The existence of life in extreme conditions, in particular in extraterrestrial environments, is certainly one of the most intriguing scientific questions of our time. In this report, we demonstrate the use of an innovative nanoscale motion sensor in life-searching experiments in Earth-bound and interplanetary missions. This technique exploits the sensitivity of nanomechanical oscillators to transduce the small fluctuations that characterize living systems. The intensity of such movements is an indication of the viability of living specimens and conveys information related to their metabolic activity. Here, we show that the nanomotion detector can assess the viability of a vast range of biological specimens and that it could be the perfect complement to conventional chemical life-detection assays. Indeed, by combining chemical and dynamical measurements, we could achieve an unprecedented depth in the characterization of life in extreme and extraterrestrial environments.nanomechanical sensors | extraterrestrial life | nanoscale fluctuations | living specimens | nanomotion detector T he existence of life in extreme conditions, in particular in extraterrestrial environments, is certainly one of the most intriguing scientific questions of our time. Indeed, the work of many scientists and organizations is focused on the discovery and on the consequent study of extremophiles and extraterrestrial organisms. The direct research for these kinds of life forms is usually conducted by deploying robotic crafts. These man-made vessels contain a suite of scientific analytical instrumentation that is specifically conceived to trace life signatures contained in the geological record. For instance, the search for life in our solar system started in 1975 with the Viking program and continues today. Future missions are planned to explore the presence of life on satellites of the giant planets, such as Europa (Jupiter) or Titan and Enceladus (Saturn). The biological instrumentation that is included in these vessels is complex, but up to now, it is mainly devoted to the chemical detection of molecules involved in living metabolism, as we know it on Earth.In this report, we show how a technique, the nanomotion detector, can be used in new life-searching instrumentation in Earth-bound and interplanetary missions. The technique exploits the sensitivity of nanomechanical sensors to transduce the small movements that characterize living systems. The intensity of such movements is an indication of the viability of the specimens and conveys information related to their metabolic activity. Here, we demonstrate that this simple technique can assess the viability of a vast range of biological specimens and that it could be the perfect complement to conventional chemical assays. Moreover, due to the simplicity of its working principle, a device based on this technology has negligible weight and requires very low electrical power, compared with other life-detector systems.Nanomechanical oscillators are extremely sensitive devices that are commonl...

Peptides have the specificity and size required to target the protein-protein interactions involved in many diseases. Some cyclic peptides have been utilised as scaffolds for peptide drugs because of their stability; however, other cyclic peptide scaffolds remain to be explored. θ-Defensins are cyclic peptides from mammals; they are characterised by a cyclic cystine ladder motif and have low haemolytic and cytotoxic activity. Here we demonstrate the potential of the cyclic cystine ladder as a scaffold for peptide drug design by introducing the integrin-binding Arg-Gly-Asp (RGD) motif into the θ-defensin RTD-1. The most active analogue had an IC50 of 18 nM for the αv β3 integrin as well as high serum stability, thus demonstrating that a desired bioactivity can be imparted to the cyclic cystine ladder. This study highlights how θ-defensins can provide a stable and conformationally restrained scaffold for bioactive epitopes in a β-strand or turn conformation. Furthermore, the symmetry of the cyclic cystine ladder presents the opportunity to design peptides with dual bioactive epitopes to increase activity and specificity.

By combining the nanomotion sensor with the rapid preparation method of blood culture pellets, we obtained an innovative, rapid and relatively accurate method for antibiotic susceptibility test directly from positive blood culture bottles, without the need for bacterial subculture.

Infectious diseases are caused by pathogenic microorganisms and are often severe. Time to fully characterize an infectious agent after sampling and to find the right antibiotic and dose are important factors in the overall success of a patient's treatment. Previous results suggest that a nanomotion detection method could be a convenient tool for reducing antibiotic sensitivity characterization time to several hours. Here, the application of the method for slow-growing bacteria is demonstrated, taking Bordetella pertussis strains as a model. A low-cost nanomotion device is able to characterize B. pertussis sensitivity against specific antibiotics within several hours, instead of days, as it is still the case with conventional growth-based techniques. It can discriminate between resistant and susceptible B. pertussis strains, based on the changes of the sensor's signal before and after the antibiotic addition. Furthermore, minimum inhibitory and bactericidal concentrations of clinically applied antibiotics are compared using both techniques and the suggested similarity is discussed.

The insurgence of newly arising, rapidly developing health threats, such as drug‐resistant bacteria and cancers, is one of the most urgent public‐health issues of modern times. This menace calls for the development of sensitive and reliable diagnostic tools to monitor the response of single cells to chemical or pharmaceutical stimuli. Recently, it has been demonstrated that all living organisms oscillate at a nanometric scale and that these oscillations stop as soon as the organisms die. These nanometric scale oscillations can be detected by depositing living cells onto a micro‐fabricated cantilever and by monitoring its displacements with an atomic force microscope‐based electronics. Such devices, named nanomotion sensors, have been employed to determine the resistance profiles of life‐threatening bacteria within minutes, to evaluate, among others, the effect of chemicals on yeast, neurons, and cancer cells. The data obtained so far demonstrate the advantages of nanomotion sensing devices in rapidly characterizing microorganism susceptibility to pharmaceutical agents. Here, we review the key aspects of this technique, presenting its major applications. and detailing its working protocols.

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Scanning electrochemical microscopy (SECM) has been widely used for the electrochemical imaging of dynamic topographical and metabolic changes in alive adherent mammalian cells. However, extracting intracellular information by SECM is challenging, since it requires redox species to travel in and out the lipid cell membrane. Herein, we present cell fixation and permeabilization approaches as an alternative tool for visualizing cell properties by SECM. With this aim, adherent cells were analyzed in the SECM feedback mode in three different conditions: (i) alive; (ii) fixed, and (iii) fixed and permeabilized. The fixation was carried out with formaldehyde and does not damage lipid membranes. Therefore, this strategy can be used for the SECM investigation of cell topography or the passive transport of the redox mediator into the cells. Additional permeabilization of the cell membrane after fixation enables the analysis of the intracellular content through the coupling of SECM with immunoassay strategies for the detection of specific biomarkers. The latter was successfully applied as an easy and fast screening approach to detect the expression of the melanoma-associated marker tyrosinase in adherent melanoma cell lines corresponding to different cancer progression stages using the SECM substrate generation–tip collection mode. The present approach is simple, fast, and reliable and can open new ways to analyze cell cultures with electrochemically based scanning probe techniques.

Abstract. Our team recently demonstrated that cantilever based devices can detect signature of life in a chemistry independent manner. In this technique, the organism of interest is attached onto a classical AFM cantilever. If alive, it induces nanometre scale oscillations of the cantilever that disappear once the organism is killed. The technique was successfully used on bacteria, yeast, vegetal and mammalian cells. In this work we demonstrate that the method can also be applied to sub-cellular organelles, such as mitochondria. Mitochondria are involved in cellular energy production and are present in most eukaryotic cells. Nowadays, it is believed that mitochondria were originally prokaryotes that colonized eukaryotic cells and that live in an endosymbiotic way ever since. Here we present that mitochondria are also animated by nanometre scale oscillations that depend on their metabolic state and that stop once they are inhibited. This observation opens novel avenues to investigate the numerous mitochondria-related diseases in humans.