Unraveling the Hook Effect: A Comprehensive Study of High Antigen Concentration Effects in Sandwich Lateral Flow Immunoassays.

Sandwich lateral flow immunoassays (LFIAs) are limited at high antigen concentrations by the hook effect, leading to a contradictory decrease in the test line (T) intensity and false-negative results. The hook effect is mainly associated with the loss of T, and research focuses on minimizing this effect. Nevertheless, the control line (C) intensity is also affected at higher analyte concentrations, undesirably influencing the T/C ratio in LFIA readers. The main aim of this work is to identify and understand these high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects. Four complementary experiments were performed: performance assessment of three different allergen LFIAs (two for hazelnut, one for peanut) over 0.075-3500 ppm, LFIAs with C only, surface plasmon resonance (SPR) binding experiments on the immobilized control antibody, and smartphone video recording of LFIAs during their development. As antigen concentrations increase, the C signal decreases before the T signal does, suggesting that distinct mechanisms underlie these intensity reductions. Reduced binding at the C occurred even in the absence of T, so the upfront T does not explain the loss of C. SPR confirmed that the C antibody favors binding with free labeled antibody compared with a labeled antibody-analyte complex, indicating that in antigen excess, binding is reduced at C before T. Finally, a smartphone-based video method was developed for dynamically monitoring the LFIA development in real time to distinguish between different concentration-dependent effects. Digitally analyzing the data allows clear differentiation of highly positive samples and false-negative samples and can indicate whether the LFIA is in the dynamic working range or at critically high concentrations. The aim of this work is to identify and understand such high antigen concentration effects in order to develop ubiquitous strategies to interpret and mitigate such effects.

A Hybrid Lab-on-a-Chip Injector System for Autonomous Carbofuran Screening.

Securing food safety standards is crucial to protect the population from health-threatening food contaminants. In the case of pesticide residues, reference procedures typically find less than 1% of tested samples being contaminated, thus indicating the necessity for new tools able to support smart and affordable prescreening. Here, we introduce a hybrid paper-lab-on-a-chip platform, which integrates on-demand injectors to perform multiple step protocols in a single disposable device. Simultaneous detection of enzymatic color response in sample and reference cells, using a regular smartphone, enabled semiquantitative detection of carbofuran, a neurotoxic and EU-banned carbamate pesticide, in a wide concentration range. The resulting evaluation procedure is generic and allows the rejection of spurious measurements based on their dynamic responses, and was effectively applied for the binary detection of carbofuran in apple extracts.

Epitaxial Graphene Sensors Combined with 3D-Printed Microfluidic Chip for Heavy Metals Detection.

In this work, we investigated the sensing performance of epitaxial graphene on Si-face 4H-SiC (EG/SiC) for liquid-phase detection of heavy metals (e.g., Pb and Cd), showing fast and stable response and low detection limit. The sensing platform proposed includes 3D-printed microfluidic devices, which incorporate all features required to connect and execute lab-on-chip (LOC) functions. The obtained results indicate that EG exhibits excellent sensing activity towards Pb and Cd ions. Several concentrations of Pb2+ solutions, ranging from 125 nM to 500 µM, were analyzed showing Langmuir correlation between signal and Pb2+ concentrations, good stability, and reproducibility over time. Upon the simultaneous presence of both metals, sensor response is dominated by Pb2+ rather than Cd2+ ions. To explain the sensing mechanisms and difference in adsorption behavior of Pb2+ and Cd2+ ions on EG in water-based solutions, we performed van-der-Waals (vdW)-corrected density functional theory (DFT) calculations and non-covalent interaction (NCI) analysis, extended charge decomposition analysis (ECDA), and topological analysis. We demonstrated that Pb2+ and Cd2+ ions act as electron-acceptors, enhancing hole conductivity of EG, due to charge transfer from graphene to metal ions, and Pb2+ ions have preferential ability to binding with graphene over cadmium. Electrochemical measurements confirmed the conductometric results, which additionally indicate that EG is more sensitive to lead than to cadmium.

Autonomous Chemical Sensing Interface for Universal Cell Phone Readout.

