Un biosensore rapido e ad alte prestazioni basato su nanoparticelle di oro ancorate su fogli di grafene per rivelare elettrochimicamente biomarkers e mutazioni nel DNA per applicazioni ambientali e cliniche

Responsabili di progetto

Roberto Pilloton, Subbiah Alwarappan

Accordo

INDIA - CSIR-expired - Council of Scientific and Industrial Research

Bando

CNR-CSIR 2016-2018

Dipartimento

Scienze del sistema terra e tecnologie per l'ambiente

Area tematica

Scienze del sistema Terra e tecnologie per l'ambiente

Stato del progetto

Nuovo

Proposta di ricerca

Biosensors are analytical devices that consists of biological sensing elements either integrated within or in close vicinity of a transducer element which transforms the selective information of the presence of an analyte of interest into a quantifiable signal. A typical biosensor architecture consists of three important features: 1) a biological recognition element which is usually immobilized on 2) the surface of a suitable transducer for converting the primary signal into a proportional change of a physical or chemical property and 3) an amplification or processing element. The design of appropriate sensor architectures for a given analyte depends on the specific demands arising from the analytical problem which has to be either minimized or solved such as selectivity, sensitivity, dynamic range, detection limit, response time, precision, reproducibility, stability and cost of the measurement. In this work, a novel carbon based material (graphene nanosheet, GN) will be employed as a platform for designing new biosensors. GN has been widely investigated with respect to all possible applications because of its unique properties such as high surface area, excellent electronic properties, thermal conductivity and strong mechanical strength. Though GN has been explored for several applications, there are only a very few reports available about GN based biosensors. For instance, large-sized GN field effect transistors (FETs) were fabricated for the electrical detection of DNA hybridization, graphene oxide (GO) was used in single-bacterium and label-free DNA sensors. In addition, electrolyte-gated graphene FETs for electrical detection of pH and protein adsorption were reported. Further, a polypyrrole (ppy)-graphene based sensor for glucose detection has been reported. Despite these biosensing applications, to the best of our knowledge, there are no graphene based biosensing devices available till date for other biosensing applications such as detection of tumor biomarkers or HIV. Herein, we propose a graphene-gold nanoparticle (GN-AuNPs)platform that will be employed for the detection of cancer biomarkers. In this work, we will intentionally perform the electrochemical deposition of AuNPs onto GN in order to demonstrate a reliable and a stable approach to immobilize various biomolecules through a noble metal on any GN based bio-device.
Enzymes, antibodies and oligonucleotide immobilisation (DNA fragments, 10-30 bases) onto our GN-AuNP platform will be tested with several electrochemical techniques includng Electrochemical Impedance Spectroscopy (EIS) for specific analytes of environmental interest (air pollution, indoor, outdoor) and/or food (mercury speciation, amphetamines, nicotine, PAH) in order to investigate the behavior of a wide range of biomolecules for future applications in the field of label free biosensors. After a wide exploration of the potentiality of the new GN-AuNP platform, it will be characterized and employed for the analysis of tumor marker human Chorionic Gonadotropin (hCG) which is naturally produced by trophoblasts of the placenta during pregnancy and is also an important marker for ophoblastic tumours. These potentially high sensitive GN-AuNPs based biosensor will provide a way to detect cancer biomarkers (such as hCG, CA 125) and DNA mutations associated with tumors. In addition, these biosensors can be also employed in the amperometric detection of biomarkers associated with Parkinson's disease and Alzheimer's disease at a lower concentration. Such type of detection at a lower concentration are crucial for the early diagnosis of the disease.
A single layer graphene will be deposed on a SiO2 substrate by chemical vapor deposition (CVD). Following this, we will then electrochemically deposit AuNPs onto these GN monolayer. Next, the GN-AuNPs will be characterized by surface probing techniques such as Raman Spectroscopy, and Transmission Electron Microscopy (TEM). In the next step, the surface of the GN-AuNPs will be immobilized with a protein layer that will capture the monoclonal Ab1 which will then function as a specific recognition moiety for specific biomarkers associated with tumors and other disease.
Graphitic oxide (GO) will be prepared from graphite powders by employing a red-ox technique. Graphene thus obtained will be characterized by Raman Spectroscopy and Transmission electron microscopy (TEM) to obtain information regarding the type of planes (sp2 or sp3) present in the carbon lattice and the number of graphene layers. AuNPs will be electrodeposed on graphene/GC (Glassy carbon) electrode from aqueous solutions of 0.5M H2SO4 containing 1.0mM HAuCl4 as precursor at a constant potential (-0.5V vs Ag|AgCl). The effects of concentration of the precursor and the deposition time on the morphology of the deposed AuNPs will be studied. In order to facilitate the direct formation of a Protein G layer on a gold electrode surface, the amine groups from a series of lysine residues in the Protein G molecule will be initially converted to thiol groups using long chain alkane thiols (LCATs). Protein-(LCATs) will be applied to the electrode surface and the quantity bound to the electrode surface will be then determined by estimating the residual protein content of the washing solution using a BCA-based standard Protein Assay Kit. The layer of Protein-LCATs will be characterized by a fluorescent assay for its degree of binding to anti-hCG monoclonal antibody (Ab1), which will be employed in our work as the capture component of a two-site sandwich immunoassay.
The ability of the immobilized Ab1 on the GN-AuNPs-Protein-(LCATs) to capture its analyte antigen (hCG) will be investigated with a fluorescent tagged hCG. The second antibody (Ab2) will be prepared and purified as an Ab2-(Biotin-Streptavaidin)-Alkaline Phosphatase conjugate.

More details and references on:

https://dl.dropboxusercontent.com/u/1424736/Biosensing/CSIR-CNR-Proposal%20Final.PDf

Obiettivi della ricerca

In this work, we will intentionally perform the electrochemical deposition of AuNPs onto GN in order to demonstrate a reliable and a stable approach to immobilize various biomolecules through a noble metal on any GN based bio-device.
Enzymes, antibodies and oligonucleotide immobilisation (DNA fragments, 10-30 bases) onto our GN-AuNP platform will be tested with several electrochemical techniques includng Electrochemical Impedance Spectroscopy (EIS) for specific analytes of environmental interest (air pollution, indoor, outdoor) and/or food (mercury speciation, amphetamines, nicotine, PAH) in order to investigate the behavior of a wide range of biomolecules for future applications in the field of label free biosensors.
After a wide exploration of the potentiality of the new GN-AuNP platform, it will be characterized and employed for the analysis of tumor marker hCG. These potentially high sensitive GNPs based biosensor will provide a way to detect cancer biomarkers (such as hCG, CA 125) and DNA mutations associated with tumors and with Parkinson's and Alzheimer's diseases at a lower concentration. Such type of detection at a lower concentration are crucial for the early diagnosis of the disease and subsequent successful treatment.

Ultimo aggiornamento: 21/05/2024