Engineered nanofluidic platforms for single molecule detection, analysis and manipulation

Since the pioneering studies on single ion-channel recordings in 1976, single molecule methods have evolved into powerful tools capable of probing biological systems with unprecedented detail. In this work, we build on the versatility of a type of nanofluidic devices, called nanopipettes, to explore novel modes of single molecule detection and manipulation with the aim of improving spatial and temporal control of biomolecules. In particular, a novel nanopore configuration is presented, where biomolecules were individually confined into a zeptoliter volume bridging two adjacent nanopores at the tip of a nanopipette. As a result of this confinement, the transport of biomolecules such as DNA and proteins was slow down by nearly three orders of magnitude, leading to an improved sensitivity and superior signal-to-noise performances compared to conventional nanopore sensing. Active ways of controlling the transport of biomolecule by combining the advantages of nanopore single-molecule sensing and Field-Effect Transistors are also presented. These hybrid platforms were fabricated in a simple two step process which integrates a gold electrode at the apex of a nanopipette. We show that these devices were effective in modulating the charge density of the nanopore and in actively switching "on" and "off" the transport of DNA through the nanopore. Finally, a nanoscale dielectrophoretic nanotweezer device has been developed for high resolution manipulation and interrogation of individual entities. Two closely spaced carbon nanoelectrodes were embedded at the apex of a nanopipette. Voltage and frequency applied to the electrodes generated a highly localized force capable of trapping and manipulating a broad range of biomolecules. These dielectrophoretic nanotweezers were suitable for probing complex biological environments and a new technique for minimally invasive single-cell nanobiopsy was established. Such study provides encouraging results on how nanopipettebased platforms can be integrated as a future tool for routinely interrogating molecules at the nanoscale.Open Acces

Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo

Insect Rearing: Potential, Challenges, and Circularity

Environmental pollution, population increase, water availability and misuse of land are inexorably driving humans to take on important challenges related to sustainability. The next future is expected to see a significant increase of food and feed demands, which determines a serious threat to well-being levels and even survival of modern societies. Within this scenario, the efficient and sustainable use of insects as protein sources has been invoked as a possible strategic solution. As a candidate for remarkable growth, insect farming promises significant benefits to agri-food industry, offering interesting opportunities for implementing circular economy. In the present work, we review selected literature on insect rearing with the aim of providing a short rigorous introduction to the field to researchers, entrepreneurs and common readers. After a general overview of the field, including a description of insect nutritional values, the review focuses on the three insect species that are seemingly set to beneficially affect aquaculture, which is the activity presently more sensitive to circularity and sustainability innovation. Once traditional and advanced insect rearing methods are described, the challenges that the field is going to tackle are suitably highlighted

On-line hemofiltration in chronic renal failure: advantages and limits

The actual dialysis therapy offers a notable long-term survival and rehabilitation, but it is still far from normalizing the patient's quality of life as well as mortality and morbidity. The most widely used dialysis therapy is an almost exclusive diffusive treatment performed with low-flux cellulose memb­ranes with a dialysis dose targeted to a urea Kt/V of 1.2 or higher. The convective treatments, which use high­flux membranes, offer proven biological superiority over diffusive treatments, which are performed with bio-incompatible, low­flux membranes. Retrospective epidemiolo­gical studies have documented a reduction of morbidity and mortality with the use of high-flux membranes, but the results of the prospective studies comparing low-flux with high-flux treatments are still con­flicting. Cardiovascular instability during treatment sessions is a potential cause of morbidity and mortality for patients on dialysis treatment. Hemofiltration (HF) is a pure convective treatment and offers the best tolerance to fluid subtraction in hemo­dynamically unstable patients. Because of its limitation in removing urea as well as high costs, HF treatment is restricted to few high risk unstable patients. The modern pre­dilution HF, performed with ultrapure on­line prepared solutions, overcomes, at least partially, the above limitation, but there is scarcity of data evaluating its long-term efficacy in stable patients

Gated single-molecule transport in double-barreled nanopores

Single-molecule methods have been rapidly developing with the appealing prospect of transforming conventional ensemble-averaged analytical techniques. However, challenges remain especially in improving detection sensitivity and controlling molecular transport. In this article, we present a direct method for the fabrication of analytical sensors that combine the advantages of nanopores and field-effect transistors for simultaneous label-free single-molecule detection and manipulation. We show that these hybrid sensors have perfectly aligned nanopores and field-effect transistor components making it possible to detect molecular events with up to near 100% synchronization. Furthermore, we show that the transport across the nanopore can be voltage-gated to switch on/off translocations in real time. Finally, surface functionalization of the gate electrode can also be used to fine tune transport properties enabling more active control over the translocation velocity and capture rates