To that end, the averaged ellipsometric angles ∆(λ) were fitted with an optical 3-layer model that yielded the SAM thicknesses of Au(111)/C9t, Au(111)/PEG-C9t, and Au(111)/(PEG-C9t)+ S protein. The average SAM thickness (d) measured on Au(111)/C9t is 2.9 nm (figure ). Samples of Au(111)/C9t feature a relatively high sample to sample variation in d, implying that it is difficult to control this parameter during the fabrication. This variation together with a relatively strong variation of the substrate's optical properties used for these measurements leads to a high relative error in the thickness determination (figure and Supporting Discussion 2). Nevertheless, the average thickness is low compared to the molecular length of C9t. As a fully extended chain, the contour length of C9t would be about 27 nm. A more realistic worm-like chain model of ss-DNA (of comparable number of nucleotides) tethered to a conducting surface predicts an end-to-end distance in the range of 10 nm to 15 nm. This estimation implies that ss-DNA molecules must be significantly tilted towards angles clearly above 70 • in Au(111)/C9t. This result is in common with the measured surface density (after blocking with MCH) that was determined to be about 0.013 molecules/nm 2 , 13 although before blocking this value might be up to an order of magnitude higher. Hence, the ss-DNA chains can tilt towards the surface and occupy the unblocked surface area, resulting in a relatively flat, but as shown by AFM, homogenous layer. The second fabrication step, i.e. the incubation with PEG-thiol, significantly altered the topography of the sensor interface. The AFM height image of Au(111) sc /PEG-C9t exhibits nano-domains and a further increased RMS roughness of 0.22 nm (figure ). The nanodomains show typical height variations of approximately 1.0 nm to 1.5 nm as illustrated in the line profile (figure ). The lateral size of the elevated domains was about 50 nm. The average SAM thickness was found to increase upon PEG incubation to approx. 6.3 nm on Au(111) sc /PEG-C9t (figure ), also the range of variation in d was reduced. It is noted that the absolute thickness increase may be slightly smaller considering that the true value and change of the film refractive index n (assumed here to be n = 1.43) cannot be known exactly in the studied system and that film thickness d and refractive index n are strongly correlated here (cf. Supporting Discussion 2). Due to this correlation, the SE data might also be interpreted in terms of an increase in n due to a densification of the layer coinciding with the increase in d, as for instance observed upon blocking of ss-DNA film with short alkanethiols. Assuming a strong increase of n from 1.36 to 1.5 upon PEG blocking would result in a lower thickening from 3.3 nm on Au(111)/C9t to 5.7 nm on Au(111)/PEG-C9t, which is still significant. Hence, the ellipsometric data clearly indicates that the blocking step induced a significant rise of the surface attached molecules while AFM indicates separation into phases with different heights. The average layer thickness still is considerably below the estimated end-to-end lengths of both C9t (10 nm to 15 nm) and crystalline, i.e. helical PEG2000 (approx. 12 nm). Hence, although the observed thickness increase is interpreted in terms of an uprising of the ss-DNA chains, the ss-DNA molecules are most likely still in a coil conformational state on the sensor interface. The difference of the layer thickness with respect to the expected length of helical PEG might be caused by either a significant tilt of the PEG chains or an amorphous conformation. Only, spectroscopic information can give clarity on this issue. IRRA spectra recorded after each preparation step show peaks as expected for the molecules immobilized on the sensor surface (figure ). Peak variations sample to sample peak ratio variations were observed which might be due to slight sample to sample variations in fabrication (figure steps I and II) or variations in the substrate reflectivities (cf. Supporting Discussion 2). The most prominent features confirming the presence of aptamer on the surface are two bands with maxima around 1080 cm -1 and 1260 cm -1 , which appear in the spectra of all samples and are assigned to symmetric and asymmetric stretching vibrations (ν s , ν as ) of (PO 2 ) -of the DNA backbone (table 1, figure ). The IR spectra of macromolecules such as DNA are featured by broad and complex bands that may be used as indicators for specific molecular properties. For instance, in transmission IR spectroscopy it was found that the hydration of DNA influences the shape of 1080 cm -1 and its shoulder at 1050 cm -1 assigned to the ν(C-O) of the sugar backbone. On the other hand the ν as (PO 2 ) -band position is known to be sensitive to both DNA conformation and hydration. Next to these molecular properties band intensities in IRRA spectra are very sensitive to molecular orientation and order of the examined moieties due to the so-called selection rules on metal interfaces. Additionally, the IR spectra of the molecules used here significantly overlap. Hence, complex variations in the IR spectra are observed with each fabrication step, which makes it difficult to assign unequivocally to specific effects. After PEG blocking on Au(111)/PEG-C9t the ethyleneglycol C-O-C mode expected in the 1110 cm -1 to 1140 cm -1 region, overlaps with the asymmetric ν as (PO 2 ) -and hence cannot be clearly identified. An enhanced intensity and broadening of the ν as (PO 2 ) -at 1265 cm -1 might imply significant reorientation of C9t after the blocking step. A series of broad bands from 1320 cm -1 to 1800 cm -1 appear on Au(111)/C9t with lower intensity and are assigned to the ring vibrations of the C9t nucleobases (thymin, guanine, adenine, cytosine) as well as ν s (-C --O and -C --N).