (about 1–2 h) due to the diffusion-limited hybridization kinetics.51,52 Magnetic separation presents a time saving replacement and allows the flexibility of applying different end point detection methods. Furthermore, nano-sized magnetic particles have advantages due to their large surface area and nanorods can be produced at any desired size and shape.34 The reason of using gold nanorods as Raman labels instead of nanospheres is that the Raman intensity of nanorods is higher than nanospheres. It is thought that using nanorods would increase the sensitivity of developed method.29
Moreover, it was demonstrated that the method can be easily used for the determination of PCR product quantitatively. The developed assay could be advantageous over other methods because the PCR product used in this assay is double strand DNA, and it is necessary to convert it into single strand for denaturation before detection. In addition, the denaturation temperature is around 95 °C and this temperature could cause an undesirable change on the magnetic and SERS labeled nanoparticles. Although there are so many works published on related developments of DNA assays, the effects of high temperature on the analysis performance are not mentioned so far.25,53 The functional stability of the nanoparticles at high temperature could give us an opportunity to perform the assay with only one hybridization step. This opportunity would reduce the analysis time and washing procedure. A sandwich assay was performed to investigate DNA interactions and it is observed that SERS spectra were not influenced by high temperatures which are required for the denaturation step of double strand DNA. The most important point of the developed method is that the hybridization process was perfomed in one step, and the real sample was detected in a short analysis time.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c1an15629b |
This journal is © The Royal Society of Chemistry 2012 |