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Space exploration is facing a new era in view of the planned missions to the Moon and Mars. The development and the in-flight validation of new technologies, including analytical and diagnostic platforms, is pivotal for exploring and inhabiting these extreme environments. In this context, biosensors and lab-on-chip devices can play an important role in many situations, such as the analysis of biological samples for assessing the impact of deep space conditions on man and other biological systems, environmental and food safety monitoring, and the search of molecular indicators of past or present life in extra-terrestrial environments. Small satellites such as CubeSats are nowadays increasingly exploited as fast and low-cost platforms for conducting in-flight technology validation. Herein, we report the development of a fully autonomous lab-on-chip platform for performing chemiluminescence-based bioassays in space. The device was designed to be hosted onboard the AstroBio CubeSat nanosatellite, with the aim of conducting its in-flight validation and evaluating the stability of (bio) molecules required for bioassays in a challenging radiation environment. An origami-like microfluidic paperbased analytical format allowed preloading all the reagents in the dried form on the paper substrate, thus simplifying device design and analytical protocols, facilitating autonomous assay execution, and enhancing the stability of reagents. The chosen approach should constitute the first step to implement a mature technology with the aim to conduct life science research in space (e.g., for evaluation the effect of deep space conditions on living organisms or searching molecular evidence of life) more easily and at lower cost than previously possible.

Three-dimensional (3D) printed electrochemical devices are increasingly used in point-of-need and point-of-care testing. They show several advantages such as simple fabrication, low cost, fast response, and excellent selectivity and sensitivity in small sample volumes. However, there are only a few examples of analytical devices combining 3D-printed electrodes with electrochemiluminescence (ECL) detection, an electrochemical detection principle widely employed in clinical chemistry analysis. Herein, a portable, 3D-printed miniaturized ECL biosensor for glucose detection has been developed, based on the luminol/H2O2 ECL system and employing a two-electrode configuration with carbon black-doped polylactic acid (PLA) electrodes. The ECL emission is obtained by means of a 1.5V AA alkaline battery and detected using a smartphone camera, thus providing easy portability of the analytical platform. The ECL system was successfully applied for sensing H2O2 and, upon coupling the luminol/H2O2 system with the enzyme glucose oxidase, for glucose detection. The incorporation of luminol and glucose oxidase in an agarose hydrogel matrix allowed to produce ECL devices preloaded with the reagents required for the assay, so that the analysis only required sample addition. The ECL biosensor showed an excellent ability to detect glucose up to 5 mmol L-1, with a limit of detection of 60 mu mol L-1. The biosensor was also used to analyse real samples (i.e., glucose saline solutions and artificial serum samples) with satisfactory results, thus suggesting its suitability for point-of-care analysis. Coupling with other oxidases could further extend the applicability of this analytical platform.

Long-duration manned space missions require that astronauts be equipped with analytical devices suitable for performing on-site clinical-chemistry analysis. In the frame of the “IN SITU Bioanalysis” project, an analytical device for measuring the steroid hormone cortisol (an indicator of chronic stress) in saliva has been developed. The device performed a Lateral Flow Immunoassay (LFIA) for cortisol employing disposable plastic cartridges produced by FDM (Fused Deposition Modeling) 3D printing technology, which encapsulated a sealed fluidic element containing the LFIA strip and the reagents required for the analysis. Detection was performed by chemiluminescence (CL) using a dedicated CL reader equipped with a cooled charge-coupled device (CCD) camera. The European Space Agency (ESA) astronaut Paolo Nespoli successfully used the “IN SITU Bioanalysis” analytical device during the mission “VITA”, a 6-months space mission onboard the International Space Station (ISS) in the framework of a dated bilateral agreement between the Italian Space Agency (ASI—Agenzia Spaziale Italiana) and NASA. The experiments proved the feasibility of CL-LFIA ultrasensitive (i.e., at the sub-ng/mL level) quantitative bioassays in space. In perspective, the “IN SITU Bioanalysis” analytical device could be adapted to the measurement of other biomarkers of diagnostic and prognostic relevance, even in a multiplexed fashion (i.e., detection of multiple biomarkers in a single analysis) to develop panels tests for diagnosis and follow-up of pathologies or monitoring health status of astronauts.

N-substituted acridine-containing 1,2-dioxetanes have been recently proposed as thermochemiluminescence (TCL) universal labels for bioanalytical applications. The TCL properties of these compounds markedly depend on the nature of substituents of the acridine ring. In the attempt to obtain new TCL probes with improved properties and stability, the push-pull approach was adopted, in which both electron withdrawing and electron donating groups are present in the acridine moiety. The results have been useful to better understand the role of the different decorations at the acridine fluorophore in modulating the photophysical properties and the activation parameters of 1,2-dioxetane labels. Moreover, the great versatility and innovation of these molecules make them extremely attractive for bioanalytical applications, in particular as labels for immune- and gene-probe assays.

Microfluidic paper analytical devices (µPADs) represent one of the most appealing trends in the development of simple and inexpensive analytical systems for diagnostic applications at the point of care (POC). Herein, we describe a smartphone-based origami µPAD for the quantitative determination of glucose in blood samples based on the glucose oxidase-catalyzed oxidation of glucose leading to hydrogen peroxide, which is then detected by means of the luminol/hexacyanoferrate(III) chemiluminescent (CL) system. By exploiting the foldable µPAD format, a two-step analytical procedure has been implemented. First, the diluted blood sample was added, and hydrogen peroxide was accumulated, then the biosensor was folded, and a transport buffer was added to bring hydrogen peroxide in contact with CL reagents, thus promoting the CL reaction. To enable POC applicability, the reagents required for the assay were preloaded in the µPAD so that no chemicals handling was required, and a 3D-printed portable device was developed for measuring the CL emission using the smartphone’s CMOS camera. The µPAD was stable for 30-day storage at room temperature and the assay, displaying a limit of detection of 10 µmol L−1, proved able to identify both hypoglycemic and hyperglycemic blood samples in less than 20 min.

Introduction Astrobiology is an interdisciplinary field covered by only a few CubeSat missions so far. Moreover, no CubeSat mission has ever mounted miniaturized technology for the purpose of searching for molecular evidences of life in space. AstroBio CubeSat (ABCS) is a 3U CubeSat selected by the European Space Agency (ESA) to be launched in spring 2022 with the Vega C maiden flight, as piggy back passenger of the ASI LARES2 mission. ABCS will host a payload assembly based on Lab-on-Chip (LoC) technology for biomarkers detection and will be deployed along a circular orbit with altitude of about 5900 km and inclination of 7°, therefore crossing the inner Van Allen belt where the radiation flux is close to its maximum. Due to the harsh environment, ABCS payload and subsystems will be likely exposed to damages and degradations of electronics and performances, thus the payload assembly and the operational architecture were designed to be as much dependable as possible. This approach should constitute the first step to implement a mature technology with the aim to check the stability of chemicals and biomolecules involved in space experiments. This work reports an overview of ABCS architecture and the approach chosen for its operational design. ABCS Architecture ABCS objective is to test in space an automatic in-situ multiparameter LoC [1], which exploits luminol injection and enzymatic bio-mimicking assays on a functionalized 3D wax-printed origami. Luminol will be transported by capillarity to reaction sites with immobilized biomolecules targets where the reactions will trigger chemiluminescence, detected by means of hydrogenated amorphous silicon (a-Si:H) photodiodes deposited on a borosilicate glass substrate and connected to a photocurrent readout board [2]. The described payload consists in an experiment board hosting the LoC and a support board containing peristaltic pumps for luminol injection, drivers for pumps, radiation field effect transistors (RADFETs) and pressure/temperature sensors. The LoC architecture allows to repeat the experiment up to six times. In addition to RADFETs, ABCS mounts an ancillary radiation dose sensor (ARDS), developed by Thales Alenia Space, with the aim to assess the radiation effects. The ARDS is able to measure different amounts of current, until its failure, depending on the dose acquired. To mitigate the effects of the expected very high flux of charged particles, an extra tungsten layer shielding was mounted on each side panel and all the main subsystems (experiment and support board, batteries and EPS board, on-board computer (OBC), telemetry, tracking and control board), were placed inside a 5 mm thick aluminium box. At the same time, to keep the temperature range (from 4°C to 20°C) and operative pressure (about 1 atm) required to allow the LoC capillarity effect and to prevent reagents degradation, the box was sealed and a thermal control system, composed by a multi-layer insulation and an active heather mounted inside the box, was implemented. ABCS Mission Design ABCS will be deployed in an approximately circular orbit at about 5900 km altitude and 70° of inclination, spending a significant amount of the orbital period within the inner Van Allen belt, very close to its radiation peak point. ABCS ground operations will be mainly performed from the School of Aerospace Engineering (SIA) Ground Station. Simulations show that SIA will have access to ABCS 4 times a day, with an average duration of about 65 minutes. For this reason, a network of radioamateurs and third part ground stations will be involved for supporting the collection of the telemetry and science data packages and possibly uplink commands. ABCS Operations The assumption we made is that ABCS should be able to perform the payload operations in a completely autonomous manner. As we know, radiation flux will most likely induce several errors on electronics and performances, causing potential mission failure due to the fact that payload operations may not start because the OBC fails to send the command to start the experiment. A possible way to reduce failure is to perform ABCS experiments where the proton flux is lower. Simulations shows that this happens when ABCS is at polar latitudes, namely outside the range [-60°; 60°]. For this reason, the payload operations, based on redundant checks and triggers, were implemented accordingly. The purpose is to automatically determine if ABCS is at a latitude useful to perform the experiments and verifying this condition by means of multiple triggers, time or position based. Each trigger is used for scheduling purposes only if the ones with higher priority are unreliable. If all the triggers are not reliable, payload operations are forced to begin, as it is better to perform eventually degraded payload operations rather than performing no payload operations at all. Conclusions ABCS is required to operate in an extremely harsh environment where radiation fluxes are likely to degrade the electronic devices. Operations should be scheduled in order to reduce the time needed to perform all the experiments. The chosen approach will lead ABCS to complete the payload operations in three orbital periods, reducing the total ionizing dose absorbed and guaranteeing the higher system reliability. Acknowledgments ABCS AstroBio-CubeSat is supported by ASI - Italian Space Agency ASI/INAF Agreement n. 2019-30-HH.0. References [1] Iannascoli, L. et al. 2020, "Astrobio cubesat: Enabling technologies for astrobiology research in space", Proceedings of the International Astronautical Congress, IAC. [2] Mirasoli M, et al. 2014. Multiwell cartridge with integrated array of amorphous silicon photosensors for chemiluminescence detection: development, characterization and comparison with cooled-CCD luminograph. Anal Bioanal Chem. Sep;406(23):5645-56.

Since the introduction of paper-based analytical devices as potential diagnostic platforms a few decades ago, huge efforts have been made in this field to develop systems suitable for meeting the requirements for the point-of-care (POC) approach. Considerable progress has been achieved in the adaptation of existing analysis methods to a paper-based format, especially considering the chemiluminescent (CL)-immunoassays-based techniques. The implementation of biospecific assays with CL detection and paper-based technology represents an ideal solution for the development of portable analytical devices for on-site applications, since the peculiarities of these features create a unique combination for fitting the POC purposes. Despite this, the scientific production is not paralleled by the diffusion of such devices into everyday life. This review aims to highlight the open issues that are responsible for this discrepancy and to find the aspects that require a focused and targeted research to make these methods really applicable in routine analysis.

The large amount of cauliflower industry waste represents an unexplored source of bioactive compounds. In this work, peptide hydrolysates from cauliflower leaves were characterized by combined bioanalytical approaches. Twelve peptide fractions were studied to evaluate unexplored biological activities by effect-based cellular bioassays. A potent inhibition of intracellular xanthine oxidase activity was observed in human vascular endothelial cells treated with one fraction, with an IC50 = 8.3±0.6 μg/ml. A different fraction significantly induced the antioxidant enzyme superoxide dismutase 1 and decreased the tumor necrosis factorα-induced VCAM-1 expression, thus leading to a significant improvement in the viability of human vascular endothelial cells. Shotgun peptidomics and bioinformatics were used to retrieve the most probable bioactive peptide sequences. Our study shows that peptides from cauliflower waste should be recycled for producing valuable products useful for the prevention of endothelial dysfunction linked to atherogenesis progression.

Point-of-care testing devices for companion diagnostic applications is a growing area in immunobiosensor technology. With the widespread diffusion of smartphones and improved enclosed photocamera technology, fast and accurate point-of-care diagnosis could be developed for delivering optical biosensing abilities to the general population. Herein, we propose a smartphone-based immunosensor employing a paper-based format coupled with thermochemiluminescence (TCL) detection, that was optimized for valproic acid (VPA) detection in blood and saliva samples. TCL is a chemical luminescence phenomenon in which photons are emitted upon thermally-induced fragmentation of a suitable molecule, with production of a moiety in its singlet electronically excited state. The latter emits a photon while decaying to its ground state. TCL peculiar characteristics combine high detectability and reagent-less nature of the measurement. A one-step competitive immunoassay for VPA detection based on vertical flow immunoassay (VFIA) format was developed, employing silica nanoparticles doped with a TCL 1,2-dioxetane derivative as a label. The VFIA sensor is a stack of paper-based layers functionalized with reagents stored in a stable form, allowing to complete the test in 12 min simply upon sample addition. By 3D printing, simple accessories were produced to turn a smartphone into a biosensing device that provides a power source for the heat shock required to trigger the TCL reaction and a sensitive camera for measuring emitted photons. The developed biosensor allowed VPA detection in blood and saliva, with limits of detection (4 and 0.05 μg mL−1 respectively) and dynamic ranges (4–300 and 0.05–20 μg mL-1) suitable for therapeutic drug monitoring. The integrated device offers an innovative analytical platform for rapid one-step biosensors exploitable in a variety of point-of-care applications.

Recent advancements in synthetic biology, organic chemistry, and computational models have allowed the application of bioluminescence in several fields, ranging from well established methods for detecting microbial contamination to in vivo imaging to track cancer and stem cells, from cell-based assays to optogenetics. Moreover, thanks to recent technological progress in miniaturized and sensitive light detectors, such as photodiodes and imaging sensors, it is possible to implement laboratory-based assays, such as cell-based and enzymatic assays, into portable analytical devices for point-of-care and on-site applications. This review highlights some recent advances in the development of whole-cell and cell-free bioluminescence biosensors with a glance on current challenges and different strategies that have been used to turn bioassays into biosensors with the required analytical performance. Critical issues and unsolved technical problems are also highlighted, to give the reader a taste of this fascinating and challenging field.

In recent years, there has been a continuously growing interest in antioxidants by both customers and food industry. The beneficial health effects of antioxidants led to their widespread use in fortified functional foods, as dietary supplements and as preservatives. A variety of analytical methods are available to evaluate the total antioxidant capacity (TAC) of food extracts and beverages. However, most of them are expensive, time-consuming, and require laboratory instrumentation. Therefore, simple, cheap, and fast portable sensors for point-of-need measurement of antioxidants in food samples are needed. Here, we describe a smartphone-based chemosensor for on-site assessment of TAC of aqueous matrices, relying on the antioxidant-induced formation of gold nanoparticles. The reaction takes place in ready-to-use analytical cartridges containing an hydrogel reaction medium preloaded with Au(III) and is monitored by using the smartphone’s CMOS camera. An analytical device including an LED-based lighting system was developed to ensure uniform and reproducible illumination of the analytical cartridge. The chemosensor permitted rapid TAC measurements of aqueous samples, including teas, herbal infusions, beverages, and extra virgin olive oil extracts, providing results that correlated with those of the reference methods for TAC assessment, e.g., oxygen radical absorbance capacity (ORAC).

Exposure to mycotoxins, which may contaminate food and feed commodities, represents a serious health risk for consumers. Ochratoxin A (OTA) is one of the most abundant and toxic mycotoxins, thus specific regulations for fixing its maximum admissible levels in foodstuff have been established. Lateral Flow ImmunoAssay (LFIA)-based devices have been proposed as screening tools to avoid OTA contamination along the whole food chain. We report a portable, user-friendly smartphone-based biosensor for the detection and quantification of OTA in wine and instant coffee, which combines the LFIA approach with chemiluminescence (CL) detection. The device employs the smartphone camera as a light detector and uses low-cost, disposable analytical cartridges containing the LFIA strip and all the necessary reagents. The analysis can be carried out at the point of need by non-specialized operators through simple manual operations. The biosensor allows OTA quantitative detection in wine and coffee samples up to 25 μg L−1 and with limits of detection of 0.3 and 0.1 μg L−1, respectively, which are below the European law-fixed limits. These results demonstrate that the developed device can be used for routine monitoring of OTA contamination, enabling rapid and reliable identification of positive samples requiring confirmatory analysis.

IntroductionAstroBio CubeSat (ABCS) is an Italian Space Agency (ASI) 3U CubeSat (100x100x340 mm) selected by European Space Agency (ESA) to be launched with the Vega C qualification maiden flight, as piggy back of the ASI LARES2 main satellite, by the end of 2020. ABCS will be deployed in an approximately circular orbit, with about 5900 km altitude and 70° of inclination. It implies that ABCS will spend a significant part of the orbital period within the internal Van Allen belt, close to its maximum. The radiation environment is characterized by a very high flux of charged particles, which have a significant effect on electronic components in terms of permanent damages due to accumulated dose effects and single events. Considering the extremely harsh space conditions, the estimated mission lifetime useful to perform the payload experiments should be defined in 3 months.ABCS Project is funded and managed by ASI in cooperation with INAF-Astrophysical Observatory of Arcetri, that will coordinate the scientific and engineering team. Partners of the projects are the School of Aerospace Engineering of Sapienza University of Rome, the University of Bologna, the University of Torino, and Kayser Italia.ABCS PayloadABCS will host a mini laboratory payload based on an innovative lab-on chip technology suitable for research in astrobiology. The objective is to test in space environments an automatic laboratory able to provide a highly integrated in-situ multiparameter platform that uses immunoassay tests exploiting chemiluminescence detection by means of on-chip a-Si:H photodiodes. The experiment will consist in a set of lateral flow immunoassays (LFIA) on nitrocellulose support where target biomolecules are immobilized in specific test areas. Reagents are deposited in a non-permanent fashion and in a dry form in the initial part (starting area) of the microfluidic path. When the reagents-delivery-system provides a volume of liquid reagent to the starting pad, capillary forces will guide the reagents through the LFIA microfluidic pathway. During the flow, liquid reagents will solubilize and transport along the path the deposited reagents, triggering specific reactions and allowing the chemiluminescence detection by the photodiodes.ABCS also mounts an ancillary radiation dose payload, to investigate the degradation of of electronic components exposed to the space environment. The device has twin components protected by established radiation screens, kindly provided by Thales Alenia Space Italia and by CESI, so that the degradation can be assessed on the basis of the difference between the observed currents.ABCS architecture and payload are based on the strong heritage gained by the research team with the ground validation of the PLEIADES (Planetary Life Explorer with Integrated Analytical Detection and Embedded Sensors) instrument, an R&D ASI project recently concluded.Enviromental challengesThe main challenges of the project are to mitigate the effects of the expected very high flux of charged particles, keeping the correct temperature (4°C/25°C) and pressure (about 1 atm) range for the payload to prevent reagents degradation. This invoked a series of technological solution to protect the payload. The pressurized environment is ensured by an inner aluminium box, hosting both the experiment and the main subsystems (batteries, on-board data handling, telemetry, tracking and control) hermetically sealed and providing shielding from radiation and charged particles. A thermal control system, including a passive control multi-layer insulation and an active heather mounted inside the pressurized box, maintain the temperature in the desired range.ConclusionABCS mission aims at evaluating the overall system functionality (delivery of reagents, mixing of chemicals, LoC characterization, detection of emitted photons, readout noise, etc.) such as the chemicals and biomolecules stability (reagents and antibodies employed in the experiment) in the extremely harsh environment.The in-orbit validation of the proposed technology would represent a significant breakthrough for autonomous execution of bio-analytical experiments in space with potential application in search for signs of life in planetary exploration missions, space biolabs without human support, health monitoring in manned missions.

