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We argue that the introduction of high-frequency, real-time biosensors into LoCs could be used to establish high-value feed-back control functionalities.

Biomarkers are nucleic acids, peptides, proteins, lipids metabolites, or other small molecules in human tissues or biological fluids whose accurate detection contributes to the prediction and determination of diseases and their status. In recent years, platforms that allow the detection of important biomarkers at the point-of-care (PoC) are moving the field of diagnostics towards personalized medicine. In order to comply with the REASSURED criteria stated by the WHO, the most efficient solution is to integrate PoC devices with smartphones. Without a doubt the smartphone camera is a “smart detector,” and almost all the optical-based methods have been integrated, including absorbance, fluorescence, microscopic bio-imaging, surface plasmon resonance, chemiluminescence, bioluminescence, and photoluminescence. In this chapter, we explain how smartphones can be used as smart detectors in diagnostic devices and we will provide an overview of recent developments of smartphone-based optical PoC devices., A.M. acknowledge FEDER/Ministerio de Ciencia, Innovación y Universidades - Agencia Estatal de Investigación/_Project MAT2017-87202-P. A.M. also acknowledge ICN2 which is funded by the CERCA programme/Generalitat de Catalunya and supported by the Severo Ochoa Centres of Excellence programme that is funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). A.M, L.H., A.I., C.P., R.A. and E.C. acknowledge Autonomous University of Barcelona (UAB) for their support. E.C. acknowledges Ministerio de Ciencia e Innovación of Spain and Fondo Social Europeo for the Fellowship PRE2018–084856 under the framework of “Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020. L. H. acknowledges China Scholarship Council which is a non-profit institute affiliated with the Ministry of Education of the P. R. China. A.I. was supported by PROBIST postdoctoral fellowship funded by European Research Council (Marie Skłodowska-Curie grant agreement No. 754510).

Biosensors and bioassays, both of which employ proteins and nucleic acids to detect specific molecular targets, have seen significant applications in both biomedical research and clinical practice. This success is largely due to the extraordinary versatility, affinity, and specificity of biomolecular recognition. Nevertheless, these receptors suffer from an inherent limitation: single, saturable binding sites exhibit a hyperbolic relationship (the "Langmuir isotherm") between target concentration and receptor occupancy, which in turn limits the sensitivity of these technologies to small variations in target concentration. To overcome this and generate more responsive biosensors and bioassays, here we have used the sequestration mechanism to improve the steepness of the input/output curves of several bioanalytical methods. As our test bed for this we employed sensors and assays against neutrophil gelatinase-associated lipocalin (NGAL), a kidney biomarker for which enhanced sensitivity will improve the monitoring of kidney injury. Specifically, by introducing sequestration we have improved the responsiveness of an electrochemical aptamer based (EAB) biosensor, and two bioassays, a paper-based "dipstick" assay and an enzyme-linked immunosorbent assay (ELISA). Doing so we have narrowed the dynamic range of these sensors and assays several-fold, thus enhancing their ability to measure small changes in target concentration. Given that introducing sequestration requires only the addition of the appropriate concentration of a high-affinity "depletant," the mechanism appears simple and easily adaptable to tuning the binding properties of the receptors employed in a wide range of biosensors and bioassays.

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., 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)”. We acknowledge also the MICROB-PREDICT project that has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 825694. Financial support from the EU Graphene Flagship Core 3 Project (No. 881603) is also acknowledged. This article reflects only the authors' view, and the European Commission is not responsible for any use that may be made of the information it contains. ICN2 is funded by the CERCA programme/Generalitat de Catalunya. ICN2 is supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). E. C. acknowledges Ministerio de Ciencia e Innovación of Spain and Fondo Social Europeo for the Fellowship PRE2018-084856 awarded under the call ‘Ayudas para contratos predoctorales para la formación de doctores, Subprograma Estatal de Formación del Programa Estatal de Promoción del Talento y su Empleabilidad en I+D+i’, under the framework of ‘Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020’. L. H. acknowledges the China Scholarship Council. L. H., E. C. and C. F.-C. acknowledge the Autonomous University of Barcelona (UAB) for their support. C. P. (ISGlobal) also acknowledges support from the Spanish Ministry of Science and Innovation and State Research Agency through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. A. I. was supported by a PROBIST postdoctoral fellowship funded by the European Research Council (Marie Skłodowska-Curie grant agreement No. 754510).

The majority of lateral flow assays (LFAs) use single-color optical labels to provide a qualitative naked-eye detection, however this detection method displays two important limitations. First, the use of a single-color label makes the LFA prone to results misinterpretation. Second, it does not allow the precise modulation of the sensitivity and dynamic range of the test. To overcome these limitations, a ratiometric approach is developed. In particular, using anti-HIgG functionalized red-fluorescent quantum dots on the conjugate pad (as target dependent labels) and blue-fluorescent nanoparticles fixed on the test line (as target independent reporters), it is possible to generate a wide color palette (blue, purple, pink, red) on the test line. It is believed that this strategy will facilitate the development of LFAs by easily adjusting their analytical properties to the needs required by the specific application., The authors acknowledge financial support from NACANCELL project PCIN-2016-066 (program Euronanomed 2). This work was also funded by the CERCA Program/Generalitat de Catalunya. The ICN2 was funded by the CERCA program/Generalitat de Catalunya. ICN2 acknowledges the support of the Spanish MINECO for the Project MAT2017-87202-P and through the Severo Ochoa Centres of Excellence Program under Grant SEV2201320295. A.I. was supported by PROBIST postdoctoral fellowship funded by European Research Council (Marie Sklodowska Curie) grant agreement No. 754510). C.P. acknowledges support from the Spanish Ministry of Science and Innovation and State Research Agency through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.

The ability to monitor molecular targets is crucial in fields ranging from healthcare to industrial processing to environmental protection. Devices employing biomolecules to achieve this goal are called biosensors. Over the last half century researchers have developed dozens of different biosensor approaches. In this chapter we analyze recent advances in the biosensing field aiming at adapting these to the problem of continuous molecular monitoring in complex sample streams, and how the merging of these sensors with lab-on-a-chip technologies would be beneficial to both. To do so we discuss (1) the components that comprise a biosensor, (2) the challenges associated with continuous molecular monitoring in complex sample streams, (3) how different sensing strategies deal with (or fail to deal with) these challenges, and (4) the implementation of these technologies into lab-on-a-chip architectures.

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 (, ICN2 is funded by CERCA programme, Generalitat de Catalunya. Grant SEV-2017-0706 funded by MCIN/AEI/10.13039/501100011033. 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)” and the research project INTCATCH 2020, Development and application of novel, integrated tools for monitoring and managing catchments supported by the European Union’s Horizon 2020 research and innovation program (Grant 689341). The authors also acknowledge the project MAT2017-87202-P funded by MCIN/AEI/10.13039/501100011033/and FEDER Una manera de hacer Europa. R.A.-D. acknowledges funding from the European Union Horizon2020 Programme under Grant No. 881603 (Graphene Flagship Core 3). A.I. was supported by PROBIST postdoctoral fellowship funded by the European Research Council (Marie Skłodowska-Curie grant agreement no. 754510).

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., We acknowledge financial support to the project AC21_2/00044 (GLEBIOASSAY), funded by Instituto de Salud Carlos III (ISCIII) and cofunded by the European Union, MICROBPREDICT project (European Union Horizon 2020 research and innovation program under grant agreement 825694), and Graphene Flagship Core 3 (European Union Horizon 2020 research and innovation program under grant agreement 881603). ICN2 is funded by CERCA programme, Generalitat de Catalunya. Grant SEV-2017-0706 is funded by MCIN/AEI/10.13039/501100011033. The authors also acknowledge the Project PID2021-124795NB-I00 funded by MCIN/AEI/10.13039/501100011033/and FEDER Una manera de hacer Europa and the project PAPYRUS: Grant PLEC2021-007972 funded by MCIN/AEI/10.13039/501100011033 and by the “European Union NextGenerationEU/PRTR”. 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 hacerfrente al impacto económico y social del COVID-19” (Ayudas CSIC-COVID-19). A.S.-T. acknowledges MINECO for the Juan de la Cierva Formación fellowship (FJC2020-043927-I). C.P. acknowledges the Marie Skłodowska-Curie Actions Individual Fellowship; this project received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement 795635. C.P. (ISGlobal) also acknowledges support from the Spanish Ministry of Science and Innovation** and State Research Agency through the “Centro de Excelencia Severo Ochoa 2019−2023” Program (CEX2018-000806-S).

Simplicity is one of the key feature for the spread of any successful technological product. Here, a method for rapid and low-cost fabrication of electrochemical biosensors is presented. This “plug, print & play” method involves inkjet-printing even in an office-like environment, without the need of highly specialized expertise or equipment, guaranteeing an ultra-fast idea to (scaled) prototype production time. The printed biosensors can be connected to a smartphone through its audio input for their impedance readout, demonstrating the validity of the system for point-of-care biosensing. Proper electrodes layout guarantees high sensitivity and is validated by finite element simulations. The introduction of a passivation method (wax printing) allowed to complete the devices fabrication process, increasing their sensitivity. Indeed, the wax allowed reducing the interference related to the parasitic currents flowing through the permeable coating of the employed substrates, which was used for the chemical sintering, thus avoiding the common thermal treatment after printing. As a case study, we used the devices to develop an electrochemical aptamer-based sensor for the rapid detection of neutrophil gelatinase-associated lipocalin (NGAL) in urine – a clinically important marker of acute kidney injury. The aptasensor platform is capable of detecting clinically relevant concentrations of NGAL with a simple and rapid smartphone readout. The developed technology may be extended in the future to continuous monitoring, taking advantage of its flexibility to integrate it in tubes, or to other diagnostic applications where cost/efficiency and rapidity of the research, development and implementation of point of care devices is a must., The MICROB-PREDICT project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 825694. This reflects only the author's view, and the European Commission is not responsible for any use that may be made of the information it contains. We thank Dr Alex Chamorro and Prof. Kevin Plaxco for the NGAL aptamers sequence used for the case study of our biosensors. E. P. N. acknowledges funding through the EU's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 754510. C.C.C.S. acknowledges funding through CAPES – PRINT (Programa Institucional de Internacionalização; grant #88887.310281/2018-00 and 88887.467442/2019-00) and Mackpesquisa-UPM. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 101008701 (Project acronym: EMERGE)

Electrochromism (EC) is a unique property of certain materials that undergo an electrochemical-induced change in colouration. During the last decades, electrochromic materials (ECMs) have been applied in a variety of technologies ranging from smart windows to information displays and energy storage devices. More recently, ECMs have attracted the attention of the (bio)sensing community thanks to their ability to combine the sensitivity of electrochemical methods with the intuitive readout of optical sensors. Although still a nascent technology, EC-based sensors are on the rise with several targets (e.g. cancer biomarkers, bacteria, metabolites and pesticides), which have already been detected by (bio)sensors using ECMs as transducers. In this review, we provide the reader with all the information to understand EC and its use in the development of EC-based biosensors., We acknowledge the MICROB-PREDICT project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 825694. Financial support from the EU Graphene Flagship Core 2 Project (No. 785219) is also acknowledged. This article reflects only the author’s view, and the European Commission is not responsible for any use that may be made of the information it contains. ICN2 is funded by the CERCA programme, Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). E.P.N. acknowledges funding through the EU’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 754510.

Water is the most important ingredient of life. Water fecal pollution threatens water quality worldwide and has direct detrimental effects on human health and the global economy. Nowadays, assessment of water fecal pollution relies on time-consuming techniques that often require well-trained personnel and highly-equipped laboratories. Therefore, faster, cheaper, and easily-used systems are needed toin situmonitor water fecal pollution. Herein, we have developed colorimetric lateral flow strips (LFS) able to detect and quantifyEscherichia colispecies in tap, river, and sewage water samples as an indicator of fecal pollution. The combination of LFS with a simple water filtration unit and a commercially available colorimetric reader enhanced the assay sensitivity and enabled more accurate quantification of bacteria concentration down to 10CFU mLin 10 minutes, yielding recovery percentages between 80% and 90% for all water samples analyzed. Overall, this system allows for monitoring and assessing water quality based onE. colispecies as a standard microbiological indicator of fecal pollution. Furthermore, we have developed a novel bioluminescent, bacteria-based method to quickly characterize the performance of a great variety of LFS materials. This new method allows evaluating the flow rate of big analytes such as bacteria through the LFS materials, as a suggestive means for selecting the appropriate materials for fabricating LFS targeting big analytes (≈2 μm). As a whole, the proposed approach can accelerate and reduce the costs of water quality monitoring and pave the way for further improvement of fecal pollution detection systems., This work was supported by the European Commission Program, H2020-WATER, INTCATCH Project (689341). We acknowledge all the INTCATCH partners for their support during the monitoring campaign in river Ter and the collection of the sewage water samples. ICN2 is supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). A. H. A. Hassan acknowledges the short-term fellowship (Grant 25347) funded by the Science and Technology Development Fund (STDF), Egypt.

Electrochemical sensors are major players in the race for improved molecular diagnostics due to their convenience, temporal resolution, manufacturing scalability, and their ability to support real-time measurements. This is evident in the ever-increasing number of health-related electrochemical sensing platforms, ranging from single-measurement point-of-care devices to wearable devices supporting immediate and continuous monitoring. In support of the need for such systems to rapidly process large data volumes, we describe here an open-source, easily customizable, multiplatform compatible program for the real-time control, processing, and visualization of electrochemical data. The software's architecture is modular and fully documented, allowing the easy customization of the code to support the processing of voltammetric (e.g., square-wave and cyclic) and chronoamperometric data. The program, which we have called Software for the Analysis and Continuous Monitoring of Electrochemical Systems (SACMES), also includes a graphical interface allowing the user to easily change analysis parameters (e.g., signal/noise processing, baseline correction) in real-time. To demonstrate the versatility of SACMES we use it here to analyze the real-time data output by (1) the electrochemical, aptamer-based measurement of a specific small-molecule target, (2) a monoclonal antibody-detecting DNA-scaffold sensor, and (3) the determination of the folding thermodynamics of an electrode-attached, redox-reporter-modified protein.

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, ICN2 was funded by the CERCA programme, Generalitat de Catalunya. ICN2 was supported by the Severo Ochoa Centres of Excellence programme and funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). 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)”. A.I. was supported by PROBIST postdoctoral fellowship funded by the European Research Council (Marie Skłodowska-Curie grant agreement no. 754510). C.P. acknowledges the Marie Skłodowska-Curie Actions Individual Fellowship; this project received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 795635. C.P. (ISGlobal) also acknowledges support from the Spanish Ministry of Science and Innovation** and State Research Agency through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. R.A.D. received financial support from the EU Graphene Flagship Core 3 Project (No. 881603)., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2018-000806-S)

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., We acknowledge financial support from the NACANCEL project PCIN-2016-066 (program Euronanomed 2). This work is also funded by the CERCA Program/Generalitat de Catalunya. The ICN2 is funded by the CERCA program/Generalitat de Catalunya. ICN2 acknowledges the support of the Spanish MINECO for the Project MAT2017-87202-P and through the Severo Ochoa Centers of Excellence Program under Grant SEV2201320295. R.A.-D. acknowledges the financial support from the EU Graphene Flagship Core 3 Project (No. 881603). A.S.-T. acknowledges the Autonomous University of Barcelona (UAB) for the possibility of performing this work inside the framework of Biotechnology Ph.D. Program. C.P. acknowledges Marie Skłodowska-Curie Actions Individual Fellowship, and this project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 795635. We also acknowledge Prof. Artur Xavier Roig from the Veterinary faculty in UAB for providing whole blood.

