A DNA-based nano-immunoassay for the label-free detection of glial fibrillary acidic protein in multicell lysates.
We have developed a quantitative approach to eventually enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells. Through DNA-directed immobilization of DNA-protein conjugates we immobilized antibodies specific for a certain protein of interest, on a complementary DNA nanoarray fabricated by means of nanografting, a nanolithography technique based on atomic force microscopy (AFM). The proof of concept was realized for glial fibrillary acidic protein (GFAP), a biomarker crucial in cell's differentiation of astrocytes, and functional to grade classification of gliomas, the most common of primary malignant brain tumors. The efficiency of the nano-immuno sensing was tested by obtaining the immobilization of purified recombinant GFAP protein at different concentration in a standard solution then in a cellular lysate. A comparison of sensitivity between our technique and conventional ELISA assays is provided at the end of the paper.


BACKGROUND
This team developed a quantitative approach to enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells, demonstrating the utility of this DNA-based nano-immunoassay in the detection of GFAP.
University of Trieste, Trieste, Italy; Nano Innovation Lab, Elettra-Sincrotrone Trieste S.C.p.A, Trieste, Italy.
Mario
M
Ganau
Nano Innovation Lab, Elettra-Sincrotrone Trieste S.C.p.A, Trieste, Italy.
Alessandro
A
Bosco
Department of Medical and Biological Sciences, University of Udine, Udine, Italy; Institute of Pathology, University Udine Hospital, Udine, Italy.
Anita
A
Palma
University of Trieste, Trieste, Italy; Nano Innovation Lab, Elettra-Sincrotrone Trieste S.C.p.A, Trieste, Italy.
Stefania
S
Corvaglia
Nano Innovation Lab, Elettra-Sincrotrone Trieste S.C.p.A, Trieste, Italy; INSTM, Trieste ST Unit, Trieste Italy.
Pietro
P
Parisse
Karlsruhe Institute of Technology DFG, Centre for Functional Nanostructures, Karlsruhe, Germany.
Ljiljana
L
Fruk
Department of Medical and Biological Sciences, University of Udine, Udine, Italy.
Antonio Paolo
AP
Beltrami
Department of Medical and Biological Sciences, University of Udine, Udine, Italy; Institute of Pathology, University Udine Hospital, Udine, Italy.
Daniela
D
Cesselli
Nano Innovation Lab, Elettra-Sincrotrone Trieste S.C.p.A, Trieste, Italy. Electronic address: loredana.casalis@elettra.trieste.it.
Loredana
L
Casalis
Nano Innovation Lab, Elettra-Sincrotrone Trieste S.C.p.A, Trieste, Italy; Department of Medical and Biological Sciences, University of Udine, Udine, Italy; Institute of Pathology, University Udine Hospital, Udine, Italy.
Giacinto
G
Scoles
D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D001253:Astrocytes / Q000276:immunology / Q000473:pathology; D015415:Biomarkers / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology / Q000302:isolation & purification*; D005910:Glioma / Q000175:diagnosis / Q000276:immunology*; D006801:Humans; D007118:Immunoassay; D018625:Microscopy, Atomic Force
D000906:Antibodies; D000941:Antigens; D015415:Biomarkers; D005904:Glial Fibrillary Acidic Protein; D004247:DNA
Atomic force microscopy; Cancer biomarkers; DNA-directed immobilization; Nanodiagnostics