Lactic acid bacteria (LAB) &ldquo, fermentates&rdquo, confer a beneficial effect on intestinal function. However, the ability of new fermentations to improve LAB broth activity in preventing pathogen-induced intestinal inflammation and barrier dysfunction has not yet been studied. The objective of this study was to determine if broths of LAB fermented with Eruca sativa or Barbarea verna seed extracts prevent gut barrier dysfunction and interleukin-8 (CXCL8) release in vitro in human intestinal Caco-2 cells infected with enterohemorrhagic Escherichia coli (EHEC) O157:H7. LAB broths were assayed for their effects on EHEC growth and on Caco-2 viability, thereafter, their biological properties were analysed in a co-culture system consisting of EHEC and Caco-2 cells. Caco-2 cells infected with EHEC significantly increased CXCL8 release, and decreased Trans-Epithelial Electrical Resistance (TEER), a barrier-integrity marker. Notably, when Caco-2 cells were treated with LAB broth enriched with E. sativa seed extract and thereafter infected, both CXCL8 expression and epithelial dysfunction reduced compared to in untreated cells. These results underline the beneficial effect of broths from LAB fermented with E. sativa seed extracts in gut barrier and inflammation after EHEC infection and reveal that these LAB broths can be used as functional bioactive compounds to regulate intestinal function.

High polyphenols levels are one of the main quality characters of Extra Virgin Olive Oil (EVOO), leading to high antioxidant activity and contributing to the EVOO health beneficial effects. Analytical methods for the rapid assessment of EVOO polyphenols content, possibly directly at the place of production or distribution, are therefore important for the commercial enhancement of this product. Unfortunately, most analytical methods for quantification of polyphenols in EVOO require laboratory instrumentation and preliminary sample extraction procedures. In this paper we describe a paper-based chemosensor based on the well-known Folin-Ciocalteu colorimetric assay for the rapid (assay time 15 min) quantitative detection of polyphenols in EVOO without any preliminary sample extraction. Use of n-propanol for diluting the EVOO sample allows its direct analysis on paper supports loaded with the Folin-Ciocalteu reagent. The color change of the paper supports is measured using a smartphone camera. A disposable analytical cartridge containing all necessary reagents, including calibration standards, and accessories for performing the assay using a Samsung S8 smartphone have been developed to perform on-site analysis. Measurement of total polyphenol content of EVOO samples gave results in good agreement with the conventional Folin-Ciocalteu assay, and the limit of detection was 30 μg gallic acid equivalents g−1 EVOO. The same approach can be employed to measure polyphenols in other oils of vegetal origin.

Chemiluminescence (CL) detection is widely employed in biosensors and miniaturized analytical devices since it offers high detectability and flexible device design (there are no geometry requirements for the measurement cell, except the ability to collect the largest fraction of emitted photons). Although the emission anisotropy phenomenon for an emitting dipole bound to the interface between two media with different refractive index is well known for fluorescence, it is still poorly investigated for CL reactions, in which the excited-state reaction products can diffuse in solution before the photon emission event. In this paper, we propose a simple method for the real-time evaluation of the CL emission anisotropy based on a radial array of optical fibers, embedded in a poly(methyl methacrylate) semicylinder and coupled with a Charge-Coupled Device (CCD) camera through a suitable interface. The polar-time evolutions of the CL emission have been studied for catalyzing enzymes immobilized onto a solid surface (heterogeneous configuration) or free in solution (homogeneous configuration). Evidence of the anisotropy phenomenon is observed, indicating that the lifetime of the excited-state products of the enzyme-catalyzed reactions is shorter than the time required for their diffusion in solution at a distance at which the CL can be considered isotropic. These results open new perspectives in the development of CL-based miniaturized analytical devices.

Thermochemiluminescence (TCL) is a potentially simple and sensitive detection principle, as the light emission is simply elicited by a thermally-triggered decomposition of a molecule to produce a singlet excited-state product. Here we report about TCL semiconductive Polymer Dots (TCL-Pdots) obtained by doping fluorescent cyano-polyphenylene vinylene (CN-PPV) Pdots with an acridin 1,2-dioxetane derivative. The TCL-Pdots showed remarkable stability over time and minimum leaching of the thermo-responsive species. Furthermore, detectability of TCL-Pdots was improved taking advantage of both the high number of 1,2-dioxetanes entrapped in each nanoparticle (about 20 molecules/Pdot) and the 5-fold enhancement of TCL emission due to energy transfer from the 1,2-dioxetane to the polymer matrix, which itself acted as an energy acceptor. Indeed, upon heating the TCL-Pdots to 110°C, the 1,2-dioxetane decomposes generating an acridanone product in its electronically excited state. The latter transfers its energy to the surrounding CN-PPV chains via Förster mechanism (ϕ(FRET) about 80%), resulting in intense yellow light emission (550 nm wavelength). We next conjugated streptavidin onto the surface of these TCL-Pdots and demonstrated their suitability for use in biological studies. In particular, we used TCL-Pdots as labels in a model non-competitive immunoassay for IgG detection, which has showed a LOD of 13 nM IgG and a dynamic range extending up to 230 nM. By combining the biocompatibility, brightness and tunability of Pdots fluorescence emission with the thermally-triggered reagentless light generation from TCL 1,2-dioxetanes, a broad panel of ultrabright TCL nanosystems could be designed for a variety of bioscience applications, even in multiplexed format.

Cosmetics analysis represents a rapidly expanding field of analytical chemistry as new cosmetic formulations are increasingly in demand on the market and the ingredients required for their production are constantly evolving. Each country applies strict legislation regarding substances in the final product that must be prohibited or regulated. To verify the compliance of cosmetics with current regulations, official analytical methods are available to reveal and quantitatively determine the analytes of interest. However, since ingredients, and the lists of regulated/prohibited substances, rapidly change, dedicated analytical methods must be developed ad hoc to fulfill the new requirements. Research focuses on finding innovative techniques that allow a rapid, inexpensive, and sensitive detection of the target analytes in cosmetics. Among the different methods proposed, immunological techniques are gaining interest, as they make it possible to carry out low-cost analyses on raw materials and finished products in a relatively short time. Indeed, immunoassays are based on the specific and selective antibody/antigen reaction, and they have been extensively applied for clinical diagnostic, alimentary quality control and environmental security purposes, and even for routine analysis. Since the complexity and variability of the matrices, as well as the great variety of compounds present in cosmetics, are analogous with those from food sources, immunological methods could also be applied successfully in this field. Indeed, this would provide a valid approach for the monitoring of industrial production chains even in developing countries, which are currently the greatest producers of cosmetics and the major exporters of raw materials. This review aims to highlight the immunological techniques proposed for cosmetics analysis, focusing on the detection of prohibited/regulated compounds, bacteria and toxins, and allergenic substances, and the identification of counterfeits.

Biosensor development exploiting various transduction principles is characterized by a strong competition to reach high detectability, portability and robustness. Nevertheless, a literature-based comparison is not possible, as different conditions are employed in each paper. Herein, we aim at evaluating which measurement, photons or electrons, yields better biosensor performance. Upon outlining an update in recent achievements to boost analytical performance, amperometry and chemiluminescence (CL)-based biosensors are directly compared employing the same biospecific reagents and analytical formats. Horseradish peroxidase (HRP) and hydrogen peroxide concentrations were directly measured, while glucose and mouse IgG were detected employing an enzyme paper-based biosensor and an immunosensor, respectively. Detectability was down to picomoles of hydrogen peroxide (4 pmol for CL and 210 pmol for amperometry) and zeptomoles of HRP (45 zmol for CL and 20 zmol for amperometry); IgG was detected down to 12 fM (CL) and 120 fM (amperometry), while glucose down to 17 μM (CL) and 40 μM (amperometry). Results showed that amperometric and CL biosensors offered similar detectability and analytical performance, with some peculiarities that suggest complementary application fields. As they generally provided slightly higher detectability and wider dynamic ranges, CL-based biosensors appear more suitable for point-of-care testing of clinical biomarkers, where detectability is crucial. Nevertheless, as high detectability in CL biosensors usually requires longer acquisition times, their rapidity will allocate electrochemical biosensors in real-time monitoring and wearable biosensors. The analytical challenge demonstrated that these biosensors have competitive and similar performance, and between photons and electrons the competition is still open.

The detection of life markers is a high priority task in the exploration of the Solar System. Biochips performing in-situ multiplex immunoassays are a very promising approach alternative to gas chromatography coupled with mass spectrometry. As part of the PLEIADES project, we present the development of a chemiluminescence-based, highly integrated analytical platform for the detection of biomarkers outside of the Earth. The PLEIADES device goes beyond the current lab-on-chip approaches that still require bulky external instrumentation for their operation. It exploits an autonomous capillary force-driven microfluidic network, an array of thin-film hydrogenated amorphous silicon photosensors, and chemiluminescence bioassays to provide highly sensitive analyte detection in a very simple and compact configuration. Adenosine triphosphate was selected as the target life marker. Three bioassay formats have been developed, namely (a) a bioluminescence assay exploiting a luciferase mutant with enhanced thermal and pH stability and (b and c) binding assays exploiting antibodies or functional nucleic acids (aptamers) as biospecific recognition elements and peroxidase or DNAzymes as chemiluminescence reporters. Preliminary results, showing limits of detection in the nanomolar range, confirm the validity of the proposed approach.

This paper reports the development of a miniaturized lab-on-glass,suitable for the on-chip detection of living cell bioluminescence and their on-chip thermal treatments. The glass substrate hosts, on one side, hydrogenated amorphous silicon diodes, working as both temperature sensors and photosensors, and, on the other side, transparent thin films acting as heating sources. The main challenge of the work is the determination of the correct fabrication recipes in order to satisfy the compatibility of different microelectronic steps. The measured uniformity of temperature distribution, sensitivity of the temperature sensors, reverse dark current and spectral response of the photosensors demonstrate the successful technological integration and the suitability of the developed lab-on-glass to control the cell temperature and detect the BL emission with high sensitivity.

During space missions, real-time monitoring of astronauts’ health status is of crucial importance and therefore there is a strong demand for simple analytical devices that astronauts can use to perform clinical chemistry analyses directly onboard. As part of the “IN SITU Bioanalysis” project, we designed a biosensor for analysing salivary levels of cortisol in astronauts, a marker of chronic stress. The biosensor is based on the Lateral Flow Immunoassay (LFIA) approach coupled with chemiluminescence (CL) detection and c o m p r i s e s a 3D-printed plastic cartridge containing a sealed fluidic element with the LFIA strip, in which the flow of sample and reagents i s activated by pressing buttons on the cartridge and sustained by exploiting capillary forces. For measurement, the photon emission i s imaged employing a CL reader based on an ultrasensitive cooled charge-coupled device (CCD) camera. The payload was designed to operate in microgravity and to withstand mechanical stress, such as take-off vibrations, and onboard depressurization events, while the microfluidics was developed considering alterations of physical phenomena occurring in microgravity, such as bubble formation, surface wettability and liquid evaporation. The biosensor, which was successfully used by the Italian astronaut Paolo Nespoli during the VITA mission (July-December 2017), demonstrated the feasibility of performing sensitive LFIA analysis of salivary cortisol down to 0.4 ng/mL directly onboard the International Space Station. It could be easily adapted for the analysis of other clinical biomarkers, thus enabling the early diagnosis of diseases and the timely activation of appropriate countermeasures.

Paper-based lateral-flow immunoassays (LFIAs) have achieved considerable commercial success and their impact in diagnostics is continuously growing. LFIA results are often obtained by visualizing by the naked eye color changes in given areas, providing a qualitative information about the presence/absence of the target analyte in the sample. However, this platform has the potential to provide ultrasensitive quantitative analysis for several applications. Indeed, LFIA is based on well-established immunological techniques, which have known in the last year great advances due to the combination of highly sensitive tracers, innovative signal amplification strategies and last-generation instrumental detectors. All these available progresses can be applied also to the LFIA platform by adapting them to a portable and miniaturized format. This possibility opens countless strategies for definitively turning the LFIA technique into an ultrasensitive quantitative method. Among the different proposals for achieving this goal, the use of enzyme-based immunoassay is very well known and widespread for routine analysis and it can represent a valid approach for improving LFIA performances. Several examples have been recently reported in literature exploiting enzymes properties and features for obtaining significative advances in this field. In this review, we aim to provide a critical overview of the recent progresses in highly sensitive LFIA detection technologies, involving the exploitation of enzyme-based amplification strategies. The features and applications of the technologies, along with future developments and challenges, are also discussed.

Great interest in new thermochemiluminescent (TCL) molecules, for example, in bioanalytical assays, has prompted the design and synthesis of a small library of more than 30 olefins to be subjected to photooxygenation, with the aim of obtaining new 1,2-dioxetane-based TCL labels with optimized properties. Fluorine atoms on the acridan system remarkably stabilize 1,2-dioxetanes when they are located in the 3- and/or 6-position (4 h and 4 i). On the other hand, 2,7-difluorinated acridan dioxetane (4 j) showed a significantly enhanced fluorescence quantum yield with respect to the unsubstituted dioxetane (4 a). Some of the synthesized olefins did not undergo singlet oxygen addition and a rationale was sought to ease the photooxygenation step, leading to the TCL dioxetanes. A chemometric approach has been adopted to exploit principal component analysis and linear discriminant analysis of the structural and electronic molecular descriptors obtained by DFT optimizations of olefins 3. This approach allows the steric and electronic parameters that govern dioxetane formation to be revealed.

The development of user-friendly devices for on site analysis and characterization of painting materials is one of the most challenging objectives in the diagnostics for cultural heritage. Thanks to the specificity of antigen–antibody reactions, immunological methods have been already successfully applied for the detection of proteins and for their localization within painting stratigraphies. Moreover, by combining the advantages of the immunological techniques with the high detectability offered by chemiluminescence detection, it has been possible to achieve good analytical performance and very low detection limits. This work was aimed at developing a portable analytical device for the detection of chicken ovalbumin (a protein found in egg tempera and in egg-based protective varnishes) in painting samples employing ready-to-use analytical cartridges and a thermoelectrically-cooled CCD camera as a chemiluminescence detector. The protein was extracted from using a simple procedure and revealed by a non-competitive immunoassay with chemiluminescence contact imaging detection. The assay was simple, fast and suitable for the detection of ovalbumin in small samples. This analytical system provided positive identification of ovalbumin in samples obtained from both fresh or artificially aged paint reconstructions and historical paintings. It might be easily employed in different contexts (such as small museums, restoration laboratories or even on site) by restorers to obtain prompt information during restoration actions, such as cleaning operations. In perspective this device could be also employed for the detection of other proteinaceous and organic painting components.

This work presents the development and testing of an integrated system for on-chip detection of thermochemiluminescent biomolecules. The activation energy of the reaction is provided by a transparent structure of thin film heaters deposited on one side of a glass substrate. Light, passing through the substrate, reaches an array of amorphous silicon differential structure deposited on the opposite side of the glass substrate. The structure is designed to perform differential current measurements between a light- shielded diode, whose current is sensitive only to temperature, and a photosensor, sensitive to both incident light and temperature. The device therefore balances the thermal variations of the photodiode current and reduces the dark-current noise. These features make the presented system very appealing as highly miniaturized micro-analytical devices for biosensing applications.

Hydrogen peroxide is an unavoidable by-product of cell metabolism, but when it is not properly managed by the body it can lead to several pathologies (e.g., premature aging, cardiovascular and neurodegenerative diseases, cancer). Several methods have been proposed for the measurement of intracellular H2O2 but none of them has proven to be selective. We developed a rapid all-in-one chemiluminescent bioassay for the quantification of H2O2 in living cells with a low limit of detection (0.15 μM). The method relies on an adamantylidene-1,2-dioxetane lipophilic probe containing an arylboronate moiety; upon reaction with H2O2 the arylboronate moiety is converted to the correspondent phenol and the molecule decomposes leading to an excited-state fragment that emits light. The probe has been successfully employed for quantifying intracellular H2O2 in living human endothelial, colon and keratinocyte cells exposed to different pro-oxidant stimuli (i.e., menadione, phorbol myristate acetate and lipopolysaccharide). Imaging experiments clearly localize the chemiluminescence emission inside the cells. Treatment of cells with antioxidant molecules leads to a dose-dependent decrease of intracellular H2O2 levels. As a proof of concept, the bioassay has been used to measure the antioxidant activity of extracts from Brassica juncea wastes, which contain glucosinolates, isothiocyanates and other antioxidant molecules.

