Viral detection using an embedded piezoresistive microcantilever sensor

Sensors and Actuators A 107 (2003) 219–224

Viral detection using an embedded piezoresistive microcantilever sensor R.L. Gunter b , W.G. Delinger b , K. Manygoats a , A. Kooser a , T.L. Porter b,∗ a

Department of Chemistry, Northern Arizona University, Flagstaff, AZ 86011, USA Department of Physics, Northern Arizona University, Flagstaff, AZ 86011, USA

b

Received 21 May 2003; received in revised form 23 July 2003; accepted 25 July 2003

Abstract The detection of aerosol-based vaccinia virus and solution-based vaccinia virus has been studied using a new type of microsensor design. In the embedded piezoresistive microcantilever (EPM) sensor, a tiny piezoresistive microcantilever is embedded or partially embedded into an active sensing material whose volumetric response to the presence of an analyte is recorded as a strain in the cantilever, and subsequent change in the resistance of the cantilever. In one set of experiments, a composite consisting of poly(ethylene oxide), or PEO, combined with vaccinia antibody was used as the active sensing material. In aerosol exposures, this sensor produced a distinct signal owing to vaccinia virus adsorption/incorporation. In addition, experiments using a piezoresistive microcantilever in direct contact with a pure antibody layer in solution were also performed to detect vaccinia virus in solution. Again, a clear and distinct analyte signals obtained. © 2003 Elsevier B.V. All rights reserved. Keywords: Vaccinia; Embedded; Piezoresistive; Microcantilever

1. Introduction Piezoresistive microcantilever-based sensors [1–6] measure the strain induced resistance change produced in the cantilever upon exposure to the analyte(s) of interest. Typically, this strain occurs when analyte moieties are adsorbed, bound to, or otherwise incorporated into a “sensing material” either coated onto the cantilever [5,6] or in which the cantilever is in direct physical contact atop the material [1,2]. In the embedded piezoresistive microcantilever (EPM) design, the cantilever is fully or partially embedded into this “sensing material” [3,4]. Upon analyte exposure, the sensing material selectively adsorbs or incorporates the analyte species, resulting in a tiny volumetric change in the sensing material (Fig. 1). This volumetric change is measured as a simple resistance change in the piezoresistive microcantilever, and thus the analyte is detected. Advantages of this design include small size (the cantilevers themselves may be only a few tens of micrometers in dimension), low cost, simple support electronics, and resistance to movement or vibration. The key element to EPM sensor design is the fabrication of an appropriate sensing material, or composite material that will swell, contract, or otherwise change in dimension

∗ Corresponding author. Tel.: +1-928-523-2540; fax: +1-928-523-1371. E-mail address: [email protected] (T.L. Porter).

0924-4247/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0924-4247(03)00379-0

upon exposure to the desired analyte. Previously, we have used common organic polymers as sensing materials to detect the presence of water vapor and many other volatile organic compounds (VOCs) such as acetone, toluene, ethanol, hexane, and others [2–4]. Here, VOC molecules partition into the polymeric materials, resulting in swelling that is easily measured by the embedded piezoresistive cantilevers. Analyte molecules that have solubility parameters closely matching that of a given polymer will readily partition into that polymer, while analyte molecules whose solubility parameter differs widely from that of the polymer will be incorporated in a correspondingly lesser amount [7–9]. Thus, arrays of sensors, each individual unit using a different polymer (with a different solubility parameter), may be used in conjunction with pattern recognition techniques to uniquely identify a wide range of analyte species [3,10–12]. Other sensing materials may also be designed that selectively change in dimension upon exposure to the desired analyte. Hydrogels may be synthesized that undergo large volumetric changes in response to changes to temperature, pH, electric field, humidity, and other factors, making these materials suitable for EPM sensors designed to record changes in these parameters [13–16]. Also, an antigen responsive hydrogel was recently synthesized [17]. Pure biological materials may also be used as sensing materials. Analyte binding to pure biological layers will also in some cases result in volumetric changes measurable by piezoresistive microcantilevers. We have recently demonstrated that

220

R.L. Gunter et al. / Sensors and Actuators A 107 (2003) 219–224

Fig. 1. Basic operation of EPM sensor. Exposure to analyte causes volumetric change in sensing material. This change is measured by embedded microcantilever.

a sensing layer may be formed by using 25 bp, thiolated single-strand DNA attached to a gold substrate. A piezoresistive microcantilever brought into contact with this layer in solution responds instantly to the addition of the complimentary DNA strand, and is selective to strands differing by 5 bp. Of further interest are sensing materials that consist of a host matrix with functionality added or incorporated so as to react to the presence of desired analytes. Here, the host material may consist of a common polymer or hydrogel, and the functionality added consists of biological molecules blended into, immobilized, or chemically bonded to the host material. In the present study, we have produced a composite sensing material by blending vaccinia polyclonal antibody with the host polymer poly(ethylene oxide) (PEO). EPM sensors were fabricated from this material, and the response to vaccinia virus in an aerosol delivery was measured. Additionally, we have performed experiments in which pure layers of vaccinia antibody were attached to a glass substrate

and used as a pure biological sensing layer in contact with a piezoresistive microcantilever.

2. Experimental For the pure vaccinia layer experiments, glass slides were first cleaned in nanopure water then treated in H2 SO4 :H2 O2 (3:1) solution for 12 h. These slides were rinsed and then annealed at 260 ◦ C for 12 h. After cooling, the glass slides were placed in a 10% aminopropylsilane (APS) in acetate buffer by volume for 3 h. The slides were rinsed and dried, then placed in an N-hydrosuccinimide (NHS) solution for 24 h. After rinsing and drying, the slides were then placed in a solution of anti-vaccinia for 96 h. These slides were imaged using intermittent contact scanning force microscopy (SFM) to confirm antibody coverage (see Fig. 2a). The binding of vaccinia virus to this antibody layer was tested by exposing

R.L. Gunter et al. / Sensors and Actuators A 107 (2003) 219–224

221

quently use the PEO only sensor as a “dosimeter” for water vapor. Vaccinia virus and polyclonal vaccinia antibody (noninfectious) was obtained from the laboratory of Bertram Jacobs (Arizona State University, Tempe, AZ). Herpes virus (Herpes Simplex Virus-1 gD (HSV-1 gD)) recombinant, non-infectious, was obtained from Fitzgerald Industries International, Concord, MA. The herpes virus was used in non-specific binding experiments. The cantilevers used in these experiments were purchased from Veeco, Inc., and typically are 305 ␮m in length, 3 ␮m thick, with a tip height of less than 2 ␮m and an aspect ratio of 2:1. The nominal resistance of these cantilevers is 2.1–2.2 k and they have a force constant of 1 N/m, a sensitivity to displacement of (1–4) × 10−6 nm−1 , and a sensitivity to force of 0.7×10−6 nN−1 . The cantilever response was recorded in real time using a Kiethley precision multimeter interfaced to a Personal computer using LabView (National Instruments).

3. Results and discussion 3.1. Pure vaccinia layer on glass

Fig. 2. (a) 2.5 ␮m×2.5 ␮m SFM image of anti-vaccinia coated glass slide. (b) 2.5 ␮m×2.5 ␮m image of anti-vaccinia slide exposed to vaccinia virus.

a functionalized glass slide to highly dilute vaccinia in solution. A dilute solution of 10 ␮l of virus was injected into a water-drop (0.2 ml) covered portion of a slide. The slide was then washed in nanopure water, dried and imaged using SFM (Fig. 2b). For vaccinia, individual virions are known to be enveloped, slightly pleomorphic, ovoid, or brick-shaped, 140–260 nm in diameter, and 220–450 nm long. In Fig. 2, we see a range of virions that vary in length from 2200 to 3600 Å, with the large cluster at the bottom left having a length of 5000 Å, probably consisting of two or more virions bound close together on the surface. For the EPM sensor experiments, a composite of the polymer PEO and polyclonal vaccinia antibody was produced. The loading of antibody into the polymer was approximately 20 wt.%. An embedded piezoresistive sensor was then fabricated by partially inserting the microcantilever into a tiny bead of the liquid composite mixture, and the assembly was allowed to dry. A second EPM was fabricated in an identical fashion, but using only PEO polymer with no antibody mixed in. A series of experiments with aerosols of water vapor, or water vapor plus virus were then performed with the two sensors side-by-side. In the first experiment, the PEO only sensor response was calibrated to the PEO + AB sensor for water vapor exposure. This allowed us to subse-

