PEMC-based Method of Measuring DNA Hybridization at Femtomolar

Sep 1, 2007 - Cover Image ... Sample Preparation-Free, Real-Time Detection of microRNA in Human Serum Using Piezoelectric Cantilever Biosensors at ...
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Anal. Chem. 2007, 79, 7392-7400

PEMC-based Method of Measuring DNA Hybridization at Femtomolar Concentration Directly in Human Serum and in the Presence of Copious Noncomplementary Strands Kishan Rijal and Raj Mutharasan*

Department of Chemical and Biological Engineering, Drexel University; 32nd and Chestnut Streets, Philadelphia, Pennsylvania 19104

Piezoelectric-excited, millimeter-sized cantilever (PEMC) sensors having high-mode resonance near 1 MHz are shown to exhibit mass change sensitivity of 1-300 ag/ Hz. Gold-coated PEMC sensors immobilized with 15-mer single-stranded DNA (ssDNA) were exposed to 10-mer complementary strands at concentrations of 1 fM, 1 pM, and 1 µM, both separately and sequentially at 0.6 mL/ min in a sample flow cell housing the sensor. Decrease in resonance frequency occurred as complementary strands hybridized to the immobilized probe DNA on the sensor surface. Hybridization in three background matrixess buffer, buffer containing 10 000 times higher noncomplementary strands, and 50% human plasmaswere successfully tested. Sensor hybridization responses to 1 fM, 1 pM, and 1 µM complementary strand were nearly the same in magnitude in all three matrixes, but the hybridization rates were different. In each case, the sensor detected the presence of 2 amol of complementary 10-mer strand. The extent of hybridization calculated from resonance frequency change did not decrease in serum. The findings suggest ssDNA can be detected at 2 amol without a sample preparation step and without the use of labeled reagents. Development of rapid DNA detection methods with minimal or no sample preparation at femtomolar concentration is important in several fields such as medical diagnostics, pathogen and biothreat agent identification, and basic research. Circulating DNA fragments have been found in serum1 and other body fluids and are believed to be symptomatic of the disease state. Ultrasensitive methods for DNA detection that do not employ amplification schemes such as polymerase chain reaction (PCR) would be useful in a hospital or point-of-care setting.2 It has been suggested that detection limit in the order of 10-18 M is required for medical diagnostic applications.3 To reach such a limit of detection, the * Corresponding author. Tel.: 895-2236. Fax: (215) 895-5837. E-mail: [email protected]. (1) Fleischhacker, M.; Schmidt, B. Biochim. Biophys. Acta: Rev. Cancer 2007, 1775, 181-232. (2) Hwang, S.; Kim, E.; Kwak, J. Anal. Chem. 2005, 77, 579-584. (3) Caruso, F.; Rodda, E.; Furlong, D. F.; Niikura, K.; Okahata, Y. Anal. Chem. 1997, 69, 2043-2049.

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conventional biosensing techniques have been combined with PCR or other amplification techniques.4-7 PCR represents the ultimate in sensitivity as a single strand is all that is needed for detection.8 But, it is limiting in terms of portability, simplicity, sensitivity to background, and contaminants.9 The majority of the methods reported for direct detection of DNA targets are at micromolar10 to nanomolar11-14 range and very rarely lower than picomolar.15 Several new and emerging methods have been reported to address these limitations. Nanostructurebased nucleic acid assays and carbon nanotubes have significantly improved the DNA-based detection limits to subatttomolar concentrations.9,16 However, they are methods rather than devices and require sample preparation and labeled reagents.5,17-20 Impressive detection limit of ∼100 aM21 and 100 fM22 have been achieved using such approaches. (4) Compton, J. Nature 1991, 350, 91-92. (5) Baeumner, A. J.; Pretz, J.; Fang, S. Anal. Chem. 2004, 76, 888-894. (6) Malek, L.; Darasch, S.; Davey, C.; Henderson, G.; Howes, M.; Lens, P.; Sooknanan, R. Clin. Chem. 1992, 38, 458-458. (7) Kievits, T.; Vangemen, B.; Vanstrijp, D.; Schukkink, R.; Dircks, M.; Adriaanse, H.; Malek, L.; Sooknanan, R.; Lens, P. J. Virol. Methods 1991, 35, 273-286. (8) Saiki, R. K.; Scharf, S.; Faloona, F.; Mullis, K. B.; Horn, G. T.; Erlich, H. A.; Arnheim, N. Science 1985, 230, 1350-1354. (9) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547-1562. (10) Huang, E.; Satjapipat, M.; Han, S. B.; Zhou, F. M. Langmuir 2001, 17, 12151224. (11) Peterlinz, K. A.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401-3402. (12) Heaton, R. J.; Peterson, A. W.; Georgiadis, R. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3701-3704. (13) Star, A.; Tu, E.; Niemann, J.; Gabriel, J. C. P.; Joiner, C. S.; Valcke, C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 921-926. (14) Csaki, A.; Moller, R.; Straube, W.; Kohler, J. M.; Fritzsche, W. Nucleic Acids Res. 2001, 29, art. no.-e81. (15) Reynolds, R. A.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 3795-3796. (16) Nam, J. M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 59325933. (17) Tokareva, I.; Hutter, E. J. Am. Chem. Soc. 2004, 126, 15784-15789. (18) Li, M.; Lin, Y. C.; Wu, C. C.; Liu, H. S. Nucleic Acids Res. 2005, 33. (19) Xu, J.; Craig, S. L. J. Am. Chem. Soc. 2005, 127, 13227-13231. (20) Kim, E. Y.; Stanton, J.; Vega, R. A.; Kunstman, K. J.; Mirkin, C. A.; Wolinsky, S. M. Nucleic Acids Res. 2006, 34. (21) Wang, J.; Polsky, R.; Merkoci, A.; Turner, K. L. Langmuir 2003, 19, 989991. (22) Jenkins, D. M.; Chami, B.; Kreuzer, M.; Presting, G.; Alvarez, A. M.; Liaw, B. Y. Anal. Chem. 2006, 78, 2314-2318. 10.1021/ac0712042 CCC: $37.00

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To date, a direct method for detecting DNA strands without labeled reagents at subpicomolar level has not been reported. Nor has it been shown that it is possible to detect DNA fragments directly in human serum without a preparation or a separation step to minimize or eliminate interference from cohabiting proteinous matter. Piezoelectric-excited millimeter-sized cantilevers (PEMC) exhibit subfemtogram mass change sensitivity,23-25 and we have found selective detection to occur with high specificity due to combined effects of flow and sensor vibration.23,24 In this paper, we show for the first time successful detection of small DNA sequences at femtomolar concentration both in buffer and in serum. PRINCIPLE OF MEASUREMENT A PEMC is a macrocantilever comprising a thin leadzirconate-titanate (PZT) layer bonded to a nonpiezoelectric glass layer of a few millimeters in length and 1 mm in width. The PZT layer serves both as an actuating and as a sensing element. When an electric field is applied across the PZT, it extends the base nonpiezoelectric layer to flex. If the electric field is alternated, the sensor experiences flexural oscillations. The sensor resonates when the excitation frequency coincides with the natural mechanical frequency of the cantilever.26,27 At resonance, the cantilever undergoes higher than normal stress and the PZT being electromechanically active exhibits a sharp change in electrical impedance. At resonance, the phase angle between the excitation voltage and the resulting current changes significantly and is measured using an impedance analyzer. The piezoelectric layer acts both as an actuating and as a sensing element, while the glass provides a surface for antibody immobilization. The sensing response is recorded by measuring changes in resonance frequency of the vibrating cantilever sensor. Recent innovations in the design of PEMC sensors have led to the discovery of a high-order resonance mode near ∼1 MHz that exhibits mass change sensitivity of 0.3-2 fg/Hz in liquid.23,24 The implication of such a high sensitivity is that detection of a few million 10-mer single-stranded DNA (ssDNA) becomes feasible as binding of them to the sensor will give rise to a frequency response that is much higher than measurement noise. MATERIALS AND METHODS PEMC Fabrication and Calibration. Description of 1-mmwide PEMC sensor fabrication was reported earlier.24 The 4-mmlong glass layer was bonded to 5-mm-long PZT such that 1.0 ( 0.1 mm of glass protruded beyond the PZT. The PZT layer alone was anchored, and the distance between the epoxy end and glass was 0.5 ( 0.1 mm. Both sides of the protruding glass area were coated with a thin polyurethane, and then a 100-nm gold layer was sputtered yielding a sensing area of 1.8 mm2. The gold film yielded predominantly (>95%) polycrystalline Au as determined by X-ray diffraction. Calibration of the sensor was carried out as previously reported.23,24 Briefly, change in resonance frequency of the sensor (23) Campbell, G. A.; Mutharasan, R. Anal. Chem. 2007, 79, 1145-1152. (24) Maraldo, D.; Rijal, K.; Campbell, G. A.; Mutharasan, R. Anal. Chem. 2007, 79, 2762-2770. (25) Campbell, G. A.; Mutharasan, R. Biosens. Bioelectron. 2006, 22, 78-85. (26) Campbell, G. A.; Mutharasan, R. Langmuir 2005, 21, 11568-11573. (27) Campbell, G. A.; Mutharasan, R. Sens. Actuators, A: Phys. 2005, 122, 326334.

