Although lifetimes and quantum yields of widely used fluorophores are often largely characterized, a systematic approach providing a rationale of their photophysical behavior on a quantitative basis is still a challenging goal. Here we combine methods rooted in the time-dependent density functional theory and fluorescence lifetime imaging microscopy to accurately determine and analyze fluorescence signatures (lifetime, quantum yield, and band peaks) of several commonly used rhodamine and pyronin dyes. We show that the radiative lifetime of rhodamines can be correlated to the charge transfer from the phenyl toward the xanthene moiety occurring upon the S(0) ← S(1) de-excitation, and to the xanthene/phenyl relative orientation assumed in the S(1) minimum structure, which in turn is variable upon the amino and the phenyl substituents. These findings encourage the synergy of experiment and theory as unique tool to design finely tuned fluorescent probes, such those conceived for modern optical sensors.

N-Benzyloxyethyl cyclic alpha-peptoids of various size were prepared and their conformational features were investigated by means of computational, spectroscopic, and X-ray crystallographic studies.

We present novel microgels as a particle-based suspension array for direct and absolute microRNA (miRNA) detection. The microgels feature a flexible molecular architecture, antifouling properties, and enhanced sensitivity with a large dynamic range of detection. Specifically, they possess a core-shell molecular architecture with two different fluorescent dyes for multiplex spectral analyses and are endowed with a fluorescent probe for miRNA detection. Encoding and detection fluorescence signals are distinguishable by nonoverlapping emission spectra. Tunable fluorescence probe conjugation and emission confinement on single microgels allow for ultrasensitive miRNA detection. Indeed, the suspension array has high selectivity and sensitivity with absolute quantification, a detection limit of 10(-15) M, a dynamic range from 10(-9) to 10(-15) M, and higher accuracy than qRT-PCR. The antifouling properties of the microgels also permit the direct measurement of miRNAs in serum, without sample pretreatment or target amplification. A multiplexed assay has been tested for a set of miRNAs chosen as cancer biomarkers.

Precision medicine is continuously demanding for novel point of care systems, potentially exploitable also for in-vivo analysis. Biosensing probes based on Lab-On-Fiber Technology have been recently developed to meet these challenges. However, devices exploiting standard label-free approaches (based on ligand/target molecule interaction) suffer from low sensitivity in all cases where the detection of small molecules at low concentrations is needed. Here we report on a platform developed through the combination of Lab-On-Fiber probes with microgels, which are directly integrated onto the resonant plasmonic nanostructure realized on the fiber tip. In response to binding events, the microgel network concentrates the target molecule and amplifies the optical response, leading to remarkable sensitivity enhancement. Moreover, by acting on the microgel degrees of freedom such as concentration and operating temperature, it is possible to control the limit of detection, tune the working range as well as the response time of the probe. These unique characteristics pave the way for advanced label-free biosensing platforms, suitably reconfigurable depending on the specific application.In biochemical sensing field, Lab-on -Fiber (LOF) based devices essentially consist on the combination of optical resonant nanostructures (typically patterned metallic slab supporting surface plasmon resonances (SPR)) and functional coating materials integrated on the optical fiber tip [1][2][3][4][5][6][7] . LOF technology is continuously leading to the development of novel biosensing probes with unique properties in term of size, weight and ease of interrogation 4,5 . In addition to point of care applications, LOF based devices seem to be particularly promising for in-vivo analysis systems, thanks to the intrinsic properties of optical fibers that make them easily integrable inside medical catheters or needles 8 . Typically, the working principle of LOF probes relies on the affinity interaction of a ligand attached to the sensor surface with the target molecule present in a liquid solution at a certain concentration. However, standard label-free approaches fail when target molecules are small, for example about a few hundreds of dalton. In that case, the ligand/analyte binding process produces a biological layer that is not thick enough for providing a local refractive index (RI) change that is optically detectable by the sensor. Analogous issues occur in such applications where the detection of larger analytes with very low limit of detection (LOD) is required. To enhance the sensitivity, gold and magnetic nanoparticles have been proposed as "molecular concentrators" able to localize multiple binding events on a single particle, and successively deliver target analyte from the solution to the sensor surface [9][10][11] . At the same time, approaches exploiting hydrogels (HGs) have been proposed 12,13 . HGs basically allow to: i) increase the analyte loading capacity by translating a conventional 2D interaction surface into a 3D volume inter...

Combination of responsive microgels and photonic resonant nanostructures represents an intriguing technological tool for realizing tunable and reconfigurable platforms, especially useful for biochemical sensing applications. Interaction of light with microgel particles during their swelling/shrinking dynamics is not trivial because of the inverse relationships between their size and refractive index. In this work, we propose a reliable analytical model describing the optical properties of closed-packed assembly of surface-attached microgels, as a function of the external stimulus applied. The relationships between the refractive index and thickness of the equivalent microgel slab are derived from experimental observations based on conventional morphological analysis. The model is first validated in the case of temperature responsive microgels integrated on a plasmonic lab-on-fiber optrode, and also implemented in the same case study for an optical responsivity optimization problem. Overall, our model can be extended to other photonic platforms and different kind of microgels, independently from the nature of the stimulus inducing their swelling.

The integration of nanostructures able to manipulate light at the optical fiber tip is bringing the optical fibers to a renewed dimension since their beginnings. The past decade has seen unprecedented advancements in the lab-onfiber technology field, pushed by the effective exploitation of nanoscale optical physics and supported by the improvement of nanofabrication techniques. In this context, here we report on a cavity enhanced lab-on-fiber optrode, which dramatically boosts the performances and widens the range of applications of the current lab-on-fiber systems, setting a new fundamental milestone along the roadmap of this technology. The "lab" integrated onto the fiber tip consists of a tunable optical cavity incorporating smart materials as the active layer. Specifically, the swelling/collapsing mechanism of multiresponsive microgels sandwiched in between gold layers induces the interplay between plasmonic resonances and cavity modes, according to optical cavity size. The combination of the optically resonant effects and microgels endows the optrode with the unique ability to work as both a sensor for detecting small molecules and a nano-opto-mechanical actuator triggered by light. In the specific case study here presented, we show that the device is able to detect glucose in solution, with a sensitivity improved by more than 1 order of magnitude compared to other state-of-the-art values. Moreover, we demonstrate that, by combining thermoplasmonics effects triggered by light coupled into the fiber and the microgel thermo-responsivity, it is possible to tune and control the optical cavity features, opening new avenues toward active nano-opto-mechanical actuators directly realized onto the fiber tip.

Integrating multi-responsive polymers such as microgels onto optical fiber tips, in a controlled fashion, enables unprecedented functionalities to Lab-on-fiber optrodes. The creation of a uniform microgel monolayer with a specific coverage factor is crucial for enhancing the probes responsivity to a pre-defined target parameter. Here we report a reliable fabrication strategy, based on the dip coating technique, for the controlled realization of microgel monolayer onto unconventional substrates, such as the optical fiber tip. The latter was previously covered by a plasmonic nanostructure to make it sensitive to superficial environment changes. Microgels have been prepared using specific Poly(N-isopropylacrylamide)-based monomers that enable bulky size changes in response to both temperature and pH variations. The formation of the microgel monolayer is efficiently controlled through the selection of suitable operating pH, temperature and concentration of particle dispersions used during the dipping procedure. The effect of each parameter has been evaluated, and the validity of our procedure is confirmed by means of both morphological and optical characterizations. We demonstrate that when the coverage factor exceeds 90%, the probe responsivity to microgels swelling/collapsing is significantly improved. Our study opens new paradigms for the development of engineered microgels assisted Lab-on-Fiber probes for biochemical applications.

In this paper, we report on a general approach for the detection of a specific tumoural biomarker directly in serum. Such detection is made possible using a protein-binding peptide selected through an improved phage display technique and then conjugated to engineered microparticles (MPs). Protein biomarkers represent an unlimited source of information for non-invasive diagnostic and prognostic tests; MP-based assays are becoming largely used in manipulation of soluble biomarkers, but their direct use in serum is hampered by the complex biomolecular environment. Our technique overcomes the current limitations as it produces a selective MP-engineered with an antifouling layer-that 'captures' the relevant protein staying impervious to the background. Our system succeeds in fishing-out the human tumour necrosis factor alpha directly in serum with a high selectivity degree. Our method could have great impact in soluble protein manipulation and detection for a wide variety of diagnostic applications.