During the last decades, the progress of miniaturized smart systems has largely expanded the field of diagnostics and has enabled the medical community to offer to patients worldwide better, faster and more accurate health services. Amongst the various principles of operations that have been introduced, optical sensors have attracted significant research effort. Two approaches have been used so far to realize devises based on optical sensors: external light sources and optical components (such as detectors), which render the system bulky and expensive, or hybrid integration that fuses silicon chips with external light sources. Both solutions have not allowed the decrease in neither the cost nor the complexity of operation, limiting the use of the respective instruments in every day practice. PYTHIA project (FP7-ICT-2007.3.6-224030) followed an alternative route to realize biosensors that could be the core of portable, user-friendly instruments to use in any size clinical center or even private praxis. The PYTHIA chip and accompanying instrument differentiated themselves from the other “mainstream” technologies, since they circumvented the problem of the external optical components by taking advantage of the monolithic integration of all active and passive optical components (sources, optical transducers and detectors) onto a single silicon chip. In addition, the design of this biosensor evolved around a detection principle introduced and proven within the project, namely the Broad-band Mach-Zehnder Interferometry (BB-MZI). BB-MZI exploits the full spectrum of the monolithically integrated light sources surpassing in that way the limitation of the single-wavelength interferometric techniques. BB-MZI and the monolithic integration of all components allowed shrinking the chip’s dimensions to <40mm2. Despite its small size, every chip contained ten independent transducers thus allowing multiplexed determination of up to 10 analytes. The PYTHIA biochips accommodated ten VIS-NIR light sources coupled to individually functionalized interferometric transducers all of them converging to a single detector for multiplexed operation. The signal recording could be realized either via the monolithically fabricated photodetector (intensity measurements; fully integrated chips), which offers the smallest possible size and cost, or via an external spectrometer (spectral changes measurements; semi-integrated chip), which counterbalances an increase in both the cost and size with improved analytical performance. The analytical capabilities of the PYTHIA device were demonstrated through detection of markers related to diagnosis/prognosis of three diseases: a) Prostate cancer, b) Multiple Endocrine Neoplasia type 2 (MEN2), and c) Retinitis Pigmentosa. For prostate cancer, total- and free-prostate specific antigen (PSA) were detected in human serum samples. For MEN2 and Retinitis Pigmentosa a panel of DNA mutations was selected. To reach its objectives, PYTHIA gathered a team with complementary skills in optoelectronic sensor chip fabrication and functionalization (NCSR “Demokritos”, Greece), optical sensor simulation and design (PhoeniX BV, Netherlands), optical sensor chip fabrication (LioniX BV, Netherlands), microfluidics and biochip encapsulation (Jobst Technologies, Germany), sensor surface characterization (Jagellonian University, Poland), read-out electronics and measuring system (VTT, Finland) and system evaluation (Biogenomica SA, Greece, and University College London, UK).
The scientific and technical achievements of the PYTHIA project are summarized to the following:
a) A new label-free detection principle that of Broad-Band Mach-Zehnder Interferometry (BB-MZI) was proposed and proved to resolve the issues of standard Single Wavelength MZI, namely the signal fading and phase ambiguity.
b) The monolithic integration of BB-MZI optoelectronic transducer with all optical components into a single chip abolished the need for external optical components (light source, photodetector) that render all optical biosensors from being portable.
c) A chip with surface area less than 40 mm2 accommodated ten BB-MZIs along with their respective light sources enabling the multiplexed determination of up to 10 analytes after appropriate biofunctionalization of each BB-MZI in a single chip.
d) A wafer-level scale process for the chip microfluidics encapsulation was developed that can further enhance its implementation to cost-efficient mass production.
e) The PYTHIA transducer’s sensing performance was evaluated with standard refractive index solutions and proven to be as highly-sensitive as other label-free optical sensing techniques.
f) The simultaneous detection of two prostate cancer markers, namely total- and free-PSA, in human serum with sensitivity comparable to that obtained with enzymes immunoassays in microtitration plates was achieved using PYTHIA chips modified with appropriate analyte-specific antibodies.
g) Detection of panels of mutations related with diagnosis/prognosis of Multiple Endocrine Neoplasia type 2 (MEN2) or Retinitis Pigmentosa with PYTHIA chips modified with oligonucleotides was demonstrated and a good discrimination between wild-type individuals and heterozygotes was achieved.