Developing a solution for a real world problem
In order to quantify the sleeping hormone melatonin in the saliva, we chose Local Surface Plasmon Resonance (LSPR), because it can even measure concentrations in the picomolar range. The LSPR phenomenon is comparable to an oscillating, subdued system which has an adjustable resonance frequency. Light in the visible wavelength that passes the gold nanostructure gets coupled into the resonance wavelengths. These spread over the plasmon along the border surface to the other medium. This effect can be detected by a local intensity loss in the spectrum. The plasmon field of LSPR is highly sensitive and has a range of 20 to 40 nm, depending on the geometry of the gold nanostructure, and then decays exponentially. If the composition of the medium changes locally at the interface to the gold nanostructure, the refractive index also changes locally. Even with a very small increase in the particle concentration, this leads to a shift in the local intensity loss to higher resonance wavelengths in the spectrum.
The heart of our sensor concept consists of a gold nanostructure on which the RZR binding domain is fixed via streptavidin and biotin. The sensor becomes highly selective due to the specific surface functionalization and highly sensitive by LSPR. In Figure 1, the basic structure of the sensor and the signal of the LSPR is shown. Figure 1 a visualizes that no RZR is bound to the DNA sequence on the gold nanoparticle. The RZR-melatonin complex is shown in figure 1b and results in a shift of the transmission spectrum. The relatively large 53 kDa RZR in the RZR melatonin complex amplifies the shift in resonance wavelength as if melatonin is measured directly.
The I. Physics Institute has performed a simulation with a fixed gold plate diameter of 160 nm with a variable hexagonal distance of 250 nm to 800 nm. The total diameter of the gold nanostructure was 600 μm. As shown in Figure 2, the simulation result for the hexagonal distance of 575 nm with its Fano resonance at the wavelength of 670 nm was very promising.
The Fano Resonance gives a particularly sharp measurement signal due to the coupling of two oscillating systems. In this case, the resonance wavelength also fulfills the Bragg condition, so that the LSPR couples with the interference pattern of the lattice structure. Based on the simulation results and the following paper, the hexagonal distances of 400 nm (type 1) and 575 nm (type 2) were used. The latter because of the possible Fano resonance and first because of a relatively pronounced local low point at the wavelength of 650 nm and because there are comparable literature readings from the paper. The following paper played an important role in the conceptual design Paper The sensor unit without surface functionalization was realized in the clean room by the Institute for Semiconductor Technology (IHT) of RWTH university. The dimensions of the sensor unit of Type1 and Type2 only differ in hexagonal distance. The gold nanostructure was vapor deposited at a thickness of 40 nm on specially prepared square (24 * 24 mm) glass wafers. First, one side of the thin glass wafer was coated with a 10 nm chromium layer, which acts as a bonding agent for gold and better dissipates electrons in the manufacturing process. On top of this, a smooth layer of photo-lacquer was applied over a rotary plate, which liquefied again by means of a very precisely steered E-beam. The negative pattern of the gold nanostructure was elaborately programmed into the e-beam guide. For mass production of the sensor chip a plunger from the semiconductor technology can be used, which is much more effective and cheaper with appropriate scaling. After the e-beam operation step, the gold layer is vapor deposited on the mask. The Photo Paint Remover dislodges the remaining photo paint while removing non-relevant gold, revealing the gold nanostructure as shown in Figure 3.
In the last step, excess chromium is etched away wet-chemically with perchloric acid using ammonium cerium nitrate. figure. 4 shows an example of a completed sensor unit after the etching step. The gold nanostructure is centrally recognized as a faint gray spot that reflects the light back colorfully.
Only when both subsystems have been properly validated, they are both merged and together form the highly sensitive system for the detection of very low concentrations of melatonin in the oral saliva. The measuring system is transferable and can also be used for other biomolecules.
For the construction of the principal measurement, the following scheme from Figure 5 applies.
Even though the gold nanostructure was successfully applied to the glass wafers and the simulation was promising, the functionality could not be proven with the Merlin Spectromter. Also, the measurement with a commercial high resolution spectrometer HR4000 Ocean Optics could not prove the phenomenon of LSPR in the sensor unit so far. The used concept of fine calibration and alignment can be further optimized in order to be able to determine the probability with the sensor unit LSPR. The measurement with the professional spectrometer was made possible by the Institute for Laser Technology (ILT) and carried out in cooperation. The sensor unit was placed on a calibratable carrier as shown in Figure 6.
The alignment with the halogen lamp or the laser together with the sensor unit and the Merlin spectrometer proved to be more challenging. The provisional test setup is shown in Figure 8.
If the functionality of the sensor unit can be validated, the shift of the resonance wavelength and the possible sensitivity are estimated using an aqueous BSA solution with a defined concentration. The functionality of the coupling between RZR binding domain and RZR melatonin complex is considered separately.