was measured in a constant temperature incubator (28 °C) following a series of dispensing and evaporating 1 µL of paraffin in hexane solution (1 fg/µL). Pure hexane was used as control. A plot of added mass versus resonance frequency change gave a straight line whose slope (in fg/Hz) is mass change sensitivity. Reagents. All reagents were purchased from Sigma, unless noted otherwise. Single-stranded thiolated 15-mer oligonucleotide probe from Bacillus 16S-rRNA sequence, HS-C6H12-5′-GGAAGAAGCTTGCTT-3′, the complementary 10-mer target 5′-AAGCAAGCTT-3′, and a stock 10-mer target of random and unknown sequence were purchased from Integrated DNA Technologies (Coralville, IA). The lyophilized DNA samples were reconstituted to a stock concentration of 65.8 µM in Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 1 M NaCl. NaCl was used to bring the hybridization temperature to desired temperature of operation (32 °C), which is 5 °C lower than the melting temperature 37 °C.23,28 In addition, NaCl reduces the anionic electrostatic repulsion between the probe and the target and increases the duplexes’ stability.29,30 1-Mercapto-6-hexanol (MCH) at 1 µM was freshly prepared in TE buffer for each experiment. Pooled normal human plasma (IPLA-2) was from Innovative Research (Southfield, MI) and was used as a model complex matrix. Probe Preparation. Thiolated ssDNA probe was supplied in disulfide form and was reduced prior to use. To each aliquot containing 250 µL of 65.8 µM thiolated probe, 3.9 mg of dithiothreitol (DTT) powder was added and reacted at room temperature for 30 min. Excess DTT was removed with Sephadex G-25 columns (Pure Biotech LLC) following the vendor-supplied protocol. The effluent, free of DTT, was diluted in TE buffer to the desired concentration (1 aM-100 nM) and used within 1 h. No systematic attempts were made to optimize surface probe concentration. However, concentrations used in this report are 1 pM, 49 pM, 500 pM, and 50 nM. Experimental Apparatus, Probe Immobilization, and Hybridization Detection. All experiments were conducted in a especially constructed flow apparatus described previously.23,24 The PEMC sensor was connected to an impedance analyzer (HP 4192A or HP 4294A) interfaced to a PC running a custom-written LabVIEW data acquisition program. Impedance, capacitance, and phase angle values of the sensor were collected at 10-30-s intervals in the frequency range of interest. A typical experiment was started by first flowing TE buffer through the sample flow cell (SFC) until a baseline resonance frequency was established (∼5-20 min). Flow rate was kept at a constant value of 0.6 mL/ min in all experiments. Once a stable baseline was established, probe solution (1 pM-50 nM; different experiments) was introduced. Upon reaching a stable resonance frequency due to chemisorption of the thiolated probe, a freshly prepared 1 µM MCH in TE buffer was pumped in to fill unoccupied Au sites and to remove any nonspecifically attached probe strands from the sensor surface.31,32 Immediately there after, sample solution (usually 2 mL) containing the target ssDNA was intro(28) Owczarzy, R.; You, Y.; Moreira, B. G.; Manthey, J. A.; Huang, L. Y.; Behlke, M. A.; Walder, J. A. Biochemistry 2004, 43, 3537-3554. (29) Biswal, S. L.; Raorane, D.; Chaiken, A.; Birecki, H.; Majumdar, A. Anal. Chem. 2006, 78, 7104-7109. (30) Owczarzy, R.; Vallone, P. M.; Gallo, F. J.; Paner, T. M.; Lane, M. J.; Benight, A. S. Biopolymers 1997, 44, 217-239. (31) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920.

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duced in a once-through flow and was put into recirculation mode as the sample approached zero in the reservoir. The test sample solutions were circulated for 30-45 min until resonance frequency of the sensor reached a constant value. Additionally, when the same sensor was reused, it was first cleaned in piranha solution (7:3 volume ratio of concentrated H2SO4 and 30% H2O2. Caution: piranha solution reacts violently with many organic materials and should be handled with great care) for 2 min, rinsed with copious amount of DI water and ethanol, and finally oven dried at 110 °C. Piranha cleaning reduced effective Au sites by 8-10% after the fourth cleaning. Hence, all sensors were recoated with gold after three uses. Because the sensors break due to handling, several PEMC sensors were used during the course of the present work. The results reported, however, were obtained using three PEMC sensors (labeled A, B, and C) whose mass change sensitivities were similar. RESULTS AND DISCUSSIONS Resonance Behavior and Sensitivity of PEMC Sensors. Each experiment reported here was repeated at least three times. Although there were three sensors, their mass change sensitivity was within 30%, ∼0.3 ( 0.1 fg/Hz at the mode present near 1 MHz, as measured by the known mass addition method.23 The resonance spectrum of each sensor was examined in air and in TE buffer to determine if suitable Q value was present. The Q value is a measure of sharpness of the peak and is defined as the ratio of resonance frequency divided by frequency width at half the peak height. The sensors A, B, and C exhibited resonance at 1007.125 (Q ) 23), 919.725 (Q ) 24), and 968.340 (Q ) 22) kHz in air, respectively, and in TE buffer, the resonance frequency decreased to 939.250 (Q ) 19), 862.320 (Q ) 20), and 899.520 (Q ) 18) kHz, respectively, due to the increased added mass.24 Although there was a ∼17% decrease in Q values, the resonance frequency value can be measured within (4 Hz in air, in stagnant liquid within (10 Hz, and in flowing buffer with a stability band of (20 Hz. PEMC-B and PEMC-C were ∼10% less sensitive than PEMC-A. Further details on the resonance spectrum and mass change sensitivity of PEMC sensors can be found in our earlier reports.23,24,33 Response to Probe Immobilization. PEMC-A sensor response to probe immobilization is shown in Figure 1. After initial stabilization, the reservoir containing 10 mL of 1 aM probe was introduced. The first 5 mL flowed in a once-through mode, and the last 5 mL was put in recirculation mode. The PEMC sensor responded, as shown in Figure 1 with a decrease in resonance frequency as the probe’s thiol group became bound to the sensor surface forming a thiolated bond.34 After a transient period of ∼13 min, the sensor reached steady state with a total resonance frequency decrease of 42 Hz. The inset in Figure 1 shows that the noise level is ∼2 Hz. At 30 min, the sample was changed to 10 aM. Again, the first 5 mL flowed in a once-through mode followed by recirculating the last 5 mL. Since the flow circuit holdup volume is 2.2 mL, 5 mL provided sufficient volume for removing the residuals. Resonance frequency decreased by an (32) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (33) Rijal, K.; Mutharasan, R. Langmuir 2007, 23, 6856-6863. (34) Love, J.; Estroff, L.; Kriebel, J.; Nuzzo, R.; Whitesides, G. Chem. Rev. 2005, 105, 1103-1169.

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Figure 1. Panel A. Gold-coated PEMC-A sensor housed in a flow cell and exposed to 10 mL of various concentrations of 15-mer thiolated ssDNA probe starting with 1 aM to 1 µM, sequentially in a recirculation mode. In the case of 1 aM, the sensor response is 42 Hz for a total target of 51 ag, indicating a mass change sensitivity of ∼1 ag/Hz. Panel B. The cumulative responses to various probe concentrations exhibit a logarithmic response. Sensor response saturates at a concentration lower than 10 nM due to fixed number of chemisorption sites on the sensor, Au .

additional 47 Hz. The process was repeated in steps of 10× in concentration until there was no measurable resonance frequency response. The response (Figure 1) suggests that the sensor surface became saturated at 100 pM. Increase of inlet concentration to 10 nM and then to 1 µM leads to no resonance frequency response, indicating that no further probe immobilization took place. The changes in resonance frequency are plotted as a function of inlet probe concentration, and the resulting curve (Figure 1B) indicates that a quantitative relationship exists between the surface and the liquid concentrations. The 10 mL of 1 aM solution contains a total of 6000 molecules and weighs 51 ag. If we assume all of them attached to the sensor, the mass change sensitivity ()mass change/resonance frequency change) is ∼1 ag/Hz. Since the sensor is 1.8 mm2, it has 1.1 × 1013 Au sites34 and can accommodate a maximum of ∼3.2 × 1011 ssDNA.35 Hence, chemisorption of 6000 strands causes a fractional surface coverage on the order of 10-8. Note that ssDNA occupies a cross-sectional area of 3.14 nm2, and the values calculated are (35) McKendry, R.; Zhang, J.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baller, M. K.; Certa, U.; Meyer, E.; Guntherodt, H.-J.; Gerber, C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9783-9788.

based on maximum packing density.35 A 10-fold increase in concentration (1-10 aM) causes a 2-fold response (47 Hz to 47 + 57 Hz) suggesting a nonlinear response to the imposed concentration change. The nonlinear response is consistent with previous work with proteins and pathogens24 and the more recent work on chemisorption of thiols33 on the sensor surface. The higher level of sensitivity obtained (∼1 ag/Hz) compared to the measured value (∼300 ag/Hz) is in part due to nonlinear sensor response (Figure 1B). The wax calibration was done with the addition of 1-5 fg to the sensor surface.23 Closer examination of the results in Figure 1B shows that 1 fM gave a sensor response of ∼370 Hz for an exposure to 51 fg of the probe. If we assume all the entering probe molecules attached to the surface, one would calculate the sensitivity as ∼138 ag/Hz, which is in closer agreement with the measured value of 300 ag/Hz. One notes in Figure 1 that, beyond 100 pM, the sensor response is saturated. It is interesting to note that 10 mL of 100 pM has ∼6 × 1011 strands, which is the approximate value of the surface capacity of the sensor for ssDNA. That is, the observed saturation response (Figure 1B) is due to the fixed number of available sites for attachment. In our earlier work with smaller thiolic compounds, a similar response was observed.33 From the results in Figure 1, it is reasonable to conclude that both mass addition method and probe chemisorption approach indicate that PEMC sensors exhibit sensitivity of subfemtogram/Hz. Sensor Response to DNA Hybridization at 1 fM, 1 pM, and 1 nM. The response to hybridization of complementary strand at the three concentrations of 1 fM, 1 pM, and 1 nM are shown in Figure 2. In these early experiments, the probe surface density was not optimized even though it is known that surface density affects hybridization efficiency and kinetics.36 After stabilizing the sensor in TE buffer, 9 mL of 1 pM probe was introduced in a recirculation mode, following an initial 5-mL flush. As soon as the probe solution entered the SFC, the resonance frequency decreased exponentially and reached steady state in ∼27 min with a total decrease of 1037 ( 21 Hz ((standard deviation (SD) over 10 min). Subsequently 10 mL of 1 µM MCH was introduced in a once-through mode that lasted ∼15 min, during which there was a further decrease of ∼305 Hz. This useful technique of MCH treatment, developed by Herne and Tarlov31 is used to reduce nonspecific adsorption of DNA molecules to the gold surface. The smaller MCH molecules compete with the probe bases that are weakly attached to the sensor surface while leaving the covalently bonded thiolated probe molecules intact. The MCH treatment also helps to extend the DNA strands away from the sensor surface and into the solution, thereby increasing the probability of interacting with the target strands.37 Additionally, the smaller MCH molecules scattered in between the larger probe DNA strands enhance the accessibility of the probe strands to the target, thereby improving the final hybridization efficiency. Once the MCH treatment was complete, 30 mL of 1 fM 10-mer complementary strand was introduced in a once-through mode without recirculation. As soon as the sample entered the analyte chamber, there was a slow decrease in resonance frequency and steady state was reached in ∼17 min. After ∼25 min of target flow and a change of -190 ( 19 Hz, the sensor resonance frequency reached (36) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 5163-5168. (37) Aqua, T.; Naaman, R.; Daube, S. S. Langmuir 2003, 19, 10573-10580.

Figure 2. Panel A. PEMC-A response to immobilization of 9 mL of 1 pM probe in TE buffer (indicated by first arrow), followed by 1 µM 1-mercaptohexanol (indicated by second arrow), and finally to various concentrations of 10-mer complementary ssDNA (third arrow). Responses are time-shifted for clarity. All experiments were done with PEMC-A, which exhibited resonance frequency in air at 1007.125 kHz. Panel B. Hybridization response to 30 mL of 1 fM, 1 pM, and 1 nM ssDNA in a once-through flow arrangement is shown. Flow rate was constant at 0.6 mL/min, and temperature was maintained at 32.1 ( 0.1 °C, which is 5 °C lower than hybridization temperature. Response to TE buffer shows zero response.

steady state. The sensor was removed, cleaned, and reused with the sequence of probe attachment and hybridization with 1 pM and 1 nM target. The complementary strand at 1 pM elicited a 345 ( 14 Hz decrease in 19 min while 1 nM caused a 540 ( 39 Hz decrease (Figure 2) in 25 min. The three probe immobilization steps in Figure 1A gave responses (1140, 1068, and 1038 Hz) that are within 9% of each other and were found to be quite reproducible on the same sensor. The response due to MCH treatment is very similar to the one reported by Calleja et al.38 The hybridization portion at each target concentration is expanded in Figure 1B. Note that even though in each case nearly the same number of probes were on the surface, a millionfold change in target concentration resulted in a change of threefold for the hybridization response (Figure 2B). Since the mass ratio of MCH to the probe ()134/5070) is 0.0264, the response to MCH (295 Hz) would suggest that ∼8.5% of the sensor surface was occupied by the probe. Given that 1.8 mm2 of sensor area has 1.1 × 1013 Au sites34 the 9 fmol (9 (38) Calleja, M.; Nordstrom, M.; Alvarez, M.; Tamayo, J.; Lechuga, L. M.; Boisen, A. Ultramicroscopy 2005, 105, 215-222.

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mL,1 pM; 5.4 × 109 molecules) of probe introduced would still leave many vacant sites and is consistent qualitatively with the observed shift with MCH. Ratio of molecular mass of target to probe ()3300/5070) is 0.65. The response ratio of hybridization to the probe response (540/1038) is 0.52, which suggests a ∼80% hybridization, if we assume all immobilized probes are accessible for hybridization and the sensor response is linear for small changes. At a lower target concentration of 1 fM and 1 pM, a similar analysis show hybridization levels as 27 and 50%, respectively. At 1 fM, the steady state was reached in ∼15 min, and thus, the amount of target exposed to the sensor was 9 amol. Considering the noise level in the response shown in Figure 2B, an even lower concentration of target would have yielded a measurable response. Sequential Addition of Complementary Target Strand. Another set of experiments were conducted to determine the response of immobilized probe to sequential addition of increasing concentrations of complementary target DNA. PEMC-B sensor (1.8 mm2) was immobilized with 10 mL of 49 pM probe that gave a response of 1,290 ( 33 Hz (Figure 3). After a TE buffer flush the sensor was treated with 1 µM MCH that resulted in a response of -240 ( 13 Hz. Sequential injection of 1 fM, 1 pM, and 1 µM complementary ssDNA samples were carried out. In each step, 2 mL of sample was loaded into a clean reservoir and was pumped into the flow circuit initially in a once-through mode. As the content in the reservoir reached close to the bottom, the outlet of the flow cell was connected to the reservoir so that the sample was in a recirculation mode. Since the flow loop volume is 2.2 mL, recirculation caused the sample to be diluted by ∼10%. The response to 1 fM took ∼20 min to reach steady state and resulted in a frequency decrease of 258 ( 11 Hz, which is slightly larger than in Figure 2. Note that probe concentration used is higher, and the target amount was lower with 2 amol. Subsequent responses to 1 pM and 1 µM caused a further decrease of 320 ( 13 and 390 ( 18 Hz, respectively. A TE buffer flush at 250 min resulted in no significant change in resonance frequency suggesting that weakly bound strands, if any, were few or none. The response to MCH in the present case was lower than in Figure 2, and we estimate probe surface coverage was 12.4% using the method described earlier. The cumulative response to the three additions was ∼931 Hz and is 0.75 of the response to probe immobilization (-1,245 Hz), which is higher than the expected response based on mass ratio, 0.65. This was not the case when the probe was immobilized at 1 pM, when the surface concentration was lower than the present case carried out at 49 pM. Repeated experiments gave response ratios of 0.72 ( 0.03 (n ) 4). The reason for this behavior is not entirely clear. But, we observe this “overresponse” whenever surface probe concentration is higher than that obtained with 1 pM. It is suggested that the sensor that is fully covered with hybridized DNA molecules may possibly entrain more fluid than the one that has low surface probe density. The entrained fluid in the dense DNA layer adds to the sensor mass, causing a larger frequency response. We observed a similar “overresponse” in serum with high surface coverage of the probe, and it is described later in this paper. If we assume a 931-Hz response to represent complete hybridization, the response to 1 fM and 1 pM may be estimated as 27 and 62% hybridization, respectively. 7396 Analytical Chemistry, Vol. 79, No. 19, October 1, 2007

Figure 3. Panel A. PEMC-B sensor prepared by first flowing 10 mL of 49 pM probe followed by 1 µM 1-mercaptohexanol. After a TE buffer flush, the sensor was exposed sequentially to 2 mL each of 1 fM, 1 pM, and 1 µM complementary ssDNA at 0.6 mL/min. The flow circuit has a holdup volume of 2.2 mL, and thus, the first 1 mL of sample flowed in a once-through mode followed by the second milliliter in recirculation mode. Panel B. Hybridization profiles are shown for the three cases. Upon exposure to 1 µM, most of the probes would be hybridized since the available target strand is far in excess of the probes on the sensor surface.

