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Staphylococcus aureus is an important pathogen that causes nosocomial infections, resulting in unacceptable morbidity and mortality rates. In this work, we proposed the construction of a nanostructured ZnO-based electrochemical immunosensor for qualitative and semiquantitative detection of S. aureus using simple methods for growing zinc oxide nanorods (ZnO NRs) on a sensor board and immobilizing the anti-S. aureus antibody on ZnO NRs through cystamine and glutaraldehyde. The immunosensor detected S. aureus in the 103-107 colony-forming unit (CFU) mL-1 range and showed a limit of detection (LoD) around 0.792 × 103 CFU mL-1. Beyond a satisfactory LoD, the developed immunosensor presented other advantages, such as high versatility for point-of-care assays and a suitable selective factor that admits the detection of the S. aureus concentration range in human hand skin after washing. Moreover, the immunosensor showed the potential to be an excellent device to control nosocomial infection by detecting the presence of S. aureus in human hand skin.
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Técnicas Biossensoriais , Infecção Hospitalar , Técnicas Eletroquímicas , Sistemas Automatizados de Assistência Junto ao Leito , Pele , Staphylococcus aureus , Óxido de Zinco , Humanos , Staphylococcus aureus/isolamento & purificação , Infecção Hospitalar/prevenção & controle , Pele/microbiologia , Técnicas Biossensoriais/métodos , Óxido de Zinco/química , Imunoensaio/métodos , Técnicas Eletroquímicas/métodos , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Mãos/microbiologia , Limite de Detecção , Nanotubos/química , Anticorpos Imobilizados/químicaRESUMO
A new conductive ink based on the addition of carbon black to a poly(vinyl alcohol) matrix is developed and investigated for electrochemical sensing and biosensing applications. The produced devices were characterized using morphological and electrochemical techniques and modified with Pd nanoparticles to enhance electrical conductivity and reaction kinetics. With the aid of chemometrics, the parameters for metal deposition were investigated and the sensor was applied to the determination of Parkinson's disease biomarkers, specifically epinephrine and α-synuclein. A linear behavior was obtained in the range 0.75 to 100 µmol L-1 of the neurotransmitter, and the device displayed a limit of detection (LOD) of 0.051 µmol L-1. The three-electrode system was then tested using samples of synthetic cerebrospinal fluid. Afterward, the device was modified with specific antibodies to quantify α-synuclein using electrochemical impedance spectroscopy. In phosphate buffer, a linear range was obtained for α-synuclein concentrations from 1.5 to 15 µg mL-1, with a calculated LOD of 0.13 µg mL-1. The proposed immunosensor was also applied to blood serum samples, and, in this case, the linear range was observed from 6.0 to 100.5 µg mL-1 of α-synuclein, with a LOD = 1.3 µg mL-1. Both linear curves attend the range for the real diagnosis, demonstrating its potential application to complex matrices.
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Técnicas Biossensoriais , Nanopartículas , Doença de Parkinson , Humanos , Doença de Parkinson/diagnóstico , alfa-Sinucleína , Técnicas Biossensoriais/métodos , ImunoensaioRESUMO
Neglected tropical diseases are those caused by infectious agents or parasites and are considered endemic in low-income populations. These diseases also have unacceptable indicators and low investment in research, drug production, and control. Tropical diseases such as leishmaniasis are some of the main causes of morbidity and mortality around the globe. Electrochemical immunosensors are promising tools for diagnostics against these diseases. One such benefit is the possibility of assisting diagnosis in isolated regions, where laboratory infrastructure is lacking. In this work, different peptides were investigated to detect antibodies against Leishmania in human and canine serum samples. The peptides evaluated (395-KKG and 395-G) have the same recognition site but differ on their solid-binding domains, which ensure affinity to spontaneously bind to either graphene oxide (GO) or graphene quantum dots (GQD). Cyclic voltammetry and differential pulse voltammetry were employed to investigate the electrochemical behavior of each assembly step and the role of each solid-binding domain coupled to its anchoring material. The graphene affinity peptide (395-G) showed better reproducibility and selectivity when coupled to GQD. Under the optimized set of experimental conditions, negative and positive human serum samples responses were distinguished based on a cut-off value of 82.5% at a 95% confidence level. The immunosensor showed selective behavior to antibodies against Mycobacterium leprae and Mycobacterium tuberculosis, which are similar antibodies and potentially sources of false positive tests. Therefore, the use of the graphene affinity peptide as a recognition site achieved outstanding performance for the detection of Leishmania antibodies.
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Técnicas Biossensoriais , Grafite , Leishmaniose , Animais , Cães , Humanos , Carbono/química , Grafite/química , Reprodutibilidade dos Testes , Imunoensaio , Peptídeos , Anticorpos , Leishmaniose/diagnósticoRESUMO
To help meet the global demand for reliable and inexpensive COVID-19 testing and environmental analysis of SARS-CoV-2, the present work reports the development and application of a highly efficient disposable electrochemical immunosensor for the detection of SARS-CoV-2 in clinical and environmental matrices. The sensor developed is composed of a screen-printed electrode (SPE) array which was constructed using conductive carbon ink printed on polyethylene terephthalate (PET) substrate made from disposable soft drink bottles. The recognition site (Spike S1 Antibody (anti-SP Ab)) was covalently immobilized on the working electrode surface, which was effectively modified with carbon black (CB) and gold nanoparticles (AuNPs). The immunosensing material was subjected to a multi-technique characterization analysis using X-ray diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) with elemental analysis via energy dispersive spectroscopy (EDS). The electrochemical characterization of the electrode surface and analytical measurements were performed using cyclic voltammetry (CV) and square-wave voltammetry (SWV). The immunosensor was easily applied for the conduct of rapid diagnoses or accurate quantitative environmental analyses by setting the incubation period to 10 min or 120 min. Under optimized conditions, the biosensor presented limits of detection (LODs) of 101 fg mL-1 and 46.2 fg mL-1 for 10 min and 120 min incubation periods, respectively; in addition, the sensor was successfully applied for SARS-CoV-2 detection and quantification in clinical and environmental samples. Considering the costs of all the raw materials required for manufacturing 200 units of the AuNP-CB/PET-SPE immunosensor, the production cost per unit is 0.29 USD.
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In this work, a conducting polymer (CP) was obtained through three electrochemical procedures to study its effect on the development of an electrochemical immunosensor for the detection of immunoglobulin G (IgG-Ag) by square wave voltammetry (SWV). The glassy carbon electrode modified with poly indol-6-carboxylic acid (6-PICA) applied the cyclic voltammetry technique presented a more homogeneous size distribution of nanowires with greater adherence allowing the direct immobilization of the antibodies (IgG-Ab) to detect the biomarker IgG-Ag. Additionally, 6-PICA presents the most stable and reproducible electrochemical response used as an analytical signal for developing a label-free electrochemical immunosensor. The different steps in obtaining the electrochemical immunosensor were characterized by FESEM, FTIR, cyclic voltammetry, electrochemical impedance spectroscopy, and SWV. Optimal conditions to improve performance, stability, and reproducibility in the immunosensing platform were achieved. The prepared immunosensor has a linear detection range of 2.0-16.0 ng·mL-1 with a low detection limit of 0.8 ng·mL-1. The immunosensing platform performance depends on the orientation of the IgG-Ab, favoring the formation of the immuno-complex with an affinity constant (Ka) of 4.32 × 109 M-1, which has great potential to be used as point of care testing (POCT) device for the rapid detection of biomarkers.
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The evaluation of serological responses to COVID-19 is crucial for population-level surveillance, developing new vaccines, and evaluating the efficacy of different immunization programs. Research and development of point-of-care test technologies remain essential to improving immunity assessment, especially for SARS-CoV-2 variants that partially evade vaccine-induced immune responses. In this work, an impedimetric biosensor based on the immobilization of the recombinant trimeric wild-type spike protein (S protein) on zinc oxide nanorods (ZnONRs) was employed for serological evaluation. We successfully assessed its applicability using serum samples from spike-based COVID-19 vaccines: ChAdOx1-S (Oxford-AstraZeneca) and BNT162b2 (Pfizer-BioNTech). Overall, the ZnONRs/ spike-modified electrode displayed accurate results for both vaccines, showing excellent potential as a tool for assessing and monitoring seroprevalence in the population. A refined outcome of this technology was achieved when the ZnO immunosensor was functionalized with the S protein from the P.1 linage (Gamma variant). Serological responses against samples from vaccinated individuals were acquired with excellent performance. Following studies based on traditional serological tests, the ZnONRs/spike immunosensor data reveal that ChAdOx1-S vaccinated individuals present significantly less antibody-mediated immunity against the Gamma variant than the BNT162b2 vaccine, highlighting the great potential of this point-of-care technology for evaluating vaccine-induced humoral immunity against different SARS-CoV-2 strains.
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COVID-19 , Vacinas , Óxido de Zinco , Humanos , Vacina BNT162 , SARS-CoV-2 , Vacinas contra COVID-19 , Estudos Soroepidemiológicos , COVID-19/diagnóstico , Anticorpos , Anticorpos AntiviraisRESUMO
Ultrasensitive electroanalytical monitoring of interleukin-6 levels in serum samples has emerged as a valuable tool for the early diagnosis of inflammatory diseases. Despite its advantages, there is a lack of strategies for the label-free voltammetric determination of cytokines. Here, a novel chitosan/genipin modified fluorine tin oxide electrode was developed providing an in-situ hydrogel formation (FTO/CSG). This platform was applied for the detection of interleukin-6, a major pro-inflammatory cytokine. Transmission electron microscopy (TEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) indicated genipin serves as an efficient green cross-linker to build the immunosensing platform (FTO/CSG/anti-IL-6). EIS showed an increase in charge transfer resistance from 326 to 1360 kΩ after the immobilization of anti-IL-6 antibodies. By square wave voltammetry, this method achieved a detection limit of 0.03 pg mL-1 with a wide linear range of 0.05-1000 pg mL-1. Additionally, it displayed a high selectivity index when tested in the presence of three inflammatory cytokines as interfering proteins: IL-12, IL-1ß, and TNF-α. The sample matrix effect showed a peak current variation lower than 5 %. The novel method was applied for the quantification of IL-6 in serum samples of septic mice. No statistical differences were observed between the standard ELISA and the proposed method using a confidence level of 95 %.
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Técnicas Biossensoriais , Quitosana , Sepse , Animais , Camundongos , Interleucina-6 , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodos , Biomarcadores , Eletrodos , Imunoensaio/métodos , Limite de DetecçãoRESUMO
Waste management is a key feature to ensure sustainable consumption and production patterns, and to combat the impacts of climate change. In this scenario, the production of biochar from different biomasses results in environmental and economic advantages. In this study, biochar was produced from sugarcane bagasse pyrolysis, to immobilize biomolecules, in order to assemble an electrochemical immunosensor to detect antibodies against SARS-CoV-2. For this, screen-printed carbon electrodes (SPCE) were modified with a dispersion of biochar and used to immobilize the receptor-binding-domain (RBD) against virus S-protein, through EDC/NHS crosslinking reaction. Under the best set of experimental conditions, negative and positive serum samples responses distinguished based on a cutoff value of 82.3 %, at a 95 % confidence level. The immunosensor showed selective behavior to antibodies against yellow fever and its performance was stable up to 7 days of storage. Therefore, biochar yielded from sugarcane bagasse is an ecofriendly material that can be used as a platform to immobilize biomolecules for construction of electrochemical biosensors.
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Técnicas Biossensoriais , COVID-19 , Saccharum , Técnicas Eletroquímicas/métodos , SARS-CoV-2 , Celulose , Imunoensaio/métodos , Eletrodos , AnticorposRESUMO
The development of immunosensors to detect antibodies or antigens has stood out in the face of traditional methods for diagnosing emerging diseases such as the one caused by the SARS-CoV-2 virus. The present study reports the construction of a simplified electrochemical immunosensor using a graphene-binding peptide applied as a recognition site to detect SARS-CoV-2 antibodies. A screen-printed electrode was used for sensor preparation by adding a solution of peptide and reduced graphene oxide (rGO). The peptide-rGO suspension was characterized by scanning electron microscopy (SEM), Raman spectroscopy, and Fourier transform infrared spectroscopy (FT-IR). The electrochemical characterization (electrochemical impedance spectroscopy-EIS, cyclic voltammetry-CV and differential pulse voltammetry-DPV) was performed on the modified electrode. The immunosensor response is based on the decrease in the faradaic signal of an electrochemical probe resulting from immunocomplex formation. Using the best set of experimental conditions, the analytic curve obtained showed a good linear regression (r2 = 0.913) and a limit of detection (LOD) of 0.77 µg mL-1 for antibody detection. The CV and EIS results proved the efficiency of device assembly. The high selectivity of the platform, which can be attributed to the peptide, was demonstrated by the decrease in the current percentage for samples with antibody against the SARS-CoV-2 S protein and the increase in the other antibodies tested. Additionally, the DPV measurements showed a clearly distinguishable response in assays against human serum samples, with sera with a response above 95% being considered negative, whereas responses below this value were considered positive. The diagnostic platform developed with specific peptides is promising and has the potential for application in the diagnosis of other infections that lead to high antibody titers.
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Técnicas Biossensoriais , COVID-19 , Grafite , Humanos , Grafite/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , SARS-CoV-2 , Espectroscopia de Infravermelho com Transformada de Fourier , Imunoensaio , COVID-19/diagnóstico , Eletrodos , Limite de Detecção , PeptídeosRESUMO
Flexible, fully printed immunosensors can meet the requirements of precision nutrition, but this demands optimized molecular architectures to reach the necessary sensitivity. Herein, we report on flexible and label-free immunosensor chips made with tree-like gold dendrites (AuDdrites) electrochemically formed by selective desorption of l-cysteine (L-cys) on (111) gold planes. Electrodeposition was used because it is scalable and cost-effective for a rapid, direct growth of Au hyperbranched dendritic structures. The 25-hydroxyvitamin D3 (25(OH)D3) metabolite was detected within 15 min with a limit of detection (LOD) of 0.03 ng mL-1. This high performance was possible due to the careful optimization of the electroactive layer and working conditions for square wave voltammetry (SWV). Electrocrystallization was manipulated by controlling the deposition potential and the molar ratio between HAuCl4 and L-cys. Metabolite detection was performed on human serum and saliva samples with adequate recovery between 97% and 100%. The immunosensors were stable and reproducible, unresponsive to interference from other molecules in human serum and saliva. They can be extended for use as wearable sensors with their mechanical flexibility and possible customization.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Calcifediol , Dendritos , Técnicas Eletroquímicas , Eletrodos , Ouro/química , Humanos , Imunoensaio , Limite de Detecção , Nanopartículas Metálicas/químicaRESUMO
A label-free nanoimmunosensor is reported based on p53/CeO2/PEDOT nanobiocomposite-decorated screen-printed gold electrodes (SPAuE) for the electrochemical detection of anti-p53 autoantibodies. CeO2 nanoparticles (NPs) were synthesized and stabilized with cyanopropyltriethoxysilane by a soft chemistry method. The nanoimmunosensing architecture was prepared by in situ electropolymerization of 3,4-ethylenedioxythiophene (EDOT) on SPAuE in the presence of CeO2 NPs. The CeO2 NPs and Ce/PEDOT/SPAuE were characterized by scanning and transmission electron microscopy, dynamic and electrophoretic light scattering, ultraviolet-visible spectrophotometry, X-ray diffraction, Fourier-transform infrared spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. Ce/PEDOT/SPAuE was biofunctionalized with p53 antigen by covalent bonding for the label-free determination of anti-p53 autoantibodies by differential pulse voltammetry. The nanobiocomposite-based nanoimmunosensor detected anti-p53 autoantibodies in a linear range from 10 to 1000 pg mL-1, with a limit of detection (LOD) of 3.2 pg mL-1. The nanoimmunosensor offered high specificity, selectivity, and long-term storage stability with great potential to detect anti-p53 autoantibodies in serum samples. Overall, incorporating organo-functional nanoparticles into polymeric matrices can provide a simple-to-assemble, rapid, and ultrasensitive approach for on-site screening of anti-p53 autoantibodies and other disease-related biomarkers with low sample volumes.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Nanocompostos , Autoanticorpos , Técnicas Biossensoriais/métodos , Compostos Bicíclicos Heterocíclicos com Pontes , Cério , Nanopartículas Metálicas/química , Nanocompostos/química , PolímerosRESUMO
BACKGROUND Malaria is a disease that affects many tropical and subtropical countries, including Brazil. The use of tests for malaria detection is one of the fundamental strategies recommended by the World Health Organization for the control and eradication of the disease. The lack of diagnostic tests leads to an increase in transmission and non-reporting cases. OBJECTIVES This work described an electrochemical immunosensor for detecting Plasmodium vivax lactate dehydrogenase antigen (Ag-PvLDH). METHODS The device has developed by immobilising egg yolk IgY antibodies (Ab-PvLDH) on a gold electrode surface using cysteamine as linker. The immunosensor fabrication was followed by differential pulse voltammetry, and contact angle measurements were performed to characterise the modified gold electrode surface. FINDINGS The results for Ag-PvLDH determination exhibit a linear response at 10-50 µg mL-1 concentration range, with a limit of detection of 455 ng mL-1. The excellent selectivity of the device was confirmed. MAIN CONCLUSIONS The developed immunosensor showed a good performance, therefore, it can be considered an alternative test to detect malaria caused by P. vivax.
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A sensitive detection of carbohydrate antigen 15-3 (CA15-3) levels may allow for early diagnosis and monitoring the treatment of breast cancer, but this can only be made in routine clinical practice if low-cost immunosensors are available. In this work, we developed a sandwich-type electrochemical immunosensor capable of rapid detection of CA15-3 with an ultra-low limit of detection (LOD) of 0.08 fg mL-1 within a wide linear concentration range from 0.1 fg mL-1 to 1 µg mL-1. The immunosensor had a matrix of a layer-by-layer film of Au nanoparticles and reduced graphene oxide (Au-rGO) co-electrodeposited on screen-printed carbon electrodes (SPCE). The high sensitivity was achieved by using secondary antibodies (Ab2) labeled with horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2) as signal amplifiers, and hydroquinone (HQ) was used as an electron mediator. The immunosensor was selective for CA15-3 in human serum and artificial saliva samples, robust, and stable to permit storage at 4 °C for more than 30 days. With its high performance, the immunosensor may be incorporated into future point-of-care (POC) devices to determine CA15-3 in distinct biological fluids, including in blood and saliva samples.
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Biomarcadores Tumorais/sangue , Técnicas Eletroquímicas/métodos , Grafite/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , Mucina-1/sangue , Anticorpos Imobilizados/imunologia , Armoracia/enzimologia , Biomarcadores Tumorais/imunologia , Ouro/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Hidroquinonas/química , Limite de Detecção , Mucina-1/imunologia , Reprodutibilidade dos Testes , Saliva/químicaRESUMO
This work describes the first method using biochar (BC) as carbonaceous platform for immunoassay application. BC is a highly functionalized material obtained through biomass pyrolysis under controlled conditions. Due to the highly functionalized surface, covalent binding between BC and biomolecules can be performed by EDC/NHS conjugation. The application of the modified electrode was done with Hantavirus, that are etiologic agents mainly transmitted by wild rodents. Among its pathologies Hantavirus Cardiopulmonary Syndrome (HCPS) arises at Americas, caused by Hantavirus Araucária and reaches 40% lethality. The diagnostic is based on the presence of specific hantavirus nucleoprotein (Np), under viremic condition or IgG2b antibodies (Ab), during first symptoms. The results presented a device sensitivity of 5.28⯵A dec-1 and a LOD of 0.14â¯ngâ¯mL-1 to the Np detection, ranging from 5.0â¯ngâ¯mL-1 to 1.0⯵gâ¯mL-1, the Ab detection works as qualitative type sensor above 200â¯ngâ¯mL-1. Both sensors were evaluated its selectivity and serum samples; selectivity against Gumboro disease, VP2 protein, and antibody IgG2a against Yellow fever disease (YF), respectively. So, the devices here proposed are promising tool suitable for both rodent and human hantavirus clinical surveys.
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Carvão Vegetal/química , Orthohantavírus/isolamento & purificação , Anticorpos Imobilizados/imunologia , Anticorpos Antivirais/imunologia , Sangue/virologia , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Orthohantavírus/química , Humanos , Imunoensaio/métodos , Imunoglobulina G/imunologia , Limite de Detecção , Reprodutibilidade dos Testes , Proteínas Virais/imunologiaRESUMO
In this research, ZnO nanorods - Au nanoparticles nanohybrids have been fabricated and employed to sensitive electrochemical strategy for the specific detection of the ovarian cancer antigen CA-125/MUC126. The microdevice was developed in our lab based on gold and silver electrodes sputtered on glass substrate. The ZnO nanorods arrays were grown on working electrode using assisted microwave hydrothermal synthesis than gold nanoparticles (Au NPs) were deposited by sputtering. The Au NPs onto ZnO nanorods surface provides a favorable platform for efficient loading of anti-CA-125 antibody via binding with cystamine and glutaraldehyde. The effective loading of the biological material (CA-125 antibody and antigen) on the matrix was observed by SEM images. The electrochemical immunosensor shows a sensitive response to ovarian cancer antigen recombinant human CA-125/MUC126 with detection of 2.5ng/µL, 100 times lower than immunoblot system. Due to high specificity, reproducibility and noteworthy stability, the developed sensor will provide a sensitive, selective and convenient approach to be used to detect CA-125/MUC126.
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Nanopartículas Metálicas , Nanotubos , Técnicas Biossensoriais , Antígeno Ca-125 , Técnicas Eletroquímicas , Feminino , Ouro , Humanos , Imunoensaio , Neoplasias Ovarianas , Reprodutibilidade dos TestesRESUMO
This work describes the preparation of an electrochemical immunosensor for ethinylestradiol (EE2) based on grafting of diazonium salt of 4-aminobenzoic acid onto a glassy carbon electrode modified with silver nanoparticles/SiO2/graphene oxide hybrid followed by covalent binding of anti-ethinylestradiol (anti-EE2) to activated carboxyl groups. A competitive immunoassay was developed for the determination of the hormone using peroxidase-labeled ethinylestradiol (HRP-EE2) and measurement of the amperometric response at -200mV in the presence of hydroquinone (HQ) as redox mediator. The calibration curve for EE2 exhibited a linear range between 0.1 and 50ng/mL (r(2)=0.996), with a detection limit of 65pg/mL. Interference studies with other hormones related with EE2 revealed the practical specificity of the developed method for the analyte. A good reproducibility, with RSD=4.5% (n=10) was also observed. The operating stability of a single bioelectrode modified with anti-EE2 was maintained at least for 15 days when it was stored at 4°C under humid conditions between measurements. The developed immunosensor was applied to the analysis of spiked urine with good results.