Introduction
Carbonaceous nanomaterials have demonstrated to be very attractive for the development of innovative electrochemical biosensors due to their unique properties [1,2]. The rational selection of the functionalizing agent of these nanostructures represents a very promising strategy to prepare tailor-made design of carbon nanostructures with specific properties [3]. This communication deals with the design of biosensors for the quantification of clinical relevance markers like hydrogen peroxide, ethanol, glucose and microRNA-21 based on the use of glassy carbon electrodes (GCE) modified with multiwalled carbon nanotubes (MWCNTs) non-covalently functionalized with avidin (Av), glucