RESUMO
The COVID-19 pandemic highlighted lateral flow immunoassay (LFIA) strips as the most known point-of-care (POC) devices enabling rapid and easy detection of relevant biomarkers by nonspecialists. However, these diagnostic tests are usually associated with the qualitative detection of the biomarker of interest. Alternatively, electrochemical-based diagnostics, especially known for diabetes care, enable quantitative determination of biomarkers. From an analytical point perspective, the combination of the two approaches might represent a step forward for the POC world: in fact, electrochemical transduction is attractive to be integrated into LFIA strips due to its simplicity, high sensitivity, fast signal generation, and cost effectiveness. In this work, a LFIA strip has been combined with an electrochemical transduction, yielding an electrochemical LFIA (eLFIA). As a proof-of-concept method, the detection of prostate-specific antigen has been carried out by combining a printed-electrochemical strip with the traditional LFIA tests. The electrochemical detection has been based on the measurement of Au ions produced from the dissolution of the gold nanoparticles previously captured on the test line. The analytical performances obtained at LFIA and eLFIA were compared, highlighting how the use of differential pulse voltammetry allowed for a lower detection limit (2.5-fold), respectively, 0.38 and 0.15 ng/mL, but increasing the time of analysis. Although the correlation between the two architectures confirmed the satisfactory agreement of outputs, this technical note has been thought to provide the reader a fair statement with regard to the strength and drawbacks about combining the two (apparently) competitor devices in a diagnostics field, namely, LFIA and electrochemical strips.
Assuntos
Nanopartículas Metálicas , Antígeno Prostático Específico , Humanos , Masculino , Ouro , Pandemias , Imunoensaio/métodos , Biomarcadores , Limite de DetecçãoRESUMO
Antigenic lateral flow immunoassays (LFIAs) rely on the non-competitive sandwich format, including a detection (labelled) antibody and a capture antibody immobilised onto the analytical membrane. When the same antibody is used for the capture and the detection (single epitope immunoassay), the saturation of analyte epitopes by the probe compromises the capture and lowers the sensitivity. Hence, several factors, including the amount of the probe, the antibody-to-label ratio, and the contact time between the probe and the analyte before reaching the capture antibody, must be adjusted. We explored different designs of experiments (full-factorial, optimal, sub-optimal models) to optimise a multiplex sandwich-type LFIA for the diagnosis and serotyping of two Southern African Territory (SAT) serotypes of the foot-and-mouth disease virus, and to evaluate the reduction of the number of experiments in the development. Both assays employed single epitope sandwich, so most influencing variables on the sensitivity were studied and individuated. We upgraded a previous device increasing the sensitivity by a factor of two and reached the visual limit of detection of 103.7 and 104.0 (TCID/mL) for SAT 1 and SAT 2, respectively. The positioning of the capture region along the LFIA strip was the most influent variable to increase the detectability. Furthermore, we confirmed that the 13-optimal DoE was the most convenient approach for designing the device.
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Vírus da Febre Aftosa , Animais , Sorogrupo , Projetos de Pesquisa , Imunoensaio , Antígenos , Anticorpos , EpitoposRESUMO
Lateral flow immunoassay (LFIA) is widely employed as point-of-care tests (POCT) for the diagnosis of infectious diseases. The accuracy of LFIA largely depends on the quality of the immunoreagents used. Typical LFIAs to reveal the immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) employ anti-human immunoglobulin (hIG) antibodies and recombinant viral antigens, which usually are unstable and poorly soluble. Broad selective bacterial proteins, such as Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) can be considered alternatives to anti-hIG to increase versatility and sensitivity of serological LFIAs because of their high binding capacity, interspecies reactivity, and robustness. We developed two colorimetric LFA devices including SpA and SpG linked to gold nanoparticles (GNP) as detectors and explored the use of a specific, stable, and soluble immunodominant fraction of the nucleocapsid protein from SARS-CoV-2 as the capturing agent. The optimal amount of SpA-GNP and SpG-GNP conjugates and the protein-to-GNP ratios were defined through a full factorial experimental design to maximize the diagnostic sensitivity of the LFIAs. The new LFA devices were applied to analyze 105 human serum samples (69 positive and 36 negatives according to reference molecular diagnostic methods). The results showed higher sensitivity (89.9%, 95% CI 82.7-97.0) and selectivity (91.7%, 82.6-100) for the SpA-based compared to the SpG-based LFA. In addition, 18 serum samples from cats and dogs living with COVID-19 patients were analyzed and 14 showed detectable levels of anti-SARS-CoV-2 antibodies, thus illustrating the flexibility of the SpA- and SpG-based LFAs.
Assuntos
COVID-19 , Nanopartículas Metálicas , Animais , Anticorpos Antivirais , COVID-19/diagnóstico , Gatos , Cães , Ouro/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , SARS-CoV-2 , Sensibilidade e EspecificidadeRESUMO
BACKGROUND AND AIMS: The oral administration of insulin has so far been precluded by gastro-intestinal enzyme degradation and poor intestinal absorption. Preliminary evidence for peptide uptake by the gut has recently been obtained, by our research group, following the administration of nanostructured lipid-carrier suspensions loaded with glargine insulin in healthy animal models. METHODS AND RESULTS: In this experimental study, glargine insulin-loaded nanostructured lipid carriers have been converted into solid oral dosage forms (tablets, capsules), that are more suitable for administration in humans and have prolonged shelf-life. The liquid and solid oral dosage forms were tested for glargine insulin uptake and glucose responsivity in healthy and streptozotocin-induced diabetic rats (6 animals in each group). A suitable composition gave redispersible solid oral dosage forms from glargine insulin-loaded carriers, using both spray-drying and freeze-drying. It was observed that the liquid and solid formulations had relevant hypoglycaemic effects in healthy rats, while only capsules were efficacious in diabetic rats; probably because of gut alterations in these animal models. Detected glargine insulinaemia was consistent with a glycaemic profile. CONCLUSION: The formulations under study showed their potential as oral glucose-lowering agents, particularly when used as capsules. However, further study is needed to produce a useful orally-active insulin preparation.
Assuntos
Glicemia/efeitos dos fármacos , Portadores de Fármacos , Hipoglicemiantes/administração & dosagem , Insulina Glargina/administração & dosagem , Lipídeos/química , Nanopartículas , Administração Oral , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Cápsulas , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Composição de Medicamentos , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/farmacocinética , Insulina Glargina/química , Insulina Glargina/farmacocinética , Masculino , Soluções Farmacêuticas , Ratos Wistar , Estreptozocina , ComprimidosRESUMO
The Lateral Flow Immunoassay (LFIA) is by far one of the most successful analytical platforms to perform the on-site detection of target substances. LFIA can be considered as a sort of lab-in-a-hand and, together with other point-of-need tests, has represented a paradigm shift from sample-to-lab to lab-to-sample aiming to improve decision making and turnaround time. The features of LFIAs made them a very attractive tool in clinical diagnostic where they can improve patient care by enabling more prompt diagnosis and treatment decisions. The rapidity, simplicity, relative cost-effectiveness, and the possibility to be used by nonskilled personnel contributed to the wide acceptance of LFIAs. As a consequence, from the detection of molecules, organisms, and (bio)markers for clinical purposes, the LFIA application has been rapidly extended to other fields, including food and feed safety, veterinary medicine, environmental control, and many others. This review aims to provide readers with a 10-years overview of applications, outlining the trends for the main application fields and the relative compounded annual growth rates. Moreover, future perspectives and challenges are discussed.
