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.
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.
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.
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.
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.
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.
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.
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.
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.
We discuss how nanomaterials can be integrated in diagnostic paper-based biosensors for the detection of proteins, nucleic acids and cells.
Abstract
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.
PDF
