IOP PUBLISHING Nanotechnology 18 (2007) 125503 (5pp)

NANOTECHNOLOGY doi:10.1088/0957-4484/18/12/125503

Antibody-based protein detection using piezoresistive cantilever arrays

Vita Dauksaite1,2 , Martin Lorentzen1,3 , Flemming Besenbacher1,3 and Jrgen Kjems1,2,4
Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, DK-8000 Aarhus C, Denmark 2 Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark 3 Department of Physics and Astronomy, University of Aarhus, DK-8000 Aarhus C, Denmark E-mail: jk@mb.au.dk
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Received 31 October 2006, in nal form 4 January 2007 Published 21 February 2007 Online at stacks.iop.org/Nano/18/125503 Abstract A piezoresistive cantilever array platform with electrical read-out was applied for protein detection using GST (glutathione-S-transferase) and GST antibodies as a model system. Sensing was performed in the static deection mode under constant ow conditions. The GST antibodies were directly immobilized on the cantilever gold surface by means of free thiol groups. The setup allowed simultaneous deection measurements with sensor and control-antibody-immobilized reference cantilevers and enabled detection of 1 ng l1 (40 nM) of GST protein, which is similar to the sensitivity reported for cantilever sensors using an optical read-out system.

1. Introduction
Proteins regulate and perform most of the biochemical processes in cells and their detection is essential in a variety of applications ranging from clinical diagnosis (Ciordia et al 2006), environmental control (Clark et al 2005) to homeland security issues (Andreotti et al 2003). At the same time it is a complex and challenging task to study proteins. Historically, biochemical methods have been widely used, but recently biosensor research has attracted considerable interest in the search for new and improved methods for monitoring proteins and their interactions with other macromolecules (Murphy 2006). Widely used biochemical methods for protein detection (Western blotting and ELISA (enzyme-linked immunosorbent assay)) (Ausubel et al 1995) are usually time- and materialconsuming and require multistep protocols. Others (e.g. uorescence) require the attachment of labels. Biophysical sensors, such as SPR (surface plasmon resonance) (Liedberg et al 1983) and QCM (quartz crystal microbalance) (Rodahl et al 1995), offer several advantages over conventional biochemical techniques, including real-time, label-free detection. Furthermore, new methods that allow for simultaneous analysis of many thousands of interactions by combining newly developed SPR imaging with protein arrays (reviewed in Boozer
4 Author to whom any correspondence should be addressed.

et al 2006) are emerging. Another recently emerged highly advantageous method is the surface-stress-based cantilever sensor (Fritz et al 2000). The adsorption of biochemical species on a functionalized surface of a microfabricated cantilever can cause surface stress and consequently induce cantilever bending. Recently, progress in the development of cantilever-based sensors for biological applications has been reported (Hansen and Thundat 2005, reviewed in Then and Ziegler 2004). The most commonly used read-out method is the optical beam deection method, in which the angular perturbation of a laser beam is measured upon bending of the cantilever. An alternative to this method is the piezoresistive read-out, where a piezoresistive material is integrated in the cantilever, a structure enabling electrical sensing of cantilever bending. The integrated piezoresistor changes its electrical resistance when the stress applied to the resistor changes (Boisen et al 2000). This approach offers several advantages over the optical detection techniques, including the simpler read-out, measurement in a non-transparent (e.g. blood) environment and the possibility of miniaturizing the sensor and incorporating it in handheld devices suitable for use in a variety of high-growth markets for detection in both liquids and gases. To detect proteins specically, highly specic binding reagents, such as antibodies, are commonly used for protein detection. A critical step in preparing any protein sensor based on antibodyantigen interactions is the creation of a
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0957-4484/07/125503+05$30.00

2007 IOP Publishing Ltd Printed in the UK

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sensing surface from immobilized antibodies. Antibodies might be fragile in handling or might lose their activity upon immobilization. Usually, antibodies are deposited on the cantilever surface by a conjugating molecule, or on a monolayer of self-assembled alkane chain molecules (SAMs). Here we apply functionalization, where the antibodies are attached directly to the gold surface via free thiol groups obtained after antibodies have been subjected to reduction. To detect antibodyantigen interactions in this study, the Cantion-NanoNord A/S nanomechanical cantilever technology platform with an in-build piezoresistor was applied (CantionNanoNord A/S). We demonstrate that the cantilever sensor is capable of sensing glutathione-S-transferase (GST) with sensitivities down to 1 ng l1 (40 nM), similar to the sensitivity reported for the optical read-out cantilever detection system. The piezo-electrical cantilever system thus provides an attractive alternative platform for protein detection in the future.

Invitrogen BenchMark Protein Ladder was used as a molecular weight marker in SDS-PAGE. 2.3. GST protein purication and SDS-PAGE The GST protein was expressed in Esherichia coli BL21(DE3) and puried according to the manufacturers instructions (Pharmacia Biotech) with a few changes. Elution was performed in reduced glutathione containing PBS, and the reduced glutathione concentration in the elution buffer was raised to 40 mM. Puried protein was stored in PBS at 20 C. SDS-PAGE was performed according to the Invitrogen gel casting instructions, using Invitrogen equipment. Samples were boiled for 5 min at 95 C prior to gel electrophoresis. For non-reduced and TCEPHCl reduced samples, loading dye without reducing agent was used, and the boiling step was omitted. 2.4. Cantilever array functionalization with free thiol-containing antibodies The polyclonal nature of the GST antibody enables it to recognize more than one epitope on GST proteins. The free thiol-containing antibodies used for cantilever functionalization were obtained from the chemical reduction of antibodies by using the reducing agent, TCEP hydrochloride (tri(2-carboxyethyl)phosphine hydrochloride, TCEPHCl). TCEPHCl is a better alternative to dithiothreitol (DTT) or 2-mercaptoethanol (ME) as a reducing agent because it is more stable and does not contain free thiol groups that can bind directly to the gold surface of the cantilever. Being a phoshine, TCEPHCl can bind to gold, but the afnity is much lower than for the thiol group (Warner et al 2000). As a consequence of the afnity of thiols for metal, they are able to displace the organic material from the gold surface (Gooding 2004). The TCEPHCl was dissolved in 100 mM PBS buffer (100 mM sodium phosphate, 150 mM sodium chloride, pH 7.5), and the sensing anti-GST antibody was used as supplied by the producer (protein concentration of around 5 mg ml1 supplied in 150 mM NaCl containing 0.02% sodium azide). The concentration of the reference goat IgG antibody (11 mg ml1 ) in PBS buffer was adjusted to be equal to the sensing antibody concentration. To reduce the antibodies, samples were incubated for 4560 min at room temperature in the presence of a three times molar excess of TCEPHCl. The reduction level was conrmed by analysing a small aliquot of the reaction mixture by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions (gure 2). CantiChip4 was cleaned by UV-ozone treatment for 15 min using a UV-tip cleaner (Bioforce Laboratories) prior to functionalization. Separate cantilevers in the array were functionalized using a CantiSpot spotter capable of delivering 100 pl droplets. Each cantilever was usually functionalized with 1015 droplets. Free thiol-containing antibodies were chemisorbed on the gold substrate for 1 h at room temperature in a home-built humidity chamber (approximately 97% relative humidity (RH) was achieved with saturated K2 SO4 solution). The functionalized cantilever array was blocked overnight at +(48) C in 100 g ml1 (0.01%) BSA solution in PBS and rinsed in PBS buffer before mounting it in the CantiLab instrument. 2

2. Materials and methods

2.1. Equipment The equipment consists of the signal reading instrument CantiLab (Cantion-NanoNord A/S), the cantilever array chip CantiChip4 (Cantion-NanoNord A/S) and the inkjet-based functionalization unit CantiSpot (Cantion-NanoNord A/S). Each of the cantilevers in the CantiChip4 is 120 m long, 50 m wide and 480 nm thick (including a 30 nm gold layer). In addition, liquid ow was regulated by a syringe pump (Kent Scientic Corporation), and sample injection was performed using the 6-port valve (Vici) attached with a 100 l sample loop. Recently, we reported the use of this system for specic SNP (single nucleotide polymorphism) detection by DNADNA hybridization (Mukhopadhyay et al 2005a) and peptide-labelled cantilevers for estrogen detection (Mukhopadhyay et al 2005b). The setup has been described in detail (Mukhopadhyay et al 2005a). A 2.5 V bridge voltage was used in all the experiments. The cantilever array measurement cell (with a volume of 1 l) was connected to a micro ow channel, which was used to introduce the sample. The functionalized CantiChip4 was placed in the CantiLab that measures electric signals. The array was equilibrated under a constant ow (10 l min1 ) of running buffer until a steady baseline was obtained (less than 1 V drift in differential measurements during 100 s). The absolute signal and temperature were recorded constantly during the experiment. The software further controlled the liquidhandling system of the setup, i.e. the syringe pump and the two-position valve system, as well as data processing and celltemperature measurements. 2.2. Reagents All PBS buffer components (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.5) were purchased from Merck, BSA, reduced glutathione and goat IgG antibody from Sigma, acrylamide/bisacrylamide for SDS-PAGE from National Diagnostics, afnity puried goat anti-GST antibody and Glutathione Sepharose 4B from Amersham Biosciences, and TCEPHCl from Pierce. The GST protein was prepared from the pGEX-GTH expression vector (Jensen et al 1995).

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Differential

Time (s)

Time (s)

Figure 1. Specic detection of GST protein. Differential signals (panel (A) and (B)) and absolute signals (panel (C) and (D)) are plotted versus time. The arrows indicate the time of injection, and patterned rectangles indicate the time period when the cantilevers are in contact with the injected running buffer (panel (A)) or analyte (panels (B), (C) and (D)). Sensor cantilevers were labelled with GST, and reference cantilevers with goat IgG antibodies. Cantilevers were equilibrated in running buffers for at least 60 min (only the period of 200 s is shown) prior to the sample injection (100 l). Control injections of the running buffer (panel (A)) and a summary of different concentrations of GST protein injections (panel (B)) are shown. The light grey line in panel (B) indicates a differential signal as a result of the control protein (BSA) injection. Absolute deection signals from 40 nM GST (panel (C)) and 400 nM GST (panel (D)) are shown. The dark colours (black and dark grey) represent deection of sensor cantilevers, and the light colours (grey and light grey) represent deection of reference cantilevers.

3. Results and discussion

3.1. Cantilever array setup Using the piezoresistive cantilever biosensor, we can sense the static surface stress that arises when the analytes (the protein) interact specically with receptor molecules immobilized on the upper cantilever surface. The baseline from single microcantilevers usually drifts during the staticmode measurements, probably as a consequence of external conditions during the experiment (i.e. temperature variations, etc). To circumvent this potential problem, it is of key importance to perform simultaneous measurements on a reference cantilever. In the present studies the CantiChip4 cantilever array contains four cantilevers, of which two cantilevers were used as biosensor surfaces and the other two as reference sensors. By measuring the differential signal between the sensor and reference cantilevers, effects such as drifts are ideally eliminated. It should be noted that in the Cantion-NanoNord A/S technology, the in-built piezoresistor 3

is protected from the buffer solutions by encapsulating it in silicon nitride, which is a good diffusion barrier (Sze 1985). To block non-specic protein adsorption to the silicon nitride bottom side of the cantilever biosensor surface or to the empty spaces on the golden upper side of the cantilever, we pre-treated the cantilevers with a 100 g ml1 (0.01%) BSA solution in a running buffer (PBS). To discriminate between specic and non-specic interactions in the best possible manner, we used chemically similar molecules to functionalize the reference cantilever. For this purpose, reference goat immunoglobulin G was prepared exactly in the same manner as the sensor GST antibody. 3.2. GST detection The sensor cantilever was functionalized with GST antibodies and the reference cantilever with goat IgG antibodies and blocked with BSA solution in running buffer. After a stable baseline was obtained, several injections of buffer were performed to verify that there was no differential signal due to buffer injection (gure 1, panel (A)). GST protein was

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Figure 2. SDS-PAGE gel was used as a method to follow the integrity of antibodies used for cantilever functionalization. A Commassie-stained 10% gel shows GST (lanes 13) and goat IgG (lanes 46) antibodies, each at a concentration of 2 mg ml1 . Antibodies were either directly loaded onto a gel (non-reduced antibodies, lanes 2 and 5) or reduced with the SDS-PAGE gel loading dye (completely reduced antibodies, lanes 1 and 4). Antibodies used for cantilever functionalization (lanes 3 and 6) have been treated according to the protocol given in section 2 (treated with TCEPHCl in PBS buffer, pH 7.5 for 45 min at room temperature) and compared to the samples described above. Lane 1: GST antibodies (completely reduced); lane 2: GST antibodies (non-reduced); lane 3: GST antibodies (TCEPHCl reduced); lane 4: goat IgG (completely reduced); lane 5: goat IgG (non-reduced); lane 6: goat IgG (TCEPHCl reduced); and lane 7: molecular weight marker.

= A Voutput / Vinput , A = 3125 N m (Cantion-NanoNord A/S, instrument handbook)), E is Youngs modulus, is Poissons ratio, and Voutput and Vinput is the output voltage and the bridge voltage over the wheatstone bridge, respectively. For estimates of Youngs modulus of 220 GPa and Poissons ratio of 0.25, a value of z = 0.8 nm V1 is obtained for the bridge voltage of 2.5 V that was used. Experiments were repeated reproducibly at least ve times. It should be mentioned that the resolution of the sensing element in our equipment is limited by the noise level, which, in terms of output voltage, is approximately 1 V (as read off from the graphs). Thus, we will not be able to detect signals below 1 V. After the GST has passed the ow cell, cantilevers were again exposed to the buffer. Finally, as a control mechanism, the BSA protein was injected at a concentration corresponding to the highest GST concentration used in the experiment. No signicant signal was obtained, indicating that the signal is GST specic. In the GST protein preparation process, the protein was eluted from the Glutathione Sepharose 4B using reduced glutathione that contains free thiol groups. This concentration is subsequently diluted >1000 times by dialysis. To investigate whether any remaining reduced glutathione from the GST protein dialysis reaction causes a signal to rise, the last dialysis buffer was diluted in the same manner as the GST protein and used for injection. Indeed, no signal was observed (results not shown), suggesting that the GST protein preparation is free from reduced glutathione, and the specic signal is caused by the GST protein.

