Anal. Chem. 2005, 77, 1601-1606
Dynamic Microcantilever Sensors for Discerning Biomolecular Interactions Fang Tian, Karolyn M. Hansen, Thomas L. Ferrell, and Thomas Thundat*
Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6123 Douglas C. Hansen
University of Dayton Research Institute, Dayton, Ohio 45469
Response of a conductive micromechanical cantilever placed in close proximity to a surface undergoing electrical excitation near the resonance frequency of the cantilever is influenced by the presence of microscopic dielectrics in the gap between the cantilever and the sample surface. The variations of the resonance response of unmodified cantilevers at gap distances below a few hundred nanometers are used to discern biomolecular differences of oligomeric nucleic acids in an array format without the use of extrinsic labels. The resonance response variation paves the way for the development of high throughput detection of biomolecular reactions, such as DNA hybridization reactions or antibody-antigen interactions without the use of external labels, in which the need is only to see the presence or absence of interaction. This dynamic method is simple, does not require immobilizing individual elements on a cantilever array, and is compatible with current generation DNA chips in which DNA spots are deposited in micro- and nanoarray format. The expanding applications of genomics and proteomics demand a fast, efficient, and high-throughput molecular interaction screening technique that can be carried out on a chip format without the use of exogenous labels. High-throughput DNA and protein chip analyses are a mature technology, but they typically utilize extrinsic labels which require expensive detection equipment to determine if the label is present after DNA-DNA hybridization or antigen-antibody binding. An urgent need exists for high-throughput detection of these biomolecular interactions without the use of external labels to exploit the success of the Human Genome project. Our work has been stimulated in part by the enormous advances in microarray development and microfabrication of MEMS systems and in part by the desire to develop an alternate technique for a high-throughput, label-free detection system for genomic and proteomic applications. The recent demonstration of labelless detection of DNA and proteins using micromachined, mass-produced cantilevers has generated great excitement for the high-throughput, label-free detection method.1-4 The microcantilever approach, however, involves the challenging task of modifying individual cantilevers * Corresponding author. Fax: +1(865) 574-6210. E-mail:
[email protected]. 10.1021/ac048602e CCC: $30.25 Published on Web 02/11/2005
© 2005 American Chemical Society
in an array and requires the development of reliable and reproducible coating techniques. Here, we present a description and demonstration of a technique that combines the advances of chip array technology with advances in the development of microcantilever arrays. Discrimination of biomolecular interaction is accomplished by noting the resonance frequency, phase angle, and amplitude response variations of an unmodified cantilever placed a few hundred nanometers above the sample surface. Electrical excitation of a cantilever beam placed a few hundred nanometers away from a conducting substrate is achieved by using a function generator to impose an alternating voltage waveform on the sample at the frequency around the resonance frequency of the cantilever. The motion of the cantilever is detected by the optical beam deflection method, and the cantilever resonance frequency is determined by noting the driving frequency corresponding to the maximum cantilever amplitude. A typical resonance frequency of 25.5 kHz is observed for a Pt/Ir-coated silicon cantilever of 200-µm length and 1 µm thickness. Electrical connection to the chip arrays was made using direct contact to gold. A chip array with discrete spots of DNA sequences was placed under an unmodified, conducting cantilever probe that was electrically grounded. The cantilever was brought in close proximity using a piezoelectric element to monitor height from the substrate. The motion of the free end of the cantilever was monitored using an optical beam as in the case of an atomic force microscope. An alternating voltage of 100 mV was applied to the sample. When the frequency of the voltage applied to the sample matches the resonance frequency of the cantilever, the cantilever begins to resonate, as evidenced by the drastic increase in the cantilever vibration amplitude. Resonance frequency, phase angle, and amplitude of cantilever resonance were recorded as the cantilever was moved laterally over various DNA sample spots on the array substrate (single stranded DNA, double stranded DNA, and mismatched DNA). (1) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Guntherodt, H.-J.; Gerber, Ch.; Gimzewski, J. K. Science 2000, 288, 316318. (2) Wu, G.; Ji, H.-F.; Hansen, K. M.; Thundat, T. G.; Datar, R. H.; Cote, R. J.; Hagan, M. F.; Chakraborty, A. K.; Majumdar, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1560-1564. (3) Wu, G.; Datar, R. H.; Hansen, K. M.; Thundat, T. G.; Cote, R. J.; Majumdar, A. Nat. Biotechnol. 2001, 19, 856-860. (4) Hansen, K. M.; Ji, H.-F.; Wu, G.; Datar, R.; Cote, R.; Majumdar, A.; Thundat, T. G. Anal. Chem. 2001, 73, 1567-1571.
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Table 1. Sequences of Thiolated Probe and Complementary Target Oligonucleotidesa probe DNA (20-mer) target DNA (20-mer complementary) 1 MM target DNA (20-mer complementary w/1 mismatch) a
1 U ) C(∆V)2 2
5′-thiol-TT AAG GTC TGG ACT GGC CTG-3′ 3′-AA TTC CAG ACC TGA CCG GAC-5′
(1)
3′-AA TTC CAG ACG TGA CCG GAC-5′
Noncomplementary bases are indicated in italics.
Using microcantilever responses of phase and amplitude variation to measure inherent electrical properties associated with biomolecular interactions, we have been able to discriminate fully complementary nucleic acid hybridization and the presence of single nucleotide polymorphism (SNPs). We propose that the selective discrimination of DNA hybridization by oscillating microcantilever probe is due to a change in dielectric properties and molecular conformation in the DNA oligomer layer and that these parameters are characteristic of discreet molecular interactions. EXPERIMENTAL SECTION Surface Functionalization with DNA. Gold-coated substrates were prepared by epitaxial growth of gold on freshly cleaved mica substrates.5 Substrates were cleaned by sequential rinsing in acetone and ethanol. Cleaned gold-coated mica chips were exposed to phosphate buffer or 5′-end-thiolated DNA oligomers (Table 1; Oligos Etc., Wilsonville, OR) of known length and nucleic acid sequence (25 µg/mL (42 µM) in sodium phosphate buffer (100 mM PO4-, 150 mM Na+), pH 7.0) in discreet spot sizes by placing 500-nL drops by hand onto the chip and allowed to incubate for 1 h in a humid atmosphere at room temperature (hereafter known as probe DNA). Probe spots were washed with an excess volume of phosphate buffer for 10 min. Target DNA aliquots (1000 nL of 20 µg/mL (33 µM) DNA solution) in phosphate buffer containing complementary or mismatched DNA of known length, and nucleic acid sequence were placed over the probe DNA spots. The spots were allowed to sit on the chip for 1 h in a humid atmosphere at room temperature. Aliquots were removed by washing sequentially with phosphate buffer, deionized water, and 100% ethanol. Samples were dried under a nitrogen stream and stored in the desiccator until analysis. Probe and target DNA sequences are presented in Table 1. Estimated densities of DNA oligonucleotides per spot on the gold-coated mica chips are in the picomole-per-square centimeter range.6 Theoretical Background. In this work, a grounded, electrically conducting cantilever is placed over a conducting sample surface, and an oscillating voltage (Vdc + Vac sin ωt) is directly applied to the sample. Because of this alternating applied voltage, the cantilever is electrically excited due to the oscillating electrostatic interaction force acting on the cantilever at frequency, ω. When the oscillating frequency of the voltage applied to the sample matches the resonance frequency of the cantilever, the cantilever enters resonance, as observed by the drastic increase in the cantilever oscillating amplitude. (5) DeRose, J. A.; Thundat, T.; Nagahara, L. A.; Lindsay, S. M. Surf. Sci. 1991, 256, 102-108. (6) Williams, T. T.; Odom, D. T.; Barton, J. K. J. Am. Chem. Soc. 2000, 122, 9048-9049.
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The cantilever-sample system can be treated as a parallel plate capacitor with an electrostatic force energy of
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where C is the local capacitance between the cantilever and the sample, and ∆V is the potential difference between the cantilever and the sample, ∆V ) ∆Vdc + Vac sin ωt. Since the cantilever is grounded, ∆Vdc will be a sum of applied dc voltage on sample Vdc and intrinsic sample potential. Thus, the force between the cantilever and the sample can be written as
F)-
∂U 1 ∂C )(∆V)2 ) ∂z 2 ∂z Fdc + Fω sin (ωt) - F2ω cos(2ωt) (2)
where ∂C/∂z is the first derivative of the cantilever/sample capacitance with respect to the separation distance between cantilever and sample. It is clear that the total force includes three different force components: the static component of Fdc ) -(1/2)(∂C/∂z)(∆Vdc2 + (1/2)Vac2), the in-phase component at frequency ω of Fω ) -(∂C/∂z)∆VdcVac, and the out-of-phase component at frequency 2ω of F2ω ) (1/4)(∂C/∂z)Vac2. With the cantilever electrically excited at frequency, ω, the observed amplitude of the cantilever motion, A, is only directly proportional to the driving force Fω.7-9 We should note that different polarities of ∆Vdc result in the same amplitude of oscillation of the cantilever; however, the phase angle between the ac voltage and the driving force Fω will differentiate those states. The phase angle φ (in radians) of a free cantilever oscillation as a function of the oscillation frequency, ω, can be expressed as
φ ) tan-1
(
mωω0
Q(k - mω2)
)
(3)
where m is the mass of cantilever, k is the spring constant, and Q is the quality factor. The cantilever resonance frequency, ω0, is related to m and k as k ) mω02. The existence of a gradient of cantilever-sample interaction forces will result in the new effective force constant of the cantilever, keff ) k + F′. F′ represents the force gradient acting on the cantilever, which is dependent on the separation distance between the cantilever and the sample. The variation in the spring constant affects the phase angle and can be represented as
φ ) tan-1
(
mωω0
)
Q(k + F′ - mω2)
(4)
Therefore, as the interaction between the cantilever and sample varies, the phase angle varies as ∆φ ) QF′/k.10,11 (7) Gil, A.; Colchero, J.; Go´mez-Herrero, J.; Baro´, A. M. Nanotechnology 2003, 14, 332-340. (8) Lambert, J.; Guthmann, C.; Saint-Jean, M. J. Appl. Phys. 2003, 93, 53695376. (9) Cherniavskaya, O.; Chen, L.; Weng, V.; Yuditsky, L.; Brus, L. E. J. Phys. Chem. B 2003, 107, 1525-1531. (10) Magonov, S. N.; Elings, V.; Whangbo, M.-H. Surf. Sci. 1997, 375, L385L391.
Figure 1. Schematic representation of the instrument showing the methods for exciting the cantilever, measuring the oscillating response and static deflection of cantilever, and controlling the cantilever-sample separation distance.
Figure 2. The resonance response of oscillating amplitude and phase lag and static deflection of the cantilever recorded simultaneously as a function of the separation distance between the cantilever and the bare gold surface. The cantilever driving frequency is 25.5 kHz. The resonance frequency of the cantilever as a function of the cantilever-sample separation distance is shown as an open circle.
Analysis of Functionalized Substrates. A schematic diagram of the instrument used in this investigation is illustrated in Figure 1. The electrically grounded unmodified conducting cantilever (Nanosensors, Switzerland, 200 µm long, 40 µm wide, 1 µm thick, spring constant 0.35 N/m, resonance frequency ∼25.5 kHz) was placed over the sample surface, where an oscillating voltage (Vdc + Vac sin ωt, where ω is ∼25.5 kHz, Vdc ) 5 V, and Vac ) 0.1 V) was directly applied. The separation distance between the sample and the cantilever was controlled by using a calibrated piezoelectric element in a linear fashion. The cantilever will be electrically excited when the frequency of this driving alternating voltage approaches the resonance frequency of the cantilever; the excitation is due to the electrostatic interaction between the cantilever and sample. The position of a laser beam reflected from the cantilever onto a four-quadrant position-sensitive detector (PSD, photodiode) was monitored. The signal from the PSD represents two components of cantilever motion: the static dc signal, which shows the static bending of the cantilever, and the ac signal, which gives the amplitude and phase angle of the oscillating cantilever. The cantilever resonance frequency is determined by noting the driving frequency (swept from 22 to 28 kHz) corresponding to the maximum cantilever amplitude. The driven dynamic amplitude, phase angle, and static deflection of the cantilever were (11) Czajkowsky, D. M.; Allen, M. J.; Elings, V.; Shao, Z. Ultramicroscopy 1998, 74, 1-5.
measured simultaneously while the cantilever approached the sample surface. We define zero separation distance as the point at which the cantilever starts to contact the sample surface. RESULTS AND DISCUSSION Results were obtained using 25.5 kHz as the cantilever driving frequency. Baseline amplitude, phase, and deflection signals of the cantilever were recorded simultaneously for cantilevers exposed to a bare gold surface as a function of separation distance between cantilever and sample surface (Figure 2). Before the cantilever contacts the sample surface, no deflection of the cantilever is observed. However, the resonance response variation of the cantilever with respect to amplitude and phase angle is observed. After sweeping the driving frequency from 22 to 28 kHz and comparing a series of amplitude response curves of the cantilever at specific separation distances, the cantilever resonance frequency is determined by noting the driving frequency corresponding to the maximum cantilever amplitude. The cantilever resonance frequency decreases as the cantilever approaches the sample surface (Figure 2). Figure 3 shows the phase angle variations between cantilever and sample as a function of separation distance. The three curves correspond to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and double-stranded DNA with an internal mismatch (dsDNA-1MM). A clear difference can be seen over separation distances. As the separation distance decreases, the Analytical Chemistry, Vol. 77, No. 6, March 15, 2005
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Figure 3. The phase variations of the cantilever as a function of the separation distance between the cantilever and the stranded DNA (ssDNA), double-stranded DNA (dsDNA), and double-stranded DNA with an internal mismatch (dsDNA-1MM), respectively. The error signals are given at some selected separation distances. The cantilever driving frequency is 25.5 kHz.
Figure 5. The resonance frequency variations of the cantilever as a function of the separation distance between the cantilever and the stranded DNA (ssDNA), double-stranded DNA (dsDNA), and doublestranded DNA with an internal mismatch (dsDNA-1MM), respectively.
between the cantilever and sample. In this study, the oscillating voltage (Vdc + Vac sin ωt) applied to sample substrate was always kept constant with a Vdc of 5 V and Vac of 0.1 V and the cantilever remained grounded. Therefore, the observed variations of oscillating amplitude of the cantilever will only be related to both ∂C/∂z and intrinsic sample surface potential. Note that the intrinsic sample surface potential is independent of the separation distance between cantilever and sample. Here the cantilever-sample system is treated as a parallel plate capacitor. The local capacitance between the cantilever and the sample can be written as Figure 4. The oscillating amplitude variations of the cantilever as a function of the separation distance between the cantilever and the stranded DNA (ssDNA), double-stranded DNA (dsDNA), and doublestranded DNA with an internal mismatch (dsDNA-1MM), respectively. The error signals are given at some selected separation distances. The cantilever driving frequency is 25.5 kHz.
difference in phase angle response increases. The results are averaged over three sample sets with nine measurements at each set. The error signals are given at selected separation distances. Figure 4 shows the variation in cantilever oscillating amplitude as a function of separation distance for ssDNA, dsDNA, and dsDNA with mismatch. Compared with Figure 3, it appears that the amplitude variation persists over a longer distance than that of the phase angle. Therefore, the observed amplitude variation is more sensitive to separation distance than that of phase-angle difference. The error signals are also given at selected separation distances. The results are averaged over three sample sets with nine measurements at each set. The variation in cantilever resonance frequency as a function of separation distance for ssDNA, dsDNA, and dsDNA with mismatch is shown in Figure 5. The resonance frequency of the cantilever shifts to lower frequencies as the cantilever approaches the surface. A clear difference can be seen as the cantilever is very close to DNA sample surfaces. Based on the discussion in Experimental Section, we know that the measured oscillating amplitude of the cantilever is directly proportional to the amplitude of the driving force Fω and, thus, proportional to ∂C/∂z, ∆Vdc, and Vac. In addition, ∆Vdc is a sum of applied dc voltage on sample Vdc and intrinsic potential difference 1604
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C ) Q/V ) 0A/z
(5)
where Q is the charge on the cantilever and sample, V is the applied voltage between the cantilever and sample, 0 is the dielectric of the medium between the cantilever and sample substrate, A is the surface area of the cantilever, and z is the separation distance between the cantilever and sample. Thus, ∂C/∂z, the first derivative of the cantilever/sample capacitance with respect to the separation distance between the cantilever and sample, is distance-dependent. The decreasing distance between the cantilever and sample will result in a higher capacitance between the cantilever and sample substrate. As result, the force between the cantilever and sample increases as the cantilever gets close to the sample surface. The existence of a force gradient will also shift the resonance frequency of the cantilever (Figure 5). In Figure 6, we present the oscillating amplitude variations of the cantilever as a function of the driving frequency at selected separation distances between the cantilever and the bare gold surface. Similar results were also observed at ssDNA, dsDNA, and dsDNA with mismatch. Not only does the resonance frequency of the cantilever shift to a lower frequency but also the amplitude of the cantilever at resonance frequency increases. In this study, the oscillating amplitude and phase angle were measured at a driving frequency of 25.5 kHz (Figures 4 and 3). As the cantilever approaches the sample surface, in the beginning, the oscillating amplitude at 25.5 kHz increases, then starts to decrease as the resonance frequency of the cantilever further shifts to a lower frequency. This explains very well the observed amplitude peaks for ssDNA, dsDNA, and dsDNA with mismatch
Figure 6. The oscillating amplitude variations of the cantilever as a function of the driving frequency at selected separation distances between the cantilever and the bare gold surface.
in Figure 4. Our results indicate that the observed variations of an oscillating cantilever at different separation distances on each DNA sample, such as phase angle in Figure 3, oscillation amplitude in Figure 4, and resonance frequency in Figure 5, are caused by the distance dependence of the capacitance between the cantilever and sample. In fact, if the sum of the voltage between the cantilever and sample, ∆Vdc, is zero, there will be no electrostatic force acting on the cantilever at frequency ω, resulting in no cantilever motion. By sweeping the dc voltage applied to the sample (Vdc) until the oscillating amplitude of cantilever vanishes, at that voltage, Vdc-0 is the same as the intrinsic sample surface potential. We propose that the observed differences of phase angle (Figure 3) and oscillating amplitude (Figure 4) at a given separation distance for ssDNA, dsDNA, and dsDNA with mismatch are caused by the surface potential variations for different DNA samples. In the future, we can measure not only the cantilever responses at frequency, ω, but also those at frequency 2ω. Moreover, the observed amplitude of cantilever motion at frequency 2ω will be related to the force, F2ω ) (1/4)(∂C/∂z)Vac2. It is clear that the capacitance can be determined directly by observing the cantilever responses as a function of the separation distance between the cantilever and sample at frequency 2ω. The variations in phase angle and oscillating amplitude are probably due to variations in the charge and conformation of the DNA species. The persistence length for dsDNA is ∼50 nm, whereas that of ssDNA is ∼0.7 nm. The single-stranded oligomers used in this study are 20 mers, ∼6.4 nm long, and are therefore in a random, coiled, and more compact conformation. Exposure of ssDNA probe films to complementary target DNA induces a double-stranded, helical, rodlike conformation, and hence, an increase in oligomer persistence length (i.e., film thickness).12,13 An increase or decrease of effective strand length alters the charge distribution of the oligomer layer along with an associated change in the dielectric properties.14,15 The presence of mismatches greatly alters the conformation, polarizability, and charge of the DNA film and results in a larger decreased resonance frequency of the cantilever as it approaches the surface. Indeed, disruptions in charge transduction through DNA oligomers due to the presence of mismatches have been confirmed16 and form the basis of an electrochemical detection assay.6,17-19 Changes in molecular conformation have also been confirmed and are the means by which DNA repair enzymes identify locations of mismatched (i.e.,
damaged) DNA.20 That we can readily discriminate a single nucleotide mismatch from ssDNA or dsDNA under ambient conditions without the need for exogenous labels, background correction, and analysis algorithms clearly distinguishes this phase/amplitude/resonance frequency microcantilever technique as a simple yet extremely effective analytical method for determining the presence of single nucleotide polymorphisms. The observed variations in phase angle and amplitude of oscillations were more long range (more than 10 times) than variations in resonance frequency. The results presented in this paper have demonstrated the efficacy of using an unmodified micromechanical probe for the discrimination of hybridized DNA and without the use of an external label. Variations in the immobilized spots of DNA were measured as the variation in the phase and the oscillating amplitude of an unmodified cantilever placed above the spot. Hybridization of fully complementary 20-mer target DNA to 20-mer immobilized probe DNA resulted in measurable phase, oscillating amplitude, and resonance frequency differences. DNA strands with 1 base mismatch (out of a 20-mer strand) yielded phase, amplitude, and resonance frequency values different from those of ssDNA and dsDNA. We propose that variations in charge density and biomolecular conformation, resulting in the variation of surface potential, are responsible for the observed differences in the cantilever response. CONCLUSIONS This dynamic microcantilever technique will pave the way for the development of a high-throughput, label-free micromechanical device for detection of molecular interactions, utilizing the wellestablished DNA array chip format. This method is superior to that of any technique in which each element in a cantilever array is modified with a different DNA sequence. Another advantage includes the use of a single cantilever probe element with sample deposited on a rotating disk, as in the case of a CD player. Alternatively, the need for scanning a single cantilever above the sample can be eliminated by using a cantilever array for simultaneous acquisition of information in a parallel manner. While the majority of detection schemes presently in use focus on the quantification of specific molecules, we have yet to explore the quantitative aspects of the phase/amplitude/resonance method. It is ideal, though, for the presence/absence detection so urgent in, for example, the Homeland Security arena. It is also ideal for initial screening of genomic and proteomic arrays, since it can eliminate the absence-of-interaction samples and allow researchers to focus on the positive interactions. Our results leave no doubt that the phase/amplitude/resonance method can easily discrimi(12) Rekesh, D.; Lyubchenko, Y.; Shlyakhtenko, L. S.; Lindsay, S. M. Biophys. J. 1996, 71, 1079-1086. (13) Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J. Biophys. J. 2000, 79, 975-981. (14) Lee, R. S.; Bone, S. Biochim. Biophys. Acta 1998, 1397, 316-324. (15) Berney, H.; West, J.; Haefele, E.; Alderman, J.; Lane, W.; Collins, J. K. Sens. Actuators, B 2000, 68, 100-108. (16) Hartwich, G.; Caruana, D. J.; de Lumley-Woodyear, T.; Wu, Y.; Campbell, C. N.; Heller, A. J. Am. Chem. Soc. 1999, 121, 10803-10812. (17) Kelley, S. O.; Barton, J. K. Science 1999, 283, 375-381. (18) Kelley, S. O.; Boon, E. M.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Nucleic Acids Res. 1999, 27, 4830-4837. (19) Boon, E. M.; Salas, J. E.; Barton, J. K. Nat. Biotechnol. 2002, 20, 282-286. (20) Wang, H.; Yang, Y.; Schofield, M. J.; Du, C. W.; Fridman, Y.; Lee, S. D.; Larson, E. D.; Drummond, J. T.; Alani, E.; Hsieh, P.; Erie, D. A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14822-14827.
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nate the presence of SNPs in an array format, and we anticipate that femtogram level of sensitivity is attainable on the basis of the current state of functionalized microcantilever detection technology. ACKNOWLEDGMENT This research was supported by the DOE Office of Biological and Environmental Research (OBER) and Environmental Man-
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agement Science Program (EMSP). Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. Department of Energy under contract number DE-AC05-0096OR22725. Received for review September 21, 2004. Accepted December 16, 2004. AC048602E
