Results

Fig.  1: Photograph of the final microfluidic cartridge: a design study showing instructions, reference positions of the immunoassay and barcodes for quality control

In the PodiTrodi project a heterogeneous technology platform for Point of Care diagnostics of tropical diseases has developed. The approach structured the system in four major research areas: (1) the bioassay and the necessary sample preparation, (2) the active microfluidic cartridge, (3) novel biosensors and (4) the instrumentation.
The bioassay represents the bio-chemical protocol for the sensitive and specific detection of the pathogen. For PodiTrodi two assays were developed in parallel for a reliable detection. For real time PCR oligonucleotides targeted to two multi-copy genes of T.cruzi were employed. A PCR reaction containing an internal control target was developed which allows to control the quality of the extraction procedure as well as for the PCR reaction. The extraction procedure was successfully tested with special alcohol-free buffers developed by CEA(see below). Under the current experimental conditions, it was possible to detect the DNA of 1 parasite diluted in 1ml of human sample. The second approach is an ELISA assay for the detection of human antibodies against T.cruzi. Synthetic genes, which encode four major peptides, were designed and expressed by E. coli. Data available in the literature showed that these peptides comprise the major T. cruzi antigens. These proteins were impregnated onto nitrocellulose membranes and challenged with Chagas-positive sera. The results show that all four chimeric proteins yielded very good detection.

 

Before the sample can be tested with the real time PCR the genetic information of the parasite needs to be extracted and purified. During the project an alcohol-free extraction for magnetic beads and silica membranes, the two major techniques for DNA extraction, has been developed. The yield of bacterial DNA purification from a blood sample is very close to the reference protocol provided by the supplier with alcohol buffers. Validation at Fiocruz (Brazil) has also shown that this protocol is efficient with T. cruzi.

Fig. 2: Photograph of the final microfluidic cartridge: explanation of the functional units of the cartridgeFor the miniaturization of the diagnostic test the whole test procedure needed to be translated and automated in a micro format. In the project an active microfluidic cartridge (see Figure 2) has been developed using a unique integrated micro pumping technology. Electrolytic gas generating enables small and powerful micro pumps at a low cost. The developed cartridge integrates a complete sample preparation from whole blood to purified DNA and two biosensors. On the real time PCR chip the extracted DNA is amplified and detected in parallel. This technique al-lows a robust and quantitative analysis of the genetic information. Secondly a nitrocellulose strip with impregnated proteins detects pathogen specific antibodies. The combination of these two techniques on one platform constitutes a completely new technology. For the control of the whole detection procedure an application specific integrated circuit (ASIC) has been developed, produced and validated with the electrolysis technology. The small chip now allows the control of micro pumps and valves with a minimum of physical infrastructure.

Fig. 3: Dual channel SAW biosensorAlthough real time PCR and ELISA are established technologies the PodiTrodi project was also exploring the potential of new biosensor technologies. A surface acoustic wave (SAW) biosensor made of LiTaO3 has been developed, which uses the interaction of surface bound molecules and a traveling acoustic wave. The piezoelectric crystal was precisely cut in order to reach a maximum frequency shift. A dual channel architecture of the sensors provides an internal reference for the measurement. For the excitation and readout of the SAW sensors an  ASIC based electronic was developed in order to obtain a picosecond time measurement accuracy. For the further enhancement of the sensitivity sensitive layers based on graphene, graphene oxide, peptide nanotubes had been developed and deposited on the surface of the SAW sensor. Successful measurements of Chagas contaminated samples with graphene coated surfaces showed potential of this new technology. Figure 3 shows a SAW sensor with attached microfluidic cell.

The reliable and comfortable operation of the test relies on a smart readout instrument. In the project a modular and portable readout instrument has been designed and fabricated (Figure 4). The instrument provides all necessary infrastructure for the correct operation of the cartridge and biosensors including an uninterruptible power supply, a multi channel optical sensor, an optimized heat flux for proper operation in harsh environments, and human-machine interface for the safe operation. The specifications of the instrument were derived from the technological necessities and end-user surveys.

Fig.  4:  Photographs of the prototype of the PodiTrodi instrument closed (left) and open with the active microfluidic cartridge (right).