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Nanomaterial-assisted PCR based on thermal generation from magnetic nanoparticles under high-frequency AC magnetic fields
Chemical Physics Letters 635 (2015) 234–240
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Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Nanomaterial-assisted PCR based on thermal generation from magnetic nanoparticles under high-frequency AC magnetic fields Toshiaki Higashi ∗ , Hiroaki Minegishi, Akinobu Echigo, Yutaka Nagaoka, Takahiro Fukuda, Ron Usami, Toru Maekawa, Tatsuro Hanajiri Bio-Nano Electronics Research Centre, Toyo University, 2100, Kujirai, Kawagoe, Saitama 350-8585, Japan
a r t i c l e
i n f o
Article history: Received 22 November 2014 In final form 25 June 2015 Available online 6 July 2015
a b s t r a c t Here the authors present a nanomaterial-assisted PCR technique based on the use of thermal generation from magnetic nanoparticles (MNPs) under AC magnetic fields. In this approach, MNPs work as internal nano thermal generators to realize PCR thermal cycling. In order to suppress the non-specific absorption of DNA synthetic enzymes, MNPs are decorated with bovine serum albumin (BSA), forming BSA/MNP complexes. Under high-frequency AC magnetic fields, these complexes work as internal nano thermal generators, thereby producing the typical temperature required for PCR thermal cycling, and perform all the reaction processes of PCR amplification in the place of conventional PCR thermal cyclers. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Polymerase chain reaction (PCR) is a standard in vitro technique that allows reproduction and amplification of specific DNA segments using DNA polymerase [1–3]. PCR methods involve the use of thermal cycling, by which DNA samples are heated or cooled to a defined series of controlled temperatures. The PCR technique has been adopted in a wide range of fields including DNA sequencing [4], pathogen detection [5] and genetic identification [6,7]. More recently, nano-scale materials have been used in PCR amplification in an approach called nanomaterial-assisted PCR (nano PCR) [8]. Such materials have unique physical and chemical properties, and various types such as single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), carbon nano-powder, metallic nanoparticles and nonmetallic nanoparticles have been used to enhance PCR efficiency [9–12]. In particular, Au nanoparticles (AuNPs) improve the specificity of PCR amplification even at low annealing temperatures, and also significantly increase PCR efficiency thanks to the favorable heat transfer properties of Au [13,14]. Meanwhile, a number of reports have conversely stated that high AuNP concentrations negatively affect PCR efficiency [15–17]. According to these reports, the interaction between Taq polymerase DNA synthetic enzymes and nano-scale materials has a significant negative influence on PCR efficiency. Its inhibiting effects are attributed to reduced enzyme activity due
∗ Corresponding author. E-mail address: [email protected] (T. Higashi). http://dx.doi.org/10.1016/j.cplett.2015.06.070 0009-2614/© 2015 Elsevier B.V. All rights reserved.
to non-specific adsorption onto nanoparticle surfaces. Like gold nanoparticles, magnetic nanoparticles (MNPs) are nano-scale and metallic. They are composed mainly of magnetite (Fe3 O4 ), and also disperse in solvents to form colloidal systems. In previous work, we focused on how MNPs affect PCR amplification. The results showed the influence without an external magnetic field and highlighted the mechanism behind the effects of superparamagnetic particle clusters on PCR efficiency based on determination of the structures of such clusters in PCR thermal cycles [18]. It was concluded that superparamagnetic nanoparticles tended to inhibit PCR amplification via non-specific adsorption of Taq polymerase on the surface of MNP clusters in the same way as gold nanoparticles. We also found that the product yield of PCR could be regulated by controlling the structure and dynamics of MNP clusters in a rotating magnetic field during PCR thermal cycles [19]. In addition, it should be noted that MNPs have favorable magnetic properties and exhibit various forms of physical behavior depending on magnetic field characteristics. Magnetic particles are classified as either ferromagnetic or paramagnetic/superparamagnetic. The former have permanent magnetic dipole moments, and tend to aggregate and form clusters [20,21]. The latter remain stable in the absence of an external magnetic field [22,23]. In dispersion systems, both particle types form chain clusters via dipole-dipole interaction in an external DC magnetic field [20–23]. Accordingly, external AC/DC magnetic fields can be applied to control the structure and magnetization of both types. When a rotating magnetic field is applied, the chain clusters turn in line with the field’s rotation [24–29]. MNPs can be utilized in biomedical application as contrast agent for magnetic resonance
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imaging (MRI) [30], as carrier of drug delivery systems (DDSs) [31] and as DNA bead array carrier for highly selective detection of genes/proteins [32–34]. Additionally, MNPs generate heat under externally applied AC magnetic fields, and particles then transform the energy of the magnetic field into thermal energy through the physical mechanisms of eddy current loss, hysteresis loss, Néel relaxation and Brown relaxation [35–38]. The transformation mechanisms depend significantly on the particle size, magnetic properties and frequency of the external magnetic fields. The thermal generation of MNPs under AC magnetic fields can produce local contactless heat with precise temperature control, and has been used to great effect agent in magnetic hyperthermia methods for cancer therapy [39–41]. In other application, Kim et al. presented magnetic nanoparticles as a external heating element integrated into micro-device platform for biochemical applications and fabricated a microheater chip based on a magnetic nanoparticles embedded poly-dimethylsiloxane (PDMS) as a molding elastomer in microfluidic devices [42]. The micro-heater chip demonstrated the precise temperature control and amplified target DNA under an AC magnetic field. Thermal generation with MNPs
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can be utilized also for in vitro nano-scale thermal generators in biochemical applications requiring thermal reactions, such as PCR thermal cycling for DNA amplification. Moreover, the nano-scale thermal generators can be realized novel nanomaterial-assisted PCR models with magnetic nanoparticles under AC magnetic fields. Accordingly, this Letter presents a novel nanomaterial-assisted PCR approach based on the use of thermal generation with magnetic nanoparticles under AC magnetic fields. In the study, heat generated from MNPs was used for in vitro internal nano thermal generators in PCR thermal cycling, and PCR amplification was demonstrated under external high-frequency AC magnetic fields. The surfaces of MNPs were decorated with bovine serum albumin (BSA) to block the non-specific adsorption of Taq polymerase, and BSA/MNP complexes were produced. Under high-frequency AC magnetic fields, BSA/MNP complexes work as internal nano thermal generators, producing the typical temperature required for PCR thermal cycling, and perform all the reaction processes of PCR amplification in the place of conventional PCR thermal cycler instruments.
Figure 1. (a) TEM image of water-based magneto rheological fluid containing superparamagnetic nanoparticles deposited on a TEM grid. (b) TEM image of a selected superparamagnetic nanoparticle area. (c) X-ray diffraction pattern of superparamagnetic nanoparticles (magnetite).
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Figure 2. (a) Schematic diagrams of BSA/MNP complex synthesis. (b) AFM image of BSA solution dispersed on a mica plate. (c) AFM image of BSA/MNP complexes.
2. Experiment details 2.1. Production and characterization of BSA/MNP complexes In the experiment reported here, BSA/MNP complexes that work as tiny thermal generators in a PCR reaction tube were produced. We previously reported that Taq polymerase was adsorbed non-specifically onto the surface of MNPs and MNP clusters, and that PCR efficiency was dramatically suppressed as a result [18]. Accordingly, BSA was used to block non-specific adsorption of Taq polymerase in this experiment. BSA is generally used as a blocking agent to prevent non-specific adsorption of antibodies and proteins
[43,44]. According to a previous report, adding BSA for PCR amplification reversed the negative effect of AuNPs on PCR, whereas BSA alone had no effect on PCR amplification [17]. BSA/MNP complexes were produced using superparamagnetic nanoparticles (EMG607, Ferrotec, Japan) and BSA (Sigma–Aldrich). The magnetic nanoparticle medium used in this study was a water-based magneto rheological fluid containing superparamagnetic nanoparticles whose surfaces were modified with a cationic surfactant to improve dispersibility. The MNPs had an average diameter of 10 nm, which was measured using a transmission electron microscope (TEM) as shown in Figure 1a and b. The particles were primarily magnetite (Fe3 O4 ), as demonstrated by the X-ray diffraction (XRD) pattern
Figure 3. (a) TEM image of BSA/MNP complexes. BSA was stained using TI blue to facilitate TEM signal detection. (b) 3D TEMT image of BSA/MNP complexes.
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Figure 4. Amplified DNA bands observed using agarose gel electrophoresis. Lane 1: control; lane 2: 10 l of 20% BSA; lane 3: 10 l of 19 wt% MNPs; lane 4: 10 l of BSA/MNP complexes.
of MNPs shown in Figure 1c. In the production of BSA/MNP complexes, BSA was dissolved in distilled water at a concentration of 20%, and 300 l of MNPs solution (19 wt%) was then combined with 3000 l of this solution. The resulting MNP and BSA solution was subsequently mixed in a water bath and incubated for 2 h at 40 ◦ C while being shaken at 140 rpm. BSA/MNP complexes were then selectively recovered from the solution using a permanent magnet, washed three times in distilled water and observed under an atomic force microscope (AFM; MFP-3D, Asylum Research). The internal structures of the complexes were analyzed using a transmission electron microscope (TEM; JEM-2200FS, JEOL) and transmission electron 3D microtomography (TEMT; JEM2100, JEOL), and the BSA was stained using the negative staining method with TI blue (Nisshin EM Co.) to facilitate TEM signal detection. The magnetization-magnetic field curve of BSA/MNP complexes was also determined with a vibrating sample magnetometer (VSM; 7407, Lake Shore Cryotronics Inc.). 2.2. PCR experiment with a thermal cycler The strain used in this experiment was Escherichia coli DH5a cultivated in a lysogeny broth (LB) medium. Cells were harvested via centrifugation and suspended in a TEN buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl), and 0.3 g of glass beads were added. The cells were broken by being shaken for 10 min on a vortex mixer at maximum speed, and nucleic acid was extracted
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via phenol/chloroform treatment and ethanol precipitation. An approximate 1500-bp segment of the 16S rRNA gene was amplified using PCR with the forward and reverse primers 5 -AGA GTT TGA TCC TGG CTC AG-3 (positions 8–27 according to E. coli numbering) and 5 -GGC TAC CTT GTT ACG ACT T-3 (positions 1510–1492) using an Ex Taq polymerase kit (TaKaRa). In the PCR with a conventional thermal cycler, thermal cycling was carried out in 0.2-ml reaction tubes in a QuickBath QB-0225A unit (ThermoGen Inc.). Amplification was performed in a 50 l mixture of 5 l of 10 × Ex Taq PCR buffer, 4 l of dNTP mixture (dATP, dTTP, dGTP and dCTP; 2.5 mM each), 1 l of forward primer, 1 l of reverse primer (10 mM each), 1 l of Ex Taq polymerase (1 U/l), 1 l of template DNA (100 ng of DNA) and 37 l of distilled water. The thermal profile for amplification began with an initial denaturation step (2 min, 94 ◦ C) followed by 25 cycles of denaturation (10 s, 94 ◦ C), annealing (15 s, 56 ◦ C) and extension (30 s, 72 ◦ C), then a final terminal extension step (2 min, 72 ◦ C). Finally, the PCR products were analyzed using agarose gel (1.2%) electrophoresis. 