RESUMO
Rapid immunochromatographic tests are frequently used to diagnose dengue due to their easy use, low cost, and fast response. A high level of accuracy is essential for rapid diagnostic tests to support their large-scale use. Thus, this systematic review aims to evaluate the accuracy of rapid dengue diagnostic tests. The investigation was run through the following databases: LILACS, Medline (Pubmed), CRD, The Cochrane Library, Trip Medical Database, and Google Scholar. To solve difficulties, two independent reviewers performed document screening and selection. ELISA assay was adopted as a reference test because of several methodologic advantages. Seventeen articles were included accordingly, reckoning 6837 participating individuals. The receiver operating characteristic (ROC) and Forest Plot were conducted to evaluate the sensitivity and specificity for each analyzed parameter (anti-dengue IgM, IgG, and NS1 antigen). The risk of bias and quality of evidence were assessed as moderate using QUADAS-2 and Grading of Recommendations Assessment, Development, and Evaluation (GRADE), respectively. The sensitivity of IgM concerning the studied tests ranged from 13.8 to 90%, while that of NS1 ranged from 14.7 to 100% (95% CI). The antibodies with NS1 presented increased sensitivity; pooled data show that the association of the three analytes bestows the best result, with a combined sensitivity of 90% (CI 95%: 87-92%) and a pooled specificity of 89% (CI 95%: 87-92%). Thus, the present review provides relevant knowledge for decision-making between available rapid diagnostic tests.
Assuntos
Testes Diagnósticos de Rotina , Testes Diagnósticos de Rotina/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoglobulina M , Curva ROC , Sensibilidade e EspecificidadeRESUMO
This work aims to investigate the possible mechanism of action of the homologue peptide Pa-MAP based on the Antarctic fish Pleuronectes americanus, through a study by electrical impedance spectroscopy (EIS) of models of bilayer lipid membranes supported (BLM-s) on solid substrates. For comparison and validation of the data obtained by EIS, we also conducted a study evaluating the human peptide LL-37, whose mechanism of action is well described in the literature: its dielectric response was found to be similar to that of Pa-MAP. The results obtained indicate that Pa-MAP has a good potential for use as a membrane-disrupting peptide and also suggest that the corresponding mechanism of action occurs according to the carpet model followed by a detergent-like effect. The addition of either one of these peptides at different concentrations resulted in a drastic decrease in the membrane's resistance, after just 1min of exposure. Additionally, it was seen that the peptides Pa-MAP and LL-37 may act on membranes with different charges, in an indication of a possible broad spectrum antimicrobial activity. These interactions with different membrane compositions have been attributed to the peptides' structure, mainly due to the presence of many hydrophobic amino acid residues, as observed by in silico studies. Here, we describe the Pa-MAP mechanism of action for the first time. Furthermore, we report the data demonstrating that EIS can be used for studies of peptide-membranes interaction, even when small changes on the surface of the electrode can be detected.
RESUMO
The objective of this systematic review is to analyze the diagnostic accuracy of rapid dengue diagnostic tests. The search was conducted in the following databases: LILACS, Medline (Pubmed), CRD, The Cochrane Library, Trip Medical Database and Google Scholar. ELISA and PCR assays were adopted as reference methods. Thirty-four articles were included in this systematic review. Receiver operating characteristic (ROC) and Forest Plot were performed to evaluate sensitivity and specificity for each parameter analyzed (NS1, IgM and IgG). The results revealed that the combined analysis of the IgM antibody with the NS1 antigen resulted in greater sensitivity than the isolated analysis of IgM. The three analytes together showed the best performance, with a combined sensitivity of 90 % (95 % CI: 89-92 %) using ELISA as a comparator. Thus, the present review provides relevant knowledge for decision-making between the available rapid diagnostic tests.
Assuntos
Anticorpos Antivirais , Dengue , Imunoglobulina M , Sensibilidade e Especificidade , Humanos , Anticorpos Antivirais/sangue , Cromatografia de Afinidade/métodos , Dengue/diagnóstico , Vírus da Dengue/imunologia , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/normas , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Curva ROC , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/sangueRESUMO
Infections caused by microorganisms are a public health problem worldwide. New biodetection systems are essential to diagnose with accuracy resulting in more effective treatment. In this work, we propose a ConA-conjugated graphene quantum dots and polypyrrole film-based biosensor for label-free detection of Candida albicans, Candida glabrata, Candida tropicalis, E. coli, B. subitilis, and S. aureus. We modified polypyrrole and graphene quantum dots (PPY-QDGs) with Concanavalin A (Con A) lectin. ConA is a glucose/mannose-specific lectin. The results showed that ConA lectin has the highest binding affinity for C. tropicalis and S. subtilis. PPY-GQDs-ConA binding profile revealed differential response for Candida spp (C. tropicalis > C. albicans > C. glabrata) and bacterial (B. subtilis > S. aureus > E. coli). The limits of detection (LOD) obtained were 1.42 CFU/mL for C. albicans, and 3.72 CFU/mL for C. glabrata. C. tropicalis yielded a LOD of 0.18 CFU/mL. The respective LODs for the evaluated bacteria were 0.39 CFU/mL for S. aureus, 0.72 CFU/mL for S. subtilis, and 2.63 CFU/mL for E. coli. The differential response obtained for the sensor can be attributed to the heterogeneous distribution of carbohydrates on the microorganism's surfaces. The proposed system based on a flexible substrate is effective for microbiological diagnosis.
Assuntos
Técnicas Biossensoriais , Concanavalina A , Grafite , Limite de Detecção , Polímeros , Pirróis , Pontos Quânticos , Grafite/química , Concanavalina A/química , Pontos Quânticos/química , Pirróis/química , Técnicas Biossensoriais/métodos , Polímeros/química , Candida/isolamento & purificação , Eletrodos , Técnicas Eletroquímicas/métodos , Escherichia coli/isolamento & purificação , Staphylococcus aureus/isolamento & purificaçãoRESUMO
E6 and E7 oncogenes are pivotal in the carcinogenic transformation in HPV infections and efficient diagnostic methods can ensure the detection and differentiation of HPV genotype. This study describes the development and validation of an electrochemical, label-free genosensor coupled with a microfluidic system for detecting the E6 and E7 oncogenes in cervical scraping samples. The nanostructuring employed was based on a cysteine and graphene quantum dots layer that provides functional groups, surface area, and interesting electrochemical properties. Biorecognition tests with cervical scraping samples showed differentiation in the voltammetric response. Low-risk HPV exhibited a lower biorecognition response, reflected in ΔI% values of 82.33 % ± 0.29 for HPV06 and 80.65 % ± 0.68 for HPV11 at a dilution of 1:100. Meanwhile, high-risk, HPV16 and HPV18, demonstrated ΔI% values of 96.65 % ± 1.27 and 93 % ± 0.026, respectively, at the same dilution. Therefore, the biorecognition intensity followed the order: HPV16 >HPV18 >HPV06 >HPV11. The limit of detection and the limit of quantification of E6E7 microfluidic LOC-Genosensor was 26 fM, and 79.6 fM. Consequently, the E6E7 biosensor is a valuable alternative for clinical HPV diagnosis, capable of detecting the potential for oncogenic progression even in the early stages of infection.
