Signal amplification for thrombin impedimetric aptasensor : sandwich protocol and use of gold-streptavidin nanoparticles

In this work, we report a highly specific amplification strategy demonstrated for the ultrasensitive biosensing of thrombin with the use of gold-streptavidin nanoparticles (strep-AuNPs) and silver reduction enhancement. The biotinylated aptamer of thrombin was immobilized onto an avidin-graphite epoxy composite (AvGEC) electrode surface by affinity interaction between biotin and avidin; electrochemical impedance measurements were performed in a solution containing the redox marker ferrocyanide/ferricyanide. The change in interfacial charge transfer resistance (Rct) experimented by the redox marker, was recorded to confirm aptamer complex formation with target protein, thrombin (Thr), in a label-free first stage. A biotinylated second thrombin aptamer, with complementary recognition properties was then used in a sandwich approach. The addition of strep-AuNPs and silver enhancement treatment led to a further increment of Rct thus obtaining significant signal amplification. The AptThrBio1-Thr-AptThrBio2 sandwich formation was inspected by confocal microcopy after incubation with streptavidin quantum dots. In order to visualize the presence of gold nanoparticles, the same silver enhancement treatment was applied to electrodes already modified with the nanoparticle-sandwich conjugate, allowing direct observation by scanning electron microscopy (SEM). Results showed high sensitivity and selectivity for thrombin detection, with an improvement from ca. 4.7 pM in a simple assay to 0.3 pM in the amplified reported scheme

Three different signal amplification strategies for the impedimetric sandwich detection of thrombin

In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM)

Aptamer-antibody sandwich assay for cytochrome c employing a MWCNT platform and the electrochemical impedance technique

We report on a sensitive aptamer-antibody interaction-based assay for cytochrome c (Cytc) using electrochemical impedance. 4-Amino benzoic acid is used for the oriented immobilization of aminated aptamers onto multi-walled carbon nanotubes on the surface of a screen-printed electrode via electrochemical grafting. Impedance was measured in a solution containing the redox system ferro/ferricyanide. The change in interfacial charge transfer resistance (Rct) experienced by the redox marker was recorded to confirm the formation of a complex between aptamer and the target (Cytc). A biotinylated antibody against cytochrome c was then used in a sandwich type of assay. The addition of streptavidin conjugated to gold nanoparticles and signal enhancement by treatment with silver led to a further increase in Rct. Under optimized conditions, a detection limit as low as 12 pM was obtained. Cross-reactivity against other serum proteins including fibrinogen, BSA and immunoglobulin G demonstrated improved selectivity

Label free aptasensor for lysozyme detection : a comparison of the analytical performance of two aptamers

This work presents a comparison of two different aptamers (COX and TRAN) for the detection of the ubiquitous protein lysozyme using aptamer-based biosensors. The detection is based on the specific recognition by the aptamer immobilized on screen printed carbon electrodes (SPCEs) via diazonium coupling reaction. The quantitative detection of lysozyme protein was achieved by electrochemical impedance spectroscopy (EIS). Very good linear ranges and detection limits for the lysozyme detection were obtained, from 0.025 to 1 μM and 725nM using aptamer COX and from 0.025 to 1 μM and 31.7nM using aptamer TRAN. The obtained results showed that the developed aptasensors exhibit good specificity, stability and reproducibility for lysozyme detection. The aptasensors were also tested in wine samples; very good recovery rates were obtained in the range from 96.4 to 102% for lysozyme detection. The recovery rates confirm the reliability and suitability of the developed method in wine matrix. The developed method could be a useful and promising platform for detection of lysozyme in different applications

A novel electrochemical aptamer-antibody sandwich assay for lysozyme detection

In this paper, we have reported a novel electrochemical aptamer-antibody based sandwich biosensor for the detection of lysozyme. In the sensing strategy, an anti-lysozyme aptamer was immobilized onto the carbon electrode surface by covalent binding via diazonium salt chemistry. After incubating with a target protein (lysozyme), a biotinylated antibody was used to complete the sandwich format. The subsequent additions of avidin-alkaline phosphatase as an enzyme label, and a 1-naphthyl phosphate substrate (1-NPP) allowed us to determine the concentration of lysozyme (Lys) via Differential Pulse Voltammetry (DPV) of the generated enzyme reaction product, 1-naphthol. Using this strategy, a wide detection range from 5 fM to 5 nM was obtained for a target lysozyme, with a detection limit of 4.3 fM. The control experiments were carried out by using bovine serum albumin (BSA), cytochrome c and casein. The results showed that the proposed biosensor had good specificity, stability and reproducibility for lysozyme analysis. In addition, the biosensor was applied for detecting lysozyme in spiked wine samples, and very good recovery rates were obtained in the range from 95.2 to 102.0% for lysozyme detection. This implies that the proposed sandwich biosensor is a promising analytical tool for the analysis of lysozyme in real samples

Aptasensores impedimétricos para la detección de trombina

En el presente trabajo se ha desarrollado el primer aptasensor (biosensor de aptámero) en nuestro grupo de investigación, Grup de Sensors i Biosensors de la Universidad Autònoma de Barcelona. En concreto se han desarrollado dos aptasensores para la detección de la proteína trombina, uno basado en la inmovilización de aptámeros por adsorción física, y otro basado en la inmovilización de aptámeros por enlace covalente mediante la reacción EDAC-NHS. El aptasensor utiliza la afinidad específica de la cadena de DNA (aptámero) por la proteína con la que interacciona. Los cambios de carga y estéricos del complejo aptámero proteína alteran la capacidad y la resistencia de transferencia interfacial de electrones en la superficie del electrodo. El principio de detección se basa en la detección de cambios de estas propiedades de interfase del electrodo con el marcador redox [Fe(CN)6]3- / [Fe(CN)6]4-, utilizando mediciones de Espectroscopia Electroquímica de Impedancia. El aptasensor basado en adsorción física del aptámero mostró una respuesta lineal a trombina en el rango de 7.5 a 75 pM y un límite de detección de 5pM, después de optimizar todas las condiciones experimentales. Posteriormente se estudió la especificidad del sistema respecto proteínas potencialmente interferentes presentes en suero sanguíneo, obteniendo cierta interferencia por parte de fibrinógeno e inmunoglobulina G, pero no por parte de albúmina. El sensor demostró ser regenerable mediante la ruptura del complejo formado entre el aptámero y la trombina con una solución de NaCl 2.0 M, aumento de la temperatura y agitación. El segundo aptasensor, basado en enlace covalente del aptámero mostró una respuesta lineal a trombina y limite de detección mejor que el anterior sensor; de 2.5 a 100 pM y 1.5 pM respectivamente. Aunque cabe destacar que este aptasensor está siendo optimizado actualmente

Aptasensors based on electrochemical impedance spectroscopy

En els últims anys, a causa de la necessitat de diàgnostics ràpids i de millores en sensat, s’han utilitzat nous elements de reconeixement en biosensors. Un tipus d’aquests nous elements de reconeixement són els aptàmers. Els aptàmers són cadenes sintètiques de ADN o ARN les quals són seleccionades in vitro i tenen la capacitat d’unir-se a proteïnes, ions, cèl.lules, fàrmacs i lligands de baix pes molecular, reconeixent les seves molècules diana amb alta afinitat i especificitat. Diversos biosensors basats en aptàmers, també anomenats aptasensors, han sigut desenvolupats recentment. D’entre totes les tècniques de transducció utilitzades en biosensors, l’Espectrocòpia Electroquímica d’Impedància ha sigut àmpliament emprada como a eina per caracteritzar la superficies de sensors i estudiar esdeveniments en el biosensat en la superficie d’elèctrodes. La característica més important que presenta aquesta tècnica és que no requereix cap espècie marcada per a la transducció, per tant, aquesta tècnica de detecció pot utilitzar-se per dissenyar protocols de detecció directa sense marcatge, evitant assajos més cars i laboriosos. El principal objectiu d’aquesta tesi doctoral va ser el desenvolupament d’aptasensors utilitzant la tècnica electroquímica d’impedància esmentada anteriorment. Per a això, diferents tipus d’elèctrodes van ser utilitzats, tals com elèctrodes de compòsit grafit-epoxi, elèctrodes de biocompòsit grafit-epoxi modificats amb molècules d’avidina i elèctrodes comercials serigrafiats de nanotubs de carboni de paret múltiple. El treball es va dividir principalmente en dues parts d'acord amb la detecció de dues proteïnes diferents. La primera part es va focalitzar en la detecció de trombina. Primer de tot, es van comparar i avaluar diversos aptasensors de detecció directa sense marcatge basat en diferents tècniques d'immobilització dels aptàmers, tals com: adsorció física humida, afinitat avidina-biotina i enllaç covalent mitjançant activació electroquímica de la superfície de l'elèctrode i mitjançant inserció electroquímica. Posteriorment, els elèctrodes de biocompòsit van ser comparats com a plataformes en genosensat i aptasensat. Amb la finalitat d'amplificar el senyal impedimètric obtingut utilitzant elèctrodes de biocompòsit, un protocol sàndwich va ser emprat incloent nanopartícules d'or modificades amb estreptavidina i tractament amplificador de plata. La segona part de l'estudi es va basar en la detecció de citocrom c. Primerament, es va realitzar un simple aptasensor de detecció directa sense marcatge per a la detecció d'aquesta proteïna utilitzant la tècnica d'immobilització d'adsorció física humida. Finalment, i amb l'objectiu d'amplificar el señal impedimètric, es va desenvolupar un assaig tipus sándwich híbrid d’aptàmer i anticòs utilitzant elèctrodes serigrafiats de nanotubs de carboni de paret múltiple. D'aquesta manera, la tesi explora i compara una àmplia gamma de procediments d'immobilització, l'ús de detecció directa sense marcatge o nanomaterial modificat amb biomolècules en diferents protocols directes o d'amplificació, i l'ús de reconeixement directe i sándwich per amplificar la sensibilitat i/o la selectivitat de l'assaig.In the recent years, due to the need for rapid diagnosis and improvements in sensing, new recognition elements are employed in biosensors. One kind of these new recognition elements are aptamers. Aptamers are synthetic strands of DNA or RNA which are selected in vitro and have the ability to bind to proteins, ions, whole cells, drugs and low molecular weight ligands recognizing their target with high affinity and specificity. Several aptamer-based biosensors, also called aptasensors, have been recently developed. Among all the transduction techniques employed in biosensors, Electrochemical Impedance Spectroscopy has widely used as a tool for characterizing sensor platforms and for studying biosensing events at the surface of the electrodes. The important feature presented by this technique is that it does not require any labelled species for the transduction; thus, this detection technique can be used for designing label-free protocols thus avoiding more expensive and time-consuming assays. The main aim of this PhD work was the development of aptasensors using the electrochemical impedance technique previously mentioned for protein detection. For that, different types of electrodes were used, such as Graphite Epoxy Composite electrodes (GECs), Avidin Graphite Epoxy Composite electrodes (AvGECs) and commercial Multi-Walled carbon nanotubes screen printed electrodes (MWCNT-SPE). The work was divided in two main parts according to the detection of the two different proteins. The first part was focused on thrombin detection. First of all, different impedimetric label-free aptasensors based on several aptamer immobilization techniques such as wet physical adsorption, avidin-biotin affinity and covalent bond via electrochemical activation of the electrode surface and via electrochemical grafting were developed and evaluated. Then, AvGECs electrodes were compared as a platform for genosensing and aptasensing. With the aim to amplying the obtained impedimetric signal using AvGECs, an aptamer sandwich protocol for thrombin detection was used including streptavidin gold-nanoparticles (Strep-AuNPs) and silver enhancement treatment. The second part of the study was based on cytochrome c detection. Firstly, a simple label-free aptasensor for the detection of this protein using a wet physical adsorption immobilization technique was performed. Finally, with the goal to amplify the impedimetric signal, a hybrid aptamer-antibody sandwich assay using MWCNT-SPE for the detection of the target protein was carried out. In this way, the thesis explores and compares a wide scope of immobilization procedures, the use of label-free or nanocomponent modified biomolecules in different direct or amplified protocols, and the use of direct recognition and sandwich alternatives to enhance sensitivity and/or selectivity of the assa

