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.

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.

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.

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.

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.

Comprehensive characterization of gold nanoparticles and their protein conjugates used as a label by hollow fiber flow field flow fractionation with photodiode array and fluorescence detectors and multiangle light scattering.

Most of lateral flow immunoassay (LFIA) devices rely on gold nanoparticles (GNP) labeled antibodies or other biospecific proteins, to achieve reagent-less color-based detection. GNP size, GNP-protein conjugation level and its stability are crucial points for the development of precise and accurate methods. In addition, the purification of the GNP-protein conjugates from unreacted protein and GNP, is necessary for adequate analytical performance of the assay. To assist the synthesis and production process of GNP and their protein conjugates, we use for the first time a non-destructive, particle separation-multi-detection approach based on miniaturized flow field flow fractionation (HF5). A separation method was developed to baseline size-separate GNP, GNP-protein, protein and GNP including BSA used as a surface coater in less than 30 minutes. Freshly synthesized GNP were first characterized and then conjugated with two different model antibodies: a mouse immunoglobulin (IgG) and a fluorescein-labeled mouse immunoglobulin (FITC-IgG). The IgG-GNP complexes were fractionated using the HF5 apparatus, able to separate IgG-GNP from free proteins by their hydrodynamic size, allowing purification of the conjugation product. Both IgG-GNPs and GNPs were characterized according to their size by the MALS detector, and according to their Surface Plasmon Resonance and spectrum by UV-Vis detection, improving the results obtained via batch characterization. This simple non-invasive approach is very useful for the LFIA development and optimization: the use of HF5-mutidetection offers a unique tool for this purpose facilitating the industrialization of the process and the relate optimization and standardization.