Toward Next Generation Lateral Flow Assays : Integration of Nanomaterials

Lateral flow assays (LFAs) are currently the most used point-of-care sensors for both diagnostic (e.g., pregnancy test, COVID-19 monitoring) and environmental (e.g., pesticides and bacterial monitoring) applications. Although the core of LFA technology was developed several decades ago, in recent years the integration of novel nanomaterials as signal transducers or receptor immobilization platforms has brought improved analytical capabilities. In this Review, we present how nanomaterial-based LFAs can address the inherent challenges of point-of-care (PoC) diagnostics such as sensitivity enhancement, lowering of detection limits, multiplexing, and quantification of analytes in complex samples. Specifically, we highlight the strategies that can synergistically solve the limitations of current LFAs and that have proven commercial feasibility. Finally, we discuss the barriers toward commercialization and the next generation of LFAs

A Programmable electrochemical Y-shaped DNA scaffold sensor for the single-step detection of antibodies and proteins in untreated biological fluids

Altres ajuts: ICN2 was funded by the CERCA programme, Generalitat de Catalunya. The authors acknowledge Consejo Superior de Investigaciones Científicas (CSIC) for the project "COVID19-122" granted in the call "Nuevas ayudas extraordinarias a proyectos de investigación en el marco de las medidas urgentes extraordinarias para hacer frente al impacto económico y social del COVID-19 (Ayudas CSIC-COVID-19)".Proteins and antibodies are key biomarkers for diagnosing and monitoring specific medical conditions. Currently, gold standard techniques used for their quantification require laborious multi-step procedures, involving high costs and slow response times. It is possible to overcome these limitations by exploiting the chemistry and programmability of DNA to design a reagentless electrochemical sensing platform. Specifically, three DNA single strands are engineered that can self-assemble into a Y-shaped DNA nanostructure that resembles one of the IgGs. In order to convert this DNA nanostructure into a responsive DNA-scaffold bioreceptor, it is modified including two recognition elements, two redox tag molecules, and a thiol group. In the absence of the target, the scaffold receptor can efficiently collide with the electrode surface and generate a strong electrochemical signal. The presence of the target induces its bivalent binding, which produces steric hindrance interactions that limit the receptor's collisional activity. In its bound state, the redox tags can therefore approach the surface at a slower rate, leading to a signal decrease that is quantitatively related to the target concentration. The Y-shape DNA scaffold sensor can detect nanomolar concentrations of antibodies and proteins in <15 min with a single-step procedure directly in untreated biological fluids

Paper-based electrophoretic bioassay : biosensing in whole blood operating via smartphone

Altres ajuts: this work is also funded by the CERCA Program/Generalitat de Catalunya.Point-of-care (PoC) tests are practical and effective diagnostic solutions for major clinical problems, ranging from the monitoring of a pandemic to recurrent or simple measurements. Although, in recent years, a great improvement in the analytical performance of such sensors has been observed, there is still a major issue that has not been properly solved: The ability to perform adequate sample treatments. The main reason is that normally sample treatments require complicated or long procedures not adequate for deployment at the PoC. In response, a sensing platform, called paperbased electrophoretic bioassay (PEB), that combines the key characteristics of a lateral flow assay (LFA) with the sample treatment capabilities of electrophoresis is developed. In particular, the ability of PEB to separate different types of particles and to detect human antibodies in untreated spiked whole blood is demonstrated. Finally, to make the platform suitable for PoC, PEB is coupled with a smartphone that controls the electrophoresis and reads the optical signal generated. It is believed that the PEB platform represents a much-needed solution for the detection of low target concentrations in complex media, solving one of the major limitations of LFA and opening opportunities for point-of-care sensors

Low-Cost, User-Friendly, All-Integrated Smartphone-Based Microplate Reader for Optical-Based Biological and Chemical Analyses

The quantitative detection of different molecular targets is of utmost importance for a variety of human activities, ranging from healthcare to environmental studies. Bioanalytical methods have been developed to solve this and to achieve the quantification of multiple targets from small volume samples. Generally, they can be divided into two different classes: point of care (PoC) and laboratory-based approaches. The former is rapid, low-cost, and user-friendly; however, the majority of the tests are semiquantitative, lacking in specificity and sensitivity. On the contrary, laboratory-based approaches provide high sensitivity and specificity, but the bulkiness of experimental instruments and complicated protocols hamper their use in resource-limited settings. In response, here we propose a smartphone-based device able to support laboratory-based optical techniques directly at the point of care. Specifically, we designed and fabricated a portable microplate reader that supports colorimetric, fluorescence, luminescence, and turbidity analyses. To demonstrate the potential of the device, we characterized its analytical performance by detecting a variety of relevant molecular targets (ranging from antibodies, toxins, drugs, and classic fluorophore dyes) and we showed how the estimated results are comparable to those obtained from a commercial microplate reader. Thanks to its low cost (< $3 00), portability (27 cm [length] × 18 cm [width] × 7 cm [height]), commercially available components, and open-source-based system, we believe it represents a valid approach to bring high-precision laboratory-based analysis at the point of care

