Figure 2: The expression of our antigens is achieved by our cell-free expression mix. This mix is based on a bacterial lysate and contains all components required for transcription and translation of the DNA sequences.

To enable the production of a protein array consisting of multiple antigens on demand, their expression is mediated by cell-free expression from a template DNA array. This expression system based on a bacterial lysate makes the need for genetically engineered organisms to produce every single antigen redundant. The protein array is generated by flushing our cell-free expression mix through the microfluidic setup. Expressing the antigens from the DNA template, the protein array is adaptable to individual requirements exhibiting the same pattern for both arrays. Our system is made up of two slides enabling the antigens to be immobilized on the opposite side of the DNA template inside the microfluidic chamber (figure 2).

Figure 3: Surface immobilization.To prevent unspecific binding of components of the cell-free expression mix to the glass slide, we established a surface that specifically binds our target proteins, the antigens.

After cell-free expression not only our desired antigens are present within the chamber, but also all other components of the cell-free mix including ribosomes, polymerases or amino acids (figure 3). All these components would bind unspecifically to an activated glass slide, thereby obstructing the binding of the antigens. We designed our DNA constructs in a way that each antigen can easily be fused to specific tags that enable targeted immobilization on a specific surface. Testing different tag systems, we found the Ni-NTA-His-tag system was working best for our purposes. A basic protocol for this specific surface was optimized by ourselves to reduce unspecific binding.

Figure 4: Optical detection method.The detection system mainly consists of a camera and an LED and is called iRIf (imaging Reflectometric Interference). Antigen-Antibody interactions can be detected label-free and in real-time. An optical output of such binding events is generated by a minimal change in the thickness of the layer on the slide right at the corresponding antigen spot.

After preparation of the DiaCHIP, a patient’s serum sample can be flushed over the protein array using the same microfluidic system. The binding of antibodies to the corresponding surface causes a minimal change in the thickness of the layer on the slide right at the corresponding antigen spot. This binding can be detected label-free and in real-time using a novel technique called iRIf (imaging Reflectometric Interference) without the need for further labeling (HIER SCHON REF?). Its core components are a camera, an LED and a few lenses. See how we reconstructed the system in a low-budget device.

Figure 5: Illustration of the perspective during a measurement.

When illustrating the basic principle of the DiaCHIP, we mainly looked at it from the side. Now it is time to explore our results and see what we actually achieved. Therefore, it is important to have in mind that you are observing the chip from the camera's position, so basically from the top (figure 6). This persepective remains the same in all the iRIf measurements we are showing in the results section.

After weeks of optimizing the different components of the DiaCHIP, we are proud to present our results. We reached the highlight of our project with the successful detection of antibodies in our own blood!