Anal. Chem. 2002, 74, 1380-1385
Programmable Delivery of DNA through a Nanopipet Liming Ying,† Andreas Bruckbauer,† Alison M. Rothery,† Yuri E. Korchev,‡ and David Klenerman*,†
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, U.K., and MRC Clinical Science Centre, Division of Medicine, Imperial College School of Medicine, London, W12 0NN, U.K.
We report the pulsed delivery of single-stranded DNA molecules through a nanopipet. The conical geometry of the pipet leads to a localized electric field, since all of the potential drop occurs in the tip region. Pulsatile delivery of DNA molecules can be achieved in an experimentally simple way with high precision by controlling the applied voltage. Single-molecule detection and fluorescence correlation spectroscopy in the nanopipet enable us to determine the number of molecules delivered. Anomalous slow diffusion of the DNA molecules in the pipet has also been observed. This nanopumping technique may have potential applications in local drug delivery and nanofabrication of biomolecules on surfaces in aqueous environments. The development of novel nanoscopic tools to deliver, manipulate, and separate molecules is an important area of research in nanotechnology. For example, interests in DNA sequencing and separation have already led to the development of novel integrated micro- and nanofluidic devices.1-6 Controlled and accurate delivery of a small number of biomolecules to a specific location is a key issue in biotechnology. In medicine, pulsatile release of drugs usually works better than continuous release, because it mimics the way that the human body naturally produces some compounds. Much previous work on achieving complex drug-release patterns has concentrated on using polymers that respond to specific stimuli, such as pH7 or temperature changes,8 electric9 or magnetic fields,10 enzymes,11 exposure to ultrasound,12 etc. Alternatively, pulsatile delivery can also be achieved using * To whom correspondence should be addressed. Tel: +44 1223 336481. Fax: +44 1223 336362. E-mail:
[email protected]. † University of Cambridge. ‡ Imperial College School of Medicine. (1) Kasianowicz, J. J.; Brandin, E.; Deamer, D. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13770-13773. (2) Vercoutere, W.; Winters-Hilt, S.; Olsen, H.; Deamer, D.; Hanssler, D.; Akeson, M. Nat. Biotechnol. 2001, 19, 248-252. (3) Howorka, S.; Cheley, S.; Bayley, H. Nat. Biotechnol. 2001, 19, 636-639. (4) Li, J.; Stein, D.; McMullan, C.; Branton, D.; Aziz, M. J.; Golovchenko, J. A. Nature 2001, 412, 166-169. (5) Bader, J. S.; Hammond, R. W.; Henck, S. A.; Deem, M. W.; Mcdermott, G. A.; Bustillo, J. M.; Simpson, J. W.; Mulhern, G. T.; Rothberg, J. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13165-13169. (6) Han J.; Craighead, H. G. Science 2000, 288, 1026-1029. (7) Siegel, R. A.; Falamarzian, M.; Firestone, B. A.; Moxley, B. C. J. Controlled Release 1988, 8, 179-182. (8) Hoffman, A. S.; Afrassiabi, A.; Dong, L. C. J. Controlled Release 1986, 4, 213-222. (9) Kwon, I. C.; Bae, Y. H.; Kim, S. W. Nature 1991, 354, 291-293.