Verification of Hybridization by Dehybridization. Doublestranded DNA can be dehybridized using strong alkali solutions such as urea or NaOH. If a hybridized DNA strand is selectively removed without destroying the probe surface, the regenerated surface can be used for further detection. For the set of probe and target oligonucleotides used in this study, dehybridization using various concentrations of NaOH was explored (n ) 4), and 0.75 M NaOH showed the most promise and was used in further experiments. After immobilizing 2 mL of 1 pM probe, it was treated with 2 mL of 1 µM MCH. In Figure 4, the response to the entire experimental sequence is shown. At 58 min, 2 mL of 1 pM complementary ssDNA was flowed in and hybridization (-667 ( 23 Hz) was observed. To dehybridize the double-stranded DNA, 0.75 M NaOH solution was then introduced, which caused an immediate and rapid change in frequency of -8,115 ( 154 Hz. The density of 0.5 M NaOH is 1.032 g/mL,39 whereas the density

Figure 4. PEMC-B sensor response to hybridization, dehybridization, and then hybridization again. Two milliliters of 1 pM probe and 1 mL of 1 µM MCH was used to prepare the sensor. First hybridization was carried out with 2 mL of 1 pM. To dehybridize, 0.75 M NaOH was used at 0.6 mL/min. Rapid decrease in resonance frequency is due to temperature increase. At 116 min, TE buffer was pumped in to flush out NaOH, followed by the introduction of 2 mL of 1 pM complementary ssDNA. Second hybridization appears not to be as efficient as the first one.

of TE buffer with 1 M NaCl is estimated as 1.038 g/mL.40 Since the density difference is small, the rapid decrease in resonance frequency is due to temperature increase as a result of release of heat of dilution. At 111 min, the flow was switched to TE buffer to remove NaOH, and the resonance frequency recovered and stabilized at 102 ( 14 Hz below the value that was present at the beginning of hybridization. That is, to the first approximation, this is interpreted as 85% dehybridization. The flow was then changed to a freshly prepared batch of 2 mL of 1 pM complementary ssDNA, which caused a decrease of 415 ( 27 Hz for the second hybridization with the same surface probe. The 38% reduction in sensor response in the second hybridization suggests that the probe surface may not have been fully regenerated. Several attempts (n ) 7) did not give any significant improvement in the second hybridization response. We conclude that the sensor response to complementary strand is due to hybridization. Response in the Presence of Copious Amounts of Noncomplementary Strands. One of the important objectives of this study is to examine the PEMC sensors’ ability to distinguish the complementary ssDNA via hybridization in the presence of copious amounts of noncomplementary DNA strands. Using the experimental protocol used earlier, the PEMC-B sensor was first exposed to 10 mL of thiolated 100 nM probe and then to 1 µM MCH. Samples (30 mL) containing mixtures of 10-mer complementary and 10-mer noncomplementary ssDNA were prepared in mole ratios of 1:0, 1:100, 1:10000, and 0:1, where the complementary ssDNA concentration was kept constant at 167 fM; the total complementary strand was constant at 5 fmol in all experiments. The sensor was cleaned after each hybridization experiment and was reimmobilized with freshly prepared thiolated (39) Weast, R. C., Ed. CRC Handbook of Chemistry and Physics, 66 ed.; CRC Press: Boca Raton, Fl, 1982. (40) Rijal, K.; Mutharasan, R. Sens. Actuators, B: Chem. 2007, 124, 237-244.

probe. The detection was done in the order of 1:100, 1:10000, 1:0, and 0:1. Panel A shows the entire experimental sequence in a time-shifted fashion for clarity, whereas panel B shows only the hybridization response from the point where the target oligonucleotide first entered the sample flow cell chamber. The immobilizations of the thiolated probe on the sensor resulted in frequency decreases of 975-1110 Hz for the four cases and are within (6% of the mean. We attribute the variance to sensor surface preparation and to the intervening piranha-cleaning step. When the sensor was exposed to sample containing all noncomplementary ssDNA (sample 0:1), there was no observable shift in resonance frequency. The other samples 1:10000, 1:100, and 1:0 caused frequency decreases of 353 ( 12, 370 ( 9, and 375 ( 11 with response times of 52, 39, and 37 min, respectively. The total responses are well within the expected variation of (6%, but the time required for reaching the maximum response was affected by the concentration of noncomplementary strands. We attribute the small variation in the steady-state response to the measurement format, which combines flow and vibration of the sensor surface. We have seen similar selective detection in the case of detecting a pathogen in the presence of copious amounts of nonpathogens23 and the detection of a pathogen in proteinous fluids.41 Hybridization Kinetics in the Presence of Copious Noncomplementary Strands. To analyze the kinetics of hybridization, we assume a first-order Langmuir kinetics. If ∆f∞ is the maximum frequency change due to hybridization, the sensor response can be represented by25

(∆f) ) (∆f∞)(1 - e-kobsτ)

(1)

where (∆f) is the resonance frequency change due to hybridization at time τ and kobs is the overall hybridization rate. The above equation can be rearranged as

(

ln

)

(∆f∞) - (∆f) (∆f∞)

) -kobsτ

(2)

The sensor frequency response can be plotted as noted in eq 2, and the overall hybridization rate constant kobs can be determined with good accuracy. kobs values obtained for the samples 1:0, 1:100, and 1:10000 were 0.047 ( 0.006 (R2 ) 0.99), 0.062 ( 0.006 (R2 ) 0.98), and 0.072 ( 0.008 min-1 (R2 ) 0.98), respectively. That is, a 35% reduction in hybridization rate was found in the presence of 10 000 times extraneous 10-mer ssDNA. This is not an unexpected result as the noncomplementary 10-mer hinders the transport of target sequence to the sensor surface. However, the constant vibration of the sensor and the continuous flow of sample allow the complementary strand to ultimately reach the sensor surface and hybridize. A similar slower response rate was observed when the target Bacillus anthracis was detected in the presence of copious nonpathogenic Bacillus cereus and Bacillus thuringiensis23 using the PEMC sensor. Hybridization in Human Plasma. In this final section, we examine hybridization in human serum. Density of human plasma (41) Campbell, G. A.; Uknalis, J.; Tu, S.-I.; Mutharasan, R. Biosens. Bioelectron. 2007, 22, 1296-1302.

