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.

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 (<$300), 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.

Paper-Based Electrophoretic Bioassay: Biosensing in Whole Blood Operating via Smartphone.

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 paper-based 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.

Experimental Comparison in Sensing Breast Cancer Mutations by Signal ON and Signal OFF Paper-Based Electroanalytical Strips.

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.

Smart Chip for Visual Detection of Bacteria Using the Electrochromic Properties of Polyaniline.

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.

Open Source Software for the Real-Time Control, Processing, and Visualization of High-Volume Electrochemical Data.

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.

An electrochemical scaffold sensor for rapid syphilis diagnosis.

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.

An electrochemical aptamer-based sensor for the rapid and convenient measurement of L-tryptophan.

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.

Quantifying Biomolecular Binding Constants using Video Paper Analytical Devices.

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.

Paper-based potentiometric ion sensing.

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.

Paper-based nanobiosensors for diagnostics.

In this review we discuss how nanomaterials can be integrated in diagnostic paper-based biosensors for the detection of proteins, nucleic acids and cells. In particular first the different types and properties of paper-based nanobiosensors and nanomaterials are briefly explained. Then several examples of their application in diagnostics of several biomarkers are reported. Finally our opinions regarding future trends in this field are discussed.

Harnessing Bioluminescent Bacteria to Develop an Enzymatic-free Enzyme-linked immunosorbent assay for the Detection of Clinically Relevant Biomarkers.

Enzyme-linked immunosorbent assay (ELISA) is the gold standard technique for measuring protein biomarkers due to its high sensitivity, specificity, and throughput. Despite its success, continuous advancements in ELISA and immunoassay formats are crucial to meet evolving global challenges and to address new analytical needs in diverse applications. To expand the capabilities and applications of immunoassays, we introduce a novel ELISA-like assay that we call Bioluminescent-bacteria-linked immunosorbent assay (BBLISA). BBLISA is an enzyme-free assay that utilizes the inner filter effect between the bioluminescent bacteriaAllivibrio fischeriand metallic nanoparticles (gold nanoparticles and gold iridium oxide nanoflowers) as molecular absorbers. Functionalizing these nanoparticles with antibodies induces their accumulation in wells upon binding to molecular targets, forming the classical immune-sandwich complex. Thanks to their ability to adsorb the light emitted by the bacteria, the nanoparticles can suppress the bioluminescence signal, allowing the rapid quantification of the target. To demonstrate the bioanalytical properties of the novel immunoassay platform, as a proof of principle, we detected two clinically relevant biomarkers (human immunoglobulin G and SARS-CoV-2 nucleoprotein) in human serum, achieving the same sensitivity and precision as the classic ELISA. We believe that BBLISA can be a promising alternative to the standard ELISA techniques, offering potential advancements in biomarker detection and analysis by combining nanomaterials with a low-cost, portable bioluminescent platform.

Not all severe malaria cases are severe: Is it time to redefine severity criteria for malaria in non-endemic regions?

BACKGROUND: The current definition of severe malaria in non-endemic areas follows WHO criteria, which mainly target children in malaria-endemic areas, potentially misclassifying cases in non-endemic regions. We assessed the performance of a modified severe malaria classification criteria within our patient cohort. METHODS: A cohort study of patients managed for malaria in a non-endemic setting (2005-2023) was analyzed. We classified patients into severe malaria (SM) using WHO 2013 criteria except for hyperparasitemia, where 2 % threshold was applied. Patients with SM were distinguished as very severe malaria (VSM) when presenting at least one of the following conditions: parasitemia >10 %, pulmonary edema, impaired consciousness, seizures, renal failure, metabolic acidosis or hyperlactatemia, shock or hypoglycemia. In patients with SM and no criteria for VSM, less severe malaria (LSM) was defined by: 2-10 % parasitemia, hyperbilirubinemia, prostration, anemia or minor bleeding. The primary composite outcome was death or the need for a life-saving intervention, as analyzed in the three comparative groups. Secondary outcome was the prevalence of co-infections. RESULTS: Among 506 patients with malaria, 176 (34.8 %) presented with SM. A total of 37 (7.3 %) patients developed a life-threatening condition, namely death (n = 4) and/or the need for life-saving interventions (n = 34). All fatalities and 33 out of the 34 life-saving interventions occurred in the VSM group. Patients in LSM group did not develop any life-threatening conditions. As to co-infections, 28 (5.5 %) patients had a community-acquired co-infection, with no differences between groups (p = 0.763). CONCLUSIONS: Severity criteria definitions would benefit from a review when assessing patients with malaria in non-endemic areas. Within the spectrum of SM, patients reclassified as LSM have a low risk of developing a life-threatening condition and present low co-infection incidence and could benefit from management out of intensive care units and a restrictive use of empirical antibiotics.

Conformational-switch biosensors as novel tools to support continuous, real-time molecular monitoring in lab-on-a-chip devices.

Recent years have seen continued expansion of the functionality of lab on a chip (LOC) devices. Indeed LOCs now provide scientists and developers with useful and versatile platforms across a myriad of chemical and biological applications. The field still fails, however, to integrate an often important element of bench-top analytics: real-time molecular measurements that can be used to "guide" a chemical response. Here we describe the analytical techniques that could provide LOCs with such real-time molecular monitoring capabilities. It appears to us that, among the approaches that are general (i.e., that are independent of the reactive or optical properties of their targets), sensing strategies relying on binding-induced conformational change of bioreceptors are most likely to succeed in such applications.

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 (R2 ≥ 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.

The sequestration mechanism as a generalizable approach to improve the sensitivity of biosensors and bioassays.

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.

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

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.

Continuous monitoring of molecular biomarkers in microfluidic devices.

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.

A plug, print & play inkjet printing and impedance-based biosensing technology operating through a smartphone for clinical diagnostics.

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.

Rapid and Efficient Detection of the SARS-CoV-2 Spike Protein Using an Electrochemical Aptamer-Based Sensor.

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.