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Investigations on antibody binding to a micro-cantilever coated with a BAM pesticide residue.

Bache M, Taboryski R, Schmid S, Aamand J, Jakobsen MH - Nanoscale Res Lett (2011)

Bottom Line: 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 system has been analyzed during repeated measurements to investigate whether the CantiLab4© system is a suited platform for a pesticide assay system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345 East, 2800 Kongens Lyngby, Denmark. Michael.bache@nanotech.dtu.dk.

ABSTRACT
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.

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Fluorescent antibody signal. (Left) An example of a clear fluorescent signal (ex. 550 nm, em. 570 nm) of Cy3 marked anti-BAM to cantilever B and C and none on A and D; (Right) Fluorescent picture of Cy5 signal (ex. 650 nm, em. 670 nm) from chip 113, showing a low background signal of Cy5 marked unspecific mouse antibody .
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Figure 4: Fluorescent antibody signal. (Left) An example of a clear fluorescent signal (ex. 550 nm, em. 570 nm) of Cy3 marked anti-BAM to cantilever B and C and none on A and D; (Right) Fluorescent picture of Cy5 signal (ex. 650 nm, em. 670 nm) from chip 113, showing a low background signal of Cy5 marked unspecific mouse antibody .

Mentions: Although the functionalization of the cantilevers (using micro-spotting and passive adsorption to the gold layer) is the source of some of the variations obtained in the differential values, it cannot fully explain the large variations of the signal. As observed by Dauksaite et al. [5], almost all experiments had a signal drift effect during the experiment varying from a few μV to several mV/h, possibly caused by a variance in the resistance of the internal resistors of the Wheatstone bridge on the chip. Another observation was a battery effect in the fluidic system causing a 0.1-1 mV signal change. To avoid this battery effect between the chip and fluidic system, the waste bottle was connected to the CantiLab4© electronic box with a gold wire (Figure 1). A weak loop switch effect of 0.01-0.004 mV was also observed when switching the fluidic loop, this was probably caused by minute changes of pH or salinity in the sample buffer resting in the sample coil. The effect immediately disappeared after one loop switch and was avoided using a continuous buffer flow. As the antibody-antigen binding is mainly controlled by electrostatic forces [9] and the bending signal is found to be very sensitive to minute changes in pH, salinity, and temperature gradients [10], we wanted to investigate whether the unspecific antibody signal was just caused by the relatively high antibody concentration (0.1 mg/ml). 2 experiments with 1/10 (0.01 mg/ml) antibody were done, but unfortunately these showed no signal when the antibody was added (data not shown). The BAM antibody was marked with Cy3 fluorochrome and the unspecific mouse IgG antibody with Cy5 to assure that the BAM antibody was attached to cantilevers B and C and no unspecific antibody was attached. All 10 experiments had a similar Cy3 signal from BAM antibody on cantilever B and C and little or no Cy3 signal on cantilever A and D. No significant amount of Cy5 marked unspecific antibody signal was found on any cantilever after the experiment. A typical Cy3 and Cy5 fluorescent signal is seen in Figure 4.


Investigations on antibody binding to a micro-cantilever coated with a BAM pesticide residue.

Bache M, Taboryski R, Schmid S, Aamand J, Jakobsen MH - Nanoscale Res Lett (2011)

Fluorescent antibody signal. (Left) An example of a clear fluorescent signal (ex. 550 nm, em. 570 nm) of Cy3 marked anti-BAM to cantilever B and C and none on A and D; (Right) Fluorescent picture of Cy5 signal (ex. 650 nm, em. 670 nm) from chip 113, showing a low background signal of Cy5 marked unspecific mouse antibody .
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3211479&req=5

Figure 4: Fluorescent antibody signal. (Left) An example of a clear fluorescent signal (ex. 550 nm, em. 570 nm) of Cy3 marked anti-BAM to cantilever B and C and none on A and D; (Right) Fluorescent picture of Cy5 signal (ex. 650 nm, em. 670 nm) from chip 113, showing a low background signal of Cy5 marked unspecific mouse antibody .
Mentions: Although the functionalization of the cantilevers (using micro-spotting and passive adsorption to the gold layer) is the source of some of the variations obtained in the differential values, it cannot fully explain the large variations of the signal. As observed by Dauksaite et al. [5], almost all experiments had a signal drift effect during the experiment varying from a few μV to several mV/h, possibly caused by a variance in the resistance of the internal resistors of the Wheatstone bridge on the chip. Another observation was a battery effect in the fluidic system causing a 0.1-1 mV signal change. To avoid this battery effect between the chip and fluidic system, the waste bottle was connected to the CantiLab4© electronic box with a gold wire (Figure 1). A weak loop switch effect of 0.01-0.004 mV was also observed when switching the fluidic loop, this was probably caused by minute changes of pH or salinity in the sample buffer resting in the sample coil. The effect immediately disappeared after one loop switch and was avoided using a continuous buffer flow. As the antibody-antigen binding is mainly controlled by electrostatic forces [9] and the bending signal is found to be very sensitive to minute changes in pH, salinity, and temperature gradients [10], we wanted to investigate whether the unspecific antibody signal was just caused by the relatively high antibody concentration (0.1 mg/ml). 2 experiments with 1/10 (0.01 mg/ml) antibody were done, but unfortunately these showed no signal when the antibody was added (data not shown). The BAM antibody was marked with Cy3 fluorochrome and the unspecific mouse IgG antibody with Cy5 to assure that the BAM antibody was attached to cantilevers B and C and no unspecific antibody was attached. All 10 experiments had a similar Cy3 signal from BAM antibody on cantilever B and C and little or no Cy3 signal on cantilever A and D. No significant amount of Cy5 marked unspecific antibody signal was found on any cantilever after the experiment. A typical Cy3 and Cy5 fluorescent signal is seen in Figure 4.

Bottom Line: 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 system has been analyzed during repeated measurements to investigate whether the CantiLab4© system is a suited platform for a pesticide assay system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345 East, 2800 Kongens Lyngby, Denmark. Michael.bache@nanotech.dtu.dk.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus