Immune biosensors

The use of optical immune biosensors based on surface plasmon resonance (SPR) for the diagnostics of human and animal diseases as well as for environmental pollution monitoring, is one of prospective directions in biosensorics. The sensitivity of immune biosensors is similar to the ELIS A-method but the simphcity of obtaining results in the real time regime and the speed of the analysis are the main advantages of the biosensor approach. Performance of optical biosensors based on SPR depends on the state of the metallic surface as well as on the density, structure and the space volume of the immobilized molecules. It was demonstrated that the application of intermediate layers between the transducer surface and the sensitive biological molecules can optimize the working characteristics of the immune biosensor [7-14]. 

The immune biosensor analysis was carried out in the SPR-4 M device produced by the Institute of Physics of Semiconductors of the Ukrainian National Academy of Sciences. SPR spectroscopy was carried out in the Kretschmann configuration using He-Ne laser ( i=632.8 nm), goniometer (G-5 M), glass prism (the angle at the basis 68°) and photodiode (FD 263). The optical contact between the prism and the metallic layer was achieved by the application of polyphenyl ether (refractive index n= 1.62). 

As the next step of investigation, 10 RID-positive (with the titer of 1 64-1 128), 18 RID-negative and 15 RID-dubious (which formed the precipitation line at 1 4 dilution) samples were identified. It was found that the immune biosensor is sensitive to Ab diluted in the serum up to 16,000 times. The change of the resonance angle varied from 700 to 1,000 angular seconds. In the samples which were identified as RID-dubious, the presence of the specific Ab was detected at the serum diluted up to 500-1,000 times. 

Fig. 8.1 The change of the resonance angle of the immune biosensor in bovine blood serum tests 1-3 - RID-positive, RID-dubious and RID-negative, respectively...

The largest differences in the resnlts were obtained in the analysis of RlD-negative samples. The sera of the vaccinated animals indnced the Ab-specific signal of the immune biosensor. 

Since the sensitivity of the immune biosensor is very high, we chose the most simple and highly efficient Ag immobilization approaches (Fig. 8.2). 

Pre-treatment of the transducer surface with PAH increased the amount of the immobilized Ag and the response of the immune biosensor was much more stable in comparison with the bare gold surface. The sensor with Ag immobihzed after pre-treatment with PAH served for 2 months versus 2-3 weeks for Ag deposited on the bare surface. Ag immobilization on the PAH layer increased the sensitivity of the immune biosensor by 15-20% in comparison with Ag immobilized on the bare surface. At the same time dodecanethiol did not affect the biosensor sensitivity but increased its service life. 

Fig. 8.2 Response of SPR immune biosensor to the introduction of the blood serum at the dilution of 1 500 in the measuring ceU which has a different transducer surface 1 - bare gold, 2 - dodecanethiol, 3 - PAH, 4 - dextran sulfate...

The main viral Ag proteins are gp51 and p24. The vaccine from the Leiconad company contains inner proteins, in particular, p24. To differentiate the sick from vaccinated cows the glycolated protein gp51 can be used. It can be immobilized on the transducer surface through the intermediate layer created from various lectins. It was found that among all the lectins tested (PLA, STA, HPA and WGA), PLA and WGA were the most suitable lectins, especially the latter. The application of WGA generates the maximal response of the immune biosensor. 

We studied the response of the immune biosensor with WGA-treated transducer surface in the analysis of the blood serum of vaccinated animals, which had Ab titer 1 128-1 256 according to the data obtained from RID test. It was found that the response of the immune biosensor to the blood serum of the sick and vaccinated animals was considerably different. In case of the non-modified transducer surface no difference was observed. 

Comparison of the Results Obtained Using the Immune Biosensor and ELISA... 

Blood serum samples from 10 sick animals in which the titer of the specific Ab was 1 256 according to RID test, were analyzed. The sensitivities of both methods (the immune biosensor and ELISA) were similar. The limit of the serum dilution for detection of the specific antibodies was 1 15,000. 

Table 8.1 Comparison of the results obtained by the immune biosensor, ELISA and PCR methods...

Similar tests were carried out with the blood serum obtained from the National Reference Laboratory for FBI (Federal Research Institute for Animal Health, Germany). In this case a pool of 21 serum samples was analyzed. The results of three methods were in agreement except for two samples for which ELISA tests were low positive, whereas the immune biosensor analysis results were negative. 

Fig. 8.3 The resonance angle of the immune biosensor in the analysis of the blood and milk serum. 1,2- blood serum, dilution 1 500, from RID-positive and RlD-negative cows, respectively 3,4 - milk serum, dilution 1 20, from RID-positive and RlD-negative cows, respectively...

Nanostructured Silicon and its Application as the Transducer in Immune Biosensors... 

Keywords Nanostructured silicon Photoluminescence Electro-conductivity Immune biosensors T2 mycotoxin... 

By controlling the structural and electronic properties of sNPS which are related to the nanocrystallite dimensions and porosity, their surface selectivity and sensitivity to different gases (nitrogen and carbon oxide, vapors of water and organic substances) can be adjusted. This approach for the effective detection of acetone, methanol and water vapor in air was described in [13-15].The minimal detectable acetone concentration was reported to be 12 pg/mL. Silicon sensors for detection of SO2 and some medicines such as penicillin were created [16-18]. sNPS were used for the development of a number of immune biosensors, particularly using the photoluminescence detection. Earlier we developed similar immune biosensors for the control of the myoglobin level in blood and for monitoring of bacterial proteins in air [19-23]. 

The experimental data presented show that sNPS can be used as transducers, which are stable for a long time after the construction of an immune biosensor. The specific immune complex formed on the sNPS surface may be registered by measuring its photoluminescence or photoconductivity. Such immune biosensors can be applied for control of T2 mycotoxin. The biosensors developed are sensitive and simple and allows for rapid analysis and analysis in field conditions. This approach may be applied for detection of any biochemical substances which can form an immune complex. Further investigations should be directed towards studying the mechanism of the biochemical signal detection by the sNPS and characterization of all the steps of analysis. 

Starodub NF, Shulyak LM, Shmyryeva OM, Pylipenko IV, Pylipenko LN, Mel nichenko MM (2009) Nanostructured silicon and its application as the transducer in immune biosensors. In Mikhalovsky SKA (ed) Biodefence advanced materials and methods for health protection, p 87 Springer - Dordrecht, Netherlands... 

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