Acoustic detection of a mutation-specific Ligase Chain Reaction based on liposome amplification.

Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation versus the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients' crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.

High-throughput extraction on a dynamic solid phase for low-abundance biomarker isolation from biological samples.

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum.

Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy.

Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically "lossy" liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.