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.).

On the Dependency of the Electromechanical Response of Rotary MEMS/NEMS on Their Embedded Flexure Hinges' Geometry.

This paper investigates how the electromechanical response of MEMS/NEMS devices changes when the geometrical characteristics of their embedded flexural hinges are modified. The research is dedicated particularly to MEMS/NEMS devices which are actuated by means of rotary comb-drives. The electromechanical behavior of a chosen rotary device is assessed by studying the rotation of the end effector, the motion of the comb-drive mobile fingers, the actuator's maximum operating voltage, and the stress sustained by the flexure when the flexure's shape, length, and width change. The results are compared with the behavior of a standard revolute joint. Outcomes demonstrate that a linear flexible beam cannot perfectly replace the revolute joint as it induces a translation that strongly facilitates the pull-in phenomenon and significantly increases the risk of ruptures of the comb-drives. On the other hand, results show how curved beams provide a motion that better resembles the revolute motion, preserving the structural integrity of the device and avoiding the pull-in phenomenon. Finally, results also show that the end effector motion approaches most precisely the revolute motion when a fine tuning of the beam's length and width is performed.

Sensitive and Compact Evanescent-Waveguide Optical Detector for Sugar Sensing in Commercial Beverages.

This work presents a compact and sensitive refractive index sensor able to evaluate the concentration of an analyte in a sample. Its working principle leverages on the changes in the optical absorption features introduced by the sample itself on the evanescent waves of a light beam. The device's high compactness is achieved by embedding the sample-light interaction site and the detector in a 1 cm2 glass substrate, thanks to microelectronics technologies. High sensitivity is obtained by employing a low-noise p-i-n hydrogenated amorphous silicon junction, whose manufacture process requires only four UV lithographic steps on a glass substrate, thus ensuring low production costs. The system's capabilities are investigated by sensing the sugar content in three commercial beverages. Sensitivities of 32, 53 and 80 pA/% and limits of detection of 47, 29 and 18 ppm are achieved. The above performance is comparable with state-of-the-art results available in the literature, where more complex optical setups, expensive instrumentation and bulky devices are used.

Gold Nanoparticles-Functionalized Cotton as Promising Flexible and Green Substrate for Impedometric VOC Detection.

This work focuses on the possible application of gold nanoparticles on flexible cotton fabric as acetone- and ethanol-sensitive substrates by means of impedance measurements. Specifically, citrate- and polyvinylpyrrolidone (PVP)-functionalized gold nanoparticles (Au NPs) were synthesized using green and well-established procedures and deposited on cotton fabric. A complete structural and morphological characterization was conducted using UV-VIS and Fourier transform infrared (FT-IR) spectroscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM). A detailed dielectric characterization of the blank substrate revealed interfacial polarization effects related to both Au NPs and their specific surface functionalization. For instance, by entirely coating the cotton fabric (i.e., by creating a more insulating matrix), PVP was found to increase the sample resistance, i.e., to decrease the electrical interconnection of Au NPs with respect to citrate functionalized sample. However, it was observed that citrate functionalization provided a uniform distribution of Au NPs, which reduced their spacing and, therefore, facilitated electron transport. Regarding the detection of volatile organic compounds (VOCs), electrochemical impedance spectroscopy (EIS) measurements showed that hydrogen bonding and the resulting proton migration impedance are instrumental in distinguishing ethanol and acetone. Such findings can pave the way for the development of VOC sensors integrated into personal protective equipment and wearable telemedicine devices. This approach may be crucial for early disease diagnosis based on nanomaterials to attain low-cost/low-end and easy-to-use detectors of breath volatiles as disease markers.

Biomolecular Monitoring Tool Based on Lab-on-Chip for Virus Detection.

Lab-on-Chip (LoC) devices for performing real-time PCR are advantageous compared to standard equipment since these systems allow to conduct in-field quick analysis. The development of LoCs, where the components for performing the nucleic acid amplification are all integrated, can be an issue. In this work, we present a LoC-PCR device where thermalization, temperature control and detection elements are all integrated on a single glass substrate named System-on-Glass (SoG) obtained using metal thin-film deposition. By using a microwell plate optically coupled with the SoG, real-time reverse transcriptase PCR of RNA extracted from both a plant and human virus has been carried out in the developed LoC-PCR device. The limit of detection and time of analysis for the detection of the two viruses by using the LoC-PCR were compared with those achieved by standard equipment. The results showed that the two systems can detect the same concentration of RNA; however, the LoC-PCR performs the analysis in half of the time compared to the standard thermocycler, with the advantage of the portability, leading to a point-of-care device for several diagnostic applications.

UV Sensor Based on Surface Acoustic Waves in ZnO/Fused Silica.

Zinc oxide (ZnO) thin films have been grown by radio frequency sputtering technique on fused silica substrates. Optical and morphological characteristics of as-grown ZnO samples were measured by various techniques; an X-ray diffraction spectrum showed that the films exhibited hexagonal wurtzite structure and were c-axis-oriented normal to the substrate surface. Scanning electron microscopy images showed the dense columnar structure of the ZnO layers, and light absorption measurements allowed us to estimate the penetration depth of the optical radiation in the 200 to 480 nm wavelength range and the ZnO band-gap. ZnO layers were used as a basic material for surface acoustic wave (SAW) delay lines consisting of two Al interdigitated transducers (IDTs) photolithographically implemented on the surface of the piezoelectric layer. The Rayleigh wave propagation characteristics were tested in darkness and under incident UV light illumination from the top surface of the ZnO layer and from the fused silica/ZnO interface. The sensor response, i.e., the wave velocity shift due to the acoustoelectric interaction between the photogenerated charge carriers and the electric potential associated with the acoustic wave, was measured for different UV power densities. The reversibility and repeatability of the sensor responses were assessed. The time response of the UV sensor showed a rise time and a recovery time of about 10 and 13 s, respectively, and a sensitivity of about 318 and 341 ppm/(mW/cm2) for top and bottom illumination, respectively. The ZnO/fused silica-based SAW UV sensors can be interrogated across the fused silica substrate thanks to its optical transparency in the UV range. The backlighting interrogation can find applications in harsh environments, as it prevents the sensing photoconductive layer from aggressive environmental effects or from any damage caused by cleaning the surface from dust which could deteriorate the sensor's performance. Moreover, since the SAW sensors, by their operating principle, are suitable for wireless reading via radio signals, the ZnO/fused-silica-based sensors have the potential to be the first choice for UV sensing in harsh environments.

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.

Thin-Film-Based Multifunctional System for Optical Detection and Thermal Treatment of Biological Samples.

In this work, we present a multifunctional Lab-on-Chip (LoC) platform based on hydrogenated amorphous silicon sensors suitable for a wide range of application in the fields of biochemical and food quality control analysis. The proposed system includes a LoC fabricated on a 5 cm × 5 cm glass substrate and a set of electronic boards for controlling the LoC functionalities. The presented Lab-on-Chip comprises light and temperature sensors, a thin film resistor acting as a heating source, and an optional thin film interferential filter suitable for fluorescence analysis. The developed electronics allows to control the thin film heater, a light source for fluorescence and absorption measurements, and the photosensors to acquire luminescent signals. All these modules are enclosed in a black metal box ensuring the portability of the whole platform. System performances have been evaluated in terms of sensor optical performances and thermal control achievements. For optical sensors, we have found a minimum number of detectable photons of 8 × 104 s-1·cm-2 at room temperature, 1.6 × 106 s-1·cm-2 in presence of fluorescence excitation source, and 2.4 × 106 s-1·cm-2 at 90 °C. From a thermal management point of view, we have obtained heating and cooling rates both equal to 2.2 °C/s, and a temperature sensor sensitivity of about 3 mV/°C even in presence of light. The achieved performances demonstrate the possibility to simultaneously use all integrated sensors and actuators, making promising the presented platform for a wide range of application fields.

Split Aptamers Immobilized on Polymer Brushes Integrated in a Lab-on-Chip System Based on an Array of Amorphous Silicon Photosensors: A Novel Sensor Assay.

Innovative materials for the integration of aptamers in Lab-on-Chip systems are important for the development of miniaturized portable devices in the field of health-care and diagnostics. Herein we highlight a general method to tailor an aptamer sequence in two subunits that are randomly immobilized into a layer of polymer brushes grown on the internal surface of microfluidic channels, optically aligned with an array of amorphous silicon photosensors for the detection of fluorescence. Our approach relies on the use of split aptamer sequences maintaining their binding affinity to the target molecule. After binding the target molecule, the fragments, separately immobilized to the brush layer, form an assembled structure that in presence of a "light switching" complex [Ru(phen)2(dppz)]2+, emit a fluorescent signal detected by the photosensors positioned underneath. The fluorescent intensity is proportional to the concentration of the target molecule. As proof of principle, we selected fragments derived from an aptamer sequence with binding affinity towards ATP. Using this assay, a limit of detection down to 0.9 µM ATP has been achieved. The sensitivity is compared with an assay where the original aptamer sequence is used. The possibility to re-use both the aptamer assays for several times is demonstrated.

On-Glass Integrated SU-8 Waveguide and Amorphous Silicon Photosensor for On-Chip Detection of Biomolecules: Feasibility Study on Hemoglobin Sensing.

An optoelectronic, integrated system-on-glass for on-chip detection of biomolecules is here presented. The system's working principle is based on the interaction, detected by a hydrogenated amorphous silicon photosensor, between a monochromatic light travelling in a SU-8 polymer optical waveguide and the biological solution under analysis. Optical simulations of the waveguide coupling to the thin-film photodiode with a specific design were carried out. A prototype was fabricated and characterized showing waveguide optical losses of about 0.6 dB/cm, a photodiode shot noise current of about 2.5 fA/Hz and responsivity of 495 mA/W at 532 nm. An electro-optical coupling test was performed on the fabricated device to validate the system. As proof of concept, hemoglobin was studied as analyte for a demonstration scenario, involving optical simulations interpolated with experimental data. The calculated detection limit of the proposed system for hemoglobin concentration in aqueous solution is around 100 ppm, in line with colorimetric methods currently on the market. These results show the effectiveness of the proposed system in biological detection applications and encourage further developments in implementing these kinds of devices in the biomedical field.

Compliant Nano-Pliers as a Biomedical Tool at the Nanoscale: Design, Simulation and Fabrication.

This paper presents the development of a multi-hinge, multi-DoF (Degrees of Freedom) nanogripper actuated by means of rotary comb drives and equipped with CSFH (Conjugate Surface Flexure Hinges), with the goal of performing complex in-plane movements at the nanoscale. The design approach, the simulation and a specifically conceived single-mask fabrication process are described in detail and the achieved results are illustrated by SEM images. The first prototype presents a total overall area of (550 × 550) µm2, an active clamping area of (2 × 4) µm2, 600 nm-wide circular curved beams as flexible hinges for its motion and an aspect ratio of about 2.5. These features allow the proposed system to grasp objects a few hundred nanometers in size.

Fluorescent Label-Free Aptasensor Integrated in a Lab-on-Chip System for the Detection of Ochratoxin A in Beer and Wheat.

This paper reports on the development of a fluorescent label-free aptamer assay integrated in a lab-on-chip (LoC) system for the detection of Ochratoxin A (OTA). The detection system relies on the integration, on a single glass substrate, of an array of amorphous silicon photosensors and a long pass interferential filter. The aptamer assay, integrated into the microfluidic network, is an aptasensor having affinity versus OTA, selected as a case study. The fluorescent molecule is a "light switch" complex [Ru(phen)2(dppz)]2+. The aptamer is directly anchored into a layer of poly(2-hydroxyethyl methacrylate) polymer brushes grown inside the channels. The fluorophore is intercalated between the base pairs of the aptamer. Upon the interaction of OTA with the aptasensor, a change of the aptamer conformation causes the release of the fluorophore, yielding a decrease of the fluorescent signal detected by the array of the amorphous silicon photosensors positioned underneath the microfluidic network. The developed LoC is a portable system capable of performing the analysis with a small volume of sample (about 10 µL) in a short time (5 min) with a limit of detection for OTA equal to 1.3 ng/mL. The LoC has been applied for the detection of OTA (5-200 ng/mL) in beer and wheat samples.

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.

Integrated Optoelectronic Device for Detection of Fluorescent Molecules.

This paper presents the development of a compact optoelectronic device suitable for on-chip detection of fluorescent molecules. In order to obtain a highly integrated device, a long-pass multi-dielectric filter has been integrated with thin-film amorphous silicon photosensors on a single glass substrate. Filter rejects the excitation light, allowing the reduction of the distance between the source and the fluorescent site and avoiding the use of external optical component. The compatibility of the technological processes determined the materials and the temporal sequence of the device fabrication. The developed device has been designed for the fluorescence detection of ruthenium complex based molecules and tested, as a proof of concept, for the detection of double-stranded DNA down to 0.5 ng. Results demonstrate the correct operation of the integrated system in both rejecting the excitation light and in detecting the fluorescent signal, demonstrating the suitability of this optoelectronic platform in practical biomedical applications.

Advances, challenges and opportunities for point-of-need screening of mycotoxins in foods and feeds.

The assurance of food and feed safety, including the identification and effective monitoring of multiple biological and chemical hazards, is a major societal challenge, given the increasing pace at which food commodities are demanded, produced and traded across the globe. Within this context, mycotoxins are globally widespread secondary fungal metabolites, which can contaminate crops either in the field or during storage and have serious human and animal health impacts such as carcinogenic, teratogenic and hepatotoxic effects. Therefore, their presence in a wide range of foods and feeds is strictly regulated, particularly in the European Union. In order to perform effective and routine monitoring of mycotoxin levels in the field prior to further processing, during transport or during processing, rapid, simple, portable and sensitive means of screening of regulated mycotoxins are in high demand. This review focuses on (1) discussing the relevance of mycotoxins and the standard approaches for their sampling and monitoring; and (2) compiling and discussing recent advances in miniaturized analytical tools for mycotoxin detection. This provides insights into current research efforts and opportunities to develop a truly integrated and fit-for-purpose analytical tool, suitable for use at critical points of the food, feed and raw material processing and distribution chains.

An All-Glass Microfluidic Network with Integrated Amorphous Silicon Photosensors for on-Chip Monitoring of Enzymatic Biochemical Assay.

A lab-on-chip system, integrating an all-glass microfluidics and on-chip optical detection, was developed and tested. The microfluidic network is etched in a glass substrate, which is then sealed with a glass cover by direct bonding. Thin film amorphous silicon photosensors have been fabricated on the sealed microfluidic substrate preventing the contamination of the micro-channels. The microfluidic network is then made accessible by opening inlets and outlets just prior to the use, ensuring the sterility of the device. The entire fabrication process relies on conventional photolithographic microfabrication techniques and is suitable for low-cost mass production of the device. The lab-on-chip system has been tested by implementing a chemiluminescent biochemical reaction. The inner channel walls of the microfluidic network are chemically functionalized with a layer of polymer brushes and horseradish peroxidase is immobilized into the coated channel. The results demonstrate the successful on-chip detection of hydrogen peroxide down to 18 µM by using luminol and 4-iodophenol as enhancer agent.

Chemiluminescence lateral flow immunoassay cartridge with integrated amorphous silicon photosensors array for human serum albumin detection in urine samples.

A novel and disposable cartridge for chemiluminescent (CL)-lateral flow immunoassay (LFIA) with integrated amorphous silicon (a-Si:H) photosensors array was developed and applied to quantitatively detect human serum albumin (HSA) in urine samples. The presented analytical method is based on an indirect competitive immunoassay using horseradish peroxidase (HRP) as a tracer, which is detected by adding the luminol/enhancer/hydrogen peroxide CL cocktail. The system comprises an array of a-Si:H photosensors deposited on a glass substrate, on which a PDMS cartridge that houses the LFIA strip and the reagents necessary for the CL immunoassay was optically coupled to obtain an integrated analytical device controlled by a portable read-out electronics. The method is simple and fast with a detection limit of 2.5 mg L-1 for HSA in urine and a dynamic range up to 850 mg L-1, which is suitable for measuring physiological levels of HSA in urine samples and their variation in different diseases (micro- and macroalbuminuria). The use of CL detection allowed accurate and objective analyte quantification in a dynamic range that extends from femtomoles to picomoles. The analytical performances of this integrated device were found to be comparable with those obtained using a charge-coupled device (CCD) as a reference off-chip detector. These results demonstrate that integrating the a-Si:H photosensors array with CL-LFIA technique provides compact, sensitive and low-cost systems for CL-based bioassays with a wide range of applications for in-field and point-of-care bioanalyses. Graphical Abstract A novel integrated portable device was developed for direct quantitative detection of human serum albumin (HSA) in urine samples, exploiting a chemiluminescence lateral flow immunoassay (LFIA). The device comprises a cartridge that holds the LFIA strip and all the reagents necessary for the analysis, an array of amorphous silicon photosensors, and a custom read-out electronics.

Thin Film Differential Photosensor for Reduction of Temperature Effects in Lab-on-Chip Applications.

This paper presents a thin film structure suitable for low-level radiation measurements in lab-on-chip systems that are subject to thermal treatments of the analyte and/or to large temperature variations. The device is the series connection of two amorphous silicon/amorphous silicon carbide heterojunctions designed to perform differential current measurements. The two diodes experience the same temperature, while only one is exposed to the incident radiation. Under these conditions, temperature and light are the common and differential mode signals, respectively. A proper electrical connection reads the differential current of the two diodes (ideally the photocurrent) as the output signal. The experimental characterization shows the benefits of the differential structure in minimizing the temperature effects with respect to a single diode operation. In particular, when the temperature varies from 23 to 50 °C, the proposed device shows a common mode rejection ratio up to 24 dB and reduces of a factor of three the error in detecting very low-intensity light signals.

Amorphous silicon p-i-n structure acting as light and temperature sensor.

In this work, we propose a multi-parametric sensor able to measure both temperature and radiation intensity, suitable to increase the level of integration and miniaturization in Lab-on-Chip applications. The device is based on amorphous silicon p-doped/intrinsic/n-doped thin film junction. The device is first characterized as radiation and temperature sensor independently. We found a maximum value of responsivity equal to 350 mA/W at 510 nm and temperature sensitivity equal to 3.2 mV/K. We then investigated the effects of the temperature variation on light intensity measurement and of the light intensity variation on the accuracy of the temperature measurement. We found that the temperature variation induces an error lower than 0.55 pW/K in the light intensity measurement at 550 nm when the diode is biased in short circuit condition, while an error below 1 K/µW results in the temperature measurement when a forward bias current higher than 25 µA/cm2 is applied.

Multiwell cartridge with integrated array of amorphous silicon photosensors for chemiluminescence detection: development, characterization and comparison with cooled-CCD luminograph.

We propose a disposable multiwell microcartridge with integrated amorphous silicon photosensors array for bio- and chemiluminescence-based bioassays, where the enzymatic reactions and the detection unit are coupled on the same glass substrate. Each well, made in a polydimethylsiloxane (PDMS) unit, hosts an enzymatic reaction that is monitored by one photosensor of the array. Photosensors were characterized in terms of their dark current background noise and response to different wavelengths of visible light in order to determine their suitability as detection devices for chemical luminescent phenomena. Calibration curves of the photosensors' response to different luminescent systems were then evaluated by using the chemiluminescent reactions catalyzed by alkaline phosphatase and horseradish peroxidase and the bioluminescent reaction catalyzed by firefly luciferase. Limits of detection in the order of attomoles for chemiluminescence enzymes and femtomoles for luciferase and sensitivities in the range between 0.007 and 0.1 pA pmol(-1) L were reached. We found that, without the need of cooling systems, the analytical performances of the proposed cartridge are comparable with those achievable with state-of-the-art thermoelectrically cooled charge-coupled device-based laboratory instrumentation. In addition, thanks to the small amount of generated output data, the proposed device allows the monitoring of long-lasting reactions with significant advantages in terms of data-storage needs, transmission bandwidth, ease of real-time signal processing and limited power consumption. Based on these results, the operation in model bioanalytical assays exploiting luminescent reactions was tested demonstrating that a-Si:H photosensors arrays, when integrated with PDMS microfluidic units, provide compact, sensitive and potentially low-cost microdevices for chemiluminescence and bioluminescence-based bioassays with a wide range of possible applications for in-field and point-of-care bio-analyses.