4. Conclusions
We have shown that mildly reduced antibodies (see section 2) directly immobilized on the gold cantilever surface can act as capture molecules for protein detection. This nding offers an alternative route for cantilever surface functionalization with antibodies, in comparison to the more commonly used immobilization by means of thiol-cleavable cross-linking molecules, or on a monolayer of self-assembled thiols (Raiteri et al 2001). Our strategy has two advantages. First, a step is omitted in the functionalization protocol compared to previous reports and, second, the capture molecules are brought closer to the cantilever surface, which will probably lead to more sensitive stress measurements. In accordance with this, we nd the sensitivity achieved for GST protein to be in the 10 nM range. This result is signicantly better than the values reported for alternative optical systems using randomly oriented antibodies, in which the sensitivity was found to be 1 M of myoglobin (Arntz et al 2003). It is important to note that both assays are controlled in the same way, including similar reference cantilevers (functionalized with control antibodies) and simultaneous measurements with control protein injection. However, since different functionalization protocols were used, it is not possible to compare the different cantilever technologies directly. The antibodies applied in this study belong to an immunoglobulin family of proteins which are composed of polypeptide chains linked by disulde bonds. Preserving the intact structure of the immunoglobulin molecule is crucial to the epitope binding abilities. We have used very mild 4

diluted in the running buffer at increasing concentrations (4, 40 and 400 nM, corresponding to 0.1; 1 and 10 g ml1 ) and equilibrated to room temperature before injection. As the injection loop volume is 100 l, receptor molecules, immobilized on the cantilever surface, were exposed to the analyte for 600 s under constant ow conditions (10 l min1 ). These conditions are in contrast to previous cantilever assay studies, in which the analyte bound under static conditions (Arntz et al 2003, Backmann et al 2005). Due to the instrument setup, an approximately 400 s delay after the injection is detected before the injected protein reaches the ow cell of CantiChip4 (indicated with an arrow in gure 1). Figure 1, panel (B) summarizes the differential signals obtained. It is observed that 40 nM of GST was detected with an average signal of 45 V, and 400 nM was detected with an average signal of 12 V, whereas 4 nM did not yield a signicant signal. A positive signal corresponds to downward bending of the cantilever, which implies compressive stress. The relationship between the endpoint deection (z) and the obtained signal (Voutput ) is derived in the appendix and given by the formula z = 3 (1 ) (l / h )2 A / E Voutput / Vinput , where l is the length of the cantilever (120 m), h is thickness of the cantilever (480 nm), A is the proportionality factor in the surface stressvoltage equation (surface stress

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reducing conditions to ensure that internal disulde bonds in the antibody are not signicantly reduced. The direct immobilization of the antibody via SH groups will bring the antibody closer to the cantilever surface, which may result in increased surface stress following analyte binding. However, the random immobilization strategy may lead to inactivation of binding for some of the antibody molecules. The obvious improvement of the experimental setup would therefore be to attach the antibodies to the cantilever surface in the specically oriented manner. At least a 500-fold improvement in the sensitivity was reported when using directed oriented IgG molecules (Backmann et al 2005). Protein detection using the electric read-out offers following advantages compared to the optical system: the potential of miniaturization and integration of the sensor in lab-on-a chip systems, elimination of the need for expensive optical components and time-consuming laser alignment, avoidance of detection artifacts due to changes in optical properties close to the cantilever surface, and possibility for detection in opaque solutions. In this setup, the protein detection is fast, real-time, label-free and suited for highthroughput upon parallelization, and because of it, such biosensors could nd a wide range of applications in a diverse number of important elds, including drug discovery, environmental pathogen detection and homeland security.

References
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Acknowledgments
This work was supported by grant no 2055-03-0004 from the Danish Research Agency to the iNANO Center at the University of Aarhus.

Appendix
For deriving the relationship between deection z and output voltage, surface stress given by the equation (CantionNanoNord A/S, instrument handbook)

=A

Voutput Vinput

(A.1)

is inserted into the Stoney equation (Stoney 1909), here with Poissons ratio taken into account, relating surface stress and radius of curvature R

Eh 2 6(1 ) R

(A.2)

and combined with the equation relating deection z and radius of curvature R for a beam of length l :

z=
The deection is then given as

l2 . 2R

(A.3)

z=

3(1 ) 2 Voutput l A . Eh 2 Vinput

(A.4)