The COVID-19 pandemic made clear how our society requires quickly available tools to address emerging healthcare issues. Diagnostic assays and devices are used every day to screen for COVID-19 positive patients, with the aim to decide the appropriate treatment and containment measures. In this context, we would have expected to see the use of the most recent diagnostic technologies worldwide, including the advanced ones such as nano-biosensors capable to provide faster, more sensitive, cheaper, and high-throughput results than the standard polymerase chain reaction and lateral flow assays. Here we discuss why that has not been the case and why all the exciting diagnostic strategies published on a daily basis in peer-reviewed journals are not yet successful in reaching the market and being implemented in the clinical practice., We acknowledge funding from the European Union Horizon2020 Programme under Grant No. 881603 (Graphene Flagship Core 3). 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)”. We acknowledge the MICROB-PREDICT Project for partially supporting the work. The MICROB-PREDICT project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant No. 825694. This reflects only the author’s view, and the European Commission is not responsible for any use that may be made of the information it contains. We also acknowledge Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) for the project MAT2017-87202-P. A.I. was supported by a PROBIST postdoctoral fellowship funded by European Research Council (Marie Skłodowska-Curie Grant No. 754510). C.C.C.S. acknowledges funding through CAPES–PRINT (Programa Institucional de Internacionalização; Grant Nos. 88887.310281/2018-00 and 88887.467442/2019-00) and Mackpesquisa-UPM. L.H. acknowledges funding through the China Scholarship Council. ICN2 is funded by the CERCA Programme/Generalitat de Catalunya and supported by the Severo Ochoa programme (MINECO Grant No. SEV-2017-0706).

The availability of sensors able to rapidly detect SARS-CoV-2 directly in biological fluids in a single step would allow performing massive diagnostic testing to track in real time and contain the spread of COVID-19. Motivated by this, here, we developed an electrochemical aptamer-based (EAB) sensor able to achieve the rapid, reagentless, and quantitative measurement of the SARS-CoV-2 spike (S) protein. First, we demonstrated the ability of the selected aptamer to undergo a binding-induced conformational change in the presence of its target using fluorescence spectroscopy. Then, we engineered the aptamer to work as a bioreceptor in the EAB platform and we demonstrated its sensitivity and specificity. Finally, to demonstrate the clinical potential of the sensor, we tested it directly in biological fluids (serum and artificial saliva), achieving the rapid (minutes) and single-step detection of the S protein in its clinical range., ICN2 was funded by the CERCA programme, Generalitat de Catalunya. ICN2 was supported by the Severo Ochoa Centres of Excellence programme and funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). 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)”. A.I. was supported by PROBIST postdoctoral fellowship funded by the European Research Council (Marie Skłodowska-Curie grant agreement no. 754510). R.A.D. received financial support from the EU Graphene Flagship Core 3 Project (No. 881603). The authors also thank Salvador Bartolomé Piñol, PhD and the Laboratori Luminescència i Espectroscòpia Biomolècules (LLEB) of the Autonomous University of Barcelona (UAB).

The ability to monitor protein biomarkers continuously and in real-time would significantly advance the precision of medicine. Current protein-detection techniques, however, including ELISA and lateral flow assays, provide only time-delayed, single-time-point measurements, limiting their ability to guide prompt responses to rapidly evolving, life-threatening conditions. In response, here we present an electrochemical aptamer-based sensor (EAB) that supports high-frequency, real-time biomarker measurements. Specifically, we have developed an electrochemical, aptamer-based (EAB) sensor against Neutrophil Gelatinase-Associated Lipocalin (NGAL), a protein that, if present in urine at levels above a threshold value, is indicative of acute renal/kidney injury (AKI). When deployed inside a urinary catheter, the resulting reagentless, wash-free sensor supports real-time, high-frequency monitoring of clinically relevant NGAL concentrations over the course of hours. By providing an "early warning system", the ability to measure levels of diagnostically relevant proteins such as NGAL in real-time could fundamentally change how we detect, monitor, and treat many important diseases.

The ease of use, low cost and quick operation of lateral flow assays (LFA) have made them some of the most common point of care biosensors in a variety of fields. However, their generally low sensitivity has limited their use for more challenging applications, where the detection of low analytic concentrations is required. Here we propose the use of soluble wax barriers to selectively and temporarily accumulate the target and label nanoparticles on top of the test line (TL). This extended internal incubation step promotes the formation of the immune-complex, generating a 51.7-fold sensitivity enhancement, considering the limit of quantification, and up to 96% signal enhancement compared to the conventional LFA for Human IgG (H-IgG) detection., We acknowledge financial support from NACANCELL project PCIN-2016-066 (program Euronanomed 2). This work is also funded by the CERCA Program/Generalitat de Catalunya. The ICN2 is funded by the CERCA programme/Generalitat de Catalunya. ICN2 acknowledges the support of the Spanish MINECO for the Project MAT 2017-87202-P and through the Severo Ochoa Centres of Excellence Program under Grant SEV2201320295. Amadeo Sena-Torralba acknowledge Autonomous University of Barcelona (UAB) for the possibility of performing this work inside the framework of Biotechnology Ph.D. Programme. Duy Ba Ngo gratefully acknowledges Petchra Pra Jom Klao Doctoral, King Mongkut's University of Technology Thonburi (KMUTT) in Thailand and Sensor Technology, School of Bioresources and Technology, KMUTT, Thailand.

The condensation of 1,1′-ferrocenedimethanol and lipoic acid yields a ferrocene derivative (FcD): 1,1′-ferrocenyl bis(methylene lipoic acid ester), a bipodant linker which was used to aggregate gold nanoparticles (AuNPs). The FcD/AuNP aggregations displayed a very well-resolved signal related to the ferrocene oxidation to ferrocenium. The aggregates were found to form an efficient transducing system for a novel concept of immunosensing based on the shifting of the ferrocene oxidation potential. αHIgG/FcD/AuNP or chicken αGIgG/FcD/AuNP bioconjugates are used along with carbon SPEs as an immunosensing platform for the detection of HIgG or GIgG as model analytes. As shown by CV and DPV, the antigens addition triggers significant anodic potential shifts (up to 75 mV) of the ferrocene oxidation peak as an immunoresponse for the Ag–Ab recognition. The results from selectivity and specificity experiments using bovine serum albumin, ovalbumin and α-lactalbumin as potentially interfering proteins to HIgG Ag showed that these potential shifts would be solely due to the Ag–Ab recognition events with excellent selectivity and specificity in the presence of up to 1000-fold of the competitive proteins.

We report the development of a tuneable plasmonic nanochain immunoassay with increased sensitivity over traditional monodisperse nanoparticle lateral flow tests. Our approach takes advantage of the unique self-assembling properties of polyamidoamine dendrimers with gold nanoparticles in aqueous media to create one-dimensional nanochains, with a distinct red to blue colour change, attributable to a longitudinal plasmon resonance, which can be readily detected by eye and a digital camera. We optimise and characterise nanochain formation and stability using UV-visible spectroscopy, transmission electron microscopy and dynamic light scattering. As a proof-of-principle we focus on the application of nanochains for point-of-care diagnostics for p24, an important biomarker of early HIV infections and successfully detect p24 with a limit of detection of 5 ng ml

The multiplexed, point-of-care measurement of specific antibodies could improve the speed with which diseases are diagnosed and their treatment initiated. To this end, we are developing E-DNA scaffold sensors, which consist of a rigid, nucleic acid “scaffold” attached on one end to an electrode and presenting both a redox reporter and an epitope on the other. In the absence of antibody, the reporter efficiently transfers electrons when interrogated electrochemically. Binding-induced steric hindrance limits movement, reducing electron transfer in a manner that is both easily measured and quantitatively related to target concentration. Previously we have used monoclonal antibodies to explore the analytical performance of E-DNA sensors, showing that they support the rapid, single-step, quantitative detection of multiple antibodies in small volume samples. Here, in contrast, we employ authentic human samples to better explore the platform’s clinical potential. Specifically, we developed E-DNA sensors targeting three HIV-specific antibodies and then compared the analytical and clinical performance of these against those of gold standard serological techniques. Doing so we find that, although the multistep amplification of an ELISA leads to a lower detection limits, the clinical sensitivity of ELISAs, E-DNA sensors and lateral-flow dipsticks are indistinguishable across our test set. It thus appears that, by merging the quantitation and multiplexing of ELISAs with the convenience and speed of dipsticks, E-DNA scaffold sensors could significantly improve on current serological practice.

Lateral-flow assays (LFAs) are quick, simple and cheap assays to analyze various samples at the point of care or in the field, making them one of the most widespread biosensors currently available. They have been successfully employed for the detection of a myriad of different targets (ranging from atoms up to whole cells) in all type of samples (including water, blood, foodstuff and environmental samples). Their operation relies on the capillary flow of the sample throughout a series of sequential pads, each with different functionalities aiming to generate a signal to indicate the absence/presence (and, in some cases, the concentration) of the analyte of interest. To have a user-friendly operation, their development requires the optimization of multiple, interconnected parameters that may overwhelm new developers. In this tutorial, we provide the readers with: (i) the basic knowledge to understand the principles governing an LFA and to take informed decisions during lateral flow strip design and fabrication, (ii) a roadmap for optimal LFA development independent of the specific application, (iii) a step-by-step example procedure for the assembly and operation of an LF strip for the detection of human IgG and (iv) an extensive troubleshooting section addressing the most frequent issues in designing, assembling and using LFAs. By changing only the receptors, the provided example procedure can easily be adapted for cost-efficient detection of a broad variety of targets., We acknowledge the MICROB-PREDICT project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 825694. Financial support from the EU Graphene Flagship Core 2 Project (No. 785219) is also acknowledged. This article reflects only the author’s view, and the European Commission is not responsible for any use that may be made of the information it contains. ICN2 is funded by the CERCA programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). C.P. acknowledges the Marie Skłodowska-Curie Actions Individual Fellowship; this project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 795635. E.C. acknowledges Ministerio de Ciencia e Innovación of Spain and Fondo Social Europeo for the Fellowship PRE2018-084856 awarded under the call ‘Ayudas para contratos predoctorales para la formación de doctores, Subprograma Estatal de Formación del Programa Estatal de Promoción del Talento y su Empleabilidad en I+D+i’, under the framework of ‘Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020’. E.P.N. acknowledges funding through the EU’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 754510.

Lateral flow assays (LFAs) are paper-based analytical devices that allow for the performance of in situ diagnostic tests with good sensitivity, specificity, repeatability and low limit of detection. The application of different kinds of nanoparticles with different properties have made LFAs compatible with different detection methods (e.g. colorimetry, photoluminescence, magnetism, heating, electroactivity), thus showing their versatility. Even though LFA technology is well-established, research on the topic is more alive than ever due to the advances in the study of nanomaterials, the implementation of smartphone technology for signal readout and the creativity in finding new strategies for signal enhancement. In this chapter, the applications of LFAs using different kind of nanoparticles as labels are compiled, demonstrating their use in a range of fields such as clinical diagnostics, environmental monitoring, food safety and veterinary. Given the relevance of nanoparticles in the performance of LFAs, a section of the chapter is dedicated to biofunctionalisation strategies.

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.

Finding fast and reliable ways to detect pathogenic bacteria is crucial for addressing serious public health issues in clinical, environmental, and food settings. Here, we present a novel assay based on the conversion of an electrochemical signal into a more convenient optical readout for the visual detection of Escherichia coli. Electropolymerizing polyaniline (PANI) on an indium tin oxide screen-printed electrode (ITO SPE), we achieved not only the desired electrochromic behavior but also a convenient way to modify the electrode surface with antibodies (taking advantage of the many amine groups of PANI). Applying a constant potential to the PANI-modified ITO SPE induces a change in their oxidation state, which in turn generates a color change on the electrode surface. The presence of E. coli on the electrode surface increases the resistance in the circuit affecting the PANI oxidation states, producing a different electrochromic response. Using this electrochromic sensor, we could measure concentrations of E. coli spanning 4 orders of magnitude with a limit of detection of 102 colony forming unit per 1 mL (CFU mL–1) by the naked eye and 101 CFU mL–1 using ImageJ software. In this work we show that merging the sensitivity of electrochemistry with the user-friendliness of an optical readout can generate a new and powerful class of biosensors, with potentially unlimited applications in a variety of fields., The ICN2 is funded by the CERCA Programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa program of the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, Grant SEV-2017-0706 and MAT2017-87202-P). C.P. acknowledges Marie Skłodowska-Curie Actions Individual Fellowship (Funding scheme, MSCA-IF-EF-RI; proposal number, 795635). The Iranian authors gratefully acknowledge the Research Council and the Center of Excellence for Nanostructures of the Sharif University of Technology, Tehran, Iran.

The ability to measure the levels of diagnostically relevant proteins, such as antibodies, directly at the point of care could significantly impact healthcare. Thus motivated, we explore here the E-DNA “scaffold” sensing platform, a rapid, convenient, single-step means to this end. These sensors comprise a rigid nucleic acid “scaffold” attached via a flexible linker to an electrode and modified on its distal end with a redox reporter and a protein binding “recognition element”. The binding of a targeted protein reduces the efficiency with which the redox reporter approaches the electrode, resulting in an easily measured signal change when the sensor is interrogated voltammetrically. Previously we have demonstrated scaffold sensors employing a range of low molecular weight haptens and linear peptides as their recognition elements. Expanding on this here we have characterized sensors employing much larger recognition elements (up to and including full length proteins) in order to (1) define the range of recognition elements suitable for use in the platform; (2) better characterize the platform’s signaling mechanism to aid its design and optimization; and (3) demonstrate the analytical performance of sensors employing full-length proteins as recognition elements. In doing so we have enlarged the range of molecular targets amenable to this rapid and convenient sensing platform.

The faster a disease can be diagnosed, the sooner effective treatment can be initiated, motivating a drive to replace standard laboratory techniques with point-of-care technologies that return answers in minutes rather than hours. Thus motivated, we describe the development of an E-DNA scaffold sensor for the rapid and convenient measurement of antibodies diagnostic of syphilis. To achieve this (and in contrast to previous sensors of this class, which relied on single, linear epitopes for detection), we utilized a near full-length antigen as the sensor's recognition element, allowing us to simultaneously display multiple epitopes. The resultant sensor is able to detect antibodies against Treponema pallidum pallidum, the causative agent of syphilis, at clinically relevant concentrations in samples in less than 10 min. Preliminary results obtained using sero-positive and sero-negative human samples suggest the clinical sensitivity and specificity of the approach compare well to current gold-standard tests, while being simple and rapid enough to deploy at the point of care.

The field of precision medicine-the possibility to accurately tailor pharmacological treatments to each specific patient-would be significantly advanced by the ability to rapidly, conveniently, and cost-effectively measure biomarkers directly at the point of care. Electrochemical aptamer-based (E-AB) sensors appear a promising approach to this end due to their low cost, ease of use, and good analytical performance in complex clinical samples. Thus motivated, we present here the development of an E-AB sensor for the measurement of the amino acid L-tryptophan, a diagnostic marker indicative of a number of metabolic and mental health disorders, in urine. The sensor employs a previously reported DNA aptamer able to recognize the complex formed between tryptophan and a rhodium-based receptor. We adopted the aptamer to the E-AB sensing platform by truncating it, causing it to undergo a binding-induced conformational change, modifying it with a redox-reporting methylene blue, and attaching it to an interrogating electrode. The resulting sensor is able to measure tryptophan concentrations in the micromolar range in minutes and readily discriminates between its target and other aromatic and non-aromatic amino acids. Using it, we demonstrate the measurement of clinically relevant tryptophan levels in synthetic urine in a process requiring only a single dilution step. The speed and convenience with which this is achieved suggest that the E-AB platform could significantly improve the ease and frequency with which metabolic diseases are monitored. Graphical Abstract.

