Hypothesis

The ball resonator optical fiber functionalized with monoclonal antibodies would allow the early and efficient detection of recombinant Vaccinia virus antigens (L1, A27,A33 and B5) in sewage water.

General plan

In order to prove this hypothesis, we engineered a non-pathogenic E.coli strain to produce recombinant proteins (L1, A27, A33, B5) of Vaccinia virus and used it to configure and calibrate the optic-fiber-based ball resonator biosensor for detection of the wild type proteins on the free-floating OPXV particles. Our expectations about the project are depicted in Figure 1.

Figure 1.> Our main goals during this project

We suggested these results based on the existing research about optic-fiber based biosensors [1,2].

To prove our concept the following experiments were performed.

Western blot experiments

The Western blot was conducted for several IPTG-induced samples of B5 1st sushi and all 4 sushi domains, L1, A27 and A33.

The expression was successful for the 3 recombinant proteins A27, A33, and L1, for B5R protein the expression was either absent or insignificant. Results from Western blots (Figure 2) indicate that proteins L1, A27 and A33 were properly folded and successfully bound to their antibodies, thus antibodies can be used for biosensor functionalization.

Figure 2.. Western blot analysis of L1 (A), A27 (B) and A33 (C) proteins.  

Specificity of the antibody functionalized biosensor

Although we were not able to express B5R protein on time, we were able to test our system that targets other orthopox antigens. We tested our system in two environments: PBS and synthetic sewage water. We compared results from these media with each other, testing optic fiber biosensor with single type monoclonal antibodies and with all three types (multiplex).

The Figure 1 demonstrates the results from ball resonator optical biosensor functionalized with single antibodies. These results were done by Aida Rakhimbekova and Baizak Kudaibergenov. There is significantly higher change in refractive index at 0.24nM concentration observed from sensors treated with target proteins. All of the sensors demonstrated that sensors treated with target proteins had

 the highest ΔRI. The ball resonator functionalized with L1 has 25mRIU difference, with A27 has 10mRIU difference, and with A33 has 15mRIU difference between ΔRI of target protein and second highest ΔRI. Therefore, we can conclude that sensors are significantly specific against target proteins.

Figure 3. Specificity of the optic fibers functionalized in detecting the target protein. a- biosensor functionalized with anti-L1 antibodies, b- biosensor functionalized with anti-A27 antibodies, c- biosensor functionalized with anti-A33 antibodies.

Concentration-dependent change (multiplex system)

We started at the 10 attoMolar concentration of proteins and increased by the power of 10, up to 10 nanomolar concentration. The sensor responses were recorded 10 minutes of immersing in a solution of concentration, and the measurements for one sensor were consecutive.

Compared to the control sensors, outputs of the optic fiber biosensors functionalized with single antibodies represented as their amplitudes, change as a function of the concentration of antigens in PBS (Figure 3). The graphs also show the average and standard deviation of three measurements, and the Limit of Detection (3y_lowest + δ_max). The sensors functionalized with single antibodies showed a logarithmically linear trend with R² > 0.90, and the range of LOD of sensors to detect vaccinia virus proteins were from 0.1pM to 1fM. This shows a good potential to detect viral antigens in low concentrations with high confidence.

On the other hand, the tests in PBS were conducted to observe if the antibodies on the surface of the sensors bind to the antigens in solutions where external interference is minimized. Then the sensors were tested in synthetic sewage water samples to detect target proteins in a more challenging and interference-rich environment, to test the capabilities of the sensor to work in medium-rich environments. The multiplex sensors showed a log-linear trend with R² > 0.92 in PBS and R² > 0.88 in sewage water samples. The Limit of Detection for multiplex sensors were in the range of 1fM and 1pM in sewage water samples, indicating that the multiplex sensors can detect target antigens in low concentrations with high confidence (Figure 5).

These results from these graphs demonstrate two things:

1. The change in concentration of antigens results in amplitude (dB) increase, proportionally. This linear trend allows us to differentiate antigen attachment from negative control (which has almost slope=0). Here, the concentration at which our system can detect antigens can be 1fM.
2. The results from the multiplex system used in PBS and synthetic sewage water demonstrate that the sensor is able to detect antigens in sewage water as good as in PBS.  
3. Moreover, the deviations in these graphs are significantly smaller than in the control one which suggests that our results are time-independent. We measured every minute throughout 10min at each concentration. While antigen concentration is stable, there is no significant difference in amplitude as time progresses.

Figure 4. Response of (a) the multiplex sensor and (b) the control optic fiber, in solutions of different concentrations of L1, A27, and A33 proteins in PBS

Figure 5. Amplitude change of multiplex sensors (a and b) and control sensor (c) as a function of the concentration of the antigens in sewage water