Minimally-invasive technologies that can sample and detect cell-free nucleic acid biomarkers from liquid biopsies have recently emerged as clinically useful for early diagnosis of a broad range of pathologies, including cancer. Although blood has been so far the most commonly interrogated body fluid, skin interstitial fluid has been mostly overlooked despite containing the same broad variety of molecular biomarkers originating from cells and surrounding blood capillaries. Minimally-invasive technologies have emerged as a method to sample this fluid in a pain-free manner and often take the form of microneedle patches. Herein, we developed microneedles that are coated with an alginate-peptide nucleic acid hybrid material for sequence-specific sampling, isolation and detection of nucleic acid biomarkers from skin interstitial fluid. Characterized by fast sampling kinetics and large sampling capacity (~6.5μL in 2 min), this platform technology also enables for the first time the detection of specific nucleic acid biomarkers either on the patch itself or in solution after light-triggered release from the hydrogel. Considering the emergence of cellfree nucleic acids in bodily fluids as clinically informative biomarkers, platform technologies that can detect them in an automated and minimally invasive fashion have great potential for personalized diagnosis and longitudinal monitoring of patient-specific disease progression.

Label-free, single-molecule sensing is anideal candidate for biomedical applications that rely on the detection of low copy numbers in small volumes and potentially complex biofluids. Among them, solid-state nanopores can be engineered to detect single molecules of charged analytes when they are electrically driven through the nanometer-sized aperture. When successfully applied to nucleic acid sensing, fast transport in the range of 10-100 nucleotides per nanosecond often precludes the use of standard nanopores for the detection of the smallest fragments. Herein, hydrogel-filled nanopores (HFN) are reported that combine quartz nanopipettes with biocompatible chemical poly(vinyl) alcohol hydrogels engineered in-house. Hydrogels were modified physically or chemically to finely tune, in a predictable manner, the transport of specific molecules. Controlling the hydrogel mesh size and chemical composition allowed us to slow DNA transport by 4 orders of magnitude and to detect fragments as small as 100 base pairs (bp) with nanopores larger than 20 nm at an ionic strength comparable to physiological conditions. Considering the emergence of cell-free nucleic acids as blood biomarkers for cancer diagnostics or prenatal testing, the successful sensing and size profiling of DNA fragments ranging from 100 bp to >1 kbp long under physiological conditions demonstrates the potential of HFNs as a new generation of powerful and easily tunable molecular diagnostics tools.

Oligonucleotide‐templated reactions are valuable tools for nucleic acid sensing both in vitro and in vivo. They are typically carried out under conditions that make any reaction in the absence of template highly unfavorable (most commonly by using a low concentration of reactants), which has a negative impact on the detection sensitivity. Herein, we report a novel platform for fluorogenic oligonucleotide‐templated reactions between peptide nucleic acid probes embedded within permeable agarose and alginate hydrogels. We demonstrate that under conditions of restricted mobility (that is, limited diffusion), non‐specific interactions between probes are prevented, thus leading to lower background signals. When applied to nucleic acid sensing, this accounts for a significant increase in sensitivity (that is, lower limit of detection). Optical nucleic acid sensors based on fluorogenic peptide nucleic acid probes embedded in permeable, physically crosslinked, alginate beads were also engineered and proved capable of detecting DNA concentrations as low as 100 pm.

Extracellular vesicle‐derived microRNA (EV‐miRNA) represent a promising cancer biomarker for disease diagnosis and monitoring. However, existing techniques to detect EV‐miRNA rely on complex, bias‐prone strategies, and preprocessing steps, making absolute quantification highly challenging. This work demonstrates the development and application of a method for quantitative and multiplex detection of EV‐miRNA, via rolling circle amplification within encoded hydrogel particles. By a one‐pot extracellular vesicle lysis and microRNA capture step, the bias and losses associated with standard RNA extraction techniques is avoided. The system offers a large dynamic range (3 orders of magnitude), ease of multiplexing, and a limit of detection down to 2.3 zmol (46 × 10−18 m), demonstrating its utility in clinical applications based on liquid biopsy tests. Furthermore, orthogonal measurements of EV concentrations coupled with the direct, absolute quantification of miRNA in biological samples results in quantitative measurements of miRNA copy numbers per volume sample, and per extracellular vesicle.