Research and medical laboratories normally rely on real-time PCR and solid phase hybridization assays to answer biological problems regarding gene expression profiling, determination of viral load in clinical samples, DNA and RNA quantification, bacterial identification, SNP genotyping and pharmaco-genomics. Both techniques are based either on non-specific or sequence-specific fluorescent reporters that generate a signal reflecting on the amount of the PCR product; detection and quantification of fluorescently labeled targets require expensive instrumentation and sophisticated algorithms in some cases. In other cases, the combination of DNA amplification with visual detection was demonstrated to be a simple and cost-effective method, but results are qualitative, which is a clear drawback for many clinical applications. In our lab, we are developing new approaches for genetic testing which retain the high sensitivity and quantitative nature of PCR combined with simple detection methodologies.
Real time digital colorimetric assay (bioPix)
We have combined an isothermal amplification method based on LAMP with smartphone-based colorimetric detection towards the development of simple and affordable platform for global diagnostics. This method, currently under patenting, can provide real time, quantitative information on the amount of nucleic acids present in crude samples without the need for purification or other sample pre-treatment steps. The concept has recently received funding (Patras Science Park) in order to bring this innovation developed within Biosenosrs’ Lab to a commercialization stage.
Acoustic detection via DNA conformation sensing
Wed are developing detection protocols for the label-free detection of DNA conformation rather than mass. The method takes advantage of the direct detection of dsDNA amplicons designed to have a specific size; this method is faster and eliminates the need for the control of the hybridization of a ss-target to a surface-immobilized probe. A considerable advantage of the method is the ability to achieve multiplexing of two or more targets by modulating their length (Sci Rep 2013).
Application to DNA testing in real samples
Our goal is to develop integrated biosensing platforms that could be used directly in real human, food or environmental samples, such as blood, urine, milk, meat, soil etc. To achieve this, we are working towards the development of biocompatible surfaces that could selectively and with high sensitivity detect the amplified DNA in a complex medium. Such an example is a surface coated with the co-polymer PEG-polylysine and is used for the selective binding of amplified DNA in the presence of lysed cells, proteins and the PCR amplification medium (i.e., primers, enzymes and triton). (Chem Comm 2017)
The acoustic methodology has been demonstrated in three different cases:
- Detection of an insecticide-resistant mutation in the ace-1 gene of Anopheles gambiae, the major malaria vector (Sci Rep 2013)
- Quantification of the change in the expression levels of the ABCA1 gene in the liver of mice which was induced by a synthetic ligand (Sci Rep 2013)
- Detection of 4 different mutations in BRCA1 and BRCA2 genes that could serve as a focused genetic screening test for breast cancer (Anal Meth 2013)
Lab-on-Chip platform for foodborne pathogens detection
The fast and efficient detection of foodborne pathogens is a scientific and technological challenge, given the need to detect as little as 1 viable cell in 25 gr of food.
We developed a micro/nano-bio system based on an acoustic wave sensor where, starting from 1 Salmonella cell in 25 ml of milk, we managed to complete the detection in 4 hrs, as opposed the 24 hrs currently taken in a food microbiology lab. We employed immuno-magnetic beads to capture cells after a short (3 h) pre-enrichment, followed by isothermal amplification (LAMP) in a microfluidic channel (½ h) and acoustic detection (½ h) in an integrated platform This work presents the first reported Lab-on-Chip platform that comprises an acoustic device as the sensing element, exhibiting impressive analytical features, namely, an acoustic limit of detection of 2 cells/μl or 3 aM of the DNA target and ability to detect in a label-free manner dsDNA amplicons in impure samples. (Biosens Bioelectron 2018) This ambitious work, funded by the EC in two follow up projects (Love Food and LoveFood2Market) is carried out in collaboration with several partners in academia (Dr A. Tserepi and Dr E.Gogolides from NCSR-Demokritos, Dr B. Depuy from Pasteur Inst. and Dr Z. Bilkova from Pardubice Un.) and industry (Jobst Technologies and Senseor).
Platform for the detection of circulating tumor DNA
Our strong interest and commitment towards the technology transfer of acoustic biosystems to clinical diagnostics is illustrated in our involvement in the development of a platform for the detection of circulating DNAs and their mutations in blood samples. The LiquBiopSens project, also funded by the EC through a Horizon20200-Innovation program, promises the delivery of a fast, direct, real time and inexpensive instrument that has the potential to same thousands of lives through the early and accurate colorectal cancer detection.