Exploiting the ubiquity of cell phones for quantitative chemical sensing imposes strong demands on interfacing devices. They should be autonomous, disposable, and integrate all necessary calibration and actuation elements. In addition, a single design should couple universally to a variety of cell phones, and operate in their default configuration. Here, we demonstrate such a concept and its implementation as a quantitative glucose meter that integrates finger pumps, unidirectional valves, calibration references, and focusing optics on a disposable device configured for universal video acquisition.

Data processing for image-based chemical sensors: unsupervised region of interest selection and background noise compensation.

Natural olfaction suggests that numerous replicas of small sensors can achieve large sensitivity. This concept of sensor redundancy can be exploited by use of optical chemical sensors whose use of image sensors enables the simultaneous measurement of several spatially distributed indicators. Digital image sensors split the framed scene into hundreds of thousands of pixels each corresponding to a portion of the sensing layer. The signal from each pixel can be regarded as an independent sensor, which leads to a highly redundant sensor array. Such redundancy can eventually be exploited to increase the signal-to-noise ratio. In this paper we report an algorithm for reduction of the noise of pixel signals. For this purpose, the algorithm processes the output of groups of pixels whose signals share the same time behavior, as is the case for signals related to the same indicator. To define these groups of pixels, unsupervised clustering, based on classification of the indicator colors, is proposed here. This approach to signal processing is tested in experiments on the chemical sensitivity of replicas of eight indicators spotted on to a plastic substrate. Results show that the groups of pixels can be defined independently of the geometrical arrangement of the sensing spots, and substantial improvement of the signal-to-noise ratio is obtained, enabling the detection of volatile compounds at any location on the distributed sensing layer.

Embedded adaptive optics for ubiquitous lab-on-a-chip readout on intact cell phones.

The evaluation of disposable lab-on-a-chip (LOC) devices on cell phones is an attractive alternative to migrate the analytical strength of LOC solutions to decentralized sensing applications. Imaging the micrometric detection areas of LOCs in contact with intact phone cameras is central to provide such capability. This work demonstrates a disposable and morphing liquid lens concept that can be integrated in LOC devices and refocuses micrometric features in the range necessary for LOC evaluation using diverse cell phone cameras. During natural evaporation, the lens focus varies adapting to different type of cameras. Standard software in the phone commands a time-lapse acquisition for best focal selection that is sufficient to capture and resolve, under ambient illumination, 50 µm features in regions larger than 500 × 500 µm(2). In this way, the present concept introduces a generic solution compatible with the use of diverse and unmodified cell phone cameras to evaluate disposable LOC devices.

Print-and-stick unibody microfluidics coupled surface plasmon resonance (SPR) chip for smartphone imaging SPR (Smart-iSRP).

The design of a smartphone imaging surface plasmon resonance (Smart-iSPR) system integrated with an affordable 3D-printed microfluidic SPR chip fabricated via a facile manufacturing approach could pave the way towards the development of miniaturized and integrated smartphone iSPR biosensors for emerging point-of-use applications. Conventional smartphone-based SPR systems using soft photolithography for the fabrication of microfluidics SPR chips are costly, labour-intensive and required a specially-equipped light-controlled environment, that is inadequate and mismatched with the consumer-based smartphone detection platform. Herein, we report the design, fabrication and testing of an innovative print-and-stick unibody microfluidics coupled SPR chip for smartphone iSPR. The 3D-printed microfluidics (∼€0.006) is assembled via an aptly-sized adhesive tape with the gold SPR sensing surface. Such a simple integrated microfluidic SPR chip with the print-and-stick configuration has a high resistance to fluid leakages at the channel-to-sensor interface with pressure up to 66.9 Pa and the tubing-to-inset interfaces with pressure up to 86.9 Pa. The smartphone iSPR platform weighs 138 g and with a dimension of around 70 × 60 × 40 mm3, and its performance was characterized using a standard Biacore® ß2-microglobulin calibration kit. The sensorgrams obtained by the smartphone iSPR show all the typical characteristics for surface functionalization, association and dissociation events. The smartphone iSPR responds linearly to ß2-microglobulin within the range of 10-200 nM (R2 = 0.986) with a limit-of-detection (LOD) of 1.5 nM. Given the miniaturized feature and simple camera-based imaging smartphone iSPR, the analytical performance is satisfactory when compared with the analytical dynamic range of 2-32 nM described in the Biacore® protocol.

