Lateral flow assays (LFA) are quick, simple and cheap assays to analyse a variety of 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 within a series of sequential pads with different functionalities aiming to generate a signal indicating the absence/presence (and, in some cases, the concentration) of the analyte of interest. In order 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: 1) the basic knowledge to understand the principles governing an LFA and to take informed decisions during lateral flow strip design and fabrication, 2) a roadmap for optimal LFA development independent of the specific application, 3) a step by step example protocol for the assembly and operation of an LF strip for the detection of Human Immunoglobulin G and 4) an extensive troubleshooting section addressing the most frequent issues in designing, assembling and using LFAs.

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.

The use of gold nanoparticles (AuNPs) as labeling carriers in combination with the enzymatic activity of the Horseradish Peroxidase (HRP) in order to achieve an improved optical lateral flow immunoassay (LFIA) performance is here presented.Briefly in a LFIA with an immune-sandwich format AuNPs are functionalized with a detection antibody already modified with HRP, obtaining an "enhanced" label. Two different detection strategies have been tested: the first one following just the red color of the AuNPs and the second one using a substrate for the HRP (3 different substrates are evaluated), which produces a darker color that enhances the intensity of the previous red color of the unmodified AuNPs. In such very simple way it is gained sensitivity (up 3

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.