After characterizing the antibody surface (Fig. 2a), a piezoresistive microcantilever was brought into gentle contact with the antibody layer. Contact was verified by an approximately 1  increase in the cantilever resistance. The system was then placed in solution (a single water drop, approximately 0.2 ml). After stabilization, a small amount (10 ␮l) of the vaccinia virus solution was added (t = 250 s). Within 50 s, a rise in the cantilever resistance of approximately 4.2  was recorded (Fig. 3). This rise in cantilever resistance is attributable to binding of the virus to the antibody layer under and around the cantilever, forcing the cantilever to bend up slightly. An identical experiment was performed, using a vaccinia antibody layer in contact

Fig. 3. Response of anti-vaccinia on glass to introduction of vaccinia virus in solution at t = 250 s. The spike at t = 80 s is the introduction of solution to the sensor element.

222

R.L. Gunter et al. / Sensors and Actuators A 107 (2003) 219–224

with a piezoresistive microcantilever in solution, but instead exposed to herpes virus. Herpes virions are large, similar in size and shape to vaccinia (or other pox viruses), but with totally different binding specificity. In this experiment, no response from the cantilever was obtained, indicating little or no non-specific binding of the herpes virus to the vaccinia antibody layer. 3.2. PEO and antibody (PEO + AB) Here, two individual sensors were used. One sensor, consisting of an EPM unit using the composite PEO + AB material, and a second EPM unit, using only PEO as the active sensing material were used. First, the two sensors were exposed side-by-side to varying amounts of water aerosol. The response of each sensor was recorded, with the PEO + AB sensor subsequently normalized and scaled to the PEO only sensor. This was accomplished in order to both use the PEO only sensor as a water dosimeter, and also to subtract off the contribution attributable to water from the PEO + AB sensor. After scaling, the sensors were exposed to an aerosol of water containing the vaccinia virus. The concentration of virus was (2.0 mg/ml of virus further diluted in 150 ml of water.) The aerosol exposures were performed with a 50/50 mix of this dilute solution combined with dry nitrogen. The response of the PEO + AB sensor minus the response of the PEO only sensor (difference signal) is indicated in Fig. 4. In this plot, the exposures began at time t = 100 s, and ended at time t = 400 s. After the aerosol exposures ended (t = 400 s), the sensors were immediately exposed to a flow of dry nitrogen. As can be seen from the figure, some decrease in the maximum peak height occurs upon nitrogen exposure, but the sensor never recovers to its original resistance owing to permanent incorporation of vaccinia virions in the composite matrix. For these exposures (Fig. 4), the total change in resistance was on the order of 20–23 , with the difference signal from the two sensors being approximately 3  (Fig. 4). This difference signal is on the order of 15% of either original

signal. Background noise for these sensors is typically 0.1  signal, including the sensor unit itself and all associated electronics. Based on these figures, an approximate signal (difference) to noise ratio of 30 may be approximated for this exposure series. Owing to the extended lateral dimensions of the polymeric sensing material in which the cantilever is embedded, an estimate of the number, or volume density, of virus particles “detected” is not possible at this time. It is possible, however, to estimate the number of particles needed to bend the cantilever an amount equal to the observed amount (2–3 ) in the present experiments. For a 2  cantilever resistance change, approximately 1 ␮m of displacement must occur. Vaccinia particles themselves are approximately 0.25 ␮m in dimension (brick shaped), which would require approximately four to six virus particles for the observed cantilever deflection under perfectly optimal conditions. Also, with a signal to noise ratio of 30, detection of a single virion is easily possible with the present electronics (a single 250 nm cantilever strain would result is a resistance change of approximately 0.5 , which is easily detectable). The type of optimal conditions described above would require that virion adsorption occur directly underneath the cantilever, deflecting the cantilever upward an amount equal to the virion dimension. It is unlikely that this scenario would occur in the present design, even if all of the active polymer material could be located directly beneath the cantilever itself (with none spilling off to the sides of the cantilever). A more practical estimate for sensitivity under nearly optimal conditions might be the detection of two to four virions bound into the sensing material. We also looked at this same effect (permanent incorporation of virions) for the PEO only sensor. It is likely that the PEO only material will also hold onto some of the virions that enter the polymer matrix in aerosol, carried along by the water particles. In Fig. 5, we show the response of the PEO only sensor to water aerosol plus antibody minus the response of the same sensor to water aerosol alone. In this experiment, exposures began at time t = 0 s, and ended at

PEO+AB (virus) - PEO+AB (water) (scaled to PEO)

Resistance Change (Ohms)

4 3 2 1 0 1

101

201

301

401

501

601

701

801

901

-1 -2 Time (s)

Fig. 4. Plot of the difference in response for the PEO + AB sensor between an aerosol of water plus vaccinia virus and an aerosol of water alone.

