The antigen used for detection of anti-tetanus antibodies is derived from the tetanus neurotoxin and commonly used for in vitro testing. For preparation of the DiaCHIP, this antigen had to be overexpressed in Escherichia coli and purified from the whole cell lysate. To verify successful purification of the antigen Western Blot analysis was performed (fig. 1). Using an antibody targeting the N-terminal His-tag, revealed a protein with a molecular weight of about 50 kDa. This correlates to the expected molecular weight of the antigen (Reference).
The purified antigen was immobilized on a Ni-NTA surface that specifically binds His-tagged proteins. Look here to read more about the establishment of this surface. As a positive control, self-purified, His-tagged GFP was immobilized at another spot. The reliable detection of anti-GFP was shown before (link). No binding event should be detected at the negative control spot that was covered with bBSA/mCherry (??).

The human serum samples we obtained have been taken before and three? weeks after a patient received a vaccination boost against tetanus. Analysis of the samples was performed using the same array set-up. Figures 2 and 3 show quotion pictures representing changes in the thickness of the slide while it was flushed with the respective samples. The negative control spots do not indicate any binding events, while the binding of anti-GFP to the positive control spot verifies that both experiments are reliable. Comparison of the antigen spots shows that we specifically detected anti-tetanus antibodies in human serum in response to vaccination. No binding event was detected by flushing the array with the sample assumed to be negative (fig. 2), whereas a distinct spot is visible after flushing with the positive sample (fig. 3). Both measurements in real-time are shown in parallel in video 1.
The increase in relative light intensity at distinct spots over the course of the experiment is visualized in figure 4 and can be correlated to the amount of antibody binding to the respective spot. This indicates that the DiaCHIP enables quantification of antibody titers in addition to detecting their presence.
Besides tetanus, other antigens of immunological relevance were taken into account. See all the results we obtained in terms of diagnostics.

We got hold of a more or less 20 years old serum of a rabbit that was immunized with GFP. This gave us the perfect opportunity to test our device with a more complex solution than antibody-dilutions.
For this experiment we used a specific Ni-NTA surface and spotted cell-free expressed GFP on the surface. For verification of correct expression of GFP with our Dia-Mix we performed a Western Blot analysis (fig. 1). With this we could prove that the cell-free expression was successful.
To obtain the specific surface an iRif slide was plasma activated and incubated with APTES and PDITC. Afterwards the Ni-NTA layer was added via incubation of the slide in Ni-NTA solution over night. The slide was blocked with blocking solution (?) and cell-free expressed GFP with a His tag ( + other GFPs?) and a positive control as well as a negative control (His-mCherry) were spotted onto the surface.
The serum of the GFP-immunized rabbit was then analysed in an iRif measurement. Binding of the GFP-antibodies present in the rabbit serum can clearly be seen, as the respective spots show a strong signal (fig. 1). A parallel measurement with serum of a rabbit that was not immunized (fig.2) does not show any binding events at the GFP spot.
With this experiment we could show that it is possible to analyse complex fluids like serum. In addition to that we demonstrated that our cell-free expressed GFP with a His-tag worked at binding partner of GFP-antibodies. Thus, His-tagged GFP was successfully expressed and immobilized on the surface, still enabling anti-GFP antibodies in a serum sample to bind.
Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.

Figure 2B shows the binding of anti-GFP to GFP as it was established before, measured with our own device. Hier müsste man dann auch noch mehr zum experiment schreiben. Microfluidic von hand. Bildkorrektur erwähnen, wie gemacht? Look here, to see how you can build your own device for label-free detection of antibody binding on a protein microarray needing two lenses, a camera and a little bit of fine feeling (??) sensitivety/sensitiveness/finesse/sure instincts.

For cell-free protein expression lots of different proteins, like RNA-Polymerase, ribosomes and other E. coli proteins, are essential. So during the process of cell-free expression all these proteins are, next to the target protein, also present in the microfluidic chamber. To get a sufficient amount of target protein, we therefore established a specific surface chemistry on the glass slide. Next to other tag systems ( link ) we established a Ni-NTA surface because it worked best for us. Using a Ni-NTA covered glass slide (figure ___) we could increase the amount of bound cell-free expressed GFP compared to an unspecific surface (figure___). In both experiments ____2,5 µg?!?____ purified His-GFP were used as positive control. A cell-free reaction performed without DNA, that is supposed to result in no target protein, served as negative control. The cell free reactions were performed for ___2?!___ hours at 37°C. The samples were spotted on the surfaces and incubated on slide o/n. In iRIf, the optical detection method we used, the slides were blocked with BSA and then flushed with anti-GFP antibodies. An increase in light intensity on the slide, where the spotted protein is located, represents a binding event with anti-GFP antibodies. Due to less unspecific binding, interaction of anti-GFP with cell free expressed His-GFP is higher on the Ni-NTA surface.