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Miniemulsion Droplets as Single Molecule Nanoreactors for Polymerase Chain Reaction Anna Musyanovych,† Volker Maila¨nder,‡ and Katharina Landfester*,† Departement of Organic Chemistry III - Macromolecular Chemistry and Organic Materials, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany, and Institute of Clinical Transfusion Medicine and Immunogenetics, Helmholtzstr. 10, 89081 Ulm, Germany Received February 3, 2005; Revised Manuscript Received May 23, 2005
Polymerase chain reaction (PCR) was successfully carried out inside stable and narrowly distributed waterin-oil nanodroplets with a size of 100-300 nm in diameter. The droplets were obtained by the miniemulsion process. Each aqueous droplet serves as a single nanoreactor for the PCR. It was found that the size of the droplets highly depends on the sonication parameters (i.e., time and amplitude) and that these parameters have a great influence on the final concentration of the PCR product. The parameters were chosen that way that conditions for single molecule chemistry were obtained, since the 3D-space is compartimentalized in small nanoreactors in each of which the same reaction takes place in a highly parallel fashion on every single DNA molecule. Polymerase chain reaction (PCR) is a common method that is used to create copies of a specific region of a desoxyribonucleic acid (DNA) sequence, to produce high enough quantities of DNA for an adequate biochemical analysis.1 A few DNA molecules, which act as templates, are rapidly amplified by PCR into many billions of molecules. In principle, one single DNA molecule is the minimum amount that is needed to perform a PCR experiment. However in this case special methods are needed. Recently, several methods were developed to reduce the amount of DNA template used. For example, Walsh and coworkers2 developed a single-tube “hanging droplet” nested reverse transcription PCR. The outer and inner primers were separated during the first round of the PCR. This was achieved by incorporating the inner primers and additional Taq DNA polymerase in a 5 µL “hanging droplet” on the underside of the reaction tube cap. Other research groups3-6 described the reaction with a single molecule DNA in waterin-oil droplets. The droplets were formed by stirring the reaction mixture with the magnetic stirrer. The sizes of final droplets were polydisperse ranging from 2 to 15 µm. Due to the large size distribution, the number of the initial template differs significantly among these droplets. This leads to the wide variation in the amount of the final product per droplet volume. Therefore, the use of more homogeneously distributed droplets is of high interest, so that only one DNA molecule will be present and a single reaction per droplet will take place without interacting with other DNA molecules. It is also of high interest to reduce the size of the droplets in order to have only a small volume per one single molecule because then more nanoreactors can be present in * To whom correspondence should be addressed. E-mail:
[email protected]. Tel.: +49(0)731 50-22870. Fax: +49(0)731 50-22883. † University of Ulm. ‡ Institute of Clinical Transfusion Medicine and Immunogenetics.
the system and therefore a high concentration of noninteracting DNA molecules. In the present work, we report a single-molecule PCR reaction performed in aqueous nanodroplets as small compartments which are obtained by employing the miniemulsion technique. Miniemulsion generally implies a method that allows one to create small stable droplets in a continuous phase by applying high shear stress.7-9 Under high shear, e.g., ultrasonication, the broadly distributed macrodroplets are broken into narrowly distributed, defined small nanodroplets. Usually homogeneous droplets in the size range between 30 and 500 nm with a narrow size distribution can be produced by the miniemulsion process. The size of the droplets mainly depends on the type and the amount of the emulsifier used in the specific system. Each droplet behaves as an independent reaction vessel and can be identified as a “nanoreactor”. One of the requirements to obtain stable miniemulsion droplets is the suppression of the Ostwald ripening mechanism. To achieve this, an osmotic pressure agent has to be introduced into the dispersed phase. For inverse miniemulsion systems (where the hydrophilic phase forms the droplets and the hydrophobic components the continuous phase), this can be one of the very hydrophilic substances which are not soluble in the continuous hydrophobic phase, e.g., salt, sugar, etc. The extremely high stability as well as the absence of the material exchange between the droplets has been illustrated by a classical color reaction such as the formation of Prussian blue in the inverse (water-in-oil) miniemulsion systems.10 Dealing with miniemulsions, the stabilization of the droplets plays a critical role. In the present work, Isopar M (a C12-C14 isoparaffinic mixture, Caldic, Germany) and light mineral oil (Aldrich, Germany) were chosen as the oil phase; Lubrizol U (polyisobutylen-succinimide pentamine, supplied from Lubrizol Ltd., U.K.) and a mixture of Tween
10.1021/bm050084+ CCC: $30.25 © 2005 American Chemical Society Published on Web 06/10/2005
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Biomacromolecules, Vol. 6, No. 4, 2005 1825
Figure 1. AFM images of the extracted genomic DNA molecules before (a) and after sonication (b) for 5 s at 10% amplitude; 286 bp DNA template molecules (c, d (magnification from c)).
80 (0.4% vol/vol), Span 80 (4.5% vol/vol), and Triton X-100 (0.05% vol/vol, all supplied from Aldrich) were used as surfactants. Preliminary experiments had shown that Isopar M containing Lubrizol U is very efficient as a continuous phase to stabilize aqueous droplets in the inverse system. The composition of the surfactant mixture containing Tween 80, Span 80, and Triton X-100 as well as their respective amount was taken according to Ghadessy and co-workers.5 The authors prepared a water-in-oil macroemulsion (size of droplets ≈ 15 µm) by stirring the light mineral oil, the mixture of surfactants, and the aqueous phase which contained the biological reagents. In our experiments, the aqueous phase of the miniemulsion consisted of 25 mM Tris‚ HCl (pH 8.4), 3.1 mM MgCl2, 62.5 mM KCl, 0.0125 mg/ mL gelatine, 7.5 µM of each primer (the sequences for the primers were as follows: forward 5′-CGGCAGCAACAGCAGGT-3 and reverse 5′-GCCAGCTGAGTCTCAGAGTG3′),11 375 µM of each dNTP, 15 units of Taq DNA polymerase (Invitrogen), and DNA template in total volume of 80 µL. It is worth mentioning that the DNA template molecule in an expanded form should fit into the nanodroplets. Extracted genomic DNA has a molecule length of at least several micrometers and could not be present in the expanded form inside the nanosize droplet. Additionally, the long molecules are very sensitive to the sonication procedure.
After sonication for 5 s at 10% amplitude, the original genomic DNA molecules were broken into small pieces of different size as can be seen from the atomic force microscopy images (Figure 1a,b). Therefore, in our experiments we used for the preparation of the miniemulsions a well defined 286 bp long DNA template,12 which has a length of about 90 nm (Figure 1c,d) and are resistant to the sonication procedure (not shown). The primer sequences were chosen in order to obtain a 135 bp PCR product so that it is different in size compared with the DNA template and can be easily discriminated by gel electrophoresis. With the aim to have a large reaction volume, the waterto-oil ratio was 1 to 2 and it was kept constant for all experiments. In such a system, the Ostwald ripening can be suppressed by addition of a high amount of a hydrophilic agent. In our case primers, dNTPs, and DNA template that were added in excess and the salts from the buffer systems served as hydrophilic agents. All PCR reactions were performed with a PCR Thermal Cycler (Bio-Rad Thermocycler). The amplification program comprised of 35 cycles with the following steps: initial denaturation at 94 °C for 4 min; 35 cycles consisting of dsDNA denaturation at 94 °C for 30 s, primer annealing at 58 °C for 45 s, primer extension at 72 °C for 30 s; and final elongation at 72 °C for 2 min. After PCR thermocycling, the content from three vials was mixed together, and the miniemulsion was broken into aqueous and oil phases by
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Figure 2. Schematic illustration of the miniemulsification procedure.
centrifugation in a microcentrifuge at 7000 rpm for 20 min. Then, the bottom aqueous layer was removed and analyzed by agarose gel electrophoresis. The gel was prepared with 1.7% of agarose (Invitrogen) in Tris Borate EDTA (TBE) 0.5 × buffer with 0.5 µg/mL of ethidium bromide (Sigma) for visualization. A 100 bp DNA ladder molecular weight marker (Invitrogen) was run on every gel to confirm the expected molecular weight of the amplification product. The running conditions were: 75 V constant for 1.5 h in TBE 0.5 × buffer. The first step of the (pre)-emulsification process was similar to that described by Dressman et al.4 Briefly, icecooled mineral oil (160 µL) containing surfactant (or a mixture of surfactants) was placed in a 2-mL round-bottom cryogenic vial (Nalgene Cryoware, Nalge Company, USA). The PCR mixture (80 µL) was gradually added (10 µL every 15 s) under constant stirring (1400 rpm). After addition of the aqueous phase, the stirring was continued for 30 min. In the second step of miniemulsification, microdroplets were broken into nanodroplets by applying ultrasonication. After the stirring process, the emulsion was divided into three PCR tubes, and each of them was sonicated separately (Sonifier Branson W450, with 1.4 mm sonication tip). The process of emulsification is schematically shown in Figure 2. Stable miniemulsions were obtained at room temperature. However, for the PCR experiment, the miniemulsion should also be stable at high (up to 95 °C) and low (4 °C) temperatures, because during the PCR the emulsion is subjected to fast changes in temperature in this range. Therefore, four miniemulsions consisting of the buffer solution as an aqueous phase and different oil phases, i.e., Isopar M with Lubrizol U, Isopar M with the mixture of surfactants, mineral oil with Lubrizol U, and mineral oil with the mixture of surfactants, were subjected to PCR thermocycles in order to choose the most stable system. At the beginning of the thermocycling process, all miniemulsions
Figure 3. Photographs obtained in light microscopic experiments for (a) a (macro)emulsion (after stirring) and (b) a miniemulsion with droplets consisting of the PCR reaction mixture and with a hydrophobic continuous phase (mineral oil) obtained by ultrasonication for 5 s at 10% amplitude.
were stable and showed macroscopically a homogeneous mixture. For a comparison of a (macro)emulsion and a miniemulsion, Figure 3 shows a photograph of light microscopy. In the case of the (macro)emulsion, large droplets are detected, in the case of the miniemulsion, the droplets are so small that they cannot be detected by light microscopy any more. Both miniemulsions with mineral oil as the continuous phase were stable, and the droplets size as detected by dynamic light scattering before and after thermocycles did not change significantly. However, in the reaction tubes containing Isopar M (with both types of stabilizers), phase separation was observed at the end of the process. This fact can be explained by the low viscosity of Isopar M (1.865 cP) compared with mineral oil (24.053 cP). Furthermore, this influences the final droplet size. The size of the aqueous compartments (as volume average) was measured by dynamic light scattering using backscatter detection (Zetasizer Nanoseries, Malvern Instrument, England). The measurements were performed with miniemulsions that were sonified for 5 s at 10% amplitude prepared with Isopar M and mineral oil and both kinds of surfactants. The obtained results are presented in Figure 4. The diameter of the miniemulsion droplets with Isopar M as continuous phase is significantly larger compared to the mineral oil. Here, the average diameter ranges from 500
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Table 1. Calculated Concentrations (Theoretical Predictions) of the PCR Reagents in Each Droplet molecules per nanocompartment average size of droplet, nm
number of droplets (in 80µl)
330 215 125
4.3 × 1012 1.5 × 1013 7.8 × 1013
fw/rev. primer
Taq polymerase
each dNTP
PCR template
per 80µl
per droplet
per 80µl
per droplet
per 80µl
per droplet
per 80µl
per droplet
3.6 × 1014
84.9 23.5 4.6
4.8 × 1012
1.13 0.31 0.06
4.5 × 1018
1.1 × 106 9.4 × 104 5.8 × 104
1.5 × 1013
3.5 1.0 0.2
Figure 4. Average diameter of the aqueous droplets after 5 s at 10% sonication. Figure 6. Electrophoresis results of PCR amplification in water-inoil miniemulsion droplets obtained with different sonication times and amplitudes. The light mineral oil with the mixture of emulsifiers (Tween 80, Span 80, Triton X-100) was used here as a continuous phase. PCR products were visualized on a 1.7% agarose gel containing ethidium bromide. Lane M: 100-bp ladder; lane 1: 300 nm droplets (sonicated for 5 s at 10%) without thermocycles; lanes 2-4: 200 nm droplets (sonicated for 10 s at 10%) after 35 PCR cycles (lane 2), and 300 nm droplets (sonicated for 5 s at 10%; lane 3), and 100 nm droplets (sonicated for 5 s at 15%; lane 4).
Figure 5. Average diameter of the aqueous droplets after 5 s at 10% sonication.
to 1200 nm, depending on the kind of the surfactant. In contrast, the size of the droplets in mineral oil is much smaller (300-600 nm) and the size distribution is narrower when using the mixture of surfactants. The influence of the sonication procedure (time and amplitude) on the droplets size was studied in a miniemulsion system containing mineral oil and a mixture of surfactants as continuous phase. The results are shown in Figure 5. With increasing sonication time and amplitude, the droplet size decreases and the droplet number increases. The distribution of the PCR reagents is strongly dependent on the number of droplets. The calculated amount of the PCR reagents in each droplet is summarized in Table 1. It should be noted that these values are average values. Especially in the case of low numbers of the PCR templates and the polymerase, the droplet size distribution and the
statistical distribution should also be considered. Both effects lead to an effective distribution so that, even in the case of one unit (either PCR template or polymerase) per droplet in average, droplets with zero units and droplets with two units will be found. As illustrated in Table 1, the limiting factor is the concentration of the Taq DNA polymerase which is already increased ten times in concentration compared to usual recipes of PCRs in solution. When the average size of droplets is around 300 nm, on average one molecule of the Taq DNA polymerase would be present in each droplet, the number of PCR template is 3 per droplet. A decrease of the droplet size to 200 nm, leads to a situation where in each droplet on average is one PCR template, but one-third of the droplets contain a Taq DNA polymerase. A further decrease of the droplet size down to about 100 nm decreases the probability of having a PCR template and at the same time the Taq DNA polymerase to about 1%. Therefore, at these concentrations, the droplets should have a diameter of about 200 nm in order to get a successful polymerase chain reaction where only one PCR template is in each droplet and also the concentration of polymerase is high enough. It should be noted that the concentration of the DNA polymerase is already much higher than in a typically performed PCR. A three times higher concentration of polymerase in
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order to have a 1:1 template to polymerase situation is difficult to achieve because the viscosity would increase too much. Therefore, under these experimental conditions, it has to be accepted that only one-third of the droplets react, but these droplets allow a single chain reaction situation. After the PCR reaction, the miniemulsions from three vials were collected in one vial and broken in two phases (oil and aqueous) by centrifugation for 20 min at 7000 rpm. Then, the aqueous phase was removed carefully and analyzed by agarose electrophoresis for the presence of PCR products. Miniemulsions stabilized by Lubrizol U were much harder to break compared to miniemulsions stabilized by the mixture of emulsifiers. Furthermore, the results of electrophoresis did not show any presence of the PCR products when Lubrizol U was used during the miniemulsion process. Even by applying the cleaning procedure using water saturated ether as described by Tawfik et al.,6 we could not recover the PCR products from the reaction mixture. We speculate that the Lubrizol U might strongly interact with the PCR components due to the chemical structure of the molecule, i.e., the succinimide pentamine or more possibly the amino groups. In contrast to the miniemulsions prepared with Lubrizol U, the analysis of the aqueous phase of the miniemulsion with the mixture of emulsifiers, a characteristic band around 135 bp was detected and confirmed the presence of PCR products (Figure 6). As it can be seen from the same figure, the amount of PCR product is higher in the 300 nm droplets (sonicated 5 s at 10%) compared to 200 nm (sonicated 10 s at 10%) and the 100 nm droplets (sonicated 5 s at 15%). It is worth mentioning that, even in the 100 nm droplets, PCR product can be detected. This confirms our theoretical prediction (Table 1). By increasing the droplet number, the possibility to have all PCR reagents in one droplet decreases, which leads to a reduced amount of the PCR product at the end of the reaction. In summary, we have demonstrated that the miniemulsion technique can be applied to prepare nanoreactors and perform PCR within these nanoreactors. By decreasing the diameter
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of the droplets, it is possible to obtain a single molecule chemistry situation in droplets with sizes smaller than 300 nm. We claim that this reaction is a first single-molecule PCR reaction performed inside miniemulsion droplets. For a better detection of the PCR product, in the future different surfactants will also chosen which do not interact with the PCR product. Further work will be focused on more detailed investigations of the system using spectroscopic measurements. References and Notes (1) Mu¨ller, H.-J. In PCR-Polymerase-Kettenreaktion; Mu¨ller, H.-J., Ed.; Spektrum Akademischer Verlag GmbH: Berlin, 2001. (2) Walsh, E. E.; Falsey, A. R.; Swinburne, I. A.; Formica, M. A. J. Med. Virol. 2001, 63, 259-263. (3) Nakano, M.; Komatsu, J.; Matsuura, S.; Takashima, K.; Katsura, S.; Mizuno, A. J. Biotechnol. 2003, 102, 117-124. (4) Dressman, D.; Yan, H.; Traverso, G.; Kinzler, K. W.; Vogelstein, B. Proc. Natl. Acad. Sci. 2003, 100 N15, 8817-8822. (5) Ghadessy, F. J.; Ong, L. J.; Holliger, P. Proc. Natl. Acad. Sci. 2001, 98 (8), 4552-4557. (6) Tawfik, D. S.; Griffiths, A. D. Nat. Biotechnol. 1998, 16, 652-656. (7) Landfester, K Miniemulsions for nanoparticle synthesis. In Colloid Chemistry; Antonietti, M., Ed.; Spinger: Heidelberg, Germany, 2003; pp 75-124. (8) Schork, F. J.; Poehlein, G. W.; Wang, S.; Reimers, J.; Rodrigues, J.; Samer, C. Colloids Surf. 1999, A 153, 39-45. (9) Landfester, K. Macromol. Symp. 2000, 150, 171-178. (10) Landfester, K. AdV. Mater. 2001, 13, 765-768. (11) Oligonucleotide primer pairs were designed using on line available resources (http://bioweb.pasteur.fr/seqanal/interfaces/eprimer3.html) to minimize the possibility of self-dimerisation that could lead to nonspecific PCR amplicons. Primers were purchased in a lyophilized form from Thermo Electron GmbH (Germany), then diluted to a final concentration of 100 µM with sterile distilled water and stored at -20 °C until use. (12) The dsDNA template (286bp) for miniemulsion-PCR is a product of a classical PCR reaction in solution and was synthesized from genomic human dsDNA (kindly provided by Dr. Kaimo Hirv, Institute of Transfusion Medicine and Immunogenetics, Ulm) using 5′-CGGCAGCAACAGCAGGT-3 as forward and 5′-CCACGCCAATCACTCTCATCT-3 as reversible primers. These primers amplify a short sequence of a housekeeping gene (PGBD.; GeneBank AccessionNo. M95623; Primers were designed as described in ref 11).
BM050084+
