# Dual aptamer-Functionalized 3D Plasmonic Metamolecule for Thrombin Sensing
## 4 Discussion  
We demonstrated that our plasmonic metamolecule reliably detected human alpha thrombin in solution at concentrations of 100 pM and above. This detection sensitivity was higher than several conventional detection methods, including DNAzyme-based colometry, carbon nanotube sensing, and capillary chromatography which operated reliably at nanomolar concentrations and above [39; 40; 41]. The sensitivity was also similar to that of previously reported high sensitivity methods, based on  
Figure 2: Detection of thrombin with plasmonic circular dichroism (CD) from the metamolecules. (**a**) CD spectrum of 100 pM metamolecule in the presence of different concentrations of thrombin. As the concentration of thrombin increases, the CD spectrum switches from right-handed (black line) to left-handed (red line). At thrombin concentrations above 100 nM (1000 nM, red arrow), the signal strength decreases as the thrombin molecules bind to each arm of the metamolecule rather than being sandwiched between two arms. To reduce the noise, all signals were measured with 10 nm spectral windows and the difference between two frequency windows (black and grey arrows) was calculated. (**b**) Normalized response-concentration data (black circles) with Hill equation fit (red line). The Hill coefficient obtained through the fit was 1.3 \(\pm\) 0.3 and K\({}_{\text{d}}\) = 1.4 nM. The normalized response values were obtained by subtracting the values at the extremes (black and grey arrows of Figure 2a) and normalizing it by the value at 10 nM.  
colorimetric assays [42], FRET assays [43], rolling circle amplification-based detection [44], or to commercially available ELISA kits. The high sensitivity in our experiments could be attributed to the strong plasmonic CD of our detection device that additionally operated in a frequency window with no naturally occurring (background) CD and to the high avidity working principle leading to cooperative binding. Our high sensitivity detection worked all the same when the position of the aptamers on the DNA origami structure was changed (Figure S3), such that the opposite CD response was generated. This proved the robustness of our method. In this context, there was room for improvement of the sensitivity, for example, by placing multiple copies of the thrombin aptamers on each arm of the structure. Additionally, there is a possibility for our structure to work in physiological buffer conditions, especially in the absence of magnesium chelator EDTA [45].  
The following are available online at [[http://www.mdpi.com/2076-3417/9/15/3006/s1](http://www.mdpi.com/2076-3417/9/15/3006/s1)]([http://www.mdpi.com/2076-3417/9/15/3006/s1](http://www.mdpi.com/2076-3417/9/15/3006/s1)). Figure S1. Strand routing diagram of our X-shaped 3D plasmonic metamolecule. Figure S2. Gel purification and characterization of AuNR conjugated melanolecules. Table S1. Parameters for the Hill equation fit of CD response curve against thrombin. Figure S3. Thrombin detection with metamolecule that locks with left-handed chirality. Figure S4. CD spectra of metamolecules in the presence of thrombin or BSA.  
[PERSON] and [PERSON] designed the experiment. [PERSON] and [PERSON] performed the experiment. [PERSON] and [PERSON] wrote the manuscript.  
[PERSON], [PERSON], and [PERSON] acknowledge the support from the European Commission through the ERC grant agreement no. 336440, ORCA, and the DFG, through the SFB1032 (A6).  
[PERSON] acknowledges [PERSON] for providing the gold nanorods.  
The authors declare no conflict of interest.