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Figure 5. PEMC-B prepared with 100 nM probe and 1 µM of MCH. Samples containing complementary and noncomplementary ssDNA in mole ratios of 1:0, 1:100, 1:10000, and 0:1 were prepared with each sample containing 5 fmol of complementary stand at 167 fM concentration. Panel A shows the entire sequence of probe immobilization and detection. Responses are time-shifted for clarity. Panel B shows the relative responses of the four cases. Note that all samples containing complementary strand give essentially the same overall frequency change at steady state. Kinetics of hybridization is slower in the presence of copious amounts of noncomplementary strands.

(∼1.025 g/cm3) is greater than TE buffer; as such, it is important to equilibrate the sensor for density effects40 with plasma before conducting hybridization studies. Hence, we formulated a “plasma buffer” consisting of equal volumes of TE buffer and human plasma. Since the solids present in the plasma clogged the experimental apparatus (tubing and valves), it was centrifuged at 4000g for 3 min and the clear fluid was recovered for use. After mixing with TE buffer, very small coagulants were formed and were visible but were not removed. A better experimental design would be to use EDTA- or heparin-serum rather than the whole plasma. The mixing dilutes the buffer salt concentration, and they are important for promoting hybridization. Therefore, the composition of plasma buffer was adjusted to make the final composition as 10 mM Tris-HCl, 1 mM EDTA, with 1 M NaCl. After obtaining a stable baseline in TE buffer, the sensor was prepared with 2 mL of 500 pM thiolated probe followed by 2 mL of 1 µM MCH to optimize the sensor surface. Response is given in Figure 6. TE buffer was reintroduced followed by the plasma buffer to equilibrate the flow cell and the sensor with the higher density plasma buffer. Flow of plasma buffer resulted in a decrease of 301 ( 9 Hz. At 92 min, TE buffer was reintroduced, and as shown in Figure 6, the resonance frequency recovered within 4 Hz of the resonance frequency value prior to the introduction of plasma buffer. This complete recovery and other experiments (n 7398 Analytical Chemistry, Vol. 79, No. 19, October 1, 2007

Figure 6. PEMC-C sensor prepared with 2 mL of 500 pM probe and 2 mL of 1 µM MCH. Plasma buffer was made consisting of 50% human plasma and 50% TE buffer with concentration (Tris-HCl, EDTA and NaCl) adjusted for dilution (see text). Introduction of plasma buffer, followed by TE buffer, gives a reversible response suggesting that plasma proteins did not attach to the sensor surface. The plasma buffer is reintroduced, and complementary ssDNA prepared in plasma buffer is then allowed to flow in sequentially at the three concentrations indicated, allowing for steady state to be reached between each sample. Panel B shows a repeat experiment with PEMC-C. The overall results are within (3%. No reduction in hybridization in plasma buffer is seen in comparison to results in TE buffer.

) 7) indicate that no plasma components adsorb permanently to the sensor prepared with the thiolated probe and MCH. This appears to be an important and fortuitous advantage in the current method. At 106 min, plasma buffer was reintroduced at 0.6 mL/ min, and the resonance frequency decreased by 305 ( 11 Hz. Samples (2 mL) of complementary strand at 1 fM, 1 pM, and 1 µM in plasma buffer were introduced sequentially using the approach used in Figure 3. As seen in Figure 6A, these three targets caused frequency decreases of 178 ( 18 Hz in 16 min, an additional 341 ( 14 Hz decrease in 21 min, and finally a change of -384 ( 16 Hz in 16 min, respectively, for the three concentrations. Similar to the results in Figure 3 and in Figure 5, the vibrating PEMC sensor showed intrinsic specificity of hybridizing complementary target strands in 50% plasma. These results suggest that purification of DNA may not be necessary for detection as long as the sample does not contain biochemical activity that destroys the probe and the target ssDNA strand. The ratio of hybridization response to probe response ()903/

Figure 8. PEMC-C sensor prepared with 49 pM probe and 1 µM MCH. Plasma buffer was introduced in a once-through flow mode as in Figures 6 and 7. After a stable resonance frequency was reached, flow was changed to TE buffer at t ) 13 min, to plasma buffer at t ) 25 min, and then to TE buffer again at t ) 36 min. The response is reversible, and recovery is within 10 Hz. Time taken to respond is ∼5 min. Given that holdup volume of the flow circuit is 2.2 mL, and the flow rate is 0.6 mL/min, the transition time indicates the time needed to homogenize the fluid environment surrounding the sensor.

Figure 7. PEMC-B prepared with 2 mL of 50 nM probe and 2 mL of 1 µM MCH. Plasma buffer (50% plasma + 50% TE buffer) is introduced at 46 min, followed by 2 mL of 1 fM complementary ssDNA in plasma buffer. Hybridization responses are 329, 838, and 671 Hz for the three concentrations. Following 1 pM addition, fresh plasma buffer is introduced to examine if there is any change in surface characteristics. After observing no response, 1 µM target is introduced. Total hybridization response is 1838 Hz, which indicates nearly 100% of the surface immobilized probes hybridized. Panel B expands the response to show the individual responses more clearly.

