APHRODITE: A Compact Lab-on-Chip Biosensor for the Real-Time Analysis of Salivary Biomarkers in Space Missions.

One of the main challenges to be faced in deep space missions is to protect the health and ensure the maximum efficiency of the crew by preparing methods of prevention and in situ diagnosis. Indeed, the hostile environment causes important health problems, ranging from muscle atrophy, osteopenia, and immunological and metabolic alterations due to microgravity, to an increased risk of cancer caused by exposure to radiation. It is, therefore, necessary to provide new methods for the real-time measurement of biomarkers suitable for deepening our knowledge of the effects of space flight on the balance of the immune system and for allowing the monitoring of the astronaut's health during long-term missions. APHRODITE will enable human space exploration because it fills this void that affects both missions in LEO and future missions to the Moon and Mars. Its scientific objectives are the design, production, testing, and in-orbit demonstration of a compact, reusable, and reconfigurable system for performing the real-time analysis of oral fluid samples in manned space missions. In the frame of this project, a crew member onboard the ISS will employ APHRODITE to measure the selected target analytes, cortisol, and dehydroepiandrosterone sulfate (DHEA-S), in oral fluid, in four (plus one additional desired session) separate experiment sessions. The paper addresses the design of the main subsystems of the analytical device and the preliminary results obtained during the first implementations of the device subsystems and testing measurements on Earth. In particular, the system design and the experiment data output of the lab-on-chip photosensors and of the front-end readout electronics are reported in detail along with preliminary chemical tests for the duplex competitive CL-immunoassay for the simultaneous detection of cortisol and DHEA-S. Different applications also on Earth are envisaged for the APHRODITE device, as it will be suitable for point-of-care testing applications (e.g., emergency medicine, bioterrorism, diagnostics in developing countries, etc.).

Microfluidic-Based Non-Invasive Wearable Biosensors for Real-Time Monitoring of Sweat Biomarkers.

Wearable biosensors are attracting great interest thanks to their high potential for providing clinical-diagnostic information in real time, exploiting non-invasive sampling of biofluids. In this context, sweat has been demonstrated to contain physiologically relevant biomarkers, even if it has not been exhaustively exploited till now. This biofluid has started to gain attention thanks to the applications offered by wearable biosensors, as it is easily collectable and can be used for continuous monitoring of some parameters. Several studies have reported electrochemical and optical biosensing strategies integrated with flexible, biocompatible, and innovative materials as platforms for biospecific recognition reactions. Furthermore, sampling systems as well as the transport of fluids by microfluidics have been implemented into portable and compact biosensors to improve the wearability of the overall analytical device. In this review, we report and discuss recent pioneering works about the development of sweat sensing technologies, focusing on opportunities and open issues that can be decisive for their applications in routine-personalized healthcare practices.

Smartphone-Based Chemiluminescence Glucose Biosensor Employing a Peroxidase-Mimicking, Guanosine-Based Self-Assembled Hydrogel.

Chemiluminescence is widely used for hydrogen peroxide detection, mainly exploiting the highly sensitive peroxidase-luminol-H2O2 system. Hydrogen peroxide plays an important role in several physiological and pathological processes and is produced by oxidases, thus providing a straightforward way to quantify these enzymes and their substrates. Recently, biomolecular self-assembled materials obtained by guanosine and its derivatives and displaying peroxidase enzyme-like catalytic activity have received great interest for hydrogen peroxide biosensing. These soft materials are highly biocompatible and can incorporate foreign substances while preserving a benign environment for biosensing events. In this work, a self-assembled guanosine-derived hydrogel containing a chemiluminescent reagent (luminol) and a catalytic cofactor (hemin) was used as a H2O2-responsive material displaying peroxidase-like activity. Once loaded with glucose oxidase, the hydrogel provided increased enzyme stability and catalytic activity even in alkaline and oxidizing conditions. By exploiting 3D printing technology, a smartphone-based portable chemiluminescence biosensor for glucose was developed. The biosensor allowed the accurate measurement of glucose in serum, including both hypo- and hyperglycemic samples, with a limit of detection of 120 µmol L-1. This approach could be applied for other oxidases, thus enabling the development of bioassays to quantify biomarkers of clinical interest at the point of care.

AstroBio-CubeSat: A lab-in-space for chemiluminescence-based astrobiology experiments.

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 paper-based 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.

Smartphone-based 3D-printed electrochemiluminescence enzyme biosensor for reagentless glucose quantification in real matrices.

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 µmol 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.

An Origami Paper-Based Biosensor for Allergen Detection by Chemiluminescence Immunoassay on Magnetic Microbeads.

Food allergies are adverse health effects that arise from specific immune responses, occurring upon exposure to given foods, even if present in traces. Egg allergy is one of the most common food allergies, mainly caused by egg white proteins, with ovalbumin being the most abundant. As allergens can also be present in foodstuff due to unintended contamination, there is a need for analytical tools that are able to rapidly detect allergens in food products at the point-of-use. Herein, we report an origami paper-based device for detecting ovalbumin in food samples, based on a competitive immunoassay with chemiluminescence detection. In this biosensor, magnetic microbeads have been employed for easy and efficient immobilization of ovalbumin on paper. Immobilized ovalbumin competes with the ovalbumin present in the sample for a limited amount of enzyme-labelled anti-ovalbumin antibody. By exploiting the origami approach, a multistep analytical procedure could be performed using reagents preloaded on paper layers, thus providing a ready-to-use immunosensing platform. The assay provided a limit of detection (LOD) of about 1 ng mL-1 for ovalbumin and, when tested on ovalbumin-spiked food matrices (chocolate chip cookies), demonstrated good assay specificity and accuracy, as compared with a commercial immunoassay kit.

