# Dual aptamer-Functionalized 3D Plasmonic Metamolecule for Thrombin Sensing
## 3 Results  
For the detection scheme, we chose plasmonic circular dichroism (CD) [31]. In CD measurements, the difference in absorption for right-handed and left-handed polarized light is often used to detect 3D structure and conformational changes of biomolecules like proteins [32] and DNA [33; 34]. Biomolecules exhibit CD in the ultraviolet region of the spectrum and usually none in the visible range. Recently developed plasmonic metamolecules [25] in contrast, generate CD at visible wavelengths, which enables detection of plasmonic CD and conformational changes of the metamolecules in an undisturbed optical region. This concept of high-sensitivity detection of RNA molecules [35] and detection of small molecules like cocaine and ATP [36] have been exploited by our research group and by other authors, in recent years. Here, we extended this approach for the detection of proteins. In our experiments, two thrombin-binding aptamers each targeting two different epitopes of human \(\alpha\)-thrombin were placed on a reconfigurable 3D plasmonic metamolecule [25] such that upon binding of the target molecule, the metamolecule switches its chiral state leading to a change in the circular dichroism (CD) signal. Our X-shaped 3D plasmonic metamolecule is based on two 10-helix DNA bundles connected in the center by two single-stranded DNA linkers (Figure 1a and Figure S1) [25]. Both bundles, or arms, carry a gold nanorod (AuNR) at the top and bottom, respectively. After assembly, the structure is initially in its \"open\" state in which its arms can freely rotate around their center (Figure 1b, open state). Two thrombin aptamers--thrombin-binding aptamer (TBA) [2] and HD22 [37] were attached at one end of each arm so that the entire structure can switch into the 'locked' state by sandwiching one thrombin molecule (Figure 1b, locked state). By design, this state is right-handed and thus exhibits a recognizable CD signal at the plasmon resonance of the metamolecule. The aspect ratio of the AuNR (\(20\times 60\) nm) was chosen to exhibit a maximum response around 700 nm. Purified and negative-stained DNA structures conjugated with two AuNRs were imaged with transmission electron microscopy (TEM) (Figure 1a, inset and Figure S2). CD spectra of the metamolecules at a concentration of 100 pM and in the absence of human \(\alpha\)-thrombin were measured first (Figure 2a, black line). These spectra show a characteristic peak-dip \"bisignate\" shape with the first peak at 705 nm (Figure 2a). This implies that the metamolecules in their relaxed (open) state are on average slightly left-handed. We attribute this to the additional electrostatic and steric repulsion originating from the two aptamers at two ends of the arms. Indeed, if the aptamers are attached onto the mirrored positions, the CD signal of the relaxed structures is right-handed (Figure S3). When thrombin was added to the solution (10 nM) the CD signal switched to a dip-peak shape of  
Figure 1: Scheme for human \(\alpha\)-thrombin sensing with the plasmonic metamolecule. (**a**) The structure consists of two 10-helix bundles each carrying two a gold nanorods (AuNRs) at the top and bottom respectively. The arms are connected by a single-stranded DNA linker (not shown) and are modified at one of their ends with one thrombin aptamer each—thrombin-binding aptamer (TBA) and HD22. Inset: TEM image of an assembled structure lying on its side (Scale bar: 50 nm). (**b**) In the absence of thrombin, the structure is in its ‘open’ state. In the presence of thrombin, the two aptamers can sandwich one target molecule to fix the structure in its ‘locked’ state with defined right-handed chirality. higher intensity, with the first dip at 693 nm (Figure 2a). Now, the metamolecules are in their locked right-handed state with thrombin molecules sandwiched by the two aptamers. After confirming that the presence of thrombin switches the CD signal of our metamolecules, we measured the gradual transition of the CD signal by ramping up the protein concentration from 10 pM to 1 \(\upmu\)M (Figure 2).