Polymers with embedded chemical indicators as an artificial olfactory mucosa.

Physiological investigations suggest that the olfactory mucosa probably plays an ancillary role in the recognition of odours introducing a sort of chromatographic separation that, together with the zonal distribution of olfactory receptors, gives place to selective spatio-temporal response patterns. It has been recently suggested that this behaviour may be simulated by chemical sensors embedded in continuous polymer layers. In this paper, in analogy to the biology of olfaction, a simple and compact platform able to separate and detect gases and vapours on the basis of their diffusion properties is proposed. In such a system, broadly selective colour indicators, such as metalloporphyrins, are embedded in continuous layers of polymers with different sorption properties. The exposure to various alcohols and amines shows that the porphyrins are mainly responsible for the recognition of the molecular family, while the occurring spatio-temporal signal patterns make possible the identification of the individual chemical species.

Evaluation of the performance of sensors based on optical imaging of a chemically sensitive layer.

Interest in the use of the optical properties of chemical indicators is growing steadily. Among the optical methods that can be used to capture changes in sensing layers, those producing images of large-area devices are particularly interesting for chemical sensor array development. Until now, few studies addressed the characterization of image sensors from the point of view of their chemical sensor application. In this paper, a method to evaluate such performance is proposed. It is based on the simultaneous measurement of absorption events in a metalloporphyrin layer with an image sensor and a quartz microbalance (QMB). Exploiting the well-known behaviour of QMB, comparison of signals enables estimation of the minimum amount of absorbed molecules that the image sensor can detect. Results indicate that at the single pixel level a standard image sensor (for example a webcam) can easily detect femtomoles of absorbed molecules. It should therefore be possible to design sensor arrays in which the pixels of images of large-area sensing layers are regarded as individual chemical sensors providing a ready and simple method for large sensor array development.

Interconnectable solid-liquid protein extraction unit and chip-based dilution for multiplexed consumer immunodiagnostics.

While consumer-focused food analysis is upcoming, the need for multiple sample preparation and handling steps is limiting. On-site and consumer-friendly analysis paradoxically still requires laboratory-based and skill-intensive sample preparation methods. Here, we present a compact, inexpensive, and novel prototype immunosensor combining sample preparation and on-chip reagent storage for multiplex allergen lateral flow immunosensing. Our comprehensive approach paves the way for personalized consumer diagnostics. The prototype allows for handheld solid-liquid extraction, pipette-free on-chip dilution, and adjustment of sample concentrations into the appropriate assay dynamic working range. The disposable and interconnectable homogenizer unit allows for the extraction and 3D-sieve based filtration of allergenic proteins from solid bakery products in 1 min. The homogenizer interconnects with a 3D-printed unibody lab-on-a-chip (ULOC) microdevice, which is used to deliver precise volumes of sample extract to a reagent reservoir. The reagent reservoir is implemented for on-chip storage of carbon nanoparticle labeled antibodies and running buffer for dilution. The handheld prototype allows for total homogenization of solid samples, solid-liquid protein extraction, 3D-printed sieve based filtration, ULOC-enabled dilution, mixing, transport, and smartphone-based detection of hazelnut and peanut allergens in solid bakery products with limited operational complexity. The multiplex lateral flow immunoassay (LFIA) detects allergens as low as 0.1 ppm in real bakery products, and the system is already consumer-operable, demonstrating its potential for future citizen science approaches. The designed system is suitable for a wide range of analytical applications outside of food safety, provided an LFIA is available.

Chemical sensitivity of self-assembled porphyrin nano-aggregates.

Nanostructured molecular assemblies may provide additional sensing properties not found in other arrangements of the same basic constituents. Among three-dimensional structures, nanotubes are particularly appealing for applications as chemical sensors, because of the potential inclusion of different guests inside the cavity or the induced modification of the skeletal interaction after analyte binding. Porphyrins are a class of compounds characterized by brilliant sensing properties, appearing also in non-ordered solid-state aggregates. In recent years, it was reported that aggregation of oppositely charged porphyrins led to the formation of self-assembled nanotubes and in this paper their sensing properties, both in solution and in the solid state, have been investigated. The interactions of porphyrin nanotubes with guest molecules have been monitored by following the changes in their UV-vis spectra. The results obtained have been exploited to build up a sensing platform based on a computer screen as a light source and a digital camera as detector. Porphyrin nanostructures exhibited an enhanced sensitivity to different compounds with respect to those shown by single porphyrin subunits. The reason for the increased sensitivity may be likely found in an additional sensing mechanism related to the modulation of the strength of the forces that keep the supramolecular ensemble together.

Autonomous lab-on-a-chip generic architecture for disposables with integrated actuation.

The integration of actuators within disposable lab-on-a-chip devices is a demanding goal that requires reliable mechanisms, systematic fabrication procedures and marginal costs compatible with single-use devices. In this work an affordable 3D printed prototype that offers a compact and modular configuration to integrate actuation in autonomous lab-on-a-chip devices is demonstrated. The proposed concept can handle multiple step preparation protocols, such as the enzyme-linked immunosorbent assay (ELISA) configuration, by integrating reagents, volume metering capabilities with performance comparable to pipettes (e.g. 2.68% error for 5 µL volume), arbitrary dilution ratio support, effective mixing and active control of the sample injection. The chosen architecture is a manifold served by multiple injectors ending in unidirectional valves, which exchange a null dead volume when idle, thus isolating reagents until they are used. Functionalization is modularly provided by a plug-in element, which together with the selection of reagents can easily repurpose the platform to diverse targets, and this work demonstrates the systematic fabrication of 6 injectors/device at a development cost of USD$ 0.55/device. The concept was tested with a commercial ELISA kit for tumor necrosis factor (TNF), a marker for infectious, inflammatory and autoimmune disorders, and its performance satisfactorily compared with the classical microplate implementation.

On the Impact of the Fabrication Method on the Performance of 3D Printed Mixers.

A wide variety of 3D printing technologies have been used for the fabrication of lab-on-a-chip (LOC) devices in recent years. Despite the large number of studies having examined the use of 3D printing technologies in microfluidic devices, the effect of the fabrication method on their performance has received little attention. In this paper, a comparison is shown between unibody-LOC micro-mixers, a particular type of monolithic design for 3D printed LOCs, fabricated in polyjet, stereolithography (SLA) and fused deposition modelling (FDM or FFF) platforms, paying particular attention to the inherent limitations of each fabrication platform and how these affect the performance of the manufactured devices.

Addressing variability in a Xenopus laevis melanophore cell line.

Xenopus laevis melanophores can be used in high-throughput screens for guanine nucleotide binding protein coupled receptor ligands and have potential as biosensors. Inherent in this immortal cell line is a substantial variability, which macroscopic evaluations disregard. Here we demonstrate a systematic way to incorporate this natural variability in the evaluations. Clusters of similar cells from a sparsely seeded cell culture are examined by imaging changes in cell appearance, pigment motility, and cumulative displacements. The time evolution of the image intensity distributions of clusters upon a pigment-dispersing stimulus is used as a signature of the cell clusters, and their behaviors are classified by multivariate analysis. Conventional image subtraction procedures are used to highlight cumulative and transitory changes in the pigment dynamics, enabling characterization of multiple aspects of the cell response from a single experiment. Additionally, a simple way to accomplish standard optical density changes at the single-cell group level is shown. The present results also provide evidence that natural cell variability arising from a cell culture can enrich the diversity of responses from pigment-containing cells assays and underscore that in conventional macroscopic evaluations these aspects are overlooked and can lead to spurious results.

Extrusion-Based 3D Printing of Microfluidic Devices for Chemical and Biomedical Applications: A Topical Review.

One of the most widespread additive manufacturing (AM) technologies is fused deposition modelling (FDM), also known as fused filament fabrication (FFF) or extrusion-based AM. The main reasons for its success are low costs, very simple machine structure, and a wide variety of available materials. However, one of the main limitations of the process is its accuracy and finishing. In spite of this, FDM is finding more and more applications, including in the world of micro-components. In this world, one of the most interesting topics is represented by microfluidic reactors for chemical and biomedical applications. The present review focusses on this research topic from a process point of view, describing at first the platforms and materials and then deepening the most relevant applications.