1380 Analytical Chemistry, Vol. 74, No. 6, March 15, 2002
microchips that incorporate micrometer-scale pumps, valves, and flow channels.13 Gene therapy, which involves the delivery of therapeutic DNA into cells, offers a good opportunity for the treatment of genetic disease.14,15 However, progress in developing effective genedelivery clinical protocols has been slow because of problems, such as safety, low efficiency and reproducibility, and high cost of existing gene delivery vectors.14,16 Recently, electrochemical routes for DNA delivery have been reported.17 The basic concept of this method is to coat the nucleic acid molecules onto the microelectrode surface and to expel them on demand under potential control.17 In fact, microelectrode systems employing micropipets have been used for decades in neurobiological research for delivering ionic species into the cell.18 However, quantitative measurements of the concentration of delivered molecules near the tip is scarce,19 because researchers in these fields are often interested only in pumping a large excess of the probe molecules into the cell. Recently, Sauer and co-workers reported the single-molecule detection in femtotips by timecorrelated single-photon counting,20-22 which is an important step toward single-molecule DNA sequencing.23,24 Nanopipets have also been used in scanning ion conductance microscopy (SICM).25-29 (10) Edelman, E. R.; Kost, J.; Bobeck, H.; Langer, R. J. Biomed. Mater. Res. 1985, 19, 67-83. (11) Fischel-Ghodsian, F.; Brown, L.; Mathiowitz, E.; Brandenburg, D.; Langer, R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 2403-2406. (12) Kost, J.; Leong, K.; Langer, R. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 76637666. (13) Santini, J. T.; Cima, M. J.; Langer, R. Nature 1999, 397, 335-338. (14) Verma, I. M.; Somia, N. Nature 1997, 389, 239-242. (15) Anderson, W. F. Nature 1998, 392, 25-30. (16) Luo, D.; Saltzman, W. Nature Biotechnol. 2000, 18, 33-37. (17) Wang, J. Electroanalysis 2001, 13, 635-638. (18) Kuffler, S. W.; Yoshikami, D. J. Physiol. (London) 1975, 244, 703-730. (19) Armstrong-James, M.; Millar, J.; Kruk, Z. L. Nature 1980, 288, 181-183. (20) Zander, C.; Drexhage, K. H.; Han, K. T.; Wolfrum, J.; Sauer, M. Chem. Phys. Lett. 1998, 286, 457-465. (21) Becker, W.; Hickl, H.; Zander, C.; Drexhage, K. H.; Sauer, M.; Siebert, S.; Wolfrum, J. Rev. Sci. Instrum. 1999, 70, 1835-1841. (22) Sauer, M.; Angerer, B.; Han, K. T.; Zander, C. Phys. Chem. Chem. Phys. 1999, 1, 2471-2477. (23) Jett, J. H.; Keller, R. A.; Martin, J. C.; Marrone, B. L.; Moyzis, R. K.; Ratliff, R. L.; Seitzinger, N. K.; Shera, E. B.; Stewart, C. C. J. Biomol. Struct. Dyn. 1989, 7, 301-309. (24) Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Johnson, M. E.; Martin, L. C.; Marrone, B. L.; Schecker, J. A.; Wilkerson, C. W.; Keller, R. A. Ber. BunsenGes. Phys. Chem. 1993, 97, 1535-1542. (25) Hansma, P. K.; Drake, B.; Marti, O.; Gould, S. A.; Prater, C. B. Science 1989, 243, 641-643. (26) Korchev, Y. E.; Bashford, C. L.; Milovanovic, M.; Vodyanoy, I.; Lab, M. J. Biophys. J. 1997, 73, 653-658. 10.1021/ac015674m CCC: $22.00
© 2002 American Chemical Society Published on Web 02/08/2002
The combination of SICM and patch-clamp techniques has demonstrated new capability of functional mapping of ion channels in vivo at high resolution.30 It is possible to deliver therapeutic DNA or proteins to a specific position on a living cell by manipulating the pipet with precise distance control. Both shear force and ion conductance are potential distance regulation methods. As a first step to use the pipet for controlled delivery, it is essential to characterize the flux of biomolecules in the nanopipet as the applied voltage is altered. In this paper, we report the programmable delivery of dyelabeled DNA molecules through a nanopipet by electrical control. Quantitative measurements using single-molecule spectroscopy and fluorescence correlation spectroscopy (FCS) enable us to determine the number of molecules delivered. EXPERIMENTAL SECTION Instrumentation. A homemade single-molecule confocal fluorescence microscope31,32 equipped with a piezoelectric nanomanipulator was used to view and position the pipet in the laser focus and also to detect the fluorescence signal from dye-labeled DNA. An argon ion laser (Ion Laser Technology 5490AWC-00) at 488 nm was used as the excitation source. The collimated linearpolarized laser beam was directed through a dichroic mirror to the inverted microscope (Nikon Diaphot 200) and focused by an oil immersion objective (Nikon Fluor 100×, NA 1.30). Fluorescence was collected by the same objective and imaged onto a 100µm pinhole to reject out-of-focus fluorescence and other background. The fluorescence was then filtered by a band-pass and a long-pass filter (Omega Optical 535AF45 and OG515) before being focused onto an avalanche photodiode, APD (EG&G SPCM AQR141). Output from the APD was coupled to a PC-implemented multichannel scalar card (EG&G MCS-Plus). A bent nanopipet was mounted to a homemade nanomanipulation system consisting of a modular focusing unit (Nikon 883320), a mechanical translation stage, and a three-axis piezoelectric translation stage (Piezosystem Jena Tritor38). The modular focusing unit and the mechanical translation stage were used for coarse positioning of the nanopipet. The piezoelectric stage was used for fine adjustment. Glass pipets with inner radii ∼50 nm were routinely fabricated using a laser-based pipet puller (Sutter Instrument P-2000). A voltage was applied to the nanopipet through two Ag/AgCl electrodes, one in the bath and the other inside the pipet, serving as the working and reference electrodes, respectively. The ion current flow through the pipet was amplified by a high impedance amplifier and monitored by an oscilloscope. The ion current was the same in the presence and absence of DNA, since the ion current is dominated by the flow of sodium and chloride ions. In addition, no ion current reduction due to partial blocking could be detected with DNA in the pipet. (27) Korchev, Y. E.; Raval, M.; Lab, M. J.; Gorelik, J.; Edwards, C. R.; Rayment, T.; Klenerman, D. Biophys. J. 2000, 78, 2675-2679. (28) Shevchuk, A. I.; Gorelik, J.; Harding, S. E.; Lab, M. J.; Klenerman, D.; Korchev, Y. E. Biophys. J. 2001, 81, 1759-1764. (29) Korchev, Y. E.; Negulyaev, Y. A.; Edwards, C. R.; Vodyanoy, I.; Lab, M. J. Nat. Cell Biol. 2000, 2, 616-619. (30) Gu, Y.; Spohr, H. A.; Gorelik, J.; Shevchuk, A.; Lab, M. J.; Harding, S. E.; Vodyanoy, I.; Klenerman, D.; Korchev, Y. E. Submitted. (31) Ying, L. M.; Wallace, M. I.; Balasubramanian, S.; Klenerman, D. J. Phys. Chem. B, 2000, 104, 5171-5178. (32) Wallace, M. I.; Ying, L. M.; Balasubramanian, S.; Klenerman, D. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5584-5589.
Materials and Experimental Procedure. A DNA sample was synthesized by Cruachem (Glasgow, U.K.) and was HPLC-purified twice. The sequence of the singly labeled DNA is 5′-CTATGCAGCCATTGTAGTCC-3′. The 3′ end was modified by Rhodamine Green (Molecular Probes). The concentration of the dye-labeled DNA was determined by UV-vis absorption at 260 nm, and the absorbance at 504 nm was used as an internal reference. The purity of the sample was better than 95% on the basis of this internal check. Tris-HCl buffer solution and NaCl were purchased from Sigma. 100 nM DNA solution was back-filled to the bent nanopipet by a microfiller (World Precision Instruments Microfil 34). Blockage of the nanopipet was avoided by filtering the solutions through a 20-nm filter. A coverglass-bottomed dish (Willco Wells GWST-1000) containing 2-3 mL of solution was used as the bath. The pipet tip was placed 5-10 µm above the dish surface. Identical buffers (10 mM Tris-HCl, 100 mM NaCl) were used both in the pipet and in the bath. For single-molecule detection in the nanopipet, 5 nM DNA solution was used, and the laser power was 0.25 mW. Fluorescence correlation measurements were performed by correlating the MCS signal in real time using our own software developed in Labview environment (National Instruments Evaluation Version 5.0). RESULTS AND DISCUSSION Figure 1 shows the schematic representation of our experimental setup and a video image of the nanopipet and fluorescence spot near the tip excited by a focused 488-nm laser beam. The spot size observed is ∼500 nm, bigger than the inner diameter of the pipet as a result of the limitation of far-field optical resolution, which is consistent with the probe volume measurement using FCS. When the laser focus was moved into the pipet, an elliptical fluorescence spot with length ∼1.5 µm was observed. When we apply a voltage across the two electrodes, the potential drop occurs almost entirely in the tip region, because of the conical geometry of the pipet. The electric field distribution along the pipet can be described by33
E(x) )
V0R0 tan θ (R0 + x tan θ)2
(1)
where x is the distance from the tip, R0 is the radius of the tip, θ is the half-cone angle of the inner wall of the pipet, and V0 is the applied potential. The pipet resistance F can also be derived as
F)
1 πξR0 tan θ
(2)
where ξ is the conductance of the solution. A 50-nm-radius pipet, with θ ∼3°, has a typical resistance of 100 MΩ in 100 mM NaCl solution according to eq 2, which is consistent with the experimental measurement. It is clear from eq 1 that the applied electric field is highly nonuniform and is located very close to the tip (the field strength drops to