R.L. Gunter et al. / Sensors and Actuators A 107 (2003) 219–224

223

PEO (virus) - PEO (water) (scaled)

Resistance Change (Ohms)

1

0.6

0.2 -0.2 1

101

201

301

401

501

601

701

801

901

-0.6

-1 Time (s)

Fig. 5. Plot of the difference in response for the PEO only sensor between an aerosol of water plus vaccinia virus and an aerosol of water alone.

time t = 300 s. After 300 s, the sensor is exposed to dry nitrogen flow. The two responses are scaled to total dose, so there is no divergence in the plots until exposure to aerosol ends, and dry nitrogen flow begins. Here, we see that some virus is indeed apparently retained by the polymer, but only enough to alter the resistance by 0.45  (after 900 s, the plot levels off). From a comparison of Figs. 4 and 5, we may postulate that either the PEO + AB material is incorporating much more of the virus when exposed, and subsequently retaining a large fraction of that virus upon drying, or the PEO only material simply expels much of the virus that it has absorbed upon drying. Non-specific binding experiments were also performed using the PEO + vaccinia antibody EPM sensors exposed to herpes virus. Here, side-by-side EPM sensors were prepared using PEO only for the first sensor and PEO plus vaccinia antibody for the second. The PEO + AB sensor was prepared in an identical fashion to the previous PEO + AB sensors described earlier. First, these sensors were exposed to dry nitrogen flow until their response was flat. Then, a 300 s exposure to water vapor alone was used to determine

the relative response of the two sensors to water vapor. A second recovery cycle with dry nitrogen was then followed by simultaneous exposure of both sensors to a solution of water plus herpes virus. For the herpes solution, 10 ␮l of herpes solution (2.1 mg/ml) was further diluted in 140 ml of DI water. The aerosol exposures were then performed with a 50/50 mix of this dilute solution combined with dry nitrogen. The herpes exposures were for 300 s. In Fig. 6, a plot of the difference in response for the PEO + AB (vaccinia) sensor between an aerosol of water plus herpes virus and an aerosol of water alone is shown. In this plot, the exposures began at t = 100 s, and ended at t = 400 s. From t = 400 s, until the end of the plots, dry nitrogen flow was re-introduced. Here, the two sensors track each other in an almost identical fashion until after the exposure ends. At this time, the PEO only sensor begins a faster recovery, while the PEO + AB sensor lags slightly behind. Also, the equilibrium (t = 800 s) value of the PEO + AB sensor has increased by approximately 0.8 . This may be explained by a small amount of herpes virus incorporation into the PEO + AB sensor (data from Fig. 5 does show that the PEO polymer will absorb small amounts

PEO+AB (non-specific virus) - PEO+AB (water) (scaled to PEO+AB)

Resistance Change (Ohms)

1 0.8 0.6 0.4 0.2 0 -0.2

1

45 89 133 177 221 265 309 353 397 441 485 529 573 617 661 705 749 793

-0.4 Time (s)

Fig. 6. Plot of the difference in response for the PEO + AB (vaccinia) sensor between an aerosol of water plus herpes virus and an aerosol of water alone.

224

R.L. Gunter et al. / Sensors and Actuators A 107 (2003) 219–224

Acknowledgements This work was supported by the National Science Foundation (DMR-9703840) and by proposition 301 funding from the state of Arizona. References

Fig. 7. SFM image (4 ␮m × 4 ␮m) of PEO film.

of virus, even though little or no antigen–antibody binding occurs). Finally, we note that the choice of PEO as a host material for incorporation of biological molecules may be unique for this class of simple organic polymers. SFM phase-contrast images (Fig. 7) of this material show an open, lamellar structure, well suited for easy penetration by large molecules such as antibodies or antigens. From the image, the PEO film is seen to be composed of close-packed individual polymer lamellae. The individual lamellae average 8.2 nm in width, and range in length from less than 1 ␮m to over several micrometers. Polymers such as PEO are known to form lamellae such as this, with the individual lamellar strands themselves composed of well ordered, transverse polymer chains folding back upon themselves. These structures are also generally accepted to be crystalline or semi-crystalline. Outside of the individual lamellae, polymer chains may terminate after some distance, or they may eventually fold back into the lamellar structure.