Ebola virus disease causes widespread and highly fatal epidemics in human populations. Today, there is still great need for point-of-care tests for diagnosis, patient management and surveillance, both during and post outbreaks. We present a point-of-care test comprising an immunochromatographic strip and a smartphone reader, which detects and semiquantifies Ebola-specific antibodies in human survivors. We developed a Sudan virus glycoprotein monoplex platform and validated it using sera from 90 human survivors and 31 local noninfected controls. The performance of the glycoprotein monoplex was 100% sensitivity and 98% specificity compared to standard whole antigen enzyme-linked immunosorbent assay (ELISA), and it was validated with freshly collected patient samples in Uganda. Moreover, we constructed a multiplex test for simultaneous detection of antibodies against three recombinant Sudan virus proteins. A pilot study comprising 15 survivors and 5 noninfected controls demonstrated sensitivity and specificity of 100% compared to standard ELISA. Finally, we developed a second multiplex subtype assay for the identification of exposure to three related EVD species: Sudan virus, Bundibugyo virus and Ebola virus (formerly Zaire) using recombinant viral glycoprotein. This multiplex test could distinguish between the host's immunity to specific viral species and identify cross-reactive immunity. These developed serological platforms consisted of capture ligands with high specificity and sensitivity, in-house developed strips and a compatible smartphone application. These platforms enabled rapid and portable testing, data storage and sharing as well as geographical tagging of the tested individuals in Uganda. This platform holds great potential as a field tool for diagnosis, vaccine development, and therapeutic evaluation.

Recently, molecular imprinted polymers (MIPs) were extensively used for separation and identification of specific molecules, replacing expensive and unstable biological receptors. Nonetheless, their application in electrochemical sensors has not been sufficiently explored. Here we report the use of a MIP as a specific receptor in a new highly sensitive tributyltin (TBT) electrochemical sensor. The sensor combines the specificity, pre-concentration capability and robustness of molecular imprinted polymer attached onto magnetic nanoparticles with the quantitative outputs of impedimetric measurements. The proposed device detects TBT in a concentration range of 5pM to 5μM with a low limit of detection (5.37pM), which is lower than the one recommended for TBT in sea water by the US Environmental Protection Agency (EPA). We believe that this new electrochemical sensor can play an important role in the monitoring of the quality of sea and fresh waters worldwide. Keywords: Electrochemical impedance spectroscopy, Pollutants, Electrochemical sensor

The fabrication, characterization, and applications of paper-based screen-printed carbon electrodes (SPCE) are discussed. In particular, microscopy images of the working electrode surface show a reproducible 3D pattern that enhances the performance of the device compared to that of a polyester-based SPCE. Gold nanoparticles and CdSe@ZnS quantum dots are detected using different electrochemical techniques. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lateral flow immunoassays (LFIA) are ideal biosensors to detect proteins, but their lack of sensitivity hinders their extensive use. We report a strategy that yields up to an 8-fold improvement in the sensitivity of a gold nanoparticles-based LFIA by changing the sizes of the pads. Theoretical flow simulations of the developed LFIA architectures are in accordance with the experimental results. © The Royal Society of Chemistry 2013., We acknowledge MEC (Madrid) for the project MAT2011-25870, the EU’s support under FP7 contract number 246513 ‘‘NADINE’’ and the NATO Science for Peace and Security Programme’s support under the project SfP 983807.

A handheld wireless optical reader for the detection of infectious diseases at the point of care is presented. The device is battery powered and interfaces with paper-based biosensors for the quantification of concentration in biological fluids using colorimetric analysis. The results are wirelessly transmitted to a smartphone. The device has been successfully tested with various concentrations of Influenza A nucleoprotein.

The design and operation of biosensors is not trivial. For instance, variation in the output signal during monitoring of analytes can not usually be controlled. Hence, if such control were possible, and could be triggered on demand, it would greatly facilitate system design and operation. Herein, we report the design of two types of voltamperometric immunosensors, in which the magnitude of the current output signal (differential pulse voltammetry [DPV]) can be increased or decreased as needed. The designed systems use monoclonal and polyclonal anti-human IgG antibodies, conjugated to monopodal ferrocene-modified gold nanoparticles that are casted onto screen-printed carbon electrodes (Ab/mFcL/AuNPs/SPCEs). Upon addition of human IgG as antigen, the systems exhibit opposite responses according to the Ab: the current decreases when monoclonal Ab is used, whereas it increases when polyclonal Ab is used. We attributed the former response to inhibition of electron-transfer (due to the formation of a protein layer), and the latter response, to a global increase in electron transfer (induced by the aggregation of gold nanoparticles). These effects were confirmed by studying a custom-made lipoic acid-based bipodal ligand, which confirmed that the increase in current is effectively induced by the aggregation of the modified nanoparticles (pAb/mFcL/AuNPs). Both sensors have large dynamic ranges, although the pAb-based one was found to be 3.3-times more sensitive. Tests of selectivity and specificity for ovalbumin, α-lactalbumin and serum bovine albumin showed that the immunosensors are highly selective and specific, even in the presence of up to 1000-fold levels of potentially competitive proteins. The limit of detection for human IgG using the pAb/mFcL/AuNP bioconjugate was estimated to be 0.85 ng/mL. The pAb/mFcL/AuNPs-based biosensor has used to determine amounts of human IgG in real sample., ICN2 acknowledges support from the Severo Ochoa Program (MINECO, Grant SEV-2013-0295). Authors wish to thank the Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya for Grant 2014 SGR 260 support and the MHESR, University of Tunis El-Manar and the LCAE laboratory for research funds and mobility grant awarded to AMM. MEC (Spain) for MAT2011-25870 grant is also acknowledged.