1335) is 0.68, is close to the theoretical value noted earlier of 0.65, and may suggest that complete hybridization occurred. Since the measured shift due to hybridization was measured in plasma buffer while the probe attachment was conducted in TE buffer, corrections to response should be made. The plasma buffer introduced a shift of ∼22% of probe + MCH response (from 1,501 to 1,804 Hz, an increase of 22%). This is due to the physical properties (density and viscosity) of the plasma buffer. Thus, correcting for the change in plasma buffer-induced response, the ratio of hybridization response to probe immobilization response ()903/[1.22 × 1,335]) is 0.55. Therefore, one would estimate that 85% hybridization was obtained. It is interesting to note that the cumulative response in plasma (-178 ( 18, -519 ( 14, and -903 ( 16 Hz) was slightly lower than the response obtained in TE buffer (-210 ( 12, -618 ( 15, and -968 ( 18 Hz) for the three concentrations of 1 fM, 1 pM, and 1 µM, but within expected experimental variance due to surface preparation, sensor sensitivity (PEMC-C vs PEMC-A) and the difference in physical properties of the two buffers. The overall hybridization rate constant (kobs) was 0.061 ( 0.005 min-1 (R2 ) 0.95) at 1 fM and is 34% lower than in TE buffer. In Figure 6 panel B, another hybridization experiment in plasma buffer is shown. Note that the responses

to probe immobilization (1348 ( 9 Hz) and to MCH (162 ( 6 Hz) are almost identical to the results in panel A. The plasma buffer response is slightly higher (312 ( 17 Hz), and the three concentrations gave frequency changes of 179 ( 6, 335 ( 7, and 382 ( 6 Hz for a total response of 896 ( 8 Hz. This represents a hybridization of ∼85% using the method used earlier. To examine if higher sensitivity can be achieved in plasma, three experiments were conducted at high probe density. A representative result is shown in Figure 7. Probe immobilization was carried out with 2 mL of 50 nM, which caused a 1557 Hz decrease in resonance frequency and is ∼16% higher than in Figure 6. Exposure to 2 mL of 1 µM MCH resulted in a further decrease of 163 Hz. Introduction of plasma buffer caused a shift of 408 Hz, representing almost 26% of probe response. It is interesting to note that the response to plasma buffer does depend on the surface density of the probe. Responses to 1 fM, 1 pM, and 1 µM were, respectively, 329 ( 9, 838 ( 11, and 671 ( 8 Hz for each of the concentration steps. The smaller response for MCH (163 Hz) compared to the earlier result in Figure 2 suggests that more of the gold sites were occupied by the probe. The response to 1 fM is also higher than the response in Figure 2 and ∼70% higher than in Figure 6. Similarly, the 1 pM and 1 µM concentrations induced a higher sensor response as well. Introduction of plasma buffer after the 1 pM target induced essentially a zero response. The cumulative response to complementary ssDNA is 1838 Hz. Correcting for plasma buffer effect, hybridization efficiency (1838/[1.26 × 1557]) is 0.94, which is higher than the theoretical value based on mass ratio, 0.75. As was noted earlier, when the surface concentration of the probe is high, the response to hybridization is higher than the theoretical mass ratio value. Our current interpretation of this variance is the entrained liquid within the surface DNA layer appears to increase the oscillating mass resulting in a larger than the expected frequency response Analytical Chemistry, Vol. 79, No. 19, October 1, 2007

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that is solely due to the hybridized ssDNA. Clearly, further study is warranted to understand the behavior of serum protein interactions with the DNA and MCH on the sensor surface. To examine the nature of plasma buffer interaction, we conducted further experiments on the PEMC-C sensor immobilized with 49 pM probe and 1 µM MCH. After a stable resonance frequency was obtained, TE buffer was replaced with plasma buffer. After a decrease of 282 Hz, the frequency stabilized and remained constant within (7 Hz over a 25-min period. Close monitoring of resonance frequency was then initiated, and the flow was changed to TE buffer at 13 min (Figure 8). The resonance frequency increased by 288 Hz in 5 min and reached a new steady-state value. At 25 min, the flow was changed to plasma buffer, and the sensor resonance frequency decreased to the original value in the same 5-min period. A second change to TE buffer resulted in the resonance frequency rising by 274 Hz, which is within 14 Hz of the value in TE buffer. The reversible response to plasma buffer and TE buffer, together with the reversible response seen in Figure 6A, suggest that serum proteins do not adsorb to the sensor surface prepared with the probe and MCH. CONCLUSIONS Gold-coated piezoelectric-excited millimeter-sized cantilever sensors exhibited responses to 15-mer ssDNA immobilization via thiol chemistry over a nine log concentration range of 1 aM to ∼1 nM. The sensor frequency response is a nonlinear function of mass change and exhibited sensitivity in the range of 1-300 ag/Hz.

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We draw three important conclusions. (1) Hybridization of 10mer ssDNA with immobilized 15-mer complementary strand was observable at as low a concentration as 1 fM. The response obtained suggests that an even lower concentration may be observable as the response to 2 amol was ∼200 Hz with a measurement noise of ∼15 Hz. (2) The extent of hybridization was not significantly affected when the complementary strand was in a mixture with 100× and 10000× higher concentration of noncomplementary 10-mer strands. The kinetics of hybridization, however, was slower by a factor of ∼2 in presence of 10000× noncomplementary strands compared to the case of pure complementary strand. (3) In 50% plasma, the extent of hybridization did not change when compared to TE buffer. In plasma samples, however, the kinetics of hybridization was ∼35% slower. Because direct detection of DNA strands can be made in plasma, the method may be useful in medical diagnostics for detecting circulating DNA. ACKNOWLEDGMENT This project was self-funded with funds from USDA 2006-5111003641 and EPA R-833007 drawn for supplies. We thank to Dan Luu for the fabrication of the flow cell and David deLesdernier for help with design of temperature control system.

Received for review June 7, 2007. Accepted July 31, 2007. AC0712042