Detecting Single Porphyrin Molecules in a Conically Shaped Synthetic

We report here the first example of abiotic resistive-pulse sensing of a molecular (as opposed to a particle or macromolecular) analyte. This was acco...
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NANO LETTERS

Detecting Single Porphyrin Molecules in a Conically Shaped Synthetic Nanopore

2005 Vol. 5, No. 9 1824-1829

Elizabeth A. Heins, Zuzanna S. Siwy, Lane A. Baker, and Charles R. Martin* Department of Chemistry and Center for Research at the Bio/Nano Interface, UniVersity of Florida, GainesVille, Florida 32611-7200 Received May 18, 2005; Revised Manuscript Received July 14, 2005

ABSTRACT We report here the first example of abiotic resistive-pulse sensing of a molecular (as opposed to a particle or macromolecular) analyte. This was accomplished by using a conically shaped nanopore prepared by the track-etch method as the sensing element. It is possible to sense the molecular analyte because the small diameter opening of the conical nanopore (∼4.5 nm) is comparable to the diameter of the analyte molecule (∼2 nm).

Introduction. There is increasing interest in using nanopores as the sensing elements in biosensors.1-15 The R-hemolysin protein nanopore is typically used, and the sensor consists of a single protein nanopore embedded within a lipid bilayer membrane.1-9 An ionic current is passed through the nanopore, and analyte species are detected as transient blocks in this current associated with translocation of the analyte through the pore. This technique has been called stochastic sensing in the literature, but is included in the more general class of sensing technologies called resistive-pulse sensing.1 Both wild-type and engineered R-hemolysin nanopores have been used to sense a wide range of analytes including metal ions,4 DNA,5,6 proteins,7 and polymers.8 Small molecule analytes can also be sensed this way, provided that the molecule can be sequestered in a larger species such as a cyclodextrin.9 Although this is a very promising sensing paradigm, it would be advantageous to eliminate the fragile lipid bilayer membrane and perhaps to replace the biological nanopore with an abiotic equivalent.10-22 A variety of approaches have been used to prepare abiotic nanopores for sensing applications, including focused ion beam etching of silicon nitride and oxide,10,11,14,15 soft lithographic techniques,13 embedded carbon nanotubes,16 and track-etched conical nanopores produced in polymeric membranes.17-22 To date, the only analytes to be sensed via the resistive-pulse approach with such abiotic nanopores are nanoparticles16,21 and large DNA molecules.10,12,13,15 Biofunctionalized gold nanotubes have been used to detect protein analytes but not via the resistivepulse method.22 We report here the first example of abiotic resistive-pulse sensing of a molecular (as opposed to a particle or macromolecular) analyte. This was accomplished * To whom correspondence should be addressed. E-mail: crmartin@ chem.ufl.edu; fax: 352-392-8206; voice: 352-392-8205. 10.1021/nl050925i CCC: $30.25 Published on Web 07/26/2005

© 2005 American Chemical Society

Scheme 1

by using a conically shaped nanopore17-22 prepared by the track-etch method23 as the sensing element. It is possible to sense this molecular analyte (Scheme 1) because the small diameter opening of the conical nanopore is comparable to the diameter of this molecule. Experimental Section. Materials. Polyimide films (Kapton-50HN, DuPont, 3 cm diameter, 12 µm thick) that had been irradiated with a single swift heavy ion of 2.2 GeV kinetic energy to create a single damage track through the film were obtained from Gesellschaft fuer Schwerionenforschung, Darmstadt, Germany. We refer to these as-received films as the “tracked” films. KCl and KI (certified A. C. S., Fisher Scientific), 4,4′,4′′,4′′′-(porphine-5,10,15,20-tetrayl)tetrakis(benzenesulfonic acid) (TPPS, the analyte, Scheme 1) (∼98.0%, Sigma), and sodium hypochlorite (13% active chloride ion, Sigma) were used as received. Solutions were prepared in 18 MΩ water (Barnstead, E-pure). Preparation and Characterization of the Conical Nanopores. Membrane samples that contained a single conically shaped nanopore were prepared by anisotropic chemical etching of the tracked films. The details of this process have been described previously.18 Briefly, this entails mounting the tracked film in a U-tube cell and placing a solution that

etches the Kapton (sodium hypochlorite) on one side and a solution that neutralizes the etchant (KI “stop” solution) on the other side. A platinum electrode was placed in each solution, and a potential of 1 V was applied during etching. The transmembrane current was initially zero, but increased after breakthrough of the etchant into the stop solution. Etching was terminated when a transmembrane current of 100 pA was obtained. This method yields a conical nanopore with the largediameter opening (base opening) facing the etch solution and small-diameter opening (tip opening) facing the stop solution. The diameter of the base opening was determined via field emission scanning electron microscopy (FESEM) (JEOL JSM-6335F). An electrochemical method discussed previously18 was used to obtain an estimate of the diameter of the tip opening, dt. Briefly, this method entails mounting the nanopore membrane between two electrolyte solutions (1 M KCl, pH ) 8), and using a Ag/AgCl electrode immersed into each solution to obtain a current-voltage curve for the nanopore. At applied transmembrane potentials between (100 mV, the current-voltage curve is linear, and the slope provides the ionic resistance (Rp) of the electrolyte-filled nanopore;18 dt is related to Rp via Rp )

4F L π dbdt

(1)

where F is the specific resistance of the electrolyte, L is the length of the nanopore (membrane thickness), and db is the diameter of the base opening determined independently via FESEM. Membranes with db ) 2 µm and dt ∼4.5 nm were used for these studies. Etching thinned the membrane from its initial thickness of 12 to 10 µm. Current-Time Recordings. The single conical nanopore membrane was mounted in a conductivity cell, and both halfcells were filled with 1 M KCl, pH ) 8.0 (phosphate buffer) solution. A Ag/AgCl electrode was immersed into each halfcell solution, and an Axopatch 200B amplifier (Molecular Devices Corporation, Union City, CA) was used to apply the desired transmembrane potential difference and measure the resulting transmembrane current. The working Ag/AgCl electrode was in the half-cell solution facing the base opening of the nanopore, and the reference Ag/AgCl electrode was in the half-cell solution facing the nanopore tip opening. The current was recorded using the voltage-clamp mode and a lowpass Bessel filter with 2 kHz bandwidth. The signal was digitized using a Digidata 1322x analog-to-digital converter (Molecular Devices Corporation) at a sampling frequency of 10 kHz. The data were recorded using pClamp 9.0 software (Molecular Devices Corporation). Positive applied transmembrane potentials (as used here) mean that the anode is in the electrolyte solution facing the base opening of the conical nanopore, and the cathode is in the solution facing the tip opening of the nanopore. Because -SO3H is a strong acid, the analyte TPPS is a tetravalent anion, and it was added to the solution facing the nanopore tip opening. As a result, at sufficiently high values of positive applied transmembrane potentials, TPPS can be driven Nano Lett., Vol. 5, No. 9, 2005

Figure 1. Current-time recordings of the conical nanopore with 1 M KCl (pH ) 8.0) solutions on both sides of the membrane. The transmembrane potential was (a) 100 mV and (b) 400 mV.

electrophoretically into the tip opening. As we will see, this results in downward current pulses, which were detected and analyzed with the QuB software package (available at www.qub.buffalo.edu/).24,25 Statistical analyses of currenttime recordings were accomplished by analyzing three or more 30-second segments of the data at each applied transmembrane potential. TPPS is an interesting test analyte because it is a model for similar porphyrins used in photodynamic treatment of tumor cells.26,27 Results and Discussion. As described above, the conical nanopore membrane is placed between two electrolyte solutions and an applied transmembrane potential is used to drive an ion current through the electrolyte-filled nanopore. An important feature of the conical nanopore is that the voltage drop caused by this ion current is focused at the nanopore tip.21 Indeed, calculations done by Lee et al. indicate that the field strength in the solution just inside the nanopore tip can be ∼106 V/m, when the total voltage drop across the nanopore membrane is only 1 V.21 A consequence of this focusing effect is that the ion current flowing through the conical nanopore is extremely sensitive to analyte species present in or near the nanopore tip.21,22 That is, there is a “sensing zone” just inside the nanopore tip, and for the nanopores used here we estimate that this sensing zone is ∼120 nm in length.28 This focusing effect makes conically shaped nanopores better suited for sensing applications than cylindrical nanopores. If no analyte is added to the buffer solutions in contact with the nanopore membrane, application of a constant transmembrane potential difference causes a steady-state ion current to flow through the pore. As would be expected, the magnitude of this current increases with the applied transmembrane potential (Figure 1).20,29 Figure 2 shows currenttime traces in the presence 60 nM TPPS (added to the solution facing the nanopore tip). At applied transmembrane potentials below ∼300 mV, steady-state current-time traces are again observed (Figure 2a). This is because the nanopore is cation permselective in the electrolyte solution used here,19,20 and the anionic TPPS is electrostatically repelled from the nanopore. In addition, the analyte pays an entropic 1825

Figure 2. Current-time recordings for the conical nanopore in the presence of 60 nM TPPS. The transmembrane potential was (a) 100 mV, (b) 400 mV, (c) 500 mV, and (d) 600 mV. Electrolyte as per Figure 2.

penalty for entering such a narrow pore.30 For these reasons, at potentials below a threshold voltage of ∼300 mV, TPPS has no effect on the current-time trace (Figure 2a). This

threshold voltage phenomenon has also been observed for DNA translocation in the R-hemolysin nanopore.30 At larger values of positive applied transmembrane potential, the electrostatic and entropic barriers can be overcome, and TPPS can be driven electrophoretically into the nanopore tip opening. Because the diameter of the TPPS molecule (∼2 nm) is comparable to the diameter of the tip opening (∼4.5 nm), TPPS experiences hindered diffusion in the nanopore tip. Indeed, theory predicts that its diffusion coefficient in the tip is ∼10 times smaller than its bulksolution value.31 Because the hydrated K+ ion is much smaller (diameter ∼0.3 nm32), its diffusion is not hindered in the nanopore tip, and charge is carried through the nanopore by these much more mobile K+ ions. However, the large and relatively immobile TPPS molecules partially occlude the pathways that the K+ ions use to carry charge through the nanopore. As a result, downward current blocks are observed (Figure 2b-d) in the presence of the analyte TPPS, resistive-pulse sensing.1 In this detection technique, the duration of an occlusion event is determined by the time it takes the analyte to translocate the nanopore sensing element.1 Although this is also true for the conical nanopore system studied here, the electric-field focusing effect discussed above means that the relevant time is the time required for the analyte to translocate the sensing zone, and not the time required for the analyte to translocate the entire length of the pore. The event-duration data were analyzed via plots of the number of times an event of a particular duration was observed versus the event duration (Figure 3).2 At the lowest value of applied transmembrane potential (400 mV), the distribution is much broader than that reported for translocation of analytes through the R-hemolysin nanopore.2 Both entropic and electrostatic interactions are expected to contribute signifi-

Figure 3. Histograms of event duration for the conical nanopore in the presence of 60 nM (a-c) and 20 nM (d) TPPS. The transmembrane potential was (a) 400 mV, (b) 500 mV, (c) 600 mV, and (d) 400 mV. 1826

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cantly to the dynamics of translocation with our system. The breadth of the distribution observed may result from these complex interactions of the highly charged TPPS molecule with the walls of the conical nanopore. At larger values of applied transmembrane potential, the event-duration distribution narrows and shifts to shorter times (e.g., Figure 3c). To gain insight into the time scales of processes associated with TPPS translocation, we have modeled the histograms of dwell-time distribution (Figure 3) using first-order kinetics between the blocked and open state.33 The histograms of blocked-state duration can be fit with the sum of two exponentials of the form A exp(-t/τ1) + B exp(-t/τ2), which suggests the existence of two types of blocked states of average duration, τ1 and τ2 (Figure 3). For all three voltages, a shorter blockage time (∼2 ms) was observed, which might correspond to bumping of TPPS molecules against the nanopore, an effect observed previously with DNA molecules and the R-hemolysin nanopore.2 The shorter blockage time could also correspond to the reorientation of molecules at the entrance of the pore, followed by translocation. The longer blockage events are due to translocation of TPPS molecules through the nanopore. The electrophoretic velocity (V) of an ion is related to the electric field strength (E ) via34 V ) |z|eE/6πηr

(2)

where z and r are the charge and radius of the ion, respectively, e is the electronic charge, and η is the viscosity of the solution. We see that ionic velocity increases with field strength, and this is why shorter duration events are observed at higher values of applied transmembrane potential. The occlusion-event duration for the 600 mV case (Figure 3c) is ∼9 ms. Additionally, the distribution of occlusion duration is independent of TPPS concentration (Figure 3a and d). The occlusion events may also be analyzed by plotting the number of times a particular current value was obtained versus the magnitude of the measured current (Figure 4).2 At applied transmembrane potentials of 400 and 500 mV, two peaks are observed. The peak at higher currents corresponds to the open state (no TPPS in the sensing zone), and the lower current peak corresponds to the partially occluded state (TPPS present in the sensing zone). We see that relative to the open state, the number of occlusion events is higher at 500 mV than at 400 mV. This is to be expected because the electrophoretic flux (J ) of an ion is related to the electric field strength via35 J ) -zFDCE/RT

(3)

where D is the diffusion coefficient and C is the concentration of the ion. Because the flux of TPPS into the nanopore increases with field strength, the number of events observed likewise increases with E. Analogous results have been observed with other nanopore systems.2,21,30,36 At the highest value of applied transmembrane potential, 600 mV, two partially occluded states are apparently Nano Lett., Vol. 5, No. 9, 2005

Figure 4. Histograms of ion current values for the conical nanopore in the presence of 60 nM TPPS. The transmembrane potential was (a) 400 mV, (b) 500 mV, and (c) 600 mV.

observed. A similar phenomenon was observed for a Ca2+gated potassium channel in the presence of high concentrations of Ca2+.37 It was suggested that this is a consequence of the very rapid pore blocking in the presence of such high Ca2+ concentrations. We believe we are seeing a related phenomenon here because, as noted above, at 600 mV we observe the smallest event durations (Figure 3). Another possible reason for observing a second level of blockage is the presence of an additional TPPS molecule in the sensing zone of the nanopore. By applying a higher transmembrane potential, a higher probability of TPPS molecules entering the nanopore is assured (eq 3).30 Therefore, at higher transmembrane potentials and higher TPPS concentrations, the probability of two TPPS molecules entering the pore at the same time is higher. This possibility is supported by a lack of the second blockage level at recordings performed in the case of 20 nM porphyrin. 1827

Figure 5. Current-time recordings for the conical nanopore with applied potential of 400 mV. The concentration of TPPS was (a) 0 nM, (b) 20 nM, and (c) 60 nM.

The distance along the x axis between the maxima in the histograms of ion current values (Figure 4) provides the change in current (∆i) between the open and partially occluded states. An applied transmembrane potential of 400 mV gave ∆i ) 20 pA, which is a 0.8% change from the 2.5 nA current of the open state. At 500 mV ∆i ) 34 pA, which is a 1% change from the 3.0 nA current of the open state. To determine if such small ∆i values are reasonable for this nanopore/analyte system, we have applied a mathematical model developed for the Coulter Counter, a device used to count and size particles.38 This model was developed for cylindrical pores,39 as used in the Coulter counter, but we have modified it to account for the conical pore geometry used here.28 This model predicts that the change in ion current for our analyte/nanopore system should be ∼1.2%, in good agreement with the experimental data. It is worth noting that ∆i values in the range of 0.1-9%, have been observed for DNA stochastic sensing with other synthetic pore systems.10,12,14 Finally, we have also begun to explore the effect of analyte concentration on the number of partial occlusion events (Figure 5). In stochastic sensing, the number of events increases with analyte concentration,1,2 and we see this with our sensor as well. For example, at a transmembrane potential of 400 mV, 103 events were observed in a 25-s time interval when the analyte TPPS concentration was 20 nM, whereas 505 events were observed in a 25-s time interval at 60 nM. However, it is clear that more work on the concentration dependence remains to be done. Conclusions. We have shown that an anionic molecule can be driven electrophoretically into the tip opening of a conical-shaped synthetic nanopore and detected via the partial current blocks that the molecule produces as it traverses the 1828

nanopore. The conical nanopore offers a number of advantages for such molecule-counting experiments relative to cylindrical nanopores. Most importantly, with the conical nanopore the voltage drop engendered by the ion current is focused to the tip of the nanopore, thus forming a sensing zone for analyte detection. In addition, the ion current through the conical nanopore can be orders of magnitude larger than that in a cylindrical nanopore with diameter equivalent to the diameter of the tip opening of the conical pore. Finally, the conical pore should be less susceptible to unwanted fouling than a cylindrical pore of comparable size. For example, we have made repetitive current-voltage curve measurements on nanopores such as those used here for a period of one month and obtained no evidence for pore blockage. The track-etch method used to prepare the conical nanopores described here also offers some advantages relative to the methods used to prepare other prototype abiotic nanopores for chemical and biochemical sensing.9,10,12,14 First, the track-etch method is a well-known and commercially practiced method for preparing nanopore membranes. Second, the chemical etch procedure used to produce the conically shaped pore requires no specialized or expensive equipment. Third, the membrane material (in this case Kapton polyimide) is mechanically strong and chemically inert. Most importantly, this material is impervious to the aqueous electrolyte solutions most commonly encountered in biosensing applications. Finally, this nanopore sensor offers some advantages relative to the R-hemolysin nanopore used for most stochasticsensing experiments. Most importantly, the fragile supported lipid bilayer membrane is eliminated and replaced with a durable polymeric membrane. This offers numerous advantages. First, because of its fragility, sensor life is prohibitively short with the bilayer membrane, and this is not the case with the polymer membrane (vide supra). Second, much larger transmembrane potentials can be applied with the polymeric membrane. Indeed, potentials as large as 10 V40 have been applied without membrane rupture. As we have shown here, this is important because the number of occlusion events observed for ionic analytes increases with applied transmembrane potential (Figure 2). Because the number of events is related to analyte concentration, it is in principle possible to detect lower concentrations of ionic analytes using higher values of applied transmembrane potential with the polymer nanopore system. An advantage of the R-hemolysin nanopore is that it can be genetically or chemically engineered to place a selective binding site at a precise location within the lumen of the pore.1 However, we have shown that conical Au nanotubes can be deposited with nanopores such as those used here,17 and this opens the door to chemical and biofunctionalization with any thiolated molecule or other chemical species. For example, we have functionalized such Au nanotubes with thiolated DNAs17 and a thiolated biotin.22 After reaction with streptavidin, the biotinylated nanotubes can be modified subsequently with nearly any biotinylated protein.22 Nano Lett., Vol. 5, No. 9, 2005

For all of these reasons, we believe that the conical nanopore/nanotube system offers real promise for preparing practical sensing devices. Acknowledgment. This work was funded by the National Science Foundation and DARPA. Supporting Information Available: Calculation of effective length of a conical nanopore and Coulter counter concept applied to a conical nanopore. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Bayley, H.; Martin, C. R. Chem. ReV. 2000, 100, 2575-2594. (2) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13770-13773. (3) Braha, O.; Gu, L.-Q.; Zhou, L.; Lu, X.; Cheley, S.; Bayley, H. Nat. Biotechnol. 2000, 18, 1005-1007. (4) Howorka, S.; Movileanu, L.; Braha, O.; Bayley, H. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12996-13001. (5) Howorka, S.; Cheley, S.; Bayley, H. Nat. Biotechnol. 2001, 19, 636639. (6) Movileanu, L.; Howorka, S.; Braha, O.; Bayley, H. Nat. Biotechnol. 2000, 18, 1091-1095. (7) Bezrukov, S. M.; Vodyanoy, I.; Brutyan, R. A.; Kasianowicz, J. J. Macromolecules 1996, 29, 8517-8522. (8) Gu, L.-Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature (London) 1999, 398, 686-690. (9) Li, J.; Gershow, M.; Stein, D.; Brandin, E.; Golovchenko, J. A. Nat. Mater. 2003, 2, 611-615. (10) Li, J.; Stein, D.; McMullan, C.; Branton, D.; Aziz, M. J.; Golovchenko, J. A. Nature (London) 2001, 412, 166-169. (11) Mara, A.; Siwy, Z.; Trautmann, C.; Wan, J.; Kamme, F. Nano Lett. 2004, 4, 497-501. (12) Saleh, O. A.; Sohn, L. L. Nano Lett. 2003, 3, 37-38. (13) Storm, A. J.; Chen, J. H.; Ling, X. S.; Zandbergen, H. W.; Dekker, C. Nat. Mater. 2003, 2, 537-540. (14) Storm, A. J.; Storm, C.; Chen, J.; Zandbergen, H.; Joanny, J.-F.; Dekker, C. Nano Lett. 2005, 5, 1193-1197. (15) Henriquez, R. R.; Ito, T.; Sun, L.; Crooks, R. M. Analyst 2004, 129, 478-482. (16) Sun, L.; Crooks, R. M. J. Am. Chem. Soc. 2000, 122, 12340-12345. (17) Harrell, C. C.; Kohli, P.; Siwy, Z.; Martin, C. R. J. Am. Chem. Soc. 2004, 126, 15646-15647.

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