RESUMO
Non-structural 1 (NS1) is a protein biomarker that can be found in blood in the early stages of dengue and related infections (Zika and Chikungunya). This study aims to develop a biosensor to selectively quantify NS1 using DNA aptamer co-immobilized on gold electrodes with 6-(ferrocenyl)hexanethiol (FCH) using electrochemical capacitive spectroscopy. This technique uses a redox probe (FCH) immobilized on the self-assembled monolayer to convert impedance into capacitance information. The developed platform was blocked with bovine serum albumin before NS1 exposure and the ratio between aptamers and FCH was optimized. The aptasensor was tested using commercial NS1 serotype 4 in phosphate-buffered saline and commercial undiluted human serum. Using the optimum applied potential provides high sensitivity (3 and 4 nF per decade) and low limit of detection (30.9 and 41.8 fg/mL) with a large linear range (10 pg to 1 µg/mL and 10 pg to 100 ng/mL, respectively). Both results exhibit a residual standard deviation value < 1%. The results suggested that this aptasensor was capable of detecting NS1 in the clinical range and can be applied to any other specific aptamer with FCH, opening the path for label-free miniaturized point-of-care devices with high sensitivity and specificity.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Dengue , Infecção por Zika virus , Zika virus , Humanos , Limite de Detecção , Aptâmeros de Nucleotídeos/química , Espectroscopia Dielétrica/métodos , Técnicas Biossensoriais/métodos , Dengue/diagnósticoRESUMO
Dengue is one of the most commonly neglected tropical diseases transmitted by Aedes aegypti infected with Dengue virus. This virus belongs to the gender Flavivirus and produces a non-structural protein 1 (NS1), which is an important biomarker found at high levels in blood in early disease stage. Therefore, this study focused on the development of an electrochemical biosensor for NS1 detection using DNA aptamers. Gold electrodes were co-immobilized with specific aptamers and 6-mercapto-1-hexanol (MCH) to obtain a self-assembled monolayer. The molar ratio between aptamers and MCH was optimized and the platform characterized by electrochemical impedance spectroscopy and atomic force microscopy. Bovine serum albumin was added in NS1 solution to stabilize it and block the surface to avoid non-specific interactions. The biosensor performance was tested with NS1 protein serotype 4 (in phosphate saline buffer and human serum) and with a solution of serotype 1 in human serum. The results showed a sensitivity of 2.9%, 2.7% and 1.7% per decade, respectively, and low limit of detection (0.05, 0.022 and 0.025 ng/mL). The platform was also tested with Envelope protein as negative control. Furthermore, the aptamer sensor was able to detect NS1 in clinical range and it is a promising candidate for a new class for miniaturized point-of-care device for different Dengue serotypes.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Dengue , Dengue/diagnóstico , Espectroscopia Dielétrica , Eletrodos , Humanos , Limite de DetecçãoRESUMO
Breast cancer remains one of the leading causes of women death. The development of more sensitive diagnostic tests, which could present a faster response, lower cost, and could promote early diagnosis would increase the chances of survival. This study reports the development and optimization of an electrochemical aptasensor for the detection of HER2 protein, a breast cancer biomarker. Two sensing platforms were developed on gold screen-printed electrodes. The first platform is composed of self-assembled monolayer (SAM) made from mixture of thiolated DNA aptamers specific for HER2 and 1-mercapto-6-hexanol (MCH), while the second one is a ternary SAM composed of the same aptamer and 1,6-hexanethiol (HDT). Both platforms were further passivated with MCH and blocked with bovine serum albumin. The biosensors were characterized using electrochemical impedance spectroscopy to detect the target protein from 1 pg/mL to 1 µg/mL in phosphate buffered saline, diluted and undiluted human serum through charge transfer resistance value. The ternary SAM architecture shows a reduction of non-specific attachment to the electrode surface due to the HDT antifouling properties. In addition, this platform exhibits 172 pg/mL as limit of detection and a sensitivity of 4.12% per decade for undiluted serum compared with SAM architecture with the 179 pg/mL and 4.32% per decade, respectively. Electrochemical aptasensors are highly promising for medical diagnostic and ternary layers could improve the limit of detection.
Assuntos
Aptâmeros de Nucleotídeos/química , Neoplasias da Mama/diagnóstico , Técnicas Eletroquímicas/instrumentação , Eletrodos , Biomarcadores Tumorais , Técnicas Biossensoriais/métodos , Neoplasias da Mama/sangue , Feminino , Genes erbB-2 , Humanos , Limite de DetecçãoRESUMO
Glucose and urea enzymatic biosensors were fabricated. One-step electrochemical immobilization process was used to produce thin polyaniline films with entrapped enzymes. Chronopotentiometric analysis, scanning electron microscopy, electrochemical impedance spectroscopy and optical reflectance spectroscopy were used to determine the structure-property relationship of the functionalized polymeric thin films. The device has a recognition stage connected to a potentiometric field-effect-transistor stage and is based on the measurement of microenvironment pH variation or locally produced ions. Optimization of biosensor fabrication and effective measurement conditions were performed. The optimized films presented sensitivity, linearity and detection range to glucose of 14.6 ± 0.4 mV/decade, 99.8% and from 10-4 M to 10-1 mol/L. Two different biosensors were produced based on the enzymatic reaction of urea with selectivity to ammonium or hydroxyl ions. For ammonium ion selective film, the sensor's parameters were 14.7 ± 0.9 mV/decade, 98.2% and from 10-5 to 10-1 mol/L. For the hydroxyl ion selective film, the same parameters were 7.4 ± 0.5 mV/decade, 98.1% and from 10-5 to 10-1 mol/L. The change in the oxidation state of the polymeric matrix explains: i) the large loss of functionality of glucose biosensor in time, ii) the conservation of functionality to the hydroxyl ions for urea biosensor and iii) the selectivity variation of the ammonium ion selective urea biosensor. The results indicate that the polymeric matrix has indeed changeable selectivity, what can be applied in different situations for biosensors production.