Liquid biopsy is a non-invasive and efficient technique for the detection of tumor biomarkers in biological fluids, which currently represents a new frontier in theranostics and precision medicine. Among mutations of the KRAS oncogene, p.G12D single nucleotide variation in the KRAS gene plays a central role in the early diagnosis and therapeutic treatment of colorectal cancer. In this context, we developed a highly sensitive magneto-genosensing assay based on PNA capture probes immobilized on magnetic microbeads. To detect the mutation of the KRAS oncogene, two capture probe sequences recognizing wild-type and p.G12D tumor DNA were used in association with a DNA signaling probe allowing for electrochemical detection using an enzyme conjugate. The assay conditions were optimized using a 32 full-factorial design, obtaining an outstanding specificity, evidenced by a remarkably lower (>95%) signal of the singly-mismatched- compared to that of fully-complementary-DNA. Ultra-high sensitivity was achieved in 10-fold diluted human plasma reaching detection limits of 818 fM for the p.G12D and 1.8 pM for the wild-type targets. The genosensing assay was tested on genomic tumor DNA samples provided by IRCCS-Regina Elena National Cancer Institute and finally integrated into a smart portable multichannel potentiostat capable of performing up to four simultaneous acquisitions. The developed magneto-genosensing assay demonstrated portability, simplicity, and high sensitivity, showing good potential as theranostic tool for personalized medicine in oncology.

A highly sensitive electrochemical magneto-genosensing assay for the specific detection of a single nucleotide variation in the KRAS oncogene in human plasma / Fortunati, Simone; Giliberti, Chiara; Giannetto, Marco; Bertucci, Alessandro; Capodaglio, Sabrina; Ricciardi, Elena; Giacomini, Patrizio; Bianchi, Valentina; Boni, Andrea; De Munari, Ilaria; Corradini, Roberto; Careri, Maria. - In: BIOSENSORS AND BIOELECTRONICS. X. - ISSN 2590-1370. - 15:(2023). [10.1016/j.biosx.2023.100404]

A highly sensitive electrochemical magneto-genosensing assay for the specific detection of a single nucleotide variation in the KRAS oncogene in human plasma

Fortunati, Simone
Funding Acquisition
;
Giliberti, Chiara
Investigation
;
Giannetto, Marco
Supervision
;
Bertucci, Alessandro
Writing – Review & Editing
;
Capodaglio, Sabrina
Formal Analysis
;
Bianchi, Valentina
Investigation
;
Boni, Andrea
Data Curation
;
De Munari, Ilaria
Data Curation
;
Corradini, Roberto
Resources
;
Careri, Maria
Supervision
2023-01-01

Abstract

Liquid biopsy is a non-invasive and efficient technique for the detection of tumor biomarkers in biological fluids, which currently represents a new frontier in theranostics and precision medicine. Among mutations of the KRAS oncogene, p.G12D single nucleotide variation in the KRAS gene plays a central role in the early diagnosis and therapeutic treatment of colorectal cancer. In this context, we developed a highly sensitive magneto-genosensing assay based on PNA capture probes immobilized on magnetic microbeads. To detect the mutation of the KRAS oncogene, two capture probe sequences recognizing wild-type and p.G12D tumor DNA were used in association with a DNA signaling probe allowing for electrochemical detection using an enzyme conjugate. The assay conditions were optimized using a 32 full-factorial design, obtaining an outstanding specificity, evidenced by a remarkably lower (>95%) signal of the singly-mismatched- compared to that of fully-complementary-DNA. Ultra-high sensitivity was achieved in 10-fold diluted human plasma reaching detection limits of 818 fM for the p.G12D and 1.8 pM for the wild-type targets. The genosensing assay was tested on genomic tumor DNA samples provided by IRCCS-Regina Elena National Cancer Institute and finally integrated into a smart portable multichannel potentiostat capable of performing up to four simultaneous acquisitions. The developed magneto-genosensing assay demonstrated portability, simplicity, and high sensitivity, showing good potential as theranostic tool for personalized medicine in oncology.
2023
A highly sensitive electrochemical magneto-genosensing assay for the specific detection of a single nucleotide variation in the KRAS oncogene in human plasma / Fortunati, Simone; Giliberti, Chiara; Giannetto, Marco; Bertucci, Alessandro; Capodaglio, Sabrina; Ricciardi, Elena; Giacomini, Patrizio; Bianchi, Valentina; Boni, Andrea; De Munari, Ilaria; Corradini, Roberto; Careri, Maria. - In: BIOSENSORS AND BIOELECTRONICS. X. - ISSN 2590-1370. - 15:(2023). [10.1016/j.biosx.2023.100404]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2959232
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