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Mãos , Testes Imunológicos , Biomarcadores , Humanos , ImunoensaioRESUMO
Paper-based lateral-flow immunoassays (LFIAs) have achieved considerable commercial success and their impact in diagnostics is continuously growing. LFIA results are often obtained by visualizing by the naked eye color changes in given areas, providing a qualitative information about the presence/absence of the target analyte in the sample. However, this platform has the potential to provide ultrasensitive quantitative analysis for several applications. Indeed, LFIA is based on well-established immunological techniques, which have known in the last year great advances due to the combination of highly sensitive tracers, innovative signal amplification strategies and last-generation instrumental detectors. All these available progresses can be applied also to the LFIA platform by adapting them to a portable and miniaturized format. This possibility opens countless strategies for definitively turning the LFIA technique into an ultrasensitive quantitative method. Among the different proposals for achieving this goal, the use of enzyme-based immunoassay is very well known and widespread for routine analysis and it can represent a valid approach for improving LFIA performances. Several examples have been recently reported in literature exploiting enzymes properties and features for obtaining significative advances in this field. In this review, we aim to provide a critical overview of the recent progresses in highly sensitive LFIA detection technologies, involving the exploitation of enzyme-based amplification strategies. The features and applications of the technologies, along with future developments and challenges, are also discussed.
Assuntos
Imunoensaio , Técnicas ImunoenzimáticasRESUMO
Multiplex lateral flow immunoassay (LFIA) is largely used for point-of-care testing to detect different pathogens or biomarkers in a single device. The increasing demand for multitargeting diagnostics requires multi-informative single tests. In this study, we demonstrated three strategies to upgrade standard multiplex LFIA to multimodal capacity. As a proof-of-concept, we applied the strategies to the differential diagnosis of Human Immunodeficiency Virus (HIV) infection, a widespread pathogen, for which conventional multiplex LFIA testing is well-established. In the new two-parameter LFIA (x2LFIA), we exploited color encoding, in which the binding of multiple targets occurs in one reactive band and the color of the probe reveals which one is present in the sample. By combining the sequential alignment of several reactive zones along the membrane of the LFIA strip and gold nanoparticles and gold nanostars for the differential visualization, in this demonstration, the x2LFIA can furnish information on HIV serotype and stage of infection in a single device. Three immunosensors were designed. The use of bioreagents as the capturing ligand anchored onto the membrane or as the detection ligand labelled with gold nanomaterials affected the performance of the x2LFIA. Higher detectability was achieved by the format involving the HIV-specific antigens as capturing agent and labelled secondary bioligands (anti-human immunoglobulins M and protein G) as the probes.
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Técnicas Biossensoriais , Colorimetria , Infecções por HIV/diagnóstico , Imunoensaio , Nanopartículas Metálicas , Ouro , HumanosRESUMO
In this study, we report the simultaneous use of gold and silver nanoparticles to set a multicolor multiplex lateral flow immunoassay (xLFIA). Silver nanoparticles (AgNPs), spherical in shape and characterized by a brilliant yellow color, were obtained by a new viable one-step synthetic protocol. AgNPs were stable over time and acceptably robust to conditions used for fabricating LFIA devices. These AgNPs were employed as a colorimetric probe in combination with two different kinds of gold nanoparticles (AuNPs) to set a visual xLFIA for detecting allergens. Surface plasmon resonance peaks of probes (AgNPs, spherical and desert rose-like AuNPs) were centered at 420, 525, and 620 nm, respectively. Therefore, the xLFIA output was easily interpreted through a "yellow magenta cyan (YMC)" color code. The prospect of the YMC xLFIA was demonstrated by simultaneously detecting three major allergens in bakery products. Antibodies directed towards casein, ovalbumin, and hazelnut allergenic proteins were individually adsorbed onto metal nanoparticles to produce three differently colored specific probes. These were inserted in a LFIA comprising three lines, each responsive for one allergen. The trichromatic xLFIA was able to detect allergenic proteins at levels as low as 0.1 mg/l and enabled the easy identification of the allergens in commercial biscuits based on the color of the probes. Graphical Abstract.
Assuntos
Alérgenos/análise , Colorimetria/métodos , Hipersensibilidade Alimentar/diagnóstico , Ouro/química , Nanopartículas Metálicas/química , Sondas Moleculares/química , Prata/químicaRESUMO
Visceral leishmaniasis (VL) is a zoonotic infectious disease with a severe impact on humans and animals. Infection is transmitted by phlebotomine sand flies, and several domestic and wild mammals act as reservoirs for the infection, so the prompt detection of infected hosts is crucial to preventing and controlling the spread of the disease and its transmission to humans. A rapid and portable tool for VL diagnosis based on the lateral flow immunoassay (LFIA) technology is described herein. The device exploits a highly specific chimeric recombinant antigen as the recognition element for capturing anti-leishmanial antibodies, and protein A labelled with gold nanoparticles as the signal reporter. The LFIA shows excellent diagnostic sensitivity (98.4%), specificity (98.9%), and agreement with serological reference methods for diagnosing canine VL. The long-term stability of the LFIA device was confirmed based on six months of storage at room temperature or 4 °C, and the qualitative response of the device was not affected by limited thermal stress. The use of the broadly specific protein A means that the LFIA can be readily adapted to diagnose VL in dogs (the main reservoir for human infection) and other mammals, thus further assisting efforts to control the spread of VL. Graphical abstract A rapid and portable diagnostic tool for visceral leishmaniasis (VL) based on lateral flow immunoassay (LFIA) technology. The presence of anti-leishmanial antibodies is revealed through the binding of these antibodies to a highly specific chimeric antigen. Employing a broadly specific signal reporter (protein A labelled with gold nanoparticles) enables the LFIA to be easily adapted to diagnose VL in different animals.
Assuntos
Imunoensaio/métodos , Leishmaniose Visceral/diagnóstico , Animais , Anticorpos/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Leishmania donovani , Limite de Detecção , Fatores de TempoRESUMO
A lateral flow immunoassay (LFIA) was developed for the determination of fumonisin mycotoxins. The fluorescence of CdSe/ZnS quantum dots (QDs), observed at excitation/emission wavelengths of 365/525 nm, is suppressed by the addition of silver nanoparticles (SNPs) or gold nanoparticles (GNPs) because SNPs overlap the excitation bands of the QDs, and GNPs overlap the emission bands. The fluorescence of the QDs is recovered upon addition of fumonisins, allowing for the sensitive detection in "positive mode" of the target mycotoxin by monitoring the changes of the QDs fluorescence intensity. The SNPs are found to be the most effective quenchers, while the use of GNPs allows for an efficient recovery of fluorescence and can be employed in the LFIA. The method was successfully applied to the fluorometric determination of fumonisins in spiked maize flour samples. The visual detection limit is at the ng·mL-1 level. This is four times lower compared to the colorimetric LFIA based on the use of the conventional gold NPs. Graphical abstract Schematic of the fluorescence quenching lateral flow immunoassay that uses fluorescent quantum dots (QD) and metal nanoparticles (NP) as the quencher: the binding of NP-labelled antibody to the antigen (purple triangle) modulates QD luminescence at the Test line, allowing for 'positive mode' detection of fumonisins. The NP accumulation at Control line assures validity of the test.
Assuntos
Fluorescência , Fumonisinas/análise , Imunoensaio/métodos , Nanopartículas Metálicas/química , Pontos Quânticos/química , Compostos de Cádmio , Ouro , Imunoensaio/normas , Limite de Detecção , Micotoxinas/análise , Compostos de Selênio , Prata , Sulfetos , Zea mays/microbiologia , Compostos de ZincoRESUMO
The point-of-care testing concept has been exploited to design and develop portable and cheap bioanalytical systems that can be used on-site by conservators. These systems employ lateral flow immunoassays to simultaneously detect two proteins (ovalbumin and collagen) in artworks. For an in-depth study on the application of these portable biosensors, both chemiluminescent and colorimetric detections were developed and compared in terms of sensitivity and feasibility. The chemiluminescent system displayed the best analytical performance (that is, two orders of magnitude lower limits of detection than the colorimetric system). To simplify its use, a disposable cartridge was designed ad hoc for this specific application. These results highlight the enormous potential of these inexpensive, easy-to-use, and minimally invasive diagnostic tools for conservators in the cultural heritage field.
Assuntos
Arte , Técnicas Biossensoriais , Cultura , Miniaturização , Colorimetria/instrumentação , Imunoensaio , Limite de Detecção , Luminescência , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
A novel and disposable cartridge for chemiluminescent (CL)-lateral flow immunoassay (LFIA) with integrated amorphous silicon (a-Si:H) photosensors array was developed and applied to quantitatively detect human serum albumin (HSA) in urine samples. The presented analytical method is based on an indirect competitive immunoassay using horseradish peroxidase (HRP) as a tracer, which is detected by adding the luminol/enhancer/hydrogen peroxide CL cocktail. The system comprises an array of a-Si:H photosensors deposited on a glass substrate, on which a PDMS cartridge that houses the LFIA strip and the reagents necessary for the CL immunoassay was optically coupled to obtain an integrated analytical device controlled by a portable read-out electronics. The method is simple and fast with a detection limit of 2.5 mg L-1 for HSA in urine and a dynamic range up to 850 mg L-1, which is suitable for measuring physiological levels of HSA in urine samples and their variation in different diseases (micro- and macroalbuminuria). The use of CL detection allowed accurate and objective analyte quantification in a dynamic range that extends from femtomoles to picomoles. The analytical performances of this integrated device were found to be comparable with those obtained using a charge-coupled device (CCD) as a reference off-chip detector. These results demonstrate that integrating the a-Si:H photosensors array with CL-LFIA technique provides compact, sensitive and low-cost systems for CL-based bioassays with a wide range of applications for in-field and point-of-care bioanalyses. Graphical Abstract A novel integrated portable device was developed for direct quantitative detection of human serum albumin (HSA) in urine samples, exploiting a chemiluminescence lateral flow immunoassay (LFIA). The device comprises a cartridge that holds the LFIA strip and all the reagents necessary for the analysis, an array of amorphous silicon photosensors, and a custom read-out electronics.
Assuntos
Albuminúria/urina , Imunoensaio/métodos , Medições Luminescentes/instrumentação , Albumina Sérica Humana/urina , Silício/química , Albuminúria/diagnóstico , Ligação Competitiva , Desenho de Equipamento , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Imunoensaio/instrumentação , Limite de Detecção , Luminol/química , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
A multiplex chemiluminescent biosensor for simple, rapid and ultrasensitive on-site quantification of aflatoxin B1 and type B-fumonisins in maize samples has been developed. The biosensor integrates a multiplex indirect competitive lateral flow immunoassay (LFIA) based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a lensless "contact" imaging configuration. The developed assay requires a simple extraction of the analytes from maize flour samples followed by their detection with a 30 min assay time. The use of chemiluminescence detection allowed accurate and objective analytes quantification, enabling simultaneous detection of type B-fumonisins and aflatoxin B1 down to 6 µg kg(-1) and 1.5 µg kg(-1), respectively, thus fulfilling the standards imposed by the legislation of European Union. Maize flour samples spiked with both analytes were subjected to multiplex analysis obtaining recoveries ranging from 80 to 115% and the coefficient of variation below 20%. Finally, analysis of naturally contaminated maize samples resulted in a good agreement between CL-LFIA and a validated confirmatory HPLC-UV and commercial ELISA kit, obtaining recoveries in the range 88-120%. The proposed CL-LFIA protocol is rapid, inexpensive, easy-to-use, and fit for the purpose of rapid screening of mycotoxins in maize flour.
Assuntos
Aflatoxina B1/análise , Técnicas Biossensoriais , Farinha/análise , Fumonisinas/análise , Luminescência , Zea mays/químicaRESUMO
BACKGROUND: Bovine herpesvirus 1 (BoHV1) is a member of the viral subfamily of Alphaherpesvirinae that infects various species, including cattle, sheep, and goats. The virus causes infectious bovine rhinotracheitis (IBR), which is included in a European list of diseases that may require control and eradication programs. The lack of confirmatory tests affects the validity of diagnostic tools, especially those used for vaccinated herds. In this study, we report the development and validation of an indirect enzyme-linked immunosorbent assay (ELISA) based on BoHV1 glycoprotein E, which was expressed as a secreted recombinant antigen in a mammalian cell system. The performance of the new rec-gE ELISA was compared with that of commercially available indirect and/or blocking ELISAs. RESULTS: The sample set included blood sera from animals from IBR-positive farms, IBR-free farms, and marker-vaccinated farms. The indirect ELISA proposed in this study is based on antibody reactivity against BoHV1 gE, and showed high sensitivity and specificity (98.41 and 99.76 %, respectively). CONCLUSIONS: The ELISA performed well, in terms of both its diagnostic sensitivity and specificity, and as a confirmatory methodology, and therefore should improve the diagnostic protocols used for IBR surveillance.
Assuntos
Anticorpos Antivirais/sangue , Doenças dos Bovinos/prevenção & controle , Ensaio de Imunoadsorção Enzimática/métodos , Rinotraqueíte Infecciosa Bovina/prevenção & controle , Vacinas Virais/imunologia , Animais , Antígenos Virais/imunologia , Bovinos , Linhagem Celular , Herpesvirus Bovino 1/imunologia , Herpesvirus Bovino 1/metabolismo , Vigilância da População , Proteínas Recombinantes , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas Virais/imunologiaRESUMO
The aim of this study was the evaluation of the binding performances and selectivity of molecularly imprinted beads prepared toward several penicillins (i) by hierarchical bulk polymerization in the pores of template-grafted silica microbeads (hMIPs) and (ii) by Pickering emulsion polymerization in the presence of template-decorated silica nanobeads (pMIPs). 6-Aminopenicillanic acid was chosen as the common fragmental mimic template. Both approaches produced micron-sized polymeric beads with good recognition properties toward the target ligands whereas the selectivity pattern appeared quite different. The polymer prepared by the Pickering emulsion approach showed binding properties similar to imprinted beads prepared by hierarchical approach. Equilibrium binding constants changed their values from 0.1-0.2 × 10(6) (hMIPs) to 0.2-0.6 × 10(6) M(-1) (pMIPs), while the binding site densities changed from 3.7-4.8 (hMIPs) to 0.3-0.55 µmol/g (pMIPs). Compared to the hierarchical polymerization, Pickering emulsion polymerization represents a more practical approach when a template mimic needs to be used.
RESUMO
Mycotoxins are toxic metabolites of molds which can contaminate food and beverages. Because of their acute and chronic toxicity, they can have harmful effects when ingested or inhaled, posing severe risks to human health. Contemporary analytical methods have the sensitivity required for contamination detection and quantification, but the direct application of these methods on real samples is not straightforward because of matrix complexity, and clean-up and preconcentration steps are needed, more and more requiring the application of highly selective solid-phase extraction materials. Molecularly imprinted polymers (MIPs) are artificial receptors mimicking the natural antibodies that are increasingly being used as a solid phase in extraction methods where selectivity towards target analytes is mandatory. In this review, the state-of-the-art about molecularly imprinted polymers as solid-phase extraction materials in mycotoxin contamination analysis will be discussed, with particular attention paid to the use of mimic molecules in the synthesis of mycotoxin-imprinted materials, to the application of these materials to food real samples, and to the development of advanced extraction methods involving molecular imprinting technology.
Assuntos
Micotoxinas , Polímeros , Humanos , Polímeros Molecularmente Impressos , Anticorpos , BebidasRESUMO
Direct pen writing offers versatile opportunities for development of low-cost tests for point-of-care applications. In this work a lateral flow immunoassay (LFIA) test was fabricated by hand "writing" immunoprobes onto hand-cut nitrocellulose strips with a commercial fountain pen. The qualitative capabilities of the test were extended by addition of a Raman reporter and consequent design and fabrication of a Surface Enhanced Resonant Raman Scattering (SERRS)-LFIA test. As proof-of-concept, dual detection of penicillin G was achieved in milk with a visual LOD of 20 ppm and a dynamic range of 0.03-97.5 ppm. Evaluation against equivalent tests performed with conventionally prepared LFIA strips showed comparable results, thus demonstrating the validity of the test. These results demonstrate the potential for further decrease in cost and consequent broader use of LFIA tests in remote regions and resource-limited environments.
RESUMO
The cross-linker methylene-bis-acrylamide is usually present in nanoMIPs obtained by solid-phase polymerization synthesis at 2 mol% concentration, with very few exceptions. Here, we studied the influence of variable amounts of methylene-bis-acrylamide in the range between 0 (no cross-linker) and 50 mol% concentration on the binding properties of rabbit IgG nanoMIPs. The binding parameters were determined by equilibrium binding experiments and the results show that the degree of cross-linking defines three distinct types of nanoMIPs: (i) those with a low degree of cross-linking, including nanoMIPs without cross-linker (0-05 mol%), showing a low binding affinity, high density of binding sites, and low selectivity; (ii) nanoMIPs with a medium degree of cross-linking (1-18 mol%), showing higher binding affinity, low density of binding sites, and high selectivity; (iii) nanoMIPs with a high degree of cross-linking (32-50 mol%), characterized by non-specific nanopolymer-ligand interactions, with low binding affinity, high density of binding sites, and no selectivity. In conclusion, the results are particularly relevant in the synthesis of high-affinity, high-selectivity nanoMIPs as they demonstrate that a significant gain in affinity and selectivity could be achieved with pre-polymerization mixtures containing quantities of cross-linker up to 10-20 mol%, well higher than those normally used in this technique.
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The lateral flow immunoassay (LFIA) technique is largely employed for the point-of-care detection of antibodies especially for revealing the immune response in serum. Visual LFIAs usually provide the qualitative yes/no detection of antibodies, while quantification requires some equipment, making the assay more expensive and complicated. To achieve visual semi-quantification, the alignment of several lines (made of the same antigen) along a LFIA strip has been proposed. The numbering of the reacting lines has been used to correlate with the quantity of some biomarkers in serum. Here, we designed the first semiquantitative LFIA for detecting antibodies and applied it to classify the immune response to SARS-CoV-2 raised by vaccination or natural infection. We used a recombinant spike receptor-binding domain (RBD) as the specific capture reagent to draw two test lines. The detection reagent was selected among three possible ligands that are able to bind to anti-spike human antibodies: the same RBD, staphylococcal protein A, and anti-human immunoglobulin G antibodies. The most convenient detector, adsorbed on gold nanoparticles, was chosen based on the highest correlation with an antibody titre of 171 human sera, measured by a reference serological method, and was the RBD (Spearman's rho = 0.84). Incorporated into the semiquantitative LFIA, it confirmed the ability to discriminate high- and low-titre samples and to classify them into two classes (Dunn's test, P < 0.05). The proposed approach enabled the semiquantification of the immune response to SARS-CoV-2 by the unaided eye observation, thus overcoming the requirement of costly and complicated equipment, and represents a general strategy for the development of semiquantitative serological LFIAs.
Assuntos
COVID-19 , Nanopartículas Metálicas , Humanos , SARS-CoV-2 , Seguimentos , Ouro , COVID-19/diagnóstico , Imunoensaio , Vacinação , Anticorpos , ImunidadeRESUMO
Natural toxin (for example mycotoxin and phycotoxin) contamination of food is of safety and economic concern, so much effort is devoted to the development of screening methods which enable the toxins to be continuously and widely monitored in food and feed. More generally speaking, rapid and non-instrumental assays for detection of a variety of food contaminants are generating ever-increasing scientific and technological interest because they enable high-throughput, economical, on-site monitoring of such contaminants. Among rapid methods for first-level screening of food contaminants, lateral-flow immunoassay (LFIA), also named immunochromatographic assay or immune-gold colloid immunoassay, has recently attracted scientific and industrial interest because of its attractive property of enabling very rapid, one-step, in-situ analysis. This review focuses on new aspects of the development and optimization of lateral-flow devices for mycotoxin and phycotoxin detection, including strategies for management of matrix interference and, particularly, for investigation of the improvements achieved by signal-enhancing strategies or by application of non-gold nanoparticle signal reporters.