11 páginas, 9 figuras., Immunosensing technology is taking advantage of the lastest developments in materials science and inparticular from the nanomaterials field. Because of their unprecedented optical tunability as well as electrical and electrochemical qualities, we are seeing significant developments in the design of novel immunoassays; various conventional optical and electrical platforms which allow for future applications in several fields are being used. Properties of nanoparticles such as light absorption and dispersion are bringing interesting immunosensing alternatives. Nanoparticles are improving the sensitivity of existing techniques used for protein detection in immunoassays based on Surface Plasmon Resonance, Quartz Crystal Microbalance, Fluorescence spectroscopy etc. Electrochemical techniques are also taking advantage of electrical properties of nanoparticles. Redox properties of metal based nanoparticles, surface impedance change and conductance changes once nanoparticles are present as labelling tags or modifiers of transducer surfaces are also improving the technology. In most of the examples nanoparticle based biosensing systems are being offered as excellent screening and superior alternatives to existing conventional strategies/assays with interest for fields in clinical analysis, food quality, safety and security., We acknowledge funding from MEC for the projects MAT2008-03079/ NAN, CSD2006-00012-‘‘NANOBIOMED’’- (Consolider-Ingenio-2010) and the JdC scholarship (A.de la Escosura-Muñiz).

A novel ultra‐low‐cost biochemical analysis platform to quantify protein dissociation binding constants and kinetics using paper microfluidics is reported. This approach marries video imaging with one of humankind's oldest materials: paper, requiring no large, expensive laboratory equipment, complex microfluidics or external power. Temporal measurements of nanoparticle–antibody conjugates binding on paper is found to follow the Langmuir Adsorption Model. This is exploited to measure a series of antibody–antigen dissociation constants on paper, showing excellent agreement with a gold‐standard benchtop interferometer. The concept is demonstrated with a camera and low‐end smartphone, 500‐fold cheaper than the reference method, and can be multiplexed to measure ten reactions in parallel. These findings will help to widen access to quantitative analytical biochemistry, for diverse applications spanning disease diagnostics, drug discovery, and environmental analysis in resource‐limited settings.

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., The authors thank MICINN (project MAT2011-25870 and TEC2011-29140-C03-03), SMS (EU FP7 project) and the Generalitat de Catalunya (project 2014SGR-524) for supporting this project.

We have developed a paper and gold nanoparticle (AuNP)-based lab-in-a-syringe (LIS) for immunosensing of biomarkers. This simple diagnostic device features simultaneous sampling and vertical-flow operation, which means that unlike typical immunosensors, it does not suffer from any delay between sampling and detection. It can handle large-volume, low-concentration samples for analysis in diverse applications (e.g. biomedical, environmental, food, etc.). Furthermore, its operating range for sample concentration can be tuned by simply changing the volume of the syringed sample, which enables on-demand limits of detection (LOD). The LIS contains two nitrocellulose pads: the conjugate pad (which captures the analyte) and the detection pad (which signals the presence of the captured analyte) both embedded into reusable plastic cartridges. We demonstrated its efficiency in detecting human IgG (HIgG) (LOD: 1.0 ng mL−1) and prostate-specific antigen (PSA) (spiked urine samples; LOD: 1.9 ng mL−1). In the field, the LIS can be used for complete on-site analysis or to obtain partially analyzed samples (AuNPs with captured analyte) for subsequent detailed testing in specialized laboratories., We acknowledge support from MINECO (Spain) under project MAT2011-25870. G. E. N. P acknowledges “Bolsista da CAPES -Processo n° 18596-12-6”, Nanobiomed Network (#AUX–PE 705/2009), UFSC and CNPq (Brazil).

This paper describes the design and fabrication of ion-sensing electrochemical paper-based analytical devices (EPADs) in which a miniaturized paper reference electrode is integrated with a small ion-selective paper electrode (ISPE) for potentiometric measurements. Ion-sensing EPADs use printed wax barriers to define electrochemical sample and reference zones. Single-layer EPADs for sensing of chloride ions include wax-defined sample and reference zones that each incorporate a Ag/AgCl electrode. In EPADs developed for other electrolytes (potassium, sodium, and calcium ions), a PVC-based ion-selective membrane is added to separate the sample zone from a paper indicator electrode. After the addition of a small volume (less than 10 μL) of sample and reference solutions to different zones, ion-sensing EPADs exhibit a linear response, over 3 orders of magnitude, in ranges of electrolyte concentrations that are relevant to a variety of applications, with a slope close to the theoretical value (59.2/z mV). Ion-selective EPADs provide a portable, inexpensive, and disposable way of measuring concentrations of electrolyte ions in aqueous solutions.

Herein, we describe the development of a new, highly efficient label for immunosensing comprising an antibody/PEGylated gold-nanoparticle (AuNP) conjugate in which the antibody molecules are bound to the AuNP surface in a fixed orientation. Our method exploits the high density of positive charges on the major plane of antibodies that exists when the pH of the solution is lower than the isoelectric point of the antibody; the antibody molecules interact with the negatively charged AuNP surface through their major plane, enabling the antigen binding sites to move freely and therefore to reach maximum accessibility. This directed ionic interaction is reinforced by the formation of a peptide bond between the amino group of the Lys residues in the antibodies and the carboxylic groups of the PEGylated-AuNP surface via EDC chemistry. Electrochemical analyses revealed that the fixed-orientation conjugate offers a limit of detection that is 1 order of magnitude lower than that of a randomly oriented label. The performance of the new conjugate as an immunosensing label was assessed for the quantitative detection of IgG in human serum. © 2013 American Chemical Society.

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.