Measurement of the signal from a single DNA molecule trapped by a nanoplasmonic structure

Transcription

1 Measurement of the signal from a single DNA molecule trapped by a nanoplasmonic structure Jung-Dae Kim, Waleed Muhammad and Yong-Gu Lee School of Mechatronics, Gwangju Institute of Science and Technology (GIST), 123 Cheomdangwagiro (Oryong-dong), Buk-gu, Gwangju, , Republic of Korea ABSTRACT Optical tweezers use focused laser to trap microobjects suspended in the medium to the focal point. They are becoming an indispensable tool in microbiology because of its ability to trap tiny biological particles so that single particle analysis is possible. However, it is still very difficult to trap particles such as DNA molecules that are smaller than the diffraction limit. Although trapping of those is possible by increasing the laser power inversely proportional to the cube of the particle diameter, such high power can cause permanent thermal damages. One of the current solutions to this problem is to intensify the local field by the use of the near-field enhancement coming from nanoplasmonic structures illuminated with lasers. Such solution allows one to use low powered laser and still be able to trap them. In this paper, we present the trapping of a single DNA molecule by the use of the strong field enhancement due to a sub-micrometer sized hole drilled on a gold plate by an e-beam milling process and the trapping is verified by the measurement of the scattering signal that comes from the trapped DNA. Keywords: Optical trapping, Optical tweezers, Nanoplasmonics, DNA molecules 1. INTRODUCTION Optical trapping is an efficient tool to trap and manipulate micro-scale objects. It has great potential in microbiological applications because of its ability to trap tiny bioparticles without damage. In 1970, Ashkin proposed that light can be used to provide non-contact radiation pressure to the microscopic objects to trap and manipulate them. Later in 1986, he demonstrated a stable, three-dimensional trap which is based on counter propagating laser beams. His experiments laid the foundation of lens based single-beam optical trap. These optical instruments are called optical tweezers [1]. The fundamental physics of optical tweezers is based on the equilibrium state of objects under tightly focused laser beams. Induced by light fields, such equilibrium is conceptually based on momentum transfer from light to the object during refraction and reflection of incident light from the objects [2]. The refraction of the laser through the dielectric object produces a gradient force perpendicular to the direction of propagation of incident laser beam while reflection of the laser field produces scattering forces on the object in the direction of incident laser beam. The object is optically trapped if the gradient forces dominate the scattering forces. Direct optical trapping of biological particles, however, has been limited to relatively large objects e.g. living cells and chromosome. After invention of the optical trapping phenomenon, a lot of research has been made to use them to trap small objects in the range of nanometers. Although trapping of nanoscale objects has been made possible by increasing the localized optical field by increasing the laser power, laser power needs to increase scaled with the third power of the particle diameter. This high power requirement can induce thermal damage to the object. Increasing the laser power also increases the scattering forces that result in the increase of possibility of pushing the object away from the stable trap. Moreover, the thermal fluctuation of the trapped objects increases with the decrease in the object size thereby decreasing the trapping time. Ashkin also trapped the nanometer scaled objects but the trapping time was very small. He showed that 1 μm particle was trapped in a focused beam (optical trap) for several minutes with less than 1 mw incident laser power however, 109 nm particles could be trapped only for 25 s even at mw of incident laser. Moreover, to trap an 85 and 38 nm particle, the incident laser beam power requirement exceeded the damage threshold of the particle and thus the particle could not be trapped. Therefore, increasing the incident laser power is not the preferred solution to trap the nano-scaled objects. * To whom correspondence should be made: lygu@gist.ac.kr Optical Trapping and Optical Micromanipulation X, edited by Kishan Dholakia, Gabriel C. Spalding, Proc. of SPIE Vol. 8810, 88100W 2013 SPIE CCC code: X/13/$18 doi: / Proc. of SPIE Vol W-1

2 A substitute to the previously developed lens-based optical trap is to use sub-wavelength metallic nanostructure to enhance the localized optical field intensity without increasing the incident laser power [3-5]. The fundamental concept behind using the metallic nanostructure is that the electromagnetic waves interact with the free electrons of the metal and they are trapped at the surface of the metal. These trapped waves are called surface plasmons [6, 7]. The combination of these surface waves and sub-wavelength nanostructures gives rise to the phenomenon of extraordinary transmission in which localized optical field intensity is enhanced even more than 100% [8]. This alternative technique to focus the incident laser beam has been studied extensively and it is found that extraordinary transmission depends upon size, shape and thickness of the metallic nanostructures [9, 10]. Recently, the use of the metallic nanostructures was employed by Mathieu et al. who demonstrated self-induced-back-action (SIBA) optical trapping of dielectric particle in which the trapped particle also plays a vital role in the optical trap. Contrary to the conventional approach of increasing the laser intensity to trap objects, SIBA mechanism can trap smaller objects at relatively lower incident laser power [11]. Using this phenomenon, 100 nm particles was optically trapped for 5 min only at 1 mw incident laser power, greatly reducing the power requirement imposed by previous approaches. The decrease in the incident laser power also results in the reduction of the photo-damage to the biological samples during the experiment. From above discussions, it is obvious that metallic nanostructures are a powerful tool to focus the laser beam to trap the nano-scaled objects. In this paper, we present the trapping mechanism of a single DNA molecule by increasing the localized optical field intensity using the nanostructure. For this purpose, a nanohole is created on a gold plate using focused ion beam (FIB). A micrometer-sized PDMS chamber with gold plate on it is prepared to store the DNA. Subsequently, a focused laser beam is delivered to the hole inscribed on the DNA carrying chamber and the scattering signal is examined by an avalanche photodiode (APD) at the end of laser path to verify the trapping of the DNA. 2.1 Preparation of DNA sample 2. EXPERIMENTAL METHOD (,) I I MMIE I MI I IN,M' LI 11 _21 Ll.fiLL» rj L. Figure 1. PCR process and gel electrophoresis In this experiment, we need large amount of DNA fragments. To get them, firstly we set up a process of Polymerase Chain Reaction (PCR). In biology, PCR is a common method to replicate a particular-sized fragment of DNA in a large quantity. For this purpose, we need several materials such as primers, DNA polymerase, 4 kinds of nucleotides and a template. Template is a long DNA strand to be replicated. Primers determine the length of PCR product, assigning starting point and end point of PCR. If we know the sequence of a template, we can design many sets of primers to get various-sized DNA fragments. DNA polymerase replicates a template between primers into a large quantity. Nucleotides are monomers of a DNA, so they are necessary to replicate new DNA fragments. During PCR, we have to change the Proc. of SPIE Vol W-2

3 temperature to separate double stranded DNA into two single stranded DNA so that DNA replication by DNA polymerase can proceed. For PCR, we have purchased λ DNA (Bioneer, Korea) to use as a template and designed set of primers. Then we set cycles of temperature changes for replication. According to the protocol provided from HiPI Super 5x PCR Master Mix reagent (ELPis Bio, Korea), cycles of PCR are performed. After the PCR, length of DNA fragment is confirmed by the gel electrophoresis. Figure 1 shows DNA amplification, DNA separation process using PCR and gel electrophoresis, respectively. Figure 1(a-e) illustrates temperature and time settings for running the PCR instrument. After the PCR, the DNA length is measured by gel electrophoresis process as shown in Fig. 1(f). Finally, we acquired the desired DNA sample for our experiment. 2.2 Experimental setup Lamp APD L6 Gold plate!mil ND filter Coverglass Piezo stage Motorized stage ) DNA C-1 L3 L4 M111 DMI CM OS camera L5 M2 Figure 2. Schematic of the nanoplasmonic system; Lens: L, Mirror: M, Dichroic mirror: DM, Neutral density: ND Figure 2 illustrates, the schematic of the nanoplasmonic system. It is based on inverted microscope geometry where the TEM 00 mode of a 1050 nm wavelength fiber laser (IPG Photonics, YLM-10) is used to deliver the optical force for the trapping mechanism. In this system, achromatic lenses L1 and L2 work as a telescope to expand the source beam by two folds. The expanded beam is reflected by mirror M1 to a second telescope composed of L3 and L4 which slightly decreases the upcoming beam. The beam expansion is required to overfill the back aperture of the objective lens to maximize the trapping force at the focus. Finally, the laser beam is focused on the specimen using a water immersion objective lens (Olympus, UPLSAPO 60XW, 1.2 NA). The resulting tightly focused spot is sent to a sample holder mounted on the three-axis piezo stage (Mad City Labs Inc., Nano-LP200) positioned on the top of a two-axis motorized stage (Thorlabs, MT1/M-Z8). The combined use of piezo and motorized platforms facilitates very good resolution of 0.4 nm to locate the laser focus at the nanohole created on the gold plate. For this sample stage, the maximum movable range is 13 mm in the x- and y-axes and 200 µm in the z-axis. A high-speed monochrome CMOS camera (Microtron, Proc. of SPIE Vol W-3

4 Eosens CL) is used to acquire the real time bright-field images of the sample. The transmitted and scattered light from the sample are captured by a condenser lens. The refracted laser beam from the gold nanohole is finally delivered onto an avalanche photodiodes (Hamamatsu, G ) mounted on the manual xy-stage after attenuating from neutral density (ND) filters. 2.3 Fabrication of nanohole on the gold plate According to the theoretical calculations for 1050 nm wavelength laser, extraordinary transmission is achieved for 100 nm thickness of gold substrate at approximately 300 nm of the drilled hole [11]. In order to verify the theoretical results, it is necessary to determine the light transmission for various sized holes on the gold substrate. For this purpose, FIB should be used to drill the various size nanoholes on the gold plate because it is difficult to fabricate the nanoholes using general lithographic processes. Figure 3 shows scanning electron microscopic (SEM) images of gold plate with various sized nanoholes created by FIB. The nanoholes were fabricated from left to right with increasing size of the hole from 210 nm to 450 nm with 10 nm increments as shown in Fig 3(a). The distance between any two holes is 15 μm. To locate the nanoholes on the microscope easily, micro-scaled cross shape was also fabricated at the four corners. Figure 3(b) shows the SEM image of the fabricated gold plate. The enlarged view of a single nanohole is shown in Fig 3(c). + o nm +. ío 450nm (a) (b) (c) Figure 3. Schematic design and SEM images of nanoholes fabricated on the gold plate 2.4 Preparation of microchamber For trapping a single DNA, it is stored in a micro chamber where the experiments are performed. This chamber is prepared using Polydimethylsiloxane (PDMS). Following procedures were undertaken. After adding the DNA solution in the chamber, the gold plate is placed on the chamber. For this purpose, Sylgard 184A and Sylgard 184B are poured on a plate with the ratio of 10 to 1 and stirred well. Bubbles created during the stirring process are removed using evaporator. After removing the bubbles, the mixture is spread on the coverglass. Then this coverglass is kept in a spin coater which spreads the PDMS evenly on the cover glass. The height of the microchamber is controlled by rpm and rotation time. The mixture is solidified on the hot plate for 30 min at 80 ºC. After this, micrometer sized PDMS section is cut from the coverglass to make the chamber. Then small amount of DNA solution is poured into the chamber. Finally, gold plate is placed on the PDMS chamber. Lastly, this sample is mounted on the sample stage. It should be noted that thickness of the chamber should be in the range of micrometers so that floating DNA would pass near to the nanostructure in the gold plate. In this way, the DNA could be trapped easily. If thickness of chamber is too large, then the DNA might float far away from the nanohole thus could not be optically trapped. Therefore, a shallow depth of chamber is desired. 3. EXPERIMENTAL RESULTS 3.1 Transmission dependence on the nanohole size In the context of light transmission through sub-wavelength apertures, the first theoretical study was proposed by Bethe in 1944 [12]. He presented a mathematical equation for light transmission from single cylindrical hole in a perfect metallic conductor sheet having zero thickness. This theory predicted that optical transmittance through the aperture increases with the increase in the aperture diameter [13]. However, he calculated the transmittance relation with respect to size of hole considering thickness of metal substrate equal to zero. In 1987, Roberts et al. considered the thickness of metallic substrate and presented the dependence of light transmission as function of hole diameter and found that it increases with increase in the hole diameter up to cut off limit [14]. Increasing the sized hole beyond cut off limit, the Proc. of SPIE Vol W-4

5 transmittance starts decreasing. We have also measured the experimental dependence of transmitted intensity for different-sized nanoholes ( nm) in 100 nm thickness of gold substrate. In our experiment, the relation between transmitted intensity and nanohole diameters follows the trend as proposed by Roberts. Figure 4 shows our experimental results for transmitted intensity vs. nanohole diameter. The transmitted intensity of incident laser increases from 210 to 450 nm hole. The transmittance trend as a function of hole diameter is found sharp among nm of hole size. For hole diameter larger than 370 nm, the transmitted intensity increases with relatively lower rate. In our experiment, drilled maximum size of hole is 450 nm. Although we could not confirm the transmittance behavior for hole diameter larger than 450 nm, however we expect that it will increase up to cut off limit Measurement of DNA trapping signal Nanohole size (nm) Figure 4. Transmission vs. nanohole size drilled on 100 nm thick gold substrate Trapping of DNA No trapping of DNA Turn on the laser mr Turn off the laser i Time (ms) Figure 5. Measurement of transmitted intensity by DNA trapping Figure 5 shows the measured transmitted intensity for DNA trapped by 1050 nm laser passing through a 380 nm nanohole. We set 1.7 mw laser power measured at the focus. After the laser is turned on, the intensity is increased from 0 to 0.2 a.u. as represented by point A. It is due to the fact that when a DNA is trapped, then more scattered light is transmitted through the nanohole and thus APD measures higher voltage. The DNA was trapped for 16 s from point A to B. After point B, sudden decrease in the intensity is observed which means that DNA is no longer trapped. We expect that DNA trapping time could be increased by increasing the incident laser power. Proc. of SPIE Vol W-5

6 4. CONCLUSIONS In this paper, we described the trapping of DNA using plasmonic nanostructures and acquired transmitted intensity signal for the trapped DNA. The DNA is spread on PDMS microchamber under the gold substrate and intensity is measured by highly sensitive APD. We could only observe the intensity signal for short time due to the quick drying of DNA media in our experimental conditions. Although we could not obtain trapping signal for longer time, we observed difference in the intensity signals in the presence and absence of DNA. In future, we will optimize the experimental conditions for longer observation of intensity signal coming from the trapped DNA. ACKNOWLEDGEMENTS This work (2012R1A2A2A ) was supported by Mid-career Researcher Program through National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) and by the Basic Research Project through a grant provided by GIST. REFERENCES [1] Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E. and Chu, S., Observation of a single-beam gradient force optical trap for dielectric particles, Opt. Lett. 11(5), (1986) [2] Ashkin, A., Forces of a single-beam gradient laser trap on a dielectric sphere in the ray optics regime, Biophys. J., 61(2), (1992) [3] Huang, L. and Martin, O. J. F., Reversal of the optical force in a plasmonic trap, Opt. Lett. 33(24), (2008) [4] Nieto-Vesperinas, M., Chaumet, P. C. and Rahmani, A., Near-field photonic forces, Phil. Trans. Math. Phys. Eng. Sci. 362(1817), (2004) [5] Okamoto, K. and Kawata, S., Radiation force exerted on subwavelength particles near a nanoaperture, Phys. Rev. Lett. 83(22), (1999) [6] Ritchie, R. H., Plasma losses by fast electrons in thin films, Phys. Rev. 106(5), (1957) [7] Barnes, W. L., Dereux, A. and Ebbesen, T. W., Surface plasmon subwavelength optics, Nature 424, (2003) [8] Garcia de Abajo, F. J., Light transmission through a single cylindrical hole in a metallic film, Opt. Express 10(25), (2002) [9] Garcia-Vidal, F. J., Moreno, E., Porto, J. A. and Martin-Moreno, L., Transmission of light through a single rectangular hole, Phys. Rev. Lett. 95(10), (2005) [10] Ishi, T., Fujikata, J., Makita, K., Baba, T. and Ohashi, K., Si nano-photodiode with a surface plasmon antenna, Jpn J. Appl. Phys. 44(12), L364 L366 (2005) [11] Mathieu, L. J., Reuven, G., Yuanjie, P., Fatima, E. and Romain, Q., Self-induced back-action optical trapping of dielectric nanoparticles, Nat. Phys. 5(12), (2009) [12] Bethe, H. A., Theory of diffraction by small holes, Phys. Rev. 66(7-8), (1944) [13] Garcia-Vidal, F. J., Martin-Moreno, L., Ebbesen, T. W. and Kuipers, L., Light passing through subwavelength apertures, Rev. Mod. Phys. 82 (1), (2010) [14] Roberts, A., Electromagnetic theory of diffraction by a circular aperture in a thick, perfectly conducting screen, J. Opt. Soc. Am. A 4(10), (1987) Proc. of SPIE Vol W-6