Focal molography

Focal molography (“molography” in short) is a biophysical method for robust and sensitive detection of biomolecular interactions without the use of additional fluorescent labels. The new method enables label-free biomolecular interaction analysis in complex biological samples. Molography widens the analytic capabilities for the investigation of biomolecular interactions in a broad range of applications, e.g. label-free trace analysis of a targeted molecule (called biomarker) in crude samples, such as blood sera or cell culture media. Contrary to refractometric methods for label-free biomolecular interaction analysis, such as surface plasmon resonance (SPR) and reflectometric interference spectroscopy (RIfS), molography allows quantification of molecular interactions in living cells in real time.
Principle.
The working principle of molography is illustrated in Figure 1. Molography is based on the interference of laser light at a special 2D nanopattern of molecular binding sites on the surface of a sensor chip, termed mologram. A mologram is a coherent assembly of binding sites on a chip that form the blueprint of a diffractive lens. The lens is illuminated by an evanescent wave as it occurs for instance upon total internal reflection of a laser beam. Biomolecules that bind to the mologram diffract laser light into a diffraction-limited focal spot, the focus of the mologram. The intensity of the focused light is measured to quantify the amount of bound molecules. The diffracted intensity in the focal spot correlates quadratically with the adsorbed mass and hence with the number of biomolecular interactions on the mologram. 
In complex biological samples, highly abundant off-target molecules readily adsorb to the surface of the sensor. Yet, this process is random and the off-target molecules do not bind to the ordered binding sites of the mologram. Thus, scattering of off-target molecules is uniform in all spatial directions and therefore off-target molecules hardly contribute to the measured light intensity in the narrow solid angle of the focal spot. In essence, focal molography forms a high-frequency spatial affinity lock-in that only measures the Fourier component of the refractive index distribution that corresponds to the binding modulation of the mologram. Since the environmental noise (temperature gradients, buffer changes and nonspecific binding of off-target molecules) is inversely proportional to the spatial frequency and therefore situated mostly at “long” spatial periods most of the noise can be rejected. This property makes molography extremely robust and renders temperature stabilization or sensor equilibration obsolete. 
History.
Christof Fattinger at Hoffmann-La-Roche in Basel already conceived the physical principles of focal molography in the 1990-ties when he investigated the bidiffractive grating-coupler. Yet it only emerged in a scientific collaboration between ETH Zürich and the Roche Innovation Center Basel in Switzerland from 2014 to 2020.
Realization: A special photolithographic method enables the synthesis molograms.
The synthesis of molograms on the sensor chip is realized with properly designed surface chemistry and reactive immersion lithography (RIL). It allows the creation of the biomolecular recognition structure of the mologram on a light-sensitive non-fouling graft copolymer layer. The copolymer layer contains photoactivatable functional groups for patterning of the recognition sites of the mologram under immersion by exposure to light. The RIL process enables tuning of surface chemistries and recognition molecules specific to the desired analytical application. 
Applications of the molography method.
The robustness of focal molography against environmental noise gives the method a platform character that enables many possible applications. They range from the investigation of a specific biomolecular interaction in basic biological research to the diagnosis of a critical health condition in an emergency.
The biological applications of molography categorize into five basic classes: