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Comprehensive characterization of molecular interactions based on nanomechanics.

Ghatkesar MK, Lang HP, Gerber C, Hegner M, Braun T - PLoS ONE (2008)

Bottom Line: Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro.This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology.The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions.

View Article: PubMed Central - PubMed

Affiliation: National Center of Competence for Research in Nanoscience, Institute of Physics, University of Basel, Basel, Switzerland.

ABSTRACT
Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions.

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Molecular model of the nano-mechanical changes on the sensor interface explaining the data.Without vesicles the pre-functionalized cantilever (AUT SAM on gold) is straight (a). Adsorption of vesicles on the cantilever surface bends the cantilever upwards driven by the interaction forces between the cantilever and the vesicles, which are flattened by this interaction (b). During the peptide injection, the melittin molecules first bind to the vesicle surface (c), and later insert into the membrane and form channels by oligomerization (d).
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pone-0003610-g003: Molecular model of the nano-mechanical changes on the sensor interface explaining the data.Without vesicles the pre-functionalized cantilever (AUT SAM on gold) is straight (a). Adsorption of vesicles on the cantilever surface bends the cantilever upwards driven by the interaction forces between the cantilever and the vesicles, which are flattened by this interaction (b). During the peptide injection, the melittin molecules first bind to the vesicle surface (c), and later insert into the membrane and form channels by oligomerization (d).

Mentions: Figure 2 shows the simultaneously measured mass adsorption and surface stress for three vesicles (500 ng/ml lipid) and two melittin (1 µM) solution injections (sections I to XI). The differential signal is shown obtained by the subtraction of the average of the negative controls from the average of the positive controls. Table 1 lists the mass and deflection changes during injection of lipid or melittin solutions. We used lipid and buffer conditions known to procure the membrane insertion and channel formation as reported previously [17]. After recording a baseline (section I), vesicles were injected with a concentration of 500 ng/ml (Fig. 3, section II). A mass increase of 6.4±0.06 ng (standard error, Table 1) is observed. The surface stress difference between the AUT functionalized top-side and the casein passivated silicon bottom side of the cantilever leads to an upward bending (towards the AUT) of the cantilever by 185±1.2 nm during vesicle adsorption. After vesicle injection buffer was flushed through the measurement chamber again (section III) before melittin (1 µM) was injected once (section IV) resulting in a mass increase of around 3.3±0.06 ng. Simultaneously the cantilever bent down by 14.6±1.1 nm. These surface stress changes are in close agreement with previously reported static mode measurements [9]. The injection sequence was complemented with two additional vesicle exposures (sections VI and VIII) and a final melittin dose (X) exhibiting the same qualitative mass and deflection changes. Every vesicle and melittin injection was terminated by a buffer wash. This procedure removes not only weakly bound molecules but also ensures that the signal is not due to the liquid rheology [21]. A summary of the mass and deflection changes is given in Table 1 and more details are available in the supplemental Table S1.


Comprehensive characterization of molecular interactions based on nanomechanics.

Ghatkesar MK, Lang HP, Gerber C, Hegner M, Braun T - PLoS ONE (2008)

Molecular model of the nano-mechanical changes on the sensor interface explaining the data.Without vesicles the pre-functionalized cantilever (AUT SAM on gold) is straight (a). Adsorption of vesicles on the cantilever surface bends the cantilever upwards driven by the interaction forces between the cantilever and the vesicles, which are flattened by this interaction (b). During the peptide injection, the melittin molecules first bind to the vesicle surface (c), and later insert into the membrane and form channels by oligomerization (d).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572191&req=5

pone-0003610-g003: Molecular model of the nano-mechanical changes on the sensor interface explaining the data.Without vesicles the pre-functionalized cantilever (AUT SAM on gold) is straight (a). Adsorption of vesicles on the cantilever surface bends the cantilever upwards driven by the interaction forces between the cantilever and the vesicles, which are flattened by this interaction (b). During the peptide injection, the melittin molecules first bind to the vesicle surface (c), and later insert into the membrane and form channels by oligomerization (d).
Mentions: Figure 2 shows the simultaneously measured mass adsorption and surface stress for three vesicles (500 ng/ml lipid) and two melittin (1 µM) solution injections (sections I to XI). The differential signal is shown obtained by the subtraction of the average of the negative controls from the average of the positive controls. Table 1 lists the mass and deflection changes during injection of lipid or melittin solutions. We used lipid and buffer conditions known to procure the membrane insertion and channel formation as reported previously [17]. After recording a baseline (section I), vesicles were injected with a concentration of 500 ng/ml (Fig. 3, section II). A mass increase of 6.4±0.06 ng (standard error, Table 1) is observed. The surface stress difference between the AUT functionalized top-side and the casein passivated silicon bottom side of the cantilever leads to an upward bending (towards the AUT) of the cantilever by 185±1.2 nm during vesicle adsorption. After vesicle injection buffer was flushed through the measurement chamber again (section III) before melittin (1 µM) was injected once (section IV) resulting in a mass increase of around 3.3±0.06 ng. Simultaneously the cantilever bent down by 14.6±1.1 nm. These surface stress changes are in close agreement with previously reported static mode measurements [9]. The injection sequence was complemented with two additional vesicle exposures (sections VI and VIII) and a final melittin dose (X) exhibiting the same qualitative mass and deflection changes. Every vesicle and melittin injection was terminated by a buffer wash. This procedure removes not only weakly bound molecules but also ensures that the signal is not due to the liquid rheology [21]. A summary of the mass and deflection changes is given in Table 1 and more details are available in the supplemental Table S1.

Bottom Line: Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro.This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology.The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions.

View Article: PubMed Central - PubMed

Affiliation: National Center of Competence for Research in Nanoscience, Institute of Physics, University of Basel, Basel, Switzerland.

ABSTRACT
Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions.

Show MeSH
Related in: MedlinePlus