2.3. PCR experiment with an induction-heating system In the PCR using BSA/MNP complexes, thermal cycling was carried out in 2-ml reaction tubes using a high-frequency induction-heating system (Easy-heat, Ameritherm Inc.) consisting of an AC power supply, a cooling unit, a condenser unit and a threeloop induction coil. The high-frequency alternating current applied to the induction coil generated an AC magnetic field whose intensity varied in accordance with the output power of the alternating current. Reaction tubes containing BSA/MNP complexes and 500 l of PCR reaction solution were placed at the center of the induction coil. The thermal profile for amplification was followed by 25 cycles of denaturation (10 s, 94 ◦ C), annealing (15 s, 56 ◦ C) and extension (30 s, 72 ◦ C). In the annealing step, the reaction tube was cooled from 94 ◦ C to 56 ◦ C using coolant water. Internal temperature distribution in the reaction tubes during the induction heating was monitored using a high-resolution infrared (IR) thermometer (testo 882, Testo AG) and a precision fiber-optic thermometer (Reflex, Neoptix Inc.). 3. Results and discussion Figure 2a shows schematic diagrams of BSA/MNP complex synthesis. Even without a magnetic field, the solvent-dispersed MNPs tend to aggregate and form tiny clusters independently, despite the belief that superparamagnetic particles do not aggregate based
Figure 5. Photos of MNPs and BSA/MNP complexes dispersed in water. (a) Dispersion state. (b) Magnetic recovery. (c) Magnetization–magnetic field curves of MNPs and BSA/MNP complexes at 25 ◦ C.
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Figure 6. (a) Schematic diagram of PCR experiments using a high-frequency induction heating system. (b) IR thermography analysis of an RCR buffer heated by BSA/MNP complexes at an induction-heating coil. (c) Temperature profiles of the dotted lines in (b).
on magnetic interaction alone. Mixing of magnetic nanoparticles and high-concentration BSA resulted in MNP cluster surface decoration with BSA pieces, and BSA/MNP complexes formed in the buffer solution. Figure 2b shows an AFM image of BSA solution dispersed on a mica plate. The molecules appear to have formed tiny round shapes. Magnetic nanoparticle clusters were decorated with numerous BSA pieces forming BSA/MNP complexes, which represents BSA decoration appearing as an incrustation on such clusters as shown in Figure 2c. The BSA/MNP complexes were also analyzed in detail using TEM and 3D TEMT, and Figure 3 shows their internal structure. It can be seen that some scattered MNP clusters were wrapped in BSA layers, and BSA/MNP complexes were formed. Video footage showing 3D TEM tomography (Supplementary Data 1) clearly indicates that some scattered MNP clusters were decorated with BSA and assembled into aggregates. The AFM and TEM results both confirm that the surfaces of MNP clusters were decorated with numerous BSA layers, and that some MNP clusters assembled with BSA forming BSA/MNP complexes as illustrated in Figure 2a. It can therefore be inferred that excess BSA decoration of MNP cluster surfaces, which causes non-specific adsorption of Taq polymerase, can suppress non-specific adsorption of Taq polymerase to low levels in PCR. The blocking effect of BSA on PCR amplification with MNPs was examined. Here, PCR amplification was performed using a conventional thermal cycler. Figure 4 shows amplified DNA bands observed using agarose gel electrophoresis. As shown in lane 2 of
Figure 4, the target DNA (1500 bp) was amplified sufficiently even when high-concentration BSA was added, and BSA had no effect on PCR amplification. The effects of MNPs and BSA/MNP complexes on PCR amplification were also examined. It was observed that the addition of high-concentration MNPs completely inhibited PCR amplification, as seen in lane 5 of Figure 4. Meanwhile, PCR amplification with BSA/MNP complexes was successful, as seen in lane 6 of Figure 4. These results indicate that BSA decoration on MNP cluster surfaces prevents non-specific adsorption of Taq polymerase, and consequently the inhibition effect of MNPs on PCR amplification can be avoided in BSA/MNP complexes. Focus was also placed on the magnetic characteristics of BSA/MNP complexes. Figure 5a shows typical photos of pristine MNPs and BSA/MNP complexes dispersed in water. When a permanent magnet was moved into the vicinity, the BSA/MNP complexes were quickly drawn by the magnetic field. Magnetic nanoparticles, which were in a dispersion state before the formation of complexes, entered condensation and precipitation states, even though pristine magnetic nanoparticles are usually in a colloidal dispersion state. The magnetic characteristics of MNPs and BSA/MNP complexes were additionally measured using a VSM. The magnetization-magnetic field curves of MNPs and BSA/MNP complexes shown in Figure 5c indicate paramagnetic characteristics for both. The saturation magnetization value for BSA/MNP complexes, 1.0 × 10−3 Wb/m2 , is higher than that for pristine MNPs, 5.4 × 10−4 Wb/m2 . These results indicate that some MNP clusters
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Figure 7. Temperature profile of BSA/MNP complexes for a single cycle of PCR.