Experimental comparison in sensing breast cancer mutations by signal on and signal off paper-based electroanalytical strips

Altres ajuts: the ICN2 is funded by the CERCA Programme/Generalitat de Catalunya.The development of paper-based electroanalytical strips as powerful diagnostic tools has gained a lot of attention within the sensor community. In particular, the detection of nucleic acids in complex matrices represents a trending topic, especially when focused toward the development of emerging technologies, such as liquid biopsy. DNA-based biosensors have been largely applied in this direction, and currently, there are two main approaches based on target/probe hybridization reported in the literature, namely Signal ON and Signal OFF. In this technical note, the two approaches are evaluated in combination with paper-based electrodes, using a single strand DNA relative to H1047R (A3140G) missense mutation in exon 20 in breast cancer as the model target. A detailed comparison among the analytical performances, detection protocol, and cost associated with the two systems is provided, highlighting the advantages and drawbacks depending on the application. The present work is aimed to a wide audience, particularly for those in the field of point-of-care, and it is intended to provide the know-how to manage with the design and development stages, and to optimize the platform for the sensing of nucleic acids using a paper-based detection method

Paper-based electroanalytical devices with an integrated, stable reference electrode

This paper describes the development of a referenced Electrochemical Paper-based Analytical Device (rEPAD) comprising a sample zone, a reference zone, and a connecting microfluidic channel that includes a central contact zone. We demonstrated that the rEPADs provide a simple system for direct and accurate voltammetric measurements that are referenced by an electrode with a constant, well-defined potential. The performance of the rEPADs is comparable to commercial electrochemical cells, and the layout can be easily integrated into systems that permit multiplexed analysis and pipette-free sampling. The cost of this portable device is sufficiently low that it could be for single-use, disposable applications, and its method of fabrication is compatible with that used for other paper-based systems.Chemistry and Chemical Biolog

Selection and characterisation of bioreceptors to develop nanoparticle-based lateral-flow immunoassays in the context of the SARS-CoV-2 outbreak

Altres ajuts: we acknowledge Consejo Superior de Investigaciones Científicas (CSIC) for the project "COVID19-122" granted in the call "Nuevas ayudas extraordinarias a proyectos de investigación en el marco de las medidas urgentes extraordinarias para hacer frente al impacto económico y social del COVID-19 (Ayudas CSIC-COVID-19)". ICN2 is funded by the CERCA programme/Generalitat de Catalunya. L. H. acknowledges the support from the Generalitat de Catalunya through the CERCA Program.This manuscript aims at raising the attention of the scientific community to the need for better characterised bioreceptors for fast development of point-of-care diagnostic devices able to support mass frequency testing. Particularly, we present the difficulties encountered in finding suitable antibodies for the development of a lateral flow assay for detecting the nucleoprotein of SARS-CoV-2

One-Step Laser Nanostructuration of Reduced Graphene Oxide Films Embedding Metal Nanoparticles for Sensing Applications

The combination of two-dimensional materials and metal nanoparticles (MNPs) allows the fabrication of novel nanocomposites with unique physical/chemical properties exploitable in high-performance smart devices and biosensing strategies. Current methods to obtain graphene-based films decorated with noble MNPs are cumbersome, poorly reproducible, and difficult to scale up. Herein, we propose a straightforward, versatile, surfactant-free, and single-step technique to produce reduced graphene oxide (rGO) conductive films integrating "naked" noble MNPs. This method relies on the instantaneous laser-induced co-reduction of graphene oxide and metal cations, resulting in highly exfoliated rGO nanosheets embedding gold, silver, and platinum NPs. The production procedure has been optimized, and the obtained nanomaterials are fully characterized; the hybrid nanosheets have been easily transferred onto lab-made screen-printed electrodes preserving their nanoarchitecture. The Au@rGO-, Ag@rGO-, and Pt@rGO-based electrodes have been challenged to detect caffeic acid, nitrite, and hydrogen peroxide in model solutions and real samples. The sensors yielded quantitative responses (R 2 ≥ 0.997) with sub-micromolar limits of detections (LODs ≤ 0.6 μM) for all the analytes, allowing accurate quantification in samples (recoveries ≥ 90%; RSD ≤ 14.8%, n = 3). This single-step protocol which requires low cost and minimal equipment will allow the fabrication of free-standing, MNP-embedded rGO films integrable into a variety of scalable smart devices and biosensors

Annexin-V/quantum dot probes for multimodal apoptosis monitoring in living cells : Improving bioanalysis using electrochemistry

There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (III) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry