| Abstract |
Although biosensors hold their roots in the early nineteen-sixties, a great increase in the
number of publications refer on this topic was recorded after the nineteen-eighties decade.
Moreover, the increasing demand for inexpensive, simplified, sensitive and reliable sensors
lead the biosensors market to a significant growth with a predicted compound annual
growth rate (CAGR) of 7.3% for the years 2020 - 2027. Biosensors are applied in a wide range
of fields including, healthcare and medical diagnostics and is expected to be positively
affected by the new trend of precision medicine and the evenly huge market of Liquid
biopsy.
Regular screening of cancerous point mutations is of great importance for efficient cancer
management and treatment selection. Although excellent techniques like next-generation
sequencing and the ultrasensitive droplet digital PCR have been developed, these techniques
are lacking in fastness, simplicity and cost-effectiveness. The work presented here focuses
on the development of new diagnostic approaches for the detection of ultralow
concentrations of cancerous point mutations in human DNA utilizing acoustic biosensors
combined with molecular amplification assays. Firstly, we present a universal acoustic
methodology that involves the direct immobilization of biotinylated DNA targets on the
sensor surface followed by liposome-based acoustic detection. Liposomes, are large
nanoparticles (here 200nm) acting as acoustic energy-dissipation signal enhancers. For the
DNA immobilization, we developed a surface chemistry composed of biotinylated-BSA and
NAv; the substrate was reproducible and was successfully validated for its specificity and
stability upon liposome and crude sample additions, respectively. While for the above we
used a standard QCM-D device and a 5 MHz QCM sensor, a novel High Fundamental
Frequency QCM (HFF-QCM) array of 24 miniaturized sensors operating at 150 MHz and a
new acoustic device were also tested. The array permitted the faster and more costeffective analysis of up to six different samples and the extraction of up to 24
measurements. The above-mentioned acoustic methodology, i.e., b-BSA/NAv and liposomes
for immobilization and detection of DNA targets using the QCM-D device, was firstly
combined with a PCR-free DNA amplification assay, the Ligase Chain Reaction (LCR). For the
LCR we used probes modified with biotin and cholesterol to produce LCR products ready for
immobilization on the NAv-coated sensor and acoustic detection. Following extended
optimization, the detection of 3.3 x103 DNA molecules carrying the BRAF V600E point
mutation was achieved. However, the overall assay suffered from some limitations
concerning the target-independent ligated by-products that occurred during the LCR, leading
to low sensitivity and decreased specificity. To overcome the problem of the low sensitivity
and specificity Allele-Specific PCR (AS-PCR) was employed as an alternative method for DNA
amplification. AS-PCR combined with acoustic detection improved the limit of detection
down to 1 copy of mt target in an excess of 104 wt molecules, otherwise with a sensitivity of
0.01%, using genomic DNA carrying the BRAF V600E point mutation. For the amplification,
we again used primers modified with biotin and cholesterol and for the acoustic detection,
we employed the 150 MHz biochip array with great success. Since the initial protocol
developed for the detection of the BRAF V600E mutation gave only qualitative results, the
assay was further optimized and applied to the analysis of KRAS G12D mutation achieving
both qualitative and quantitative results with a sensitivity of 0.05%. Finally, the assay was validated for the detection of both point mutations in real FFPE-tissue and plasma samples
obtained from melanoma, colorectal and lung and cancer patients. The obtained results
were compared with those recorded from the standard methods used for tissue and liquid
biopsy i.e., Sanger sequencing and droplet-digital PCR.
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