Synthesis of Silica-Coated Magnetic Nanoparticles and Application in the Detection of Pathogenic Viruses

Magnetic Fe 3 O 4 nanoparticles were prepared by coprecipitation and then coated with silica. These Fe 3 O 4 /SiO 2 nanoparticles consisted of a 10–15 nm magnetic core and a silica shell of 2–5 nm thickness. The superparamagnetic property of the Fe 3 O 4 /SiO 2 particles with the magnetization of 42.5 emu/g was confirmed by vibrating sample magnetometer (VSM). We further optimized buffers with these Fe 3 O 4 /SiO 2 nanoparticles to isolate genomic DNA of hepatitis virus type B (HBV) and of Epstein-Barr virus (EBV) for detection of the viruses based on polymerase chain reaction (PCR) amplification of a 434 bp fragment of S gene specific for HBV and 250 bp fragment of nuclear antigen encoding gene specific for EBV. The purification efficiency of DNA of both HBV and EBV using obtained Fe 3 O 4 /SiO 2 nanoparticles was superior to that obtained with commercialized Fe 3 O 4 /SiO 2 microparticles, as indicated by (i) brighter PCR-amplified bands for both HBV and EBV and (ii) higher sensitivity in PCR-based detection of EBV load (copies/mL). The time required for DNA isolation using Fe 3 O 4 /SiO 2 nanoparticles was significantly reduced as the particles were attracted to magnets more quickly (15–20 s) than the commercialized microparticles (2-3 min).


Introduction
Isolation of nucleic acids from clinical samples is an essential step in diagnostics, such as in the detection of pathogenic viruses and bacteria using the polymerase chain reaction (PCR), paternity testing, DNA fingerprinting for crime detection, and DNA sequencing. The nucleic acid isolation method based on interaction with silica, created by Boom et al. in 1990 [1], is currently the most commonly used. Recently, micrometer-size silica-coated magnetic beads have been developed by different groups [2,3] and biotech companies such as Roche Diagnostics, Life Technologies, Beckman Coulter, and Promega to improve the efficiency of purification and save working time because the purification procedures could be performed automatically. This development is very useful for enrichment of nucleic acids in clinical samples with a low copy number of the pathogen, tuberculosis bacterium in sputum, for example, thereby increasing the sensitivity of detection using PCR method [4]. In comparison to the micrometer-size silica-coated magnetic beads, silica-coated magnetic nanoparticles have larger total surface area, thus could be more functional in purification of DNA from samples. However, the synthesis of silicacoated magnetite nanoparticles for specific applications in nucleic acid purification of viruses in blood, which is a very important step in the diagnosis of viruses in specimen, is an emerging area. We therefore present a method for synthesizing the SiO 2 -coated Fe 3 O 4 magnetic nanoparticles and use the particles in the isolation of DNA of Hepatitis virus type B (HBV) and Epstein-Barr virus (EBV) from several real serum samples. These viruses are commonly found in blood infection and the cause of hepatitis (HBV) and particular cancers and lymphomas (EBV) [4,5].

Synthesis of Silica-Coated Magnetic
Nanoparticles. Magnetic Fe 3 O 4 nanoparticles were synthesized by using coprecipitation from iron (III) chloride and iron (II) chloride solutions with the assistance of aqueous ammonia solution as described elsewhere [6]. The synthesized magnetic nanoparticles were washed several times with alcohol and then distilled water until pH 7.0. Fe 3 O 4 /SiO 2 nanoparticles were prepared by coating magnetic nanoparticles with silica. 100 mL of the suspension of prepared magnetic nanoparticles (containing 1 g magnetite) was stored in a flask. 50 mL of the 10% solution of aqueous tetraethylorthosilicate (TEOS) was added to the flask together with 70 mL alcohol and mixed using an overhead stirrer. The pH of the suspension was adjusted to 9.0 with NaOH. The flask was then heated to 90 ∘ C and stirred at this temperature for 6 h. After cooling to room temperature (RT), the suspension was washed twice with alcohol and six times with distilled water. The final suspension volume was adjusted to 100 mL with water.
For further experiments in nucleic acid isolation, Fe 3 O 4 /SiO 2 nanoparticles were stored in 100 mM Tris-HCl buffer (pH 7.0). For each DNA isolation reaction from 200 L serum sample, 50 L of Fe 3 O 4 /SiO 2 nanoparticles at a concentration of 25 mg/mL was used.

Primer Design and Cloning of Plasmids
Harbouring Specific Genes for HBV and EBV as Standards for PCR. The primers for HBV (HBSF: 5 -CTTTCATCCTGCTGCTATGCCT; HBSR: 5 -AGGGTTCAAATGTATACCCAAAGACA-3 ) were designed based on previous work by Abe et al. [7] and Nghia et al. [8], for the amplification of a 434 bp specific fragment of gene for HBV. Sets of primers for nested PCR to detect EBV (EBVexF: 5 -TGGAAACCCGTCACTCTC-3 ; EBVexR: 5 -AATGGCATAGGTGGAATG-3 ; EBVinF: TGTTGGAAACCCGTCACTCTC-3 ; EBVinR: 5 -GGG-TAATGGCATAGGTGGAATG-3 ) were designed based on the modification of previous primer design studies by van Baarle et al. [5], which was based on the conserved sequence of genes encoding the nuclear antigens EBNA-2 that generated a DNA product of 250 bp (named as EBV-250). The PCR primers (EBVF3: 5 -GGAACCTGGTCA-TCCTTGC-3 ; EBFR3: 5 -ACGTGCATGGACCGGTTA-AT-3 ; EBV Taqman probe: 5 -(FAM)-CGCAGGCACTCG-TACTGCTCGCT-(TAMRA)-3 ) for real-time PCR using TaqMan probe (Roche Diagnostics) to measure EBV virus load were designed previously [9] based on the sequence encoding nonglycosylated membrane protein BNRF1 p143 and generating a PCR product of 74 bp named as EBV-74. The 434 bp for HBV and 250 bp and 74 bp for EBV were cloned into pGEM-T/A plasmid (Promega). The plasmids harbouring the specific 434 bp bands for HBV were named as pGEM-HBV. The ones harbouring the specific 250 bp and 74 bands for EBV were named as pGEM-EBV-250 and pGEM-EBV-74, respectively. The cloned plasmids were then purified and used as a template for sequencing their specific inserted genes for HBV (HBV-434) and EBV (EBV-250, EBV-74). The sequences were compared for homology to the abovementioned standard genes for HBV and EBV (as posted on the international gene bank). The plasmids were diluted into 10-fold serial concentrations ranging from 4 × 10 9 to 4 × 10 2 copies/mL.  allowing removal of the washing solution. This step was repeated with WB2. In this case, WB2 must be completely removed and residual ethanol evaporated by air drying. (iv) Elution using EB: MagnaBot 96 was switched off and 100 L EB was added in to each well for mixing by pipetting 5 times. The suspension was incubated at 65 ∘ C for 3 min. The solution containing DNA was eluted after applying MagnaBot 96 to attract the nanoparticles. For recovery of standard DNA plasmids, the same protocol was intentionally applied. Isolated DNA concentration was measured and calculated by absorbance at 260 nm using a NanoDrop spectrophotometer (Thermo Scientific). The samples were used immediately or stored at −80 ∘ C for PCR detection of specific DNA bands for EBV and HBV.

Polymerase Chain Reaction to Detect HBV and EBV.
Purified DNA of HBV from serum samples were used as a template for PCR detection of HBV, as described by Nghia et al. [8]. Amplification of 250 bp specific fragments for EBV was optimized in this work under 35 cycles of the following conditions: 94 ∘ C for 30 s, 52 ∘ C (initial 5 cycles) and 56 ∘ C (following 25 cycles) for 45 s, and 72 ∘ C for 60 s. Real-time PCR to quantitatively measure the EBV virus load through amplification of 74 bp specific for EBV was performed following Niesters et al. [9] using 45 cycles of the following conditions: 95 ∘ C for 15 s and 60 ∘ C for 1 min.

Properties of Silica-Coated
Magnetic Nanoparticles. Figure 1 shows the TEM image of Fe 3 O 4 /SiO 2 nanoparticles. From this image it can be seen that Fe 3 O 4 /SiO 2 nanoparticles were formed by 10-15 nm diameter of seed and the surrounding layer has a thickness of about 2-5 nm. Figure 2 shows the FTIR spectra of prepared  Figure 3 shows the magnetic curves of Fe 3 O 4 and Fe 3 O 4 /SiO 2 nanoparticles measured at room temperature. Both of them show superparamagnetic property (i.e., no remanence effect) with high saturation magnetization of

Purification of DNA of Hepatitis Virus Type B (HBV) Using Silica-Coated Magnetic Nanoparticles and Optimized
Buffers. Before testing the DNA purification procedure with real serum samples, we measured the efficiency of DNA recovery of the Fe 3 O 4 /SiO 2 nanoparticles and the optimized buffers using standard pure pGEM-HBV plasmid at 10fold diluted concentrations ranging from 4 × 10 9 copies/mL to 4 × 10 2 copies/mL. The enriched DNA solutions were used as templates for amplification of 434 bp fragment of gene specific for HBV. As shown in Figure 4, from left to right, we could detect bands of about 434 bp with reducing intensities proportional to reducing concentrations from 4 × 10 9 copies/mL to 4 × 10 2 copies/mL (Figure 4, lane [1][2][3][4][5][6][7][8]. This result indicates that Fe 3 O 4 /SiO 2 nanoparticles and the optimized buffer could successfully enrich DNA from solution and that the purified DNA was qualified for further PCR-based detection of HBV at a sensitivity of 4 × 10 2 copies/mL. We then used Fe 3 O 4 /SiO 2 nanoparticles and the buffers to isolate DNA of HBV in six real serum samples (one negative 434 bp for HBV in samples in lanes 2, 4, and 6. Meanwhile, no band was observed in the sample in lane 5. The data indicates that six real serum samples had different concentrations of virus copies, of which the sample in lane 6 had the highest virus load. Our data were in good agreement with those confirmed by the hospital where the samples were collected. In parallel, we performed similar experiments with these six serum samples using the commercialized silica-coated magnetic microparticles Dynabeads Myone Silane (short name: Dynabeads, Life Technologies). As shown in Figure 5, clear bands of 434 bp for HBV were observed in the samples in lane 2 , 4 , and 6 . However, intensities of those bands were weaker compared to those in the same samples in lanes 2, 4, and 6 obtained in the case of Fe 3 O 4 /SiO 2 nanoparticles. We could not observe the specific PCR-amplified bands in the samples in lanes 1 and 3 , possibly due to the low levels of purified template DNA obtained when using Dynabeads. We conclude then that Fe 3 O 4 /SiO 2 nanoparticles may be more efficient than Dynabeads in DNA isolation of HBV from serum. PCR based on highly specific Taqman probe dually labeled with FAM at 5 -end and TAMRA at 3 -end was performed with samples 7 and 10 to quantify the EBV virus load. As shown in the amplification chart of Figure 7, the negative control had no amplification curve while the positive control had an amplification curve occurring earlier ( = 15.65).

Purification of DNA of Epstein-Barr Viruses (EBV) Using Silica-Coated Magnetic Nanoparticles and Optimized Buffers.
All standards together with samples 7 and 10 purified by both particles had amplification curves. The linear regression line of standards had 2 = 0.996 value, indicating that the reliability of the qualitative measurement for EBV virus load was high. The concentration of EBV in sample 7 using DNA purified by Fe 3 O 4 /SiO 2 nanoparticles (= 7.17 × 10 3 copies/mL), although this was much lower than that in sample 10 (= 1.18 × 10 6 copies/mL), but was not too low because it fell in the range of standards. Nanoparticles are therefore suitable for isolating DNA at such low virus concentration. The result in Table 1 indicates that higher concentrations of EBV (copies/mL) in both samples were measured with using Fe 3 O 4 /SiO 2 nanoparticles to purify DNA compared to those with using Dynabeads. The increase in DNA isolation efficiency by Fe 3 O 4 /SiO 2 nanoparticles is likely due to a larger total surface of silica-coated magnetic nanoparticles. During the process of DNA isolation, we have found that the time required for magnets to attract completely the Dynabeads from solution was much longer, about 2-3 min, compared to 15-20 s for Fe 3 O 4 /SiO 2 nanoparticles. This phenomenon is probably also due to the fact that Fe 3 O 4 /SiO 2 nanoparticles have a larger total surface area compared to that of the Dynabeads.

Conclusion
Our study demonstrates that Fe 3 O 4 /SiO 2 nanoparticles and the optimized buffers can isolate genomic DNA of two types of viruses, HBV and EBV, for further PCR-based detection of the viruses in serum samples. The obtained primary data indicates that Fe 3 O 4 /SiO 2 nanoparticles provided better sensitivity and were time saving in detection of HBV and EBV, compared to that of the commercialized silica-coated magnetic microparticles. Further experiments on nucleic acid isolation of other pathogenic viruses infected in blood using the Fe 3 O 4 /SiO 2 nanoparticles are in progress.