were wrapped in BSA, and as a result the magnetic moments of individual MNPs were aligned with the BSA/MNP complexes. BSA/MNP complex formation improved magnetization characteristics, and thermal generation efficiency in the AC magnetic field also increased. In the high-frequency induction heating method, optimal coil design realize the thermal uniformity and thermal efficiency of the workpiece [45]. In general, a multi-turn single-place-type coil is used to heat one part at a time in high-frequency AC magnetic fields. Here, a three-loop copper-tube solenoid coil (inner diameter: 30 mm) designed especially for this experiment was used. Figure 6a shows a schematic diagram of the PCR experiments with a high-frequency induction heating system. AC magnetic field intensity varied in accordance with the output power of the alternating current, and the frequency of the AC magnetic field was fixed at 395 kHz throughout the experiment. In the AC magnetic field applied, a PCR reaction solution with BSA/MNP complexes was heated based on thermal generation from BSA/MNP complexes. When the magnetic field intensity reached 33.68 kA/m, the PCR solution warmed from room temperature to 94 ◦ C at a rate of 0.35 ◦ C/s. Thermal generation from magnetic particles with a diameter of less than 20 nm is caused by the delay in Néel relaxation and Brown relaxation of magnetic moments under AC magnetic fields [35–38]. The heating characteristics and relative contributions of these two relaxation types depends on particle sizes and dispersion states [37,38]. Temperature distribution was also monitored using a high-resolution infrared (IR) thermometer as shown in Figure 6b. The IR thermograms show a top view of the liquid surface in the reaction tube maintaining typical reaction temperatures in PCR. Figure 6c also shows the temperature along each of the dotted lines in (b). The temperature inside the reaction tube
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was uniformly maintained for each reaction temperature. In order to maintain precisely uniform temperature, sophisticated manual control of magnetic field intensity was performed in response to temperature undershoots or overshoots. Finally, PCR thermal cycling was performed using heat generated from BSA/MNP complexes under high-frequency magnetic fields. Figure 7 shows the PCR thermal profiles of a PCR solution heated using such thermal generation for a single cycle of the PCR process. The PCR thermal profiles were controlled and maintained precisely with varying AC magnetic field intensity in accordance with the profile shown in Figure 7. In the first denaturation step, the PCR solution was heated from room temperature to 94 ◦ C and maintained at this temperature for 10 sec. In the annealing step, the reaction tube was cooled using coolant water from 94 ◦ C to 54 ◦ C at a rate of −0.65 ◦ C/s. The annealing temperature was then maintained at 54 ◦ C for 15 s. In the extension step, the PCR solution was heated again from 54 ◦ C to 72 ◦ C and was maintained at this temperature for 30 s. After 25 cycles, PCR products were analyzed using agarose gel electrophoresis. Figure 8a shows amplified DNA bands observed via this method, and Figure 8b shows the product yield as determined using the InGenius system (Syngene). Target DNA was fully amplified by heat generation from BSA/MNPs complexes under high frequency magnetic fields, with a product yield of 59.3%. These results indicate that thermal generation from BSA/MNP complexes functioned as a type of internal nano heat production, controlled the typical temperature required for PCR thermal cycling, and realized PCR amplification in the same way as conventional thermal cyclers. However, the product yield with this method was still lower than that achieved using a thermal cycler. This is because a small quantity of Taq polymerase is adsorbed non-specifically onto the surfaces of uncomplexed MNPs clusters under AC magnetic fields. In addition, the activity of synthetic enzymes is also reduced by overshoot in the high temperature region during the induction heating, thereby reducing the product yield compared with thermal cycler products. In order to improve the detection sensitivity of this method, it is necessary to control the temperature of the induction heating more precisely and to suppress the non-specific adsorption of synthetic enzymes more completely. Biocompatible polymers such as 2-methacryloyloxyethyl phosphorylcholine (MPC) are useful for preventing more non-specific adsorption of Taq polymerase, and are expected to improve PCR efficiency [46,47]. The implementation of further thermal cycles using novel magnetic nanoparticle media such as iron containing carbon nanoparticles (Fe@C nanoparticles) [48] or carbon nanotubes (Fe@CNTs) [49] under AC magnetic fields is also seen as a promising area for future studies. Heat generated from MNPs can be utilized as a nano thermal source in microchannel reactors, and supports the use of DNA testing devices operating under AC magnetic fields.
Figure 8. (a) Amplified DNA bands observed using agarose gel electrophoresis (M: Hi-Low DNA maker; 1: control DNA amplified using a conventional thermal cycler; 2: DNA obtained using the BSA/MNP complex method under high-frequency AC magnetic fields). (b) Comparison of PCR efficiency for the BSA/MNP complex method with conventional thermal cycler methods.
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4. Conclusion This Letter has presented a nanomaterial-assisted PCR technique based on thermal generation with magnetic nanoparticles under AC magnetic fields. Heat generated from MNPs was used for internal nano thermal generators in PCR thermal cycling, and PCR amplification under external high-frequency AC magnetic fields was demonstrated. BSA/MNP complexes were produced and verified as suppressing non-specific adsorption of Taq polymerase as well as preventing the inhibition effect of MNPs on PCR amplification. Under high-frequency AC magnetic fields, BSA/MNP complexes generated heat through Néel relaxation and Brown relaxation. The heat generated from BSA/MNP complexes warmed a PCR solution to the typical temperature required for PCR thermal cycling and allowed precise control to maintain the temperature based on variations in AC magnetic field intensity. Under highfrequency AC magnetic fields, the complexes realized PCR thermal cycling and the target DNA was amplified in the same way as with a conventional thermal cycler. This suggests that MNPs can be utilized as thermal generators in a PCR solution instead of conventional PCR thermal cyclers. The proposed approach is expected to contribute significantly to the development of micro PCR devices for rapid DNA examination.
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Acknowledgments This research has been supported in part since April 2011 by a Grant for the Programme for the Strategic Research Foundation at Private Universities (S1101017) organized by Japan’s Ministry of Education, Culture, Sports, Science and Technology (MEXT), and since April 2012 by a Grant-in-Aid for Scientific Research (24560861) organized by the Japanese Society for the Promotion of Science (JSPS).
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Nanomaterial-assisted PCR based on thermal generation from magnetic nanoparticles under high-frequency AC magnetic fields Toshiaki Higashi ∗ , Hiroaki Minegishi, Akinobu Echigo, Yutaka Nagaoka, Takahiro Fukuda, Ron Usami, Toru Maekawa, Tatsuro Hanajiri Bio-Nano Electronics Research Centre, Toyo University, 2100, Kujirai, Kawagoe, Saitama 350-8585, Japan
a r t i c l e
i n f o
Article history: Received 22 November 2014 In final form 25 June 2015 Available online 6 July 2015
a b s t r a c t Here the authors present a nanomaterial-assisted PCR technique based on the use of thermal generation from magnetic nanoparticles (MNPs) under AC magnetic fields. In this approach, MNPs work as internal nano thermal generators to realize PCR thermal cycling. In order to suppress the non-specific absorption of DNA synthetic enzymes, MNPs are decorated with bovine serum albumin (BSA), forming BSA/MNP complexes. Under high-frequency AC magnetic fields, these complexes work as internal nano thermal generators, thereby producing the typical temperature required for PCR thermal cycling, and perform all the reaction processes of PCR amplification in the place of conventional PCR thermal cyclers. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Polymerase chain reaction (PCR) is a standard in vitro technique that allows reproduction and amplification of specific DNA segments using DNA polymerase [1–3]. PCR methods involve the use of thermal cycling, by which DNA samples are heated or cooled to a defined series of controlled temperatures. The PCR technique has been adopted in a wide range of fields including DNA sequencing [4], pathogen detection [5] and genetic identification [6,7]. More recently, nano-scale materials have been used in PCR amplification in an approach called nanomaterial-assisted PCR (nano PCR) [8]. Such materials have unique physical and chemical properties, and various types such as single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), carbon nano-powder, metallic nanoparticles and nonmetallic nanoparticles have been used to enhance PCR efficiency [9–12]. In particular, Au nanoparticles (AuNPs) improve the specificity of PCR amplification even at low annealing temperatures, and also significantly increase PCR efficiency thanks to the favorable heat transfer properties of Au [13,14]. Meanwhile, a number of reports have conversely stated that high AuNP concentrations negatively affect PCR efficiency [15–17]. According to these reports, the interaction between Taq polymerase DNA synthetic enzymes and nano-scale materials has a significant negative influence on PCR efficiency. Its inhibiting effects are attributed to reduced enzyme activity due
∗ Corresponding author. E-mail address: [email protected] (T. Higashi). http://dx.doi.org/10.1016/j.cplett.2015.06.070 0009-2614/© 2015 Elsevier B.V. All rights reserved.
to non-specific adsorption onto nanoparticle surfaces. Like gold nanoparticles, magnetic nanoparticles (MNPs) are nano-scale and metallic. They are composed mainly of magnetite (Fe3 O4 ), and also disperse in solvents to form colloidal systems. In previous work, we focused on how MNPs affect PCR amplification. The results showed the influence without an external magnetic field and highlighted the mechanism behind the effects of superparamagnetic particle clusters on PCR efficiency based on determination of the structures of such clusters in PCR thermal cycles [18]. It was concluded that superparamagnetic nanoparticles tended to inhibit PCR amplification via non-specific adsorption of Taq polymerase on the surface of MNP clusters in the same way as gold nanoparticles. We also found that the product yield of PCR could be regulated by controlling the structure and dynamics of MNP clusters in a rotating magnetic field during PCR thermal cycles [19]. In addition, it should be noted that MNPs have favorable magnetic properties and exhibit various forms of physical behavior depending on magnetic field characteristics. Magnetic particles are classified as either ferromagnetic or paramagnetic/superparamagnetic. The former have permanent magnetic dipole moments, and tend to aggregate and form clusters [20,21]. The latter remain stable in the absence of an external magnetic field [22,23]. In dispersion systems, both particle types form chain clusters via dipole-dipole interaction in an external DC magnetic field [20–23]. Accordingly, external AC/DC magnetic fields can be applied to control the structure and magnetization of both types. When a rotating magnetic field is applied, the chain clusters turn in line with the field’s rotation [24–29]. MNPs can be utilized in biomedical application as contrast agent for magnetic resonance
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imaging (MRI) [30], as carrier of drug delivery systems (DDSs) [31] and as DNA bead array carrier for highly selective detection of genes/proteins [32–34]. Additionally, MNPs generate heat under externally applied AC magnetic fields, and particles then transform the energy of the magnetic field into thermal energy through the physical mechanisms of eddy current loss, hysteresis loss, Néel relaxation and Brown relaxation [35–38]. The transformation mechanisms depend significantly on the particle size, magnetic properties and frequency of the external magnetic fields. The thermal generation of MNPs under AC magnetic fields can produce local contactless heat with precise temperature control, and has been used to great effect agent in magnetic hyperthermia methods for cancer therapy [39–41]. In other application, Kim et al. presented magnetic nanoparticles as a external heating element integrated into micro-device platform for biochemical applications and fabricated a microheater chip based on a magnetic nanoparticles embedded poly-dimethylsiloxane (PDMS) as a molding elastomer in microfluidic devices [42]. The micro-heater chip demonstrated the precise temperature control and amplified target DNA under an AC magnetic field. Thermal generation with MNPs
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can be utilized also for in vitro nano-scale thermal generators in biochemical applications requiring thermal reactions, such as PCR thermal cycling for DNA amplification. Moreover, the nano-scale thermal generators can be realized novel nanomaterial-assisted PCR models with magnetic nanoparticles under AC magnetic fields. Accordingly, this Letter presents a novel nanomaterial-assisted PCR approach based on the use of thermal generation with magnetic nanoparticles under AC magnetic fields. In the study, heat generated from MNPs was used for in vitro internal nano thermal generators in PCR thermal cycling, and PCR amplification was demonstrated under external high-frequency AC magnetic fields. The surfaces of MNPs were decorated with bovine serum albumin (BSA) to block the non-specific adsorption of Taq polymerase, and BSA/MNP complexes were produced. Under high-frequency AC magnetic fields, BSA/MNP complexes work as internal nano thermal generators, producing the typical temperature required for PCR thermal cycling, and perform all the reaction processes of PCR amplification in the place of conventional PCR thermal cycler instruments.
Figure 1. (a) TEM image of water-based magneto rheological fluid containing superparamagnetic nanoparticles deposited on a TEM grid. (b) TEM image of a selected superparamagnetic nanoparticle area. (c) X-ray diffraction pattern of superparamagnetic nanoparticles (magnetite).
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Figure 2. (a) Schematic diagrams of BSA/MNP complex synthesis. (b) AFM image of BSA solution dispersed on a mica plate. (c) AFM image of BSA/MNP complexes.
2. Experiment details 2.1. Production and characterization of BSA/MNP complexes In the experiment reported here, BSA/MNP complexes that work as tiny thermal generators in a PCR reaction tube were produced. We previously reported that Taq polymerase was adsorbed non-specifically onto the surface of MNPs and MNP clusters, and that PCR efficiency was dramatically suppressed as a result [18]. Accordingly, BSA was used to block non-specific adsorption of Taq polymerase in this experiment. BSA is generally used as a blocking agent to prevent non-specific adsorption of antibodies and proteins
[43,44]. According to a previous report, adding BSA for PCR amplification reversed the negative effect of AuNPs on PCR, whereas BSA alone had no effect on PCR amplification [17]. BSA/MNP complexes were produced using superparamagnetic nanoparticles (EMG607, Ferrotec, Japan) and BSA (Sigma–Aldrich). The magnetic nanoparticle medium used in this study was a water-based magneto rheological fluid containing superparamagnetic nanoparticles whose surfaces were modified with a cationic surfactant to improve dispersibility. The MNPs had an average diameter of 10 nm, which was measured using a transmission electron microscope (TEM) as shown in Figure 1a and b. The particles were primarily magnetite (Fe3 O4 ), as demonstrated by the X-ray diffraction (XRD) pattern
Figure 3. (a) TEM image of BSA/MNP complexes. BSA was stained using TI blue to facilitate TEM signal detection. (b) 3D TEMT image of BSA/MNP complexes.
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Figure 4. Amplified DNA bands observed using agarose gel electrophoresis. Lane 1: control; lane 2: 10 l of 20% BSA; lane 3: 10 l of 19 wt% MNPs; lane 4: 10 l of BSA/MNP complexes.
of MNPs shown in Figure 1c. In the production of BSA/MNP complexes, BSA was dissolved in distilled water at a concentration of 20%, and 300 l of MNPs solution (19 wt%) was then combined with 3000 l of this solution. The resulting MNP and BSA solution was subsequently mixed in a water bath and incubated for 2 h at 40 ◦ C while being shaken at 140 rpm. BSA/MNP complexes were then selectively recovered from the solution using a permanent magnet, washed three times in distilled water and observed under an atomic force microscope (AFM; MFP-3D, Asylum Research). The internal structures of the complexes were analyzed using a transmission electron microscope (TEM; JEM-2200FS, JEOL) and transmission electron 3D microtomography (TEMT; JEM2100, JEOL), and the BSA was stained using the negative staining method with TI blue (Nisshin EM Co.) to facilitate TEM signal detection. The magnetization-magnetic field curve of BSA/MNP complexes was also determined with a vibrating sample magnetometer (VSM; 7407, Lake Shore Cryotronics Inc.). 2.2. PCR experiment with a thermal cycler The strain used in this experiment was Escherichia coli DH5a cultivated in a lysogeny broth (LB) medium. Cells were harvested via centrifugation and suspended in a TEN buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl), and 0.3 g of glass beads were added. The cells were broken by being shaken for 10 min on a vortex mixer at maximum speed, and nucleic acid was extracted
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via phenol/chloroform treatment and ethanol precipitation. An approximate 1500-bp segment of the 16S rRNA gene was amplified using PCR with the forward and reverse primers 5 -AGA GTT TGA TCC TGG CTC AG-3 (positions 8–27 according to E. coli numbering) and 5 -GGC TAC CTT GTT ACG ACT T-3 (positions 1510–1492) using an Ex Taq polymerase kit (TaKaRa). In the PCR with a conventional thermal cycler, thermal cycling was carried out in 0.2-ml reaction tubes in a QuickBath QB-0225A unit (ThermoGen Inc.). Amplification was performed in a 50 l mixture of 5 l of 10 × Ex Taq PCR buffer, 4 l of dNTP mixture (dATP, dTTP, dGTP and dCTP; 2.5 mM each), 1 l of forward primer, 1 l of reverse primer (10 mM each), 1 l of Ex Taq polymerase (1 U/l), 1 l of template DNA (100 ng of DNA) and 37 l of distilled water. The thermal profile for amplification began with an initial denaturation step (2 min, 94 ◦ C) followed by 25 cycles of denaturation (10 s, 94 ◦ C), annealing (15 s, 56 ◦ C) and extension (30 s, 72 ◦ C), then a final terminal extension step (2 min, 72 ◦ C). Finally, the PCR products were analyzed using agarose gel (1.2%) electrophoresis. 2.3. PCR experiment with an induction-heating system In the PCR using BSA/MNP complexes, thermal cycling was carried out in 2-ml reaction tubes using a high-frequency induction-heating system (Easy-heat, Ameritherm Inc.) consisting of an AC power supply, a cooling unit, a condenser unit and a threeloop induction coil. The high-frequency alternating current applied to the induction coil generated an AC magnetic field whose intensity varied in accordance with the output power of the alternating current. Reaction tubes containing BSA/MNP complexes and 500 l of PCR reaction solution were placed at the center of the induction coil. The thermal profile for amplification was followed by 25 cycles of denaturation (10 s, 94 ◦ C), annealing (15 s, 56 ◦ C) and extension (30 s, 72 ◦ C). In the annealing step, the reaction tube was cooled from 94 ◦ C to 56 ◦ C using coolant water. Internal temperature distribution in the reaction tubes during the induction heating was monitored using a high-resolution infrared (IR) thermometer (testo 882, Testo AG) and a precision fiber-optic thermometer (Reflex, Neoptix Inc.). 3. Results and discussion Figure 2a shows schematic diagrams of BSA/MNP complex synthesis. Even without a magnetic field, the solvent-dispersed MNPs tend to aggregate and form tiny clusters independently, despite the belief that superparamagnetic particles do not aggregate based
Figure 5. Photos of MNPs and BSA/MNP complexes dispersed in water. (a) Dispersion state. (b) Magnetic recovery. (c) Magnetization–magnetic field curves of MNPs and BSA/MNP complexes at 25 ◦ C.
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Figure 6. (a) Schematic diagram of PCR experiments using a high-frequency induction heating system. (b) IR thermography analysis of an RCR buffer heated by BSA/MNP complexes at an induction-heating coil. (c) Temperature profiles of the dotted lines in (b).
on magnetic interaction alone. Mixing of magnetic nanoparticles and high-concentration BSA resulted in MNP cluster surface decoration with BSA pieces, and BSA/MNP complexes formed in the buffer solution. Figure 2b shows an AFM image of BSA solution dispersed on a mica plate. The molecules appear to have formed tiny round shapes. Magnetic nanoparticle clusters were decorated with numerous BSA pieces forming BSA/MNP complexes, which represents BSA decoration appearing as an incrustation on such clusters as shown in Figure 2c. The BSA/MNP complexes were also analyzed in detail using TEM and 3D TEMT, and Figure 3 shows their internal structure. It can be seen that some scattered MNP clusters were wrapped in BSA layers, and BSA/MNP complexes were formed. Video footage showing 3D TEM tomography (Supplementary Data 1) clearly indicates that some scattered MNP clusters were decorated with BSA and assembled into aggregates. The AFM and TEM results both confirm that the surfaces of MNP clusters were decorated with numerous BSA layers, and that some MNP clusters assembled with BSA forming BSA/MNP complexes as illustrated in Figure 2a. It can therefore be inferred that excess BSA decoration of MNP cluster surfaces, which causes non-specific adsorption of Taq polymerase, can suppress non-specific adsorption of Taq polymerase to low levels in PCR. The blocking effect of BSA on PCR amplification with MNPs was examined. Here, PCR amplification was performed using a conventional thermal cycler. Figure 4 shows amplified DNA bands observed using agarose gel electrophoresis. As shown in lane 2 of
Figure 4, the target DNA (1500 bp) was amplified sufficiently even when high-concentration BSA was added, and BSA had no effect on PCR amplification. The effects of MNPs and BSA/MNP complexes on PCR amplification were also examined. It was observed that the addition of high-concentration MNPs completely inhibited PCR amplification, as seen in lane 5 of Figure 4. Meanwhile, PCR amplification with BSA/MNP complexes was successful, as seen in lane 6 of Figure 4. These results indicate that BSA decoration on MNP cluster surfaces prevents non-specific adsorption of Taq polymerase, and consequently the inhibition effect of MNPs on PCR amplification can be avoided in BSA/MNP complexes. Focus was also placed on the magnetic characteristics of BSA/MNP complexes. Figure 5a shows typical photos of pristine MNPs and BSA/MNP complexes dispersed in water. When a permanent magnet was moved into the vicinity, the BSA/MNP complexes were quickly drawn by the magnetic field. Magnetic nanoparticles, which were in a dispersion state before the formation of complexes, entered condensation and precipitation states, even though pristine magnetic nanoparticles are usually in a colloidal dispersion state. The magnetic characteristics of MNPs and BSA/MNP complexes were additionally measured using a VSM. The magnetization-magnetic field curves of MNPs and BSA/MNP complexes shown in Figure 5c indicate paramagnetic characteristics for both. The saturation magnetization value for BSA/MNP complexes, 1.0 × 10−3 Wb/m2 , is higher than that for pristine MNPs, 5.4 × 10−4 Wb/m2 . These results indicate that some MNP clusters
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Figure 7. Temperature profile of BSA/MNP complexes for a single cycle of PCR.
were wrapped in BSA, and as a result the magnetic moments of individual MNPs were aligned with the BSA/MNP complexes. BSA/MNP complex formation improved magnetization characteristics, and thermal generation efficiency in the AC magnetic field also increased. In the high-frequency induction heating method, optimal coil design realize the thermal uniformity and thermal efficiency of the workpiece [45]. In general, a multi-turn single-place-type coil is used to heat one part at a time in high-frequency AC magnetic fields. Here, a three-loop copper-tube solenoid coil (inner diameter: 30 mm) designed especially for this experiment was used. Figure 6a shows a schematic diagram of the PCR experiments with a high-frequency induction heating system. AC magnetic field intensity varied in accordance with the output power of the alternating current, and the frequency of the AC magnetic field was fixed at 395 kHz throughout the experiment. In the AC magnetic field applied, a PCR reaction solution with BSA/MNP complexes was heated based on thermal generation from BSA/MNP complexes. When the magnetic field intensity reached 33.68 kA/m, the PCR solution warmed from room temperature to 94 ◦ C at a rate of 0.35 ◦ C/s. Thermal generation from magnetic particles with a diameter of less than 20 nm is caused by the delay in Néel relaxation and Brown relaxation of magnetic moments under AC magnetic fields [35–38]. The heating characteristics and relative contributions of these two relaxation types depends on particle sizes and dispersion states [37,38]. Temperature distribution was also monitored using a high-resolution infrared (IR) thermometer as shown in Figure 6b. The IR thermograms show a top view of the liquid surface in the reaction tube maintaining typical reaction temperatures in PCR. Figure 6c also shows the temperature along each of the dotted lines in (b). The temperature inside the reaction tube
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was uniformly maintained for each reaction temperature. In order to maintain precisely uniform temperature, sophisticated manual control of magnetic field intensity was performed in response to temperature undershoots or overshoots. Finally, PCR thermal cycling was performed using heat generated from BSA/MNP complexes under high-frequency magnetic fields. Figure 7 shows the PCR thermal profiles of a PCR solution heated using such thermal generation for a single cycle of the PCR process. The PCR thermal profiles were controlled and maintained precisely with varying AC magnetic field intensity in accordance with the profile shown in Figure 7. In the first denaturation step, the PCR solution was heated from room temperature to 94 ◦ C and maintained at this temperature for 10 sec. In the annealing step, the reaction tube was cooled using coolant water from 94 ◦ C to 54 ◦ C at a rate of −0.65 ◦ C/s. The annealing temperature was then maintained at 54 ◦ C for 15 s. In the extension step, the PCR solution was heated again from 54 ◦ C to 72 ◦ C and was maintained at this temperature for 30 s. After 25 cycles, PCR products were analyzed using agarose gel electrophoresis. Figure 8a shows amplified DNA bands observed via this method, and Figure 8b shows the product yield as determined using the InGenius system (Syngene). Target DNA was fully amplified by heat generation from BSA/MNPs complexes under high frequency magnetic fields, with a product yield of 59.3%. These results indicate that thermal generation from BSA/MNP complexes functioned as a type of internal nano heat production, controlled the typical temperature required for PCR thermal cycling, and realized PCR amplification in the same way as conventional thermal cyclers. However, the product yield with this method was still lower than that achieved using a thermal cycler. This is because a small quantity of Taq polymerase is adsorbed non-specifically onto the surfaces of uncomplexed MNPs clusters under AC magnetic fields. In addition, the activity of synthetic enzymes is also reduced by overshoot in the high temperature region during the induction heating, thereby reducing the product yield compared with thermal cycler products. In order to improve the detection sensitivity of this method, it is necessary to control the temperature of the induction heating more precisely and to suppress the non-specific adsorption of synthetic enzymes more completely. Biocompatible polymers such as 2-methacryloyloxyethyl phosphorylcholine (MPC) are useful for preventing more non-specific adsorption of Taq polymerase, and are expected to improve PCR efficiency [46,47]. The implementation of further thermal cycles using novel magnetic nanoparticle media such as iron containing carbon nanoparticles (Fe@C nanoparticles) [48] or carbon nanotubes (Fe@CNTs) [49] under AC magnetic fields is also seen as a promising area for future studies. Heat generated from MNPs can be utilized as a nano thermal source in microchannel reactors, and supports the use of DNA testing devices operating under AC magnetic fields.
Figure 8. (a) Amplified DNA bands observed using agarose gel electrophoresis (M: Hi-Low DNA maker; 1: control DNA amplified using a conventional thermal cycler; 2: DNA obtained using the BSA/MNP complex method under high-frequency AC magnetic fields). (b) Comparison of PCR efficiency for the BSA/MNP complex method with conventional thermal cycler methods.
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4. Conclusion This Letter has presented a nanomaterial-assisted PCR technique based on thermal generation with magnetic nanoparticles under AC magnetic fields. Heat generated from MNPs was used for internal nano thermal generators in PCR thermal cycling, and PCR amplification under external high-frequency AC magnetic fields was demonstrated. BSA/MNP complexes were produced and verified as suppressing non-specific adsorption of Taq polymerase as well as preventing the inhibition effect of MNPs on PCR amplification. Under high-frequency AC magnetic fields, BSA/MNP complexes generated heat through Néel relaxation and Brown relaxation. The heat generated from BSA/MNP complexes warmed a PCR solution to the typical temperature required for PCR thermal cycling and allowed precise control to maintain the temperature based on variations in AC magnetic field intensity. Under highfrequency AC magnetic fields, the complexes realized PCR thermal cycling and the target DNA was amplified in the same way as with a conventional thermal cycler. This suggests that MNPs can be utilized as thermal generators in a PCR solution instead of conventional PCR thermal cyclers. The proposed approach is expected to contribute significantly to the development of micro PCR devices for rapid DNA examination.
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Acknowledgments This research has been supported in part since April 2011 by a Grant for the Programme for the Strategic Research Foundation at Private Universities (S1101017) organized by Japan’s Ministry of Education, Culture, Sports, Science and Technology (MEXT), and since April 2012 by a Grant-in-Aid for Scientific Research (24560861) organized by the Japanese Society for the Promotion of Science (JSPS).
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