RESUMO
Systemic sclerosis (SSc) is a chronic, autoimmune disease that primarily affects connective tissue. SSc can be classified into limited cutaneous (lSSc) and diffuse cutaneous (dSSc). Oncostatin M receptor (sOSMR) is an important inflammatory biomarker expressed in the serum of patients with autoimmune diseases. A nanoengineered immunosensor surface was developed. The biosensor was composed of a conductive layer of polypyrrole, electrodeposited gold nanoparticles, and sOSMR protein for anti-human OSMR monoclonal antibody biorecognition. The electrochemical response evaluated by cyclic voltammetry and electrochemical impedance spectroscopy indicated the detection of the target analyte present in clinical samples from lSSc and dSSc patients. The voltammetric anodic shift for lSSc specimens was 82.7% ± 0.9-93.6% ± 3.2, and dSSc specimens was 118.7 ± 2.6 to 379.6 ± 2.6, revealing a differential diagnostic character for SSc subtypes. The sensor platform was adapted for identifying sOSMR, using anti-OSMR antibodies as bioreceptors. With a linear response range estimated from 0.005 to 500 pg mL-1 and a limit of detection of 0.42 pg mL-1, the sensing strategy demonstrated high sensitivity in identifying the human OSMR protein in clinical samples. The proposed biosensor is a promising and innovative tool for SSc-related biomarker research.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Escleroderma Sistêmico , Humanos , Autoanticorpos , Biomarcadores , Ouro , Imunoensaio , Polímeros , Pirróis , Receptores de Oncostatina M , Escleroderma Sistêmico/diagnóstico , Técnicas EletroquímicasRESUMO
This article developed a novel electrochemical immunosensor for the specific detection of aflatoxin B1 (AFB1). Amino-functionalized iron oxide nanoparticles (Fe3 O4 -NH2 ) were synthesized. Fe3 O4 -NH2 were chemically bound on self-assembly monolayers (SAMs) of mercaptobenzoic acid (MBA). Finally, polyclonal antibodies (pAb) were immobilized on Fe3 O4 -NH2 -MBA. The sensor system was evaluated through atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). A reduction in the anodic and cathodic peak currents was observed after the assembly of the sensor platform. The charge transfer resistance (Rct ) was increased due to the electrically insulating bioconjugates. Then, the specific interaction between the sensor platform and AFB1 blocks the electron transfer of the [Fe(CN)6 ]3-/4- redox pair. The nanoimmunosensor showed a linear response range estimated from 0.5 to 30 µg/mL with a limit of detection (LOD) of 9.47 µg/mL and a limit of quantification (LOQ) of 28.72 µg/mL for AFB1 identification in a purified sample. In addition, a LOD of 3.79 µg/mL, a LOQ of 11.48 µg/mL, and a regression coefficient of 0.9891 were estimated for biodetection tests on peanut samples. The proposed immunosensor represents a simple alternative, successfully applied in detecting AFB1 in peanuts, and therefore, represents a valuable tool for ensuring food safety.
Assuntos
Arachis , Técnicas Biossensoriais , Aflatoxina B1/análise , Aflatoxina B1/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Imunoensaio , Limite de Detecção , Ouro/químicaRESUMO
Acute promyelocytic leukemia (APL) in children is associated with a favorable initial prognosis. However, minimal residual disease (MRD) follow-up remains poorly defined, and relapse cases are concerning due to their recurrent nature. Thus, we report two electrochemical flexible genosensors based on polypyrrole (PPy) and graphene quantum dots (GQDs) for label-free PML-RARα oncogene detection. Atomic force microscopy (AFM), scanning electron microscope (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used to characterize the technological biosensor development. M7 and APLB oligonucleotide sequences were used as bioreceptors to detect oncogenic segments on chromosomes 15 and 17, respectively. AFM characterization revealed heterogeneous topographical surfaces with maximum height peaks for sensor layers when tested with positive patient samples. APLB/Genosensor exhibited a percentage change in anode peak current (ΔI) of 423 %. M7/Genosensor exhibited a ΔI of 61.44 % for more concentrated cDNA samples. The described behavior is associated with the biospecific recognition of the proposed biosensors. Limits of detection (LOD) of 0.214 pM and 0.677 pM were obtained for APLB/Genosensor and M7/Genosensor, respectively. The limits of quantification (LOQ) of 0.648 pM and 2.05 pM were estimated for APLB/Genosensor and M7/Genosensor, respectively. The genosensors showed reproducibility with a relative standard deviation of 7.12 % for APLB and 1.18 % for M7 and high repeatability (9.89 % for APLB and 1.51 % for M7). In addition, genetic tools could identify the PML-RARα oncogene in purified samples, plasmids, and clinical specimens from pediatric patients diagnosed with APL with high bioanalytical performance. Therefore, biosensors represent a valuable alternative for the clinical diagnosis of APL and monitoring of MRD with an impact on public health.
Assuntos
Grafite , Leucemia Promielocítica Aguda , Pontos Quânticos , Humanos , Criança , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/genética , Polímeros , Pirróis , Reprodutibilidade dos TestesRESUMO
Carbohydrate recognition is clearly present throughout nature, playing a major role in the initial attachment of one biological entity to another. The important question is whether these prevalent interactions could provide a real suitable alternative to the use of antibodies or nucleic acid for detection and identification. Currently, examples of carbohydrates being employed in biological detection systems are limited. The challenges of using carbohydrate recognition for detection mainly come from the weak affinity of carbohydrate-protein interactions, the lack of versatile carbohydrate scaffolds with well-defined structures, and the less developed high-information-content, real-time, and label-free assay technology. In this review, we focus on discussing the characteristics of carbohydrate-protein interactions in nature and the methods for carbohydrate immobilization based on surface coupling chemistry in terms of their general applicability for developing carbohydrate- and lectin-based label-free sensors. Furthermore, examples of innovative design of multivalent carbohydrate-protein interactions for sensor applications are given. We limit our review to show the feasibility of carbohydrate and lectin as recognition elements for label-free sensor development in several representative cases to formulate a flexible platform for their use as recognition elements for real-world biosensor applications.
Assuntos
Técnicas Biossensoriais/métodos , Carboidratos/química , Proteínas/química , Animais , Metabolismo dos Carboidratos , Humanos , Ligação Proteica , Proteínas/metabolismoRESUMO
Zika virus (ZIKV) infection is associated with the Guillain-Barré syndrome, and when non-vector congenital transmission occurs, fetal brain abnormalities are expected. After ZIKV infection, the blood, breast milk, and other body fluids contain low viral loads. Their detection is challenging as it requires the processing of larger input volumes of the clinical samples. Pre-enrichment is a valuable strategy to increase the analyte concentration. Therefore, the authors propose the use of a hierarchal composite polyaniline-(electrospun nanofiber) hydrogel mat (ENM) for the simultaneous enrichment and impedimetric sensing of ZIKV viral particles. The electrospinning conditions of polyvinyl alcohol and alginate, including blend formulation, were optimized through a factorial design. Disintegration and gelatinization were controlled via cross-linking to improve the hydrogel properties. Hierarchization was achieved by in situ chemical deposition of conductive polyaniline. The carboxyl groups of the ENM were used for the covalent immobilization of anti-ZIKV polyclonal antibodies used in the specific recognition of ZIKV within the medium of Vero cell culture. The specific capture and desorption of virions were studied at different pHs. ENMs were characterized by scanning electron microscopy and FTIR. Atomic force microscopy along with UV-vis and electrochemical impedance spectroscopies was used to monitor the antibody immobilization, ZIKV capture, and elution processes. Our results show that 14.2 mg (0.25 cm3) of ENM can capture 38.7 ± 2.5 µg of ZIKV with a desorption rate of 99.97% (38.29 ± 2.7 µg ZIKV), which is reusable for at least three times. Therefore, the capture capacity (micrograms of ZIKV captured per milligram of ENM) of polyaniline-hierarchized mats was 2.72 µg ZIKV/mg. The impedance LOD value was determined to be 2.76 µg of ZIKV particles (approximately 6.6 × 103 PFU/mL). As a result, we present a fast small-scale purification system that can simultaneously monitor ZIKV electrochemically and optically.
Assuntos
Alginatos/química , Compostos de Anilina/química , Técnicas Biossensoriais/métodos , Nanofibras/química , Carga Viral/métodos , Zika virus/isolamento & purificação , Animais , Anticorpos Imobilizados/imunologia , Anticorpos Antivirais/imunologia , Sangue/virologia , Chlorocebus aethiops , Técnicas Eletroquímicas , Hidrogéis/química , Imunoensaio/métodos , Limite de Detecção , Células Vero , Zika virus/imunologiaRESUMO
The present research refers to elaborating a new label-free electrochemical biosensor used to detect the BCR/ABL fusion gene. We used a hybrid nanocomposite composed of chitosan and zinc oxide nanoparticles (Chit-ZnONP) immobilized on a polypyrrole (PPy) film. DNA segments were covalently immobilized, allowing biomolecular recognition. Atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used to evaluate the assembly stages of the biosensor. The biosensor's analytical performance was investigated using recombinant plasmids containing the target oncogene and clinical samples from patients with chronic myeloid leukemia (CML). A limit of detection (LOD) of 1.34 fM, limit of quantification (LOQ) of 4.08 fM, and sensitivity of 34.03 µA fM-1 cm2 were calculated for the BCR/ABL fusion oncogene. The sensing system exhibited high specificity, selectivity, and reproducibility with a standard deviation (SD) of 4.21%. Additionally, a linear response range was observed between 138.80 aM to 13.88 pM with a regression coefficient of 0.96. Also, the biosensor shows easy operationalization and fast analytical response, contributing to the early cancer diagnosis. The proposed nanostructured device is an alternative for the genetic identification BCR/ABL fusion gene.
Assuntos
Técnicas Biossensoriais , Leucemia Mielogênica Crônica BCR-ABL Positiva , Nanocompostos , Técnicas Biossensoriais/métodos , DNA/genética , Técnicas Eletroquímicas/métodos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Nanocompostos/química , Polímeros/química , Pirróis , Reprodutibilidade dos TestesRESUMO
The increasing number of multidrug resistance microorganisms is an alarming threat, and their rapid detection is essential to prevent nosocomial, foodborne, or waterborne infections. Many peptides derived from the venom of wasp Synoeca surinama have antimicrobial activity against Gram-positive and Gram-negative bacteria. Synoeca-MP, an antimicrobial peptide (AMP) from mastoparan family, seems to increase bacterial membrane permeability, promoting cytotoxicity and membrane disruption. Here Synoeca-MP was evaluated as biorecognition element tethered over chitosan-coated magnetic nanoparticles (Fe3O4-Chit). The transducing layer of the biosensor was developed from the self-assembling of 4-mercaptobenzoic acid (4-MBA) monolayer onto gold substrate. Atomic force microscopy (AFM) analyses confirmed the biointeraction between AMP and different pathogens membranes. The fabrication and performance of the biosensing assembly were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Detection of Enterococcus faecalis (G+), Klebsiella pneumoniae (G-), Pseudomonas aeruginosa (G-), and Candida tropicalis was assessed in a recognition range from 101 to 105 CFU.mL-1. An instrumental limit of detection of 10 CFU.mL-1 was obtained for each specimen. However, the device presented a preferential selectivity towards Gram-negative bacteria. The proposed biosensor is a sensitive, fast, and straightforward platform for microbial detection in aqueous samples, envisaged for environmental monitoring applications.
Assuntos
Técnicas Biossensoriais , Nanopartículas de Magnetita , Antibacterianos/farmacologia , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Ouro/química , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Peptídeos e Proteínas de Sinalização Intercelular , Nanopartículas de Magnetita/química , Venenos de VespasRESUMO
Bacterial and fungal infections are challenging due to their low susceptibility and resistance to antimicrobial drugs. For this reason, antimicrobial peptides (AMP) emerge as excellent alternatives to overcome these problems. At the same time, their active insertion into the cell wall of microorganisms can be availed for biorecognition applications in biosensing platforms. Temporin-PTA (T-PTA) is an AMP found in the skin secretions of the Malaysian fire frog Hylarana picturata, which presents antibacterial activity against MRSA, Escherichia coli, and Bacillus subtilis. In this work, T-PTA was explored as an innovative sensing layer aiming for the electrochemical differentiation of Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus subtilis, Enterococcus faecalis, Candida albicans, and C. tropicalis based on the structural differences of their membranes. The biosensor was analyzed through electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). In this approach, the different structural features of each microorganism resulted in different adherence degrees and, therefore, different electrochemical responses. The transducing layer was fabricated by the self-assembling of a 4-mercaptobenzoic acid (MBA) monolayer and gold-capped magnetic nanoparticles (Fe3O4@Au) implemented to improve the electrical signal of the biointeraction. We found that each interaction, expressed in variations of electron transfer resistance and anodic peak current, demonstrated a singular response from which the platform can discriminate all different microorganisms. We found expressive sensitivity towards Gram-negative species, especially K. pneumoniae. A detection limit of 101 CFU.mL-1 and a linear range of 101 to 105 CFU.mL-1 were obtained. The T-PTA biosensor platform is a promising and effective tool for microbial identification.
Assuntos
Técnicas Biossensoriais , Peptídeos Catiônicos Antimicrobianos/química , Técnicas Biossensoriais/métodos , Eletrodos , Ouro/químicaRESUMO
BACKGROUND: In human malaria, the naturally-acquired immune response can result in either the elimination of the parasite or a persistent response mediated by cytokines that leads to immunopathology. The cytokines are responsible for all the symptoms, pathological alterations and the outcome of the infection depends on the reciprocal regulation of the pro and anti-inflammatory cytokines. IL-10 and IFN-gamma are able to mediate this process and their production can be affected by single nucleotide polymorphisms (SNPs) on gene of these cytokines. In this study, the relationship between cytokine IL-10/IFN-gamma levels, parasitaemia, and their gene polymorphisms was examined and the participation of pro-inflammatory and regulatory balance during a natural immune response in Plasmodium vivax-infected individuals was observed. METHODS: The serum levels of the cytokines IL-4, IL-12, IFN-gamma and IL-10 from 132 patients were evaluated by indirect enzyme-linked immunosorbent assays (ELISA). The polymorphism at position +874 of the IFN-gamma gene was identified by allele-specific polymerase chain reaction (ASO-PCR) method, and the polymorphism at position -1082 of the IL-10 gene was analysed by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). RESULTS: The levels of a pro- (IFN-gamma) and an anti-inflammatory cytokine (IL-10) were significantly higher in P. vivax-infected individuals as compared to healthy controls. The IFN-gamma levels in primoinfected patients were significantly higher than in patients who had suffered only one and more than one previous episode. The mutant alleles of both IFN-gamma and IL-10 genes were more frequent than the wild allele. In the case of the IFNG+874 polymorphism (IFN-gamma) the frequencies of the mutant (A) and wild (T) alleles were 70.13% and 29.87%, respectively. Similar frequencies were recorded in IL-10-1082, with the mutant (A) allele returning a frequency of 70.78%, and the wild (G) allele a frequency of 29.22%. The frequencies of the alleles associated with reduced production of both IFN-gamma and IL-10 were high, but this effect was only observed in the production of IFN-gamma. CONCLUSIONS: This study has shown evidence of reciprocal regulation of the levels of IL-10 and IFN-gamma cytokines in P. vivax malaria, which is not altered by the presence of polymorphism in the IL-10 gene.
Assuntos
Interferon gama/sangue , Interleucina-10/sangue , Malária Vivax/imunologia , Adolescente , Adulto , Idoso , Alelos , Sangue/imunologia , Sangue/parasitologia , Criança , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Humanos , Interferon gama/genética , Interferons , Interleucina-10/genética , Masculino , Pessoa de Meia-Idade , Parasitemia/imunologia , Plasmodium vivax/imunologia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Regiões Promotoras Genéticas , Adulto JovemRESUMO
Considering the low sensitivity of cytological exams and high costs of the molecular methods, the development of diagnostic tests for effective diagnosis of HPV infections is a priority. In this work, biosensor composed of polypyrrole (PPy) films and gold nanoparticles (AuNPs) was obtained for specific detection of HPV genotypes. The biosensor was developed by using flexible electrodes based on polyethylene terephthalate (PET) strips coated with indium tin oxide (ITO). Polymeric films and AuNPs were obtained by electrosynthesis. Oligonucleotides sequences modified with functional amino groups were designed to recognize HPV gene families strictly. The modified oligonucleotides were chemically immobilized on the nanostructured platform. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used for the analysis of the electrode modification and monitoring of molecular hybridization. Electrochemical changes were observed after exposure of the biosensors to plasmid samples and cervical specimens. The biosensor based on the BSH16 probe showed a linear concentration range for target HPV16 gene detection of 100 pg µL-1 to 1 fg µL-1. A limit of detection (LOD) of 0.89 pg µL-1 and limit of quantification (LOQ) of 2.70 pg µL-1 were obtained, with a regression coefficient of 0.98. Screening tests on cervical specimens were performed to evaluate the sensibility and specificity for HPV and its viral family. The expression of a biomarker for tumorigenesis (p53 gene) was also monitored. In this work, a flexible system has been successfully developed for label-free detection of HPV families and p53 gene monitoring with high specificity, selectivity, and sensitivity.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Infecções por Papillomavirus , Técnicas Eletroquímicas , Eletrodos , Ouro , Humanos , Limite de Detecção , Infecções por Papillomavirus/diagnóstico , Polímeros , PirróisRESUMO
Biosensors are pre-prepared diagnostic devices composed of at least one biological probe. These devices are envisaged for the practical identification of specific targets of microbiological interest. In recent years, the use of narrow-specific probes such as lectins has been proven to distinguish bacteria and glycoproteins based on their superficial glycomic pattern. For instance, Concanavalin A is a carbohydrate-binding lectin indicated as a narrow-specific biological probe for Gram-negative bacteria. As a drawback, Gram-positive bacteria are frequently overlooked from lectin-based biosensing studies because their identification results in low resolution and overlapped signals. In this work, the authors explore the effect that platform nanostructuration has over the electrochemical response of ConA-based platforms constructed for bacterial detection; one is formed of chitosan-capped magnetic nanoparticles, and another is composed of gold nanoparticle-decorated magnetic nanoparticles. The biosensing platforms were characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) as a function of bacterial concentration. Our results show that probe-target interaction causes variations in the electrical responses of nanostructured transducers. Moreover, the association of gold nanoparticles to magnetic nanoparticles resulted in an electrical enhancement capable of overcoming low resolution and overlapping Gram-positive identification. Both platforms attained a limit of detection of 10 ° CFU mL-1, which is useful for water analyses and sanitation concerns, where low CFU mL-1 are always expected. Although both platforms were able to detect Gram-negative bacteria, Gram-positives were only correctly differentiated by the gold nanoparticle-decorated magnetic nanoparticles, thus demonstrating the positive influence of hierarchically nanostructured platforms.
Assuntos
Técnicas Biossensoriais , Concanavalina A , Bactérias Gram-Positivas , Técnicas Biossensoriais/métodos , Concanavalina A/farmacologia , Ouro , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/efeitos dos fármacos , Nanopartículas Metálicas , TransdutoresRESUMO
The projection of new biosensing technologies for genetic identification of SARS-COV-2 is essential in the face of a pandemic scenario. For this reason, the current research aims to develop a label-free flexible biodevice applicable to COVID-19. A nanostructured platform made of polypyrrole (PPy) and gold nanoparticles (GNP) was designed for interfacing the electrochemical signal in miniaturized electrodes of tin-doped indium oxide (ITO). Oligonucleotide primer was chemically immobilized on the flexible transducers for the biorecognition of the nucleocapsid protein (N) gene. Methodological protocols based on cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) were used to characterize the nanotechnological apparatus. The biosensor's electrochemical performance was evaluated using the SARS-CoV-2 genome and biological samples of cDNA from patients infected with retrovirus at various disease stages. It is inferred that the analytical tool was able to distinguish the expression of SARS-CoV-2 in patients diagnosed with COVID-19 in the early, intermediate and late stages. The biosensor exhibited high selectivity by not recognizing the biological target in samples from patients not infected with SARS-CoV-2. The proposed sensor obtained a linear response range estimated from 800 to 4000 copies µL-1 with a regression coefficient of 0.99, and a detection limit of 258.01 copies µL-1. Therefore, the electrochemical biosensor based on flexible electrode technology represents a promising trend for sensitive molecular analysis of etiologic agent with fast and simple operationalization. In addition to early genetic diagnosis, the biomolecular assay may help to monitor the progression of COVID-19 infection in a novel manner.
Assuntos
Técnicas Biossensoriais , COVID-19 , Nanopartículas Metálicas , Anticorpos Imobilizados , Técnicas Eletroquímicas , Eletrodos , Ouro , Humanos , Limite de Detecção , Microeletrodos , Polímeros , Pirróis , SARS-CoV-2RESUMO
Fungi stand out as primary pathogens present in healthcare-acquired infections, presenting an increased number of cases even using appropriate antifungal therapy. Candida spp. is a predominant microorganism among several fungal pathogens present in the healthcare setting. Candidemia and candidiasis are fungal infections responsible for high morbidity and mortality among ill patients in hospitals. It is noticeable that prolonged hospital stays lead to a higher economic impact and increased risk for developing secondary fungal or even bacterial infections. New fast and sensitive approaches for the detection of Candida species is highly required. Electrochemical biosensors are an excellent alternative to conventional techniques by combining fast analyte detection, low cost, and the possibility of miniaturization. Lectins are carbohydrate-binding proteins with the capability to reach out to the microorganism cell wall. In this work, we proposed the development of an impedimetric biosensor for Candida spp. based on Concanavalin A (ConA) and wheat germ agglutinin (WGA) as recognition agents of the yeast cells. Atomic force microscopy images indicate changes in the biosensor surface after assembly of the molecules and exposure to fungal samples. Electrochemical impedance spectroscopy results revealed a proportional increase of charge transfer resistance (RCT) as fungal CFU increased, where four Candida species were evaluated (Candida krusei, Candida tropicalis, Candida parapsilosis and Candida albicans). The biosensor is useful to differentiate Candida spp. with a detection limit between 102 to 106 CFU mL-1. The obtained biosensor appears as an innovative candidate for the detection and differentiation of pathogenic Candida spp.
Assuntos
Técnicas Biossensoriais , Candida , Antifúngicos , Diferenciação Celular , Humanos , Lectinas , Testes de Sensibilidade Microbiana , PichiaRESUMO
The human papillomavirus (HPV) is one of the main sexually transmitted pathogens that infect the anogenital epithelium and mucous membranes. HPV genotypes can be classified as high and low oncogenic risk, with infection by the former resulting in cervical cancer in approximately 100 % of the cases. In this work, we developed an ultrasensitive electrochemical biosensor for the detection and identification of different HPV genotypes. A nanostructured platform based on a matrix of polyaniline (PANI) containing gold nanoparticles (AuNps) was designed for the chemical immobilization of a DNA probe capable of recognizing different HPV types. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) were used to characterize the genosensor. The impedimetric responses indicate that the proposed sensor was able to detect HPV (types 6, 11, 16, 31, 33, 45, and 58) in cervical specimens (cDNA samples). We obtained different profiles of electrochemical responses for the high and low-risk HPV genotypes. By adopting a three-dimensional quantitative analysis of impedance response variables, it was possible to identify the existence of a pattern of association for samples of high oncogenic risk, which may lead to the differential diagnosis of HPV. The biosensor demonstrated an excellent analytical performance for the detection of HPV genotypes with high sensibility and selectivity. The genosensor exhibited a linear range of response in the 1 pg µL-1 to 100 pg µL-1 range. Besides, a limit of detection (LOD) of 2.74 pg µL-1 and 7.43 pg µL-1 was obtained for HPV11 and HPV16, respectively, with regression coefficients of 99.88 % and 99.47 %. Thus, the proposed sensor may serve as a good prognostic indicator for patients infected with papillomavirus.
Assuntos
Alphapapillomavirus/isolamento & purificação , Técnicas Biossensoriais/métodos , Colo do Útero/virologia , Nanopartículas Metálicas/química , Infecções por Papillomavirus/diagnóstico , Alphapapillomavirus/genética , Colo do Útero/patologia , DNA Viral/isolamento & purificação , Diagnóstico Diferencial , Estudos de Viabilidade , Feminino , Técnicas de Genotipagem/métodos , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura , Microeletrodos , Microscopia de Força Atômica , Sondas Moleculares/química , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Prognóstico , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologiaRESUMO
Arboviruses have been emerging as a significant global health problem due to the recurrent epidemics. Arboviruses require the development of new diagnostic devices due to the nonspecific clinical manifestations. Herein, we report a biosensor based on cysteine (Cys), zinc oxide nanoparticles (ZnONp), and Concanavalin A (ConA) lectin to differentiate between arboviruses infections. ConA is capable of interacting with the saccharide components of the viral capsid. In this study, we evaluated the reproducibility, sensitivity, and specificity of the sensor for the virus of Dengue type 2 (DENV2), Zika (ZIKV), Chikungunya (CHIKV), and Yellow fever (YFV). Atomic force microscopy measurements confirmed the electrode surface modification and revealed a heterogeneous topography during the biorecognition process. Cyclic voltammetry (CV) and impedance spectroscopy (EIS) were used to characterize the biosensor. The blockage of the oxidation-reduction process is related to the formation of Cys-ZnONp-ConA system on the electroactive area and its subsequent interaction with viral glycoproteins. The sensor exhibited a linear response to different concentrations of the studied arboviruses. Our study demonstrates that ConA lectin recognizes the structural glycoproteins of the DENV2, ZIKV, CHIKV, and YFV. DENV2 is the most structurally similar to ZIKV. Our results have shown that the impedimetric response correlates with the structural glycoproteins, as follow: DENV2 (18.6â¯kΩ)â¯>â¯ZIKV (14.6â¯kΩ)â¯>â¯CHIKV (6.86â¯kΩ)â¯>â¯YFV (5.98â¯kΩ). The homologous structural regions contribute to ConA-arboviruses recognition. Our results demonstrate the use of the proposed system for the development of biosensors for arboviruses infections.