The point-of-care testing concept has been exploited to design and develop portable and cheap bioanalytical systems that can be used on-site by conservators. These systems employ lateral flow immunoassays to simultaneously detect two proteins (ovalbumin and collagen) in artworks. For an in-depth study on the application of these portable biosensors, both chemiluminescent and colorimetric detections were developed and compared in terms of sensitivity and feasibility. The chemiluminescent system displayed the best analytical performance (that is, two orders of magnitude lower limits of detection than the colorimetric system). To simplify its use, a disposable cartridge was designed ad hoc for this specific application. These results highlight the enormous potential of these inexpensive, easy-to-use, and minimally invasive diagnostic tools for conservators in the cultural heritage field.

Long-duration space missions pose important health concerns for astronauts, especially regarding the adverse effects of microgravity and exposure to high-energy cosmic rays. The long-term maintenance of crew health and performance mainly relies on prevention, early diagnoses, condition management, and medical interventions in situ. In-flight biosensor diagnostic devices and medical procedures must use few resources and operate in a microgravity environment, which complicates the collection and management of biological samples. Moreover, the biosensors must be certified for in-flight operation according to strict design and safety regulations. Herein, we report on the state of the art and recent advances in biosensing diagnostic instrumentation for monitoring astronauts' health during long-duration space missions, including portable and wearable biosensors. We discuss perspectives on new-format biosensors in autonomous space clinics. We also describe our own work in developing biosensing devices for non-invasively diagnosing space-related diseases, and how they are used in long-duration missions. Finally, we discuss the benefits of space exploration for Earth-based medicine.

The present paper describes the development of an integrated lab-on-chip, in which viral DNA amplification with real-time on-chip detection is carried out under constant temperature of 65 °C. The lab-on-chip is composed of a disposable 10-μL polydimethylsiloxane reaction chamber which is thermally and optically coupled to a glass substrate that hosts a thin-film metallic resistive heater and thin-film amorphous silicon diodes which act as temperature and radiation sensors. A loop-mediated isothermal amplification (LAMP) technique was optimized to specifically amplify parvovirus B19 DNA and coupled with Bioluminescent Assay in Real Time (BART) technology to provide real-time detection of target DNA. The experimental results demonstrate the ability of the proposed device to discriminate among different concentrations of viral DNA with an excellent agreement with standard off-chip methods.

On-chip optical detection of chemiluminescent reactions is presented. The device is based on the integration of thin film hydrogenated amorphous silicon photosensors on a functionalized glass substrate ensuring both a good optical coupling and an optimal separation between biological or chemical reagents and the sensing elements. The sensor has been characterized and optimized using the chemiluminescent system composed by the enzyme horseradish peroxidase (HRP) and luminol/peroxide/enhancer cocktail. The detectability of HRP is at the attomole level with a sensitivity of 1.46 fA/fg. Experiments, involving the detection of immobilized bio-specific probes on the functionalized surface have been performed both in bulk and microfluidics regime, proving the ability of the system to effectively detect chemiluminescent reactions and their kinetics. In particular, results achieved using conventional polydimethylsiloxane microfluidics for samples and reagents handling confirmed the good detection capabilities of the proposed system.

Doped organically modified silica nanoparticles (ORMOSIL NPs) with luminescent molecules represent a potent approach to signal amplification in biomolecule labeling. Herein, we report the synthesis of new ORMOSIL NPs incorporating thermochemiluminescent (TCL) 1,2-dioxetane derivatives to prepare TCL labels for ultrasensitive immunoassay, displaying a detectability comparable to those offered by other conventional luminescence-based systems. Amino-functionalized ORMOSIL NPs were synthesized for inclusion of acridine-containing 1,2-dioxetane derivatives with a fluorescence energy acceptor. The doped ORMOSIL NPs were further functionalized with biotin for binding to streptavidin-labeled species to be used as universal detection reagents for immunoassays. A quantitative non-competitive immunoassay for streptavidin has been developed by immobilizing anti-streptavidin antibody to capture streptavidin, then the antibody-bound streptavidin was detected by the biotinylated TCL ORMOSIL NPs. The analytical performance was similar to that obtained by chemiluminescent (CL) detection using horseradish peroxidase (HRP) as label, being the limits of detection 2.5-3.8 and 0.8 ng mL(-1) for TCL and CL detection, respectively. In addition, since the TCL emission is simply initiated by thermolysis of the label, chemical reagents were not required, thus allowing reagentless detection with a simplification of the analytical protocols. A compact mini dark box device based on the use of a cooled charge-coupled device (CCD) and a miniaturized heater has been developed and used to quantify the light emission after heat decomposition of the label at a temperature of 90-120 °C. These characteristics make TCL-doped ORMOSIL NPs ideal universal nanoprobes for ultrasensitive bioassays such as immuno- and DNA-based assay.