Simultaneous Multianalyte Detection with aNanometer-Scale Pore

John J. Kasianowicz,* Sarah E. Henrickson, Howard H. Weetall, and Baldwin Robertson

NIST, Biotechnology Division, ACSL 227/A251, Gaithersburg, Maryland 20899-8313

It was recently shown that naturally occurring, geneticallyengineered or chemically modified channels can be usedto detect analytes in solution. We demonstrate here thatthe overall range of analytes that can be detected by singlenanometer-scale pores is expanded using a potentiallysimpler system. Instead of attaching recognition elementsto a channel, they are covalently linked to polymers thatotherwise thread through a nanometer-scale pore. Be-cause the rate of unbound polymer entering the pore isproportional to its concentration in the bulk, the bindingof analyte to the polymer alters the latter’s ability to threadthrough the pore, and the signal that results from indi-vidual polymer translocation is unique to the polymertype; the method permits multianalyte detection andquantitation. We demonstrate here that two differentproteins can be simultaneously detected with this tech-nique.

Ion channels are proteinaceous nanometer-scale pores inphospholipid bilayer membranes. They play diverse roles in cellsand organelles including the transport of specific ions andmacromolecules, conduction of signals within and between cells,and antibiotic activity against organisms.1-10 Some ion channelsare elements of cellular sensors: they transduce the concentrationof an analyte into a change in channel conductance and, hence,in the transmembrane potential.1

The ability to measure picoampere currents permits trace andultratrace chemical analysis. This low detection limit can beenhanced further by the use of nanometer-scale pores that cangate ∼1-100 pA ionic current by the binding of single moleculesto them. As a result, relatively simple amperometric measurementsprovide single-molecule detection capability.

Until recently, only highly specific analyte types (e.g., neuro-transmitters,1 anesthetics,11 and small ions4,12,13), which bind tosome naturally occurring channels, could be detected usingchannels. However, with the goal of using channels as transducersfor analyte detection, recognition sites have been geneticallyengineered inside the channel,14,15 adjacent to the channel’smouth,16 or chemically linked to locations outside the channel’slumen.17-19

We demonstrate here that two different proteins can besimultaneously detected with a simpler system. Instead of attach-ing the recognition elements to a channel, they are covalentlylinked to polymers that thread through a nanometer-scale pore.Because the signal that results from such a translocation is uniqueto the polymer type and the binding of analyte to the polymeralters the latter’s ability to traverse the pore, the sensor shouldpermit simultaneous multianalyte detection and quantitation fora wide range of molecules.

The potential for using channels in generalized analyticalapplications was realized when the stochastic current fluctuationsinduced in a solitary R-hemolysin (RHL) channel by the reversiblebinding of hydrogen ions4,12,13 and deuterium ions13 were observed.Frequency analysis of the analyte-induced stochastic current noiseprovided a direct measurement of the binding constant and thekinetic rate constants (kon and koff) for the fastest diffusion-controlled chemical reactions in solution. In those studies, aspectral analysis of the current fluctuations permitted the identi-fication of two isotopically different species that bind to thechannel.

Ion channels have either been genetically engineered orotherwise modified to bind specific types of analytes.14-17 As wasshown with the reversible binding of hydrogen and deuterium

* Corresponding author: (office) 301-975-5853; (fax) 301-330-3447; (e-mail)john.kasianowicz@nist.gov.(1) Hille, B. Ionic channels of excitable membranes, 2nd ed.; Sinauer Assoc.:

Sunderland, MA, 1992.(2) Tsien, R. W.; Tsien, R. Y. Annu Rev. Cell Biol. 1990, 6, 716-760.(3) Crowley, K. S.; Liao, S.; Worrell, V. E.; Reinhart, G. D.; Johnson, A. E. Cell

1994, 78, 461-471. Simon, S. M.; Blobel, G. Cell 1991, 65, 371-380(4) Prod′hom B.; Pietrobon D.; Hess P. Nature (London) 1987, 329, 243-

246.(5) Blachly-Dyson, E.; Peng, S. Z.; Colombini, M.; Forte, M. Science 1990, 247,

1233-1236.(6) Garcia, L. R.; Molineux, I. J. J. Bacteriol. 1996, 178, 6921-6929.(7) Gene Transfer in the Environment; Levy, S. B., Miller, R. V., Eds.; McGraw-

Hill: New York, 1989.(8) Young, R. Microbiol. Rev. 1992, 56, 430-481.(9) Leippe, M. Parasitol. Today 1997, 13, 178-183.

(10) Jakes, K. S.; Kienker, P. K.; Slatin, S. L.; Finkelstein, A. Proc. Natl. Acad.Sci. U.S.A. 1998, 95, 4321-4326.

(11) Barann, M.; Wenningmann, I.; Dilger, J. P. Toxicol. Lett. 1998, 101, 155-161.

(12) Bezrukov, S. M.; Kasianowicz, J. J. Phys. Rev. Lett. 1993, 70, 2352-2355.(13) Kasianowicz, J. J.; Bezrukov, S. M. Biophys. J. 1995, 69, 94-105.(14) Walker, B.; Kasianowicz, J. J.; Krishnasastry, M.; Bayley, H. Protein Eng.

1994, 7, 655-662. Kasianowicz, J.; Walker, B.; Krishnasastry, M.; Bayley,H. MRS Symp. 1994, 330, 217-223.

(15) Braha, O.; Walker, B.; Cheley, S.; Kasianowicz, J. J.; Hobaugh, M. R.; Song,L.; Gouaux, J. E.; Bayley, H. Chem. Biol. 1997, 4, 497-505.

(16) Kasianowicz, J. J.; Burden, D. L.; Han, L.; Cheley, S.; Bayley, H. Biophys. J.1999, 76, 837-845.

(17) Van Wie, B. J.; Davis, W. C.; Moffett, D. F.; Koch, A. R.; Silber, M.; Reiken,S. R.; Sutisna, H. U.S. Patent 5,736,342, 1998.

(18) Gu, L. Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature (London)1999, 398, 686-690.

(19) Cornell B. A.; Braach-Maksvytis V. L. B.; King L. G.; Osman P. D. J.; RaguseB.; Wieczorek, L.; Pace, R. J. Nature (London) 1997, 387, 580-583.

Anal. Chem. 2001, 73, 2268-2272

2268 Analytical Chemistry, Vol. 73, No. 10, May 15, 2001 10.1021/ac000958c Not subject to U.S. Copyright. Publ. 2001 Am. Chem. Soc.Published on Web 04/11/2001

ions to the wild-type RHL channel,12,13 engineered RHL mutantsexhibited analyte-induced single-channel current fluctuations thatidentified the chemical species of interest.15,16

Experiments using channels in supported bilayer membranesas a sensor were recently reported.19 This kind of robust settingfor channels is significantly more stable than a planar lipid bilayerand appears suitable for measuring analyte concentration inrelatively harsh environments. However, because that system usesa large number of channels, the characteristic kinetic informationcontained in the stochastic reactions between a single moleculeand a solitary channel4,12,13,16,18,20 is lost.

In addition, the ability of several molecules that bind to theRHL channel was recently reported.18,20,21 Among these moleculesis â-cyclodextrin, which has a cavity that binds organic ligands.The latter effort may eventually help circumvent the problem ofengineering binding sites for such ligands into a channel’sstructure, but it did not address the problem of making a trulyrobust sensor with RHL.

We show here that analyte quantitation can be accomplishedusing polymers that thread through a single nanometer-scale pore.By placing the analyte recognition site on a pore-permeant polymerinstead of the nanopore, this single-molecule detection methodhas the advantage of being easily reprogrammed to detect differentspecies (e.g., by replacing one class of pore-permeant polymerwith another). It also permits simultaneous detection and quan-titation of multiple analytes and should be realizable with robust,artificial nanopores, if they become available.

SENSOR MODELA generalized method of measuring analyte concentration can

be accomplished using a polymer that threads through a singlenanometer-scale pore. When an individual polymer is driventhrough a nanopore, it will sterically block the pore’s ionicconductance20-24 (Figure 1, top). The average repetition rate ofthe polymer entering the pore is directly proportional to theconcentration of the polymer in the bulk solution.23 Analyte boundto the polymer will cause the latter either to become pore-impermeant (model I; Figure 1, middle) or to occlude the porefor a time that is commensurate with the mean lifetime of theanalyte-polymer complex (model II; Figure 1, bottom). Each caseleads to a distinct and measurable effect on polymer-inducednanopore conductance changes.

EXPERIMENTAL SECTIONWe are using the ion channel formed by Staphylococcus aureus

RHL as a model nanopore for sensor applications12-16,20-23 andbiotinylated single-stranded DNA (bT-DNA) as the pore-permeantpolymer. The method of reconstituting single RHL channels intosolvent-free planar lipid bilayer membranes is described in detailelsewhere.13 Briefly, a membrane of diphytanoylphosphatidylcho-line (Avanti Polar Lipids, Birmingham, AL) is formed on a small

orifice (∼50-100 µm diameter) in a Teflon partition (∼17 µmthick) that separates two identical Teflon chambers. Each chambercontains ∼1.5 mL of electrolyte solution (1 M KCl, Mallinckrodt,Paris, KY; 10 mM HEPES, Calbiochem, La Jolla, CA; pH 7.5). Lessthan 1 µg of RHL is added to one chamber, herein called cis, andexcess protein is immediately removed after a conductanceincrease heralds the formation of a single channel.

A potential of -120 mV is applied across the membrane viatwo Ag-AgCl electrodes, and the current is converted to voltageusing an Axopatch amplifier with a high-impedance headstage(Axon Instruments, Foster City, CA). This signal was digitizedusing a National Instruments AT-MIO16 A/D board (Austin, TX),an Axon Instruments Digidata 1200B, or a Digidata 1320Ainterface. The data were acquired using software either from ourlaboratory or from Axon Instruments and subsequently analyzedusing in-house software. A negative potential drives anions fromthe cis to the trans chamber. Unless otherwise noted, DNAhomopolymers (Midland Certified Reagants), NeutrAvidin (Pierce,Rockford, IL), StreptAvidin (Pierce), or sheep anti-bromodeox-yuridine polyclonal antibody (Fitzgerald Industries, Int., Inc.,Concord, MA) are added to the cis chamber. The biotinylatedhomooligonucleotides were synthesized by Midland Certified

(20) Bezrukov, S. M.; Vodyanoy, I.; Brutyan, R. A.; Kasianowicz, J. J. Macromol-ecules 1996, 29, 8517-8522.

(21) Bezrukov, S. M.; Kasianowicz, J. J. Eur. Biophys. J. 1997, 6, 241-246.(22) Kasianowicz, J. J.; Brandin, E.; Branton, D.;Deamer, D. W. Proc. Natl. Acad.

Sci. U.S.A. 1996, 93, 13770-13773.(23) Henrickson, S. E.; Misakian, M.; Robertson, B.; Kasianowicz, J. J. Phys. Rev.

Lett. 2000, 85, 3057-3060.(24) Akeson, M.; Branton, D.; Kasianowicz, J. J.; Brandin, E.; Deamer, D. W.

Biophys. J. 1999, 77, 3227-3233.

Figure 1. Analytical sensor based on nanometer-scale pores andpolymers. In the absence of analyte, polymers with analyte bindingsites covalently linked to them partition freely into the pore and causeshort-lived blockades (i.e., downward transients) in the ionic current(top). The frequency of blockades is proportional to the free polymerconcentration. The polymer’s physical properties change when it isbound to analyte, rendering the polymer either undetectable by thepore (model I) or able to occlude the pore for a time commensuratewith the mean time the analyte is bound to the polymer (i.e., 1/koff;model II). If the system exhibits model I behavior, the analyteconcentration is deduced from the decrease in the number of polymer-induced blockades per unit time. In a model II system, the analyteconcentration is determined from the mean time to the first long-livednanopore current blockade that occurs after the electric field (whichis indicated by the + and - symbols) is applied.

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Reagants using 5′-bT-phosphoramidite (Glen Research, Sterling,VA) and standard solid-phase oligodeoxyribonucleotide synthetic,deprotection, and purification techniques.

RESULTS AND DISCUSSIONSingle-Channel Current in the Absence and Presence of

DNA. Previous experiments in our laboratory showed that single-stranded DNA and RNA cause transient blockades in the ioniccurrent that flows through a solitary RHL channel.22,23 Two linesof experimental evidence demonstrated that the current blockadesare caused by the translocation of polynucleotide moleculesthrough the pore. First, the lifetime of the polymer-inducedchannel current blockades is proportional to the polynucleotide’slength. Second, polymerase chain reaction experiments demon-strated that single-stranded, but not blunt-ended double-strandedDNA (i.e., without overhangs) traverses the pore.

The current through a single RHL channel is large andquiescent (Figure 2, leftmost recording). Adding relatively shortbiotinylated single-stranded DNA (10-nucleotide-long poly(deoxy-adenylic acid) biotinylated at the 5′-end; bT-poly(dA)10, Figure 2,top middle) or relatively long bT-poly(dA)50 (Figure 2, bottommiddle) to the aqueous phase bathing one side of the channelcauses transient decreases in the open channel current.

Analyte Alters Polymer-Induced Nanopore Current Block-ades. Avidin binds strongly to biotin. Subsequent addition ofexcess avidin (i.e., a concentration much greater than that of thebT-poly(dA)) causes the polymer-induced current blockades todisappear if the polynucleotide is relatively short (e.g., bT-poly-(dA)10, Figure 2, top right). In contrast, excess avidin complexedto the longer bT-ssDNA occludes the channel for times that are

much longer than occlusion by bT-poly(dA)50 (Figure 2, bottomright). Results qualitatively similar to both of these outcomes areobtained with relatively short and long strands of bT-poly(dC)(poly(deoxycytidilic acid)) (four trials; data not shown).

Avidin25-27 itself does not cause single-channel current block-ades, because it is too large to partition into the RHL channel’spore 20,21,28 and remain there for a time comparable to or greaterthan the electronic detector’s bandwidth (i.e., the very short-livedchannel blockades caused by the random collision of avidin withthe pore’s entrance apparently occur on a time scale that is muchfaster than the current amplifiers’ temporal resolution). However,avidin alters the ability of biotinylated homopolymers of ssDNAto traverse the RHL channel (Figure 2) as suggested in the sensormodels illustrated in Figure 1.

Quantitative Analysis. The sensor systems described herecan be used to quantitate analyte concentration and to characterizethe type of analyte that binds to the polymer. The number ofpolynucleotide-induced current blockades per unit time is pro-portional to the free polymer concentration.23 Thus, it follows fora model I system (Figure 1, middle), analyte binding to freepolymer will decrease the number of current transients per unittime. The concentration of bound polymer can be determined fromthe decrease in the time-averaged frequency of transient currentblockades.

By the principle of mass action, for a completely irreversiblereaction between analyte and sensing polymer, the blockade rateshould decrease linearly with analyte concentration. If the reactionis reversible, as the free polymer concentration approaches zero,the blockade rate will decrease asymptotically.

Avidin reduces the frequency of current blockades caused bybT-poly(dA)10 in a graded manner (Figure 3). The results suggestthat, under the conditions used here, the reaction between avidinand biotin attached to a short polynucleotide does not proceedwith a 4:1 (biotin/avidin) stoichiometry. The initial slope of theblockade rate versus avidin concentration is ∼2-fold less steepthan one would predict if all four binding sites per avidin moleculewere equally accessible to biotin. Nevertheless, as is shown inFigure 3, a calibration determines empirically the stoichiometrybetween the analyte and polymer. With this information, theanalyte concentration can easily be determined.

For a model II detector, the analyte concentration is deducedfrom the mean time that it takes the nanopore to be first occludedby the analyte-polymer complex after the electric field is applied.This was verified experimentally by monitoring the kinetics ofRHL channel occlusion by the complex of avidin-bT-poly(dA)50

(data not shown).Alternative Detector Schemes. The method described here

could be used to detect any analyte that alters a polymer’s abilityto partition into or completely traverse the pore. A model I sensorcould also work in the reverse of the manner illustrated in Figure1. For example, if an analyte converts a pore-impermeant polymerinto a pore-permeant form, then the number of blockades per unittime will increase with analyte concentration, (i.e., exactly opposite

(25) Pugliese, L.; Coda, A.; Malcovati, M.; Bolognesi, M. J. Mol. Biol. 1993,231, 698-710.

(26) Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L. Proc. Natl. Acad. SciU.S.A. 1993, 90, 5076-5080.

(27) Green, N. M. Adv. Protein Chem. 1975, 29, 85-133.(28) Song, L.; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J. E.

Science 1996, 274, 1859-1866.

Figure 2. Experimental verification of the two sensor modelsillustrated in Figure 1. The leftmost recording shows the step increasein current that flows through a single RHL ion channel in response toan applied potential of -120 mV. The middle recordings demonstratethat adding 250 nM short (10-mer) or 30 nM long (50-mer) single-stranded 5′-biotinylated poly(dA) causes transient current blockades.Most of these transient blockades correspond to the transport of anindividual DNA strand through the channel.22,23 Adding excess avidin(2.6 µM for bT-(dA)10 or 670 nM for bT-(dA)50) to bind virtually all ofthe bT-poly(dA) caused the polymer-induced current blockades eitherto disappear or to remain until the potential was reversed. The dottedlines indicate the zero current level. Both effects were observed morethan 10 times each in experiments with single channels reconstitutedinto fresh lipid membranes.

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to that suggested in Figure 1, middle, and shown in Figure 3).Although not strictly analogous, a similar principle was demon-strated by the cleavage of RNA homopolymers (e.g., poly(U)) intomore numerous and shorter polymers by ribonuclease A.22

A model II sensor could also be used to detect a wide varietyof analytes if the applied potential across the pore is sufficientlylow that the analyte-polymer complex is not rapidly torn asunderby the electrical force that drives the sensing polymer into thepore. In this case, the rate constants for the association anddissociation of an analyte with a recognition site on a polymer,kon and koff, could be directly measured because the potential holdsthe complex in the pore until dissociation of the analyte andsensing polymer occurs. Specifically, the pore will be partiallyoccluded by the analyte-polymer complex for a mean time t ∼1/koff. This additional kinetic information may help reduce errorcaused by false positive signals.

Multianalyte Detection. Because single-stranded DNA ho-mopolymers thread through the RHL channel at ∼1-10 µs/base,and at relatively low concentration of short polymers, the channelis almost always polymer-free (Figure 2, top middle recording).Thus, the probability that two polymers would simultaneously

occupy the pore is virtually nil. It follows that a model I nanoporesensor can accommodate a large number of polymer types thatproduce unique blockade patterns. For example, the transientblockades induced by identical length homopolymers of poly(dT)(poly(deoxythymidilic acid)), poly(dC), or poly(dA) are easilydistinguished from each other on the basis of their distinctivecurrent blockade patterns or different lifetimes (Figure 4). Thus,in the absence of analytes, a model I sensor with N differenthomopolymers (differing either by nucleotide type or contourlength, or both), each designed to bind a specific analyte, willproduce an ensemble of N unique current blockade patterns.Adding an analyte that binds to one of the polymers will cause areduction in the number of transient blockades caused by itscorresponding polymer, as was shown for one analyte and itscorresponding polymer (Figure 2).

The single-channel recordings in Figure 5 demonstrate proof-of-concept for multianalyte detection with this nanopore sensorillustrated in Figure 1. Specifically, the results show that twodifferent polymers (a relatively short strand of 5′-biotinylated-poly-(dC) and a longer strand of 5′- bromodeoxyuridine-poly(dT)) withunique current blockade signatures and with unique analytebinding sites can be simultaneously detected. Moreover, theblockades caused by the shorter bT-poly(dC) polymer disappearafter the addition of an excess concentration of the analyte thatbinds to it (i.e., avidin). Subsequent addition of a second analyte(i.e., an R-BRDU antibody) that binds to the longer polymer causesthe second polymer to occlude the pore for time intervals thatare much longer than the blockades caused by the polymer itself(i.e., model II behavior). Obviously, if more than two analytes areto be detected, an ensemble of short polymers that only exhibitmodel I behavior should be used.

Although the signals caused by the polymers in Figures 4 and5 are easy to differentiate by eye, a wide variety of statisticalmeasures (e.g., mean, variance, mean lifetime, autocorrelation or

Figure 3. Measuring analyte concentration by detecting the rate ofblocking of single-channel ionic current by pore-permeant polymers.In the absence of polymer, a steady current flows through a singleRHL channel (Figure 2, leftmost recording). Adding 400 nM single-stranded DNA (bT-poly(dA)10) to the cis side causes transientblockades of the current (Figure 3, upper left current recording).Increasing the analyte concentration (NeutrAvidin) decreases thefrequency of channel blocking because the analyte binds to thebiotinylated polymer and prevents it from entering the pore. Theblocking rate is proportional to the unbound polymer concentration.23

The solid curve is a fit function and can be used as a calibration curvefor the sensor. The resolution of an analyte concentration measure-ment by the sensor, e.g., at 120 nM, is just the inverse slope (∼1.5nM/count for a 1-min average) multiplied by (1 count. This equals(1% of reading and is the minimum resolution for a 1-min average.Similarly the resolution is (5% at 20 nM and at 240 nM. This definesthe analyte measurement range for 400 nM polymer. The range ischanged by changing the polymer concentration. The resolution canbe improved 10-fold by averaging for 10 min. The results shown hererepresent the mean and standard deviation for the blockade rates offive experiments on a single RHL channel. The error bars representthe standard deviation of four consecutive measurements performedon the same single channel at the same analyte concentration.Virtually identical results were observed for at least five out of fivesimilarly prepared experiments with different membranes containingsingle RHL channels. The small tail in the blockade rate at high avidinconcentration may be due to a relatively small amount (∼10%) ofthe single-stranded DNA not being biotinylated.

Figure 4. Transport of different DNA homopolymers through theRHL channel, Transport causes unique single-channel current block-ade signatures that could be used to simultaneously detect multipleanalytes with one pore (for a model I sensor). Transient blockadesinduced by 100-nucleotide-long homopolymers of poly(dT), poly(dC),or poly(dA) have markedly different lifetimes. In addition, poly(dT)-induced blockades are clearly distinguishable from those caused byeither poly(dA) or poly(dC) because of the pronounced double-steppattern. Homopolymers of poly(dG) were not studied because theyare essentially insoluble, difficult to produce, and form more compli-cated structures than do homopolymers of dT, dC, and dA. Thecharacteristic current blockade patterns and relative blockade lifetimeswere observed in more than 10 different experiments for each polymertype. They were also observed for shorter lengths of each of thesehomopolymers.

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spectral analysis12,13,20-24 of the current blockades, etc.) could beused to automatically distinguish current blockades caused bydifferent types of polymers. One of the challenges is to design alarge number of suitable and unique polymers that will enablecombinatorial levels of analyte detection with the sensor systemsdescribed herein.

SUMMARYWe have demonstrated that a single nanoscale pore can provide

the basis for a novel sensing scheme that locates analyte bindingsites on pore-permeant polymers. This method offers severalimportant advantages over detection schemes that affix therecognition sites directly to an ion channel. First, the two sensormechanisms illustrated in Figure 1 are more flexible in the typeand number of analytes that can be simultaneously measured (e.g.,the specificity and/or concentration range of the sensor can bechanged by replacing the polymer). Second, the analytes to bedetected need not fit inside the pore or inside an even smallermolecular adapter,18 thus permitting the detection of large

macromolecules. Third, the sensor can be used to simultaneouslydetect multiple analytes. Fourth, this method utilizes a singlenanopore and detects individual molecules, which preserves thekinetic information of the reaction between the analytes and thesensing polymers. Finally, the method described here is notrestricted to the use of biological channels. It will work withartificial nanopores, if they become available.

ACKNOWLEDGMENT

We thank Mr. Sean Lee for writing some of the computerprograms used in the data acquisition. Supported in part by theNIST Advanced Technology Program and the National Academyof Sciences/National Research Council (J.J.K.).

Received for review August 14, 2000. Accepted March 1,2001.

AC000958C

Figure 5. Simultaneous detection of two analytes with two different polymers and a single nanopore. The upper leftmost single-channelrecording shows the step increase in current that flows through a single RHL ion channel in response to an applied potential equal to -120 mVin the absence of single-stranded DNA and analytes. Adding ∼400 nM bT-poly(dC)10 to the cis chamber causes transient current blockades.The leftmost expanded view shows one of the characteristic poly(dC)10 events. Subsequently adding ∼400 nM BRDU-poly(dT)50 (i.e.,5′-bromodeoxyuridine-poly(dT)50-3′) to the cis chamber increases the total number of blockades. The central expanded view shows thatcharacteristic poly(dT)50 events, with the double-step pattern, are now clearly present in addition to the poly(dC)10 events. Adding ∼600 nMstreptavidin eliminates virtually all the bT-poly(dC)10 events, shown by the reduction in event frequency and in the rightmost expanded view,which is expected for a model I sensor (Figure 1). Adding ∼240 nM R-BRDU polyclonal antibody (Fitzgerald Industries) causes single-channelcurrent blockades that are much longer lived than those induced by BRDU-poly(dT)50, many remaining until the potential is reversed, as expectedfor a model II sensor (Figure 1).

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