4. Conclusions Sensors based on piezoresistive microcantilever technology may be used in the detection of biological molecules, including viruses such as vaccinia. We have detected the presence of vaccinia both in solution, and in aerosol deliveries using EPM sensors. In EPM sensors, a host material may be used that incorporates species that will bind to the analytes to be detected. The choice of host material may include polymers, hydrogels, or other materials. In the case of PEO as a host material, the swelling of the PEO host owing to water vapor or aerosol must be subtracted from the overall signal in order to detect the analyte.

[1] T.L. Porter, M.P. Eastman, D.L. Pace, M. Bradley, Scanning 22 (2000) 1. [2] T.L. Porter, M.P. Eastman, D.L. Pace, M. Bradley, Sens. Actuators A88 (2001) 47. [3] T.L. Porter, An Embedded Piezoresistive Microcantilever Based Sensor, Scanning Probe Microscopy, Sensors and Nanostructures, Las Vegas, NV, 2002. [4] T.L. Porter, M.P. Eastman, C. Macomber, W.G. Delinger, R. Zhine, Ultramicroscopy, in press. [5] T. Thundat, R.J. Warmack, G.Y. Chen, D.P. Allison, Appl. Phys. Lett. 64 (21) (1994) 2894. [6] E.A. Wachter, T. Thundat, Rev. Sci. Instrum. 66 (6) (1995) 3662. [7] B.J. Doleman, M.C. Longeran, E.J. Severin, T.P. Vaid, N.S. Lewis, Anal. Chem. 70 (1998) 4177. [8] B.J. Doleman, R.D. Sanner, E.J. Severin, R.H. Grubbs, N.S. Lewis, Anal. Chem. 70 (1998) 2560. [9] M.P. Eastman, R.C. Hughes, G. Yelton, A.J. Ricco, S.V. Patel, M.W. Jenkins, J. Electrochem. Soc. 146 (10) (1999) 3907. [10] M.K. Baller, H.P. Lang, J. Fritz, C. Gerber, J.K. Gimzewski, U. Drechsler, H. Rothuizen, M. Despont, P. Vettiger, F.M. Battiston, J.P. Ramseyer, P. Fornaro, E. Meyer, H.-J. Guntheodt, Ultramicroscopy 82 (2000) 1. [11] A.R. Hopkins, N.S. Lewis, Anal. Chem. 73 (2001) 884. [12] H.P. Lang, M.K. Baller, R. Berger, C. Gerber, J.K. Gimzewski, F.M. Battiston, P. Fornaro, J.P. Ramseyer, E. Meyer, H.-J. Guntheodt, Anal. Chim. Acta 393 (1999) 59. [13] E. Kokufuta, Y.-Q. Zhang, T. Tanaka, Nature 351 (1991) 302. [14] Y. Osada, H. Okuzaki, H. Hori, Nature 355 (1992) 242. [15] T. Tanaka, Phys. Rev. Lett. 40 (1978) 820. [16] Y. Hirokawa, T. Tanaka, J. Chem. Phys. 81 (1984) 6379. [17] T. Miyata, N. Asami, T. Uragami, Nature 399 (1999) 766.

Biographies Timothy Porter is a professor of physics at Northern Arizona University. Dr. Porter obtained his PhD degree from Arizona State University, Tempe, AZ. Dr. Porter’s interests include composite materials, biophysics, SFM, and time of flight mass spectrometry. William Delinger is a professor of physics at Northern Arizona University. Dr. Delinger obtained his PhD degree from the University of Iowa, Iowa City, IA. Dr. Delinger’s interests include electronic instrumentation, computer data acquisition and control, and sublimation of ices. Robert Gunter is a graduate student in physics at Northern Arizona University. Ara Kooser is a graduate student in chemistry at Northern Arizona University. Kevin Manygoats is a research specialist in chemistry at Northern Arizona University.