Patterning Highly Conducting Conjugated Polymer Electrodes for Soft and Flexible Microelectrochemical Devices.

There is a need for soft actuators in various biomedical applications to manipulate delicate objects such as cells and tissues. Soft actuators are able to adapt to any shape and limit the stress applied to delicate objects. Conjugated polymer (CP) actuators, especially in the so-called trilayer configuration, are interesting candidates for driving such micromanipulators. However, challenges involved in patterning the electrodes in a trilayer with individual contact have prevented further development of soft micromanipulators based on CP actuators. To allow such patterning, two printing-based patterning techniques have been developed. First, an oxidant layer is printed using either syringe-based printing or microcontact printing, followed by vapor-phase polymerization of the CP. Submillimeter patterns with electronic conductivities of 800 S·cm-1 are obtained. Next, laser ablation is used to cleanly cut the final device structures including the printed patterns, resulting in fingers with individually controllable digits and miniaturized hands. The methods presented in this paper will enable integration of patterned electrically active CP layers in many types of complex three-dimensional structures.

Towards autonomous lab-on-a-chip devices for cell phone biosensing.

Modern cell phones are a ubiquitous resource with a residual capacity to accommodate chemical sensing and biosensing capabilities. From the different approaches explored to capitalize on such resource, the use of autonomous disposable lab-on-a-chip (LOC) devices-conceived as only accessories to complement cell phones-underscores the possibility to entirely retain cell phones' ubiquity for distributed biosensing. The technology and principles exploited for autonomous LOC devices are here selected and reviewed focusing on their potential to serve cell phone readout configurations. Together with this requirement, the central aspects of cell phones' resources that determine their potential for analytical detection are examined. The conversion of these LOC concepts into universal architectures that are readable on unaccessorized phones is discussed within this context.

Generation of biochemical response patterns of different substances using a whole cell assay with multiple signaling pathways.

Distinctive generation of biochemical response patterns of eight different substances, using an assay based on pigment containing cells, was demonstrated. Xenopus laevis melanophores, transfected with human beta(2)-adrenergic receptor, were seeded in a 96 well microplate and used to generate individual biochemical images through a two transient measuring protocol that contributes to highlight the response signatures of the agents. Adequate signal processing creates distinctive patterns in a time-concentration response space suitable for substance classification. The concept of biochemical images is introduced here. The assays were evaluated both with a standard microplate reader and with a computer screen photo-assisted technique (CSPT) yielding similar results. Since CSPT platforms only demand standard computer sets and web cameras as measuring setup, applications for these kind of assays outside main-laboratories were discussed.

Frog melanophores cultured on fluorescent microbeads: biomimic-based biosensing.

Melanophores are pigmented cells in lower vertebrates capable of quick color changes and thereby suitable as whole cell biosensors. In the frog dermis skin layer, the large and dark pigmented melanophore surrounds a core of other pigmented cells. Upon hormonal stimulation the black-brown pigment organelles will redistribute within the melanophore, and thereby cover or uncover the core, making complex color changes possible in the dermis. Previously, melanophores have only been cultured on flat surfaces. Here we mimic the three dimensional biological geometry in the frog dermis by culturing melanophores on fluorescent plastic microbeads. To demonstrate biosensing we use the hormones melatonin and alpha-melanocyte stimulating hormone (alpha-MSH) as lightening or darkening stimuli, respectively. Cellular responses were successfully demonstrated on single cell level by fluorescence microscopy, and in cell suspension by a fluorescence microplate reader and a previously demonstrated computer screen photo-assisted technique. The demonstrated principle is the first step towards "single well/multiple read-out" biosensor arrays based on suspensions of different selective-responding melanophores, each cultured on microbeads with distinctive spectral characteristics. By applying small amount of a clinical sample, or a candidate substance in early drug screening, to a single well containing combinations of melanophores on beads, multiple parameter read-outs will be possible.