Atomic Force Microscopy becomes an invaluable technique for the study of biological systems at a nanometric scale [1, 2]. During the past decade, it has been increasingly used to image and manipulate biomolecules and cell surfaces in air or in situ. Development of dynamic modes of operation adapted for the imaging of soft samples has significantly contributed to overcome the usual issue of damage done to these biological objects by the scanning tip .
Applications To Biology
Most of the experiments concern the observation of adsorbed biological molecules, DNA or cells to study their individual conformation or their assembly in molecular layers. DNA is the most commonly studied molecule and numerous papers relate experiments concerning its adsorption on various substrates observed in different media . However, few articles are concerned with AFM studies on real biosystems with direct applications such as DNA microarrays . Deoxyribonucleic acid (DNA) chips are major tools in the biomedical field for DNA diagnosis, gene monitoring and genome sequencing . These are structured in an array of single-stranded DNA chains called probes, covalently bonded on a substrate, which are able to recognize complementary single-stranded DNA chains called targets, by hybridization. In fact, the sensitivity of these objects depends on the molecular organization of the immobilized probes on the substrate. In most cases, sensitivity is evaluated after biological recognition by measuring the amount of hybridized targets with fluorescent spectroscopy or radioactivity analysis. Then, it appears necessary to characterize the chip at a molecular scale before hybridization. The present work reports an AFM characterization of a DNA chip preparation from the probe immobilization to hybridization.
We consider a model chip presenting two separate areas composed of two different 25-mer oligonucleotide probes. The first area is made up of an oligonucleotide presenting a sequence which perfectly matches one part of the selected target. For this reason, it is called 25-C (C for complementary). The second area is formed with a sequence which is totally different to the 25-C sequence.
Since no hybridization can occur with the target, it is called 25-NC (NC for non-complementary). The target is a long strand of 1500 base-paired DNA with 25 bases matching the 25-C probe [7, 8].
Before And After Hybridization
AFM is well adapted to characterize the chip before and after hybridization at a molecular scale. Probe molecules are observed as granular structures of 1 - 1.5 nm in height (the top image in fig. 1) oblong in shape with lateral dimensions broadened by the dilatation tip effect. After hybridization, individual target molecules lying on the substrate can be identified by AFM on the chip even with the probe topography visible on the substrate (the bottom image in fig. 1). Moreover, a precise measurement of the height along the fragment lets us recognize the hybridized region along the target molecule. All target molecules globally present a height of 1 nm, but also reach 2 nm on a small region corresponding to a double strand height and covering the measured 25-C probe length (cross sections in fig. 1) .
A Sequence Dependent Organisation
Larger scale images give us interesting information concerning the molecular organization of the ss-DNA on the surface . On this chip, two different morphologies are reported depending on the area. An homogeneous distribution of ss-DNA is observed on the 25C region (fig. 2a), whereas a lacy structure appears on the 25NC area with numerous holes, typically of 100 nm in diameter (fig. 2b). This particular molecular organization is induced by different physico-chemical properties probably related to a sequence effect. It was shown that the spreading of droplets containing the probes, and subsequently the repartition of probe molecules on the surface, strongly depend on the nature of the sequences .
Special AFM Experimental Conditions
On this biological system, contact mode and hard tapping are not appropriate and only soft tapping can be used. AFM conditions under an attractive regime is deliberately chosen, thus limiting direct contact with the surface. In our experiment, we can distinguish between a single strand and a double strand DNA molecule by measuring a doubling of the height (1 to 2 nm). It is not so obvious because the great interaction between the tip and a soft surface induces some flattening of the biological molecule. In our system, minor interaction changes occur when scanning from probes to targets because DNA targets lie on a DNA "carpet", unlike other studies where DNA fragments lie on different stiff substrates.
The paper describes the study of a DNA-chip by AFM at a molecular scale. With appropriate experimental conditions and depending on the interaction between DNA targets and the chip, AFM allows probes and targets to be identified and the hybridized region along the target to be recognized. We observe a different probe organization depending on the sequence which could influence the following hybridation step and then the selectivity of the chip. This demonstrates that AFM is an appropriate technique to follow the molecular organization of probes and targets not only on DNA chip but also on any bio-chip.
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