Aptasensores impedimétricos para la detección de trombina

En el presente trabajo se ha desarrollado el primer aptasensor (biosensor de aptámero) en nuestro grupo de investigación, Grup de Sensors i Biosensors de la Universidad Autònoma de Barcelona. En concreto se han desarrollado dos aptasensores para la detección de la proteína trombina, uno basado en la inmovilización de aptámeros por adsorción física, y otro basado en la inmovilización de aptámeros por enlace covalente mediante la reacción EDAC-NHS. El aptasensor utiliza la afinidad específica de la cadena de DNA (aptámero) por la proteína con la que interacciona. Los cambios de carga y estéricos del complejo aptámero proteína alteran la capacidad y la resistencia de transferencia interfacial de electrones en la superficie del electrodo. El principio de detección se basa en la detección de cambios de estas propiedades de interfase del electrodo con el marcador redox [Fe(CN)6]3- / [Fe(CN)6]4-, utilizando mediciones de Espectroscopia Electroquímica de Impedancia. El aptasensor basado en adsorción física del aptámero mostró una respuesta lineal a trombina en el rango de 7.5 a 75 pM y un límite de detección de 5pM, después de optimizar todas las condiciones experimentales. Posteriormente se estudió la especificidad del sistema respecto proteínas potencialmente interferentes presentes en suero sanguíneo, obteniendo cierta interferencia por parte de fibrinógeno e inmunoglobulina G, pero no por parte de albúmina. El sensor demostró ser regenerable mediante la ruptura del complejo formado entre el aptámero y la trombina con una solución de NaCl 2.0 M, aumento de la temperatura y agitación. El segundo aptasensor, basado en enlace covalente del aptámero mostró una respuesta lineal a trombina y limite de detección mejor que el anterior sensor; de 2.5 a 100 pM y 1.5 pM respectivamente. Aunque cabe destacar que este aptasensor está siendo optimizado actualmente

Three different signal amplification strategies for the impedimetric sandwich detection of thrombin

In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM)

Signal amplification for thrombin impedimetric aptasensor : sandwich protocol and use of gold-streptavidin nanoparticles

In this work, we report a highly specific amplification strategy demonstrated for the ultrasensitive biosensing of thrombin with the use of gold-streptavidin nanoparticles (strep-AuNPs) and silver reduction enhancement. The biotinylated aptamer of thrombin was immobilized onto an avidin-graphite epoxy composite (AvGEC) electrode surface by affinity interaction between biotin and avidin; electrochemical impedance measurements were performed in a solution containing the redox marker ferrocyanide/ferricyanide. The change in interfacial charge transfer resistance (Rct) experimented by the redox marker, was recorded to confirm aptamer complex formation with target protein, thrombin (Thr), in a label-free first stage. A biotinylated second thrombin aptamer, with complementary recognition properties was then used in a sandwich approach. The addition of strep-AuNPs and silver enhancement treatment led to a further increment of Rct thus obtaining significant signal amplification. The AptThrBio1-Thr-AptThrBio2 sandwich formation was inspected by confocal microcopy after incubation with streptavidin quantum dots. In order to visualize the presence of gold nanoparticles, the same silver enhancement treatment was applied to electrodes already modified with the nanoparticle-sandwich conjugate, allowing direct observation by scanning electron microscopy (SEM). Results showed high sensitivity and selectivity for thrombin detection, with an improvement from ca. 4.7 pM in a simple assay to 0.3 pM in the amplified reported scheme