Figure 1: Western Blot analysis of purified tetanus antigen.

Figure 2: Quotion picture of iRIf measurement of human serum sample taken before vaccination.

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 (??).

Figure 3: Quotion picture of iRIf measurement of human serum sample taken three weeks after vaccination.

PLATZHALTER! Figure 4: Binding curve of the tetanus measurements.

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 could successfully show that cell-free expression of GFP-His results in functional GFP proteins that are bound by GFP-antibodies during an iRIf measurent. The DNA coding for GFP fused to a 10x His-tag was added to the DiaMIX and expression took place for 2h. The DiaMIX containing cell-free expressed GFP was then spotted on an iRIf slide with a NiNTA (fig.1) surface by hand. The spots were incubated on the slide for ? hours.

Figure 1: Specific NiNTA surface on iRIf glass.

This specific surface allows binding of the expressed GFP via the His-tag while all the other proteins inside the DiaMIX are not bound to the surface. This step was followed by the blocking and washing protocol (link?). The iRIf slide was then flushed with a GFP-antibody solution, to analyse the binding of GFP antibodies to the cell-free expressed GFP. As a positive control we spotted a purified GFP-10His onto the surface as well as DiaMIX that did not contain any DNA as negative control (fig.2)

Figure 2: The two upper left spot are the cell-free expressed GFP proteins spotted on the NiNTA surface. The spot below shows the positive control, GFP-10-His expressed in E. coli, purified and spotted onto the NiNTA surface. On the right sied you can see the negative control spots, where DiaMIX without any DNA was spotted o the slide.

The measurement shows high signal for the cell-free expressed GFP and the positive control. There is no unspecific binding observable at the negative spots, proving that the NiNTA surface is indeed highly specific.

Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.

To test our device we aimed at reproducing measurements that we were previously able to perform in our commercial device. For this reason we focused on using the two antibodies: anti-GFP and polyclonal anti-rabbit. GFP and rabbit derived anti-HCV (hepatitis C virus) antibodies were used as antigens in this experiment. Note that the anti-HCV antibodies only served as binding partners for the anti-rabbit antibodies, since no HCV proteins were used in this experiment. The antigens were spotted onto an iRIf slide whose binding layer consisted of an APTES/PDITC surface. The spots on the slide were made by pipetting 3,5 µl (1 mg/ml) rabbit-anti-HCV protein and 3,5 µl (1 mg/ml) GFP onto the slide and incubated over Night. After incubation, the slide was blocked for 30 min in BSA solution. A Canon 50D camera was used to record the measurement and was set to take one picture every 5 seconds. The exposure time was set so that the pixels in the image were approx. 80% of maximum light saturation before the solution was flown onto the chip. The antibody solutions were pipetted into the flow-chamber without the use of any microfluidics device. Instead a syringe was loaded with 660 µl [5 µg/ml] anti-GFP solution (diluted in PBS) and connected to the input tube of our device. The content of the syringe was then slowly released into the binding chamber of the device by gently dispensing the solution from the syringe. When the whole volume ran over the chip, the process was repeated with 660 µl [5 µg/ml] anti-rabbit antibody in the same way. The injection of both solutions took approximately 45 minutes.

Figure 3: Quotient picture of the same measurement with favourable light conditions

Video 1 shows the results of the measurement. Both binding of anti-GFP and anti-rabbit to the corresponding antigen spots could be observed. Due to the fact that the experiment was performed on our demonstration device which is build with a transparent casing, fluctuations in surrounding light had a strong, detrimental influence on the measurement quality. To minimize the influence of the surrounding light being unstable, the resulting pictures had to be averaged over 10 pictures each. This in turn lead to a more stable light situation, however the signal strengh dropped as a consequence. Figure XY shows a quotient picture during the measurement where the light situation was temporarily appropriate. The problem of surrounding light scattering into the device can of course be overcome using a non-transparent casing.

Figure 1: Cell-free expressed His-GFP spotted on a Ni-NTA slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified GFP-His, the negative control was a sample of cell-free reaction performed without DNA.

Figure 2: Cell-free expressed His-GFP spotted on a PDITC slide. Cell-free expression was performed for 2 hours at 37°C. The positive control was purified GFP-His, the negative control was a sample of cell-free reaction performed without DNA.

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. After testing several tag systems we established a Ni-NTA surface because it worked best for us. Using a Ni-NTA covered glass slide (fig. 1) we could increase the amount of bound cell-free expressed GFP compared to an unspecific surface (fig. 2). In both experiments ____2,5 µg?!?____ self purified GFP-His was 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 pipetted by hand 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.

Our DiaMIX was prepared based on a protocol of ??? (Ref.). It is a very complex system containing many different enzymes and chemicals for special purposes. An overview of cell-free expression systems and its components can be found on methodology page .
To investigate the efficiency of our self-prepared expression system we performed an experiment comparing it with a commercially available kit. In order to obtain reliable results we used the exact same vector, containing a sequence encoding GFP. Like this we were able to trace the amount of expressed GFP using a plate reader over a period of two hours. As a negative control, both mixes were treated equally in a second sample but did not contain DNA.
The evaluation is shown in Figure 1 and is based on triplicates. The data was normalized to the mean value of the measurement of air. The uncertainty was calculated using the standard deviation.
picture; Figure 1: Cell-free expression of GFP using the DiaMIX and a commercial kit. The reaction was performed in a volume of 50µl each and monitored every minute at 37°C over a period of two hours. Excitation at 480nm, emission at 520nm.
Both expression systems were shown to successfully express the applied vector. There is a clear enhancement of light emission observable at 520nm for both mixes in comparison with the respective negative control. The expression of GFP using the DiaMIX seems to be slightly better than by using the commercial kit. Still, one has to mention the actual purpose of the commercial kit to express linear templates. However, as the basis protocol for our mix was optimized for circular templates, we used a circular vector for the experiment.
Furthermore, as already shown above, we successfully verified the expression of GFP with our own cell-free mix in an iRIf measurement.
To obtain these final results, a lot of optimization and testing had to be done. If you want to know more about how we established the DiaMIX for the DiaCHIP, you can find detailed information about the most important experiments on our results page .