Singlet oxygen-based photoelectrochemical detection of single-point mutations in the KRAS oncogene.

Single nucleotide point mutations in the KRAS oncogene occur frequently in human cancers, rendering them intriguing targets for diagnosis, early detection and personalized treatment. Current detection methods are based on polymerase chain reaction, sometimes combined with next-generation sequencing, which can be expensive, complex and have limited availability. Here, we propose a novel singlet oxygen (1O2)-based photoelectrochemical detection methodology for single-point mutations, using KRAS mutations as a case study. This detection method combines the use of a sandwich assay, magnetic beads and robust chemical photosensitizers, that need only air and light to produce 1O2, to ensure high specificity and sensitivity. We demonstrate that hybridization of the sandwich hybrid at high temperatures enables discrimination between mutated and wild-type sequences with a detection rate of up to 93.9%. Additionally, the presence of background DNA sequences derived from human cell-line DNA, not containing the mutation of interest, did not result in a signal, highlighting the specificity of the methodology. A limit of detection as low as 112 pM (1.25 ng/mL) was achieved without employing any amplification techniques. The developed 1O2-based photoelectrochemical methodology exhibits unique features, including rapidity, ease of use, and affordability, highlighting its immense potential in the field of nucleic acid-based diagnostics.

Highly flexible and accurate serial picoinjection in droplets by combined pressure and flow rate control.

Picoinjection is a robust method for reagent addition into microfluidic droplets and has enabled the implementation of numerous multistep droplet assays. Although serial picoinjectors allow to screen many conditions in one run by injecting different combinations of reagents, their use is limited because it is complex to accurately control each injector independently. Here, we present a novel method for flexible, individual picoinjector control that allows to inject a predefined range of volumes by controlling the flow rate of the injector as well as turning off injection by setting the equilibrium pressure, which resulted in a stable interface of the injector liquid with the main microfluidic channel. Robust setting of the equilibrium pressure of an injector was achieved by applying accurate (R2 > 0.94) linear models between the injector and oil pressure in real-time. To illustrate the flexibility of this method, we performed several proof-of-concepts using 1, 2 or 3 picoinjectors loaded with fluorescent dyes. We successfully demonstrated picoinjection approaches using time-invariant settings, in which an injector setting was applied for prolonged times, and time-variant picoinjection, in which the injector settings were continuously varied in order to sweep the injected volumes, both resulting in monodisperse (CV < 3.4%) droplet libraries with different but reproducible fluorescent intensities. To illustrate the potential of the technology for fast compound concentration screenings, we studied the effect of a concentration range of a detergent on single-cell lysis. We anticipate that this robust and versatile methodology will make the serial picoinjection technology more accessible to researchers, allowing its wide implementation in numerous life science applications.