Investigations on antibody binding to a micro-cantilever coated with a BAM pesticide residue
© Bache et al; licensee Springer. 2011
Received: 21 October 2010
Accepted: 16 May 2011
Published: 16 May 2011
The attachment of an antibody to an antigen-coated cantilever has been investigated by repeated experiments, using a cantilever-based detection system by Cantion A/S. The stress induced by the binding of a pesticide residue BAM (2,6 dichlorobenzamide) immobilized on a cantilever surface to anti-BAM antibody is measured using the CantiLab4© system from Cantion A/S with four gold-coated cantilevers and piezo resistive readout. The detection mechanism is in principle label-free, but fluorescent-marked antibodies have been used to subsequently verify the binding on the cantilever surface. The bending and increase in mass of each cantilever has also been investigated using a light interferometer and a Doppler Vibrometer. The system has been analyzed during repeated measurements to investigate whether the CantiLab4© system is a suited platform for a pesticide assay system.
During the last 10 years an increasing number of water wells have been polluted by pesticides or its break down products. BAM is among the most frequent found pesticide residues in European groundwater. As pesticide analysis of drinking water is currently being done by laboratory analysis, an in-line sensor will therefore be beneficial for water quality monitoring. Cantilever-based assays for pesticide detection has been reported [1, 2], but few description of repeated measurements using cantilever-based detection systems are available. As a central principle of a possible cantilever-based competitive assay, we have tested the binding of a BAM antibody to a cantilever surface passive coated with a BAM ovalbumine conjugate. In a working assay, the BAM molecules in a water sample would compete with BAM attached to a cantilever surface for the binding to anti-BAM monoclonal antibodies, similar to a BAM ELISA described by Bruun et al . The binding of anti-BAM antibodies to the surface of the cantilever will change the surface stress, causing bending of the cantilever. The bending is then detected by a change in resistance of the imbedded piezoelectric layer in the cantilever [4–6]. To investigate whether the system is suited as a transducer for a pesticide bio-assay, the variance of the cantilever bending signal during 10 antibody binding experiments was analyzed. The mechanical properties of the cantilevers were also monitored by measuring the cantilever bending profile, cantilever mass/stiffness, and antibody fluorescent signal. This was repeated on the clean cantilevers, after the cantilevers were functionalization with antigens, and after the antibody was added.
Materials and methods
A cantilever system CantiChip4® from NanoNord/Cantion A/S was chosen for the assay. The bending of the cantilever causes a proportional change in voltage between the piezo layer in the cantilever and a fixed resistor embedded in the chip measured via a Wheatstone bridge setup. The system consists of four silicon-based cantilevers with integrated piezo resistive readout. All four cantilevers are 120 μm length × 50 μm width × 0.45 μm thickness, coated with a 40-nm gold layer, electrically grounded, and flip chip bonded to a contact pad. The CantiChip4® is inserted in the CantiLab4© that converts the voltage signal to proprietary recording software . The functionalization of each cantilever was done using a micro-spotter from Cantion A/S with a piezo electric controlled pin head (GESIM Sub-Micro liter Piezoelectric Dispenser A010-006 SPIP) in a xyz stage setup monitored via a camera and a PC interface. A 2,6 dichlorobenzamide hapten (BAM hapten EQ0031) and ovalbumine conjugate was synthesized following Bruun et al . The BAM ovalbumine conjugate was dialyzed 3× in 1× PBS buffer, and diluted to 0.75 mg/ml of ovalbumine in 1× PBS. The BAM-ovalbumine conjugate was determined to contain 5 U BAM/ovalbumine via a UV-Visual spectrophotometer method and was tested positive for BAM via an ELISA .
In order to verify the binding of antibodies to the cantilever surface and control for unspecific binding, a set of fluorescent pictures of Cy5 and Cy3 signal were taken after spotting and antibody attachment. An optical surface profilometer (Polytech TMS-100), based on light interference, was used to analyze the absolute bending of the cantilevers on five experiments. To analyze the mass/stiffness values, a laser-based vibrometer with a piezo actuator (Doppler Vibrometer Polytech MSA 500) was used on eight experiments . All chemicals used in the assay were purchased via Sigma Aldrich Denmark; only new glassware was used and rinsed in Milli-Q water to avoid any unwanted effect from surfactants.
Results and discussion
Although three chips were discarded during the 20 experiments, the Cantion chips were able to perform a continuous voltage readout lasting several days. The Cantion chips could also be re-used following a rinsing protocol. This opens up the possibility of regeneration of the surface chemistry by repeated assays, using only one sensor in an automated system. However, the system was not found suitable as a platform for a pesticide bio-assay in its current form, as the quality of the differential signal was not repeatable. The fluorescent pictures of anti-BAM showed repeated attachment only to the BAM functionalized cantilever surfaces, and no binding of unspecific Cy5 marked antibody. The signal variation is therefore unlikely to be caused only by variations in the cantilever functionalization step. The variations are more likely caused by minute changes in buffer pH, temperature, and salinity, as this affects the electromagnetic field surrounding the cantilever (caused by the 2.5 V tension in the cantilever piezo layer). The very large antibody concentrations needed to obtain a differential signal on the system is believed to be the cause of the signal from the unspecific antibody as it interacted with the cantilever surface, but this could not be proved in the experiments.
This study is financed by the Danish consortium SENSOWAQ in collaboration with GEUS (Grant no. 2104-06-0006 from the Danish Council for Strategic Research).
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