Luminescent Aptamer-Based Bioassays for Sensitive Detection of Food Allergens.

The presence of hidden allergens in food products, often due to unintended contamination along the food supply chain (production, transformation, processing, and transport), has raised the urgent need for rapid and reliable analytical methods for detecting trace levels of such species in food products. Indeed, food allergens represent a high-risk factor for allergic subjects due to potentially life-threatening adverse reactions. Portable biosensors based on immunoassays have already been developed as rapid, sensitive, selective, and low-cost analytical platforms that can replace analyses with traditional bench-top instrumentation. Recently, aptamers have attracted great interest as alternative biorecognition molecules for bioassays, since they can bind a variety of targets with high specificity and selectivity, and they enable the development of assays exploiting a variety of transduction and detection technologies. In particular, aptasensors based on luminescence detection have been proposed, taking advantage of the development of ultrasensitive tracers and enhancers. This review aims to summarize and discuss recent efforts in the field of food allergen analysis using aptamer-based bioassays with luminescence detection.

Bioluminescence Sensing in 3D Spherical Microtissues for Multiple Bioactivity Analysis of Environmental Samples.

The development of predictive in vitro sensing tools able to provide rapid information on the different bioactivities of a sample is of pivotal importance, not only to monitor environmental toxicants, but also to understand their mechanisms of action on diverse molecular pathways. This mechanistic understanding is highly important for the characterization of toxicological hazards, and for the risk assessment of chemicals and environmental samples such as surface waters and effluents. Prompted by this need, we developed and optimized a straightforward bioluminescent multiplexed assay which enables the measurement of four bioactivities, selected for their relevance from a toxicological perspective, in bioluminescent microtissues. The assay was developed to monitor inflammatory, antioxidant, and toxic activity, and the presence of heavy metals, and was successfully applied to the analysis of river water samples, showing potential applicability for environmental analyses. The assay, which does not require advanced equipment, can be easily implemented in general laboratories equipped with basic cell culture facilities and a luminometer.

Portable light detectors for bioluminescence biosensing applications: A comprehensive review from the analytical chemist's perspective.

Bioluminescence, that is the emission of light in living organisms, has been extensively explored and applied for diverse bioanalytical applications, spanning from molecular imaging to biosensing. The unprecedented technological evolution of portable light detectors opened new possibilities to implement bioluminescence detection into miniaturized devices. We are witnessing a number of applications, including DNA sequencing, reporter gene assays, DNA amplification for point-of care and point-of need analyses relying on BL. Several photon detectors are currently available for measuring low light emission, such as photomultiplier tubes (PMT), charge-coupled devices (CCD), complementary metal oxide semiconductors (CMOS), single photon avalanche diodes (SPADs), silicon photomultipliers (SiPMs) and smartphone-integrated CMOS. Each technology has pros and cons and several issues, such as temperature dependence of the instrumental specific noise, the power supply, imaging capability and ease of integration, should be considered in the selection of the most appropriate detector for the selected BL application. These issues will be critically discussed from the perspective of the analytical chemist together with relevant examples from the literature with the goal of helping the reader in the selection and use of the most suitable detector for the selected application and to introduce non familiar readers into this exciting field.

Smartphone-Based Chemiluminescent Origami µPAD for the Rapid Assessment of Glucose Blood Levels.

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.

A Smartphone-Based Chemosensor to Evaluate Antioxidants in Agri-Food Matrices by In Situ AuNP Formation.

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).

Paper-Based Immunosensors with Bio-Chemiluminescence Detection.

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.

Recent Advancements in Enzyme-Based Lateral Flow Immunoassays.

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.

Smartphone biosensor for point-of-need chemiluminescence detection of ochratoxin A in wine and coffee.

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.

Bioluminescence goes portable: recent advances in whole-cell and cell-free bioluminescence biosensors.

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.

Precision medicine, bioanalytics and nanomaterials: toward a new generation of personalized portable diagnostics.

The customization of disease treatment focused on genetic, environmental and lifestyle factors of individual patients, including tailored medical decisions and treatments, is identified as precision medicine. This approach involves the combination of various aspects such as the collection and processing of a large amount of data, the selection of optimized and personalized drug dosage for each patient and the development of selective and reliable analytical tools for the monitoring of clinical, genetic and environmental parameters. In this context, miniaturized, compact and ultrasensitive bioanalytical devices play a crucial role for achieving the goals of personalized medicine. In this review, the latest analytical technologies suitable for providing portable and easy-to-use diagnostic tools in clinical settings will be discussed, highlighting new opportunities arising from nanotechnologies, offering peculiar perspectives and opportunities for precision medicine.

A challenge in biosensors: Is it better to measure a photon or an electron for ultrasensitive detection?

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.

Integrated chemiluminescence-based lab-on-chip for detection of life markers in extraterrestrial environments.

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.

Chemiluminescence-based biosensor for monitoring astronauts' health status during space missions: Results from the International Space Station.

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 comprises a 3D-printed plastic cartridge containing a sealed fluidic element with the LFIA strip, in which the flow of sample and reagents is activated by pressing buttons on the cartridge and sustained by exploiting capillary forces. For measurement, the photon emission is 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.

Miniaturized Biosensors to Preserve and Monitor Cultural Heritage: from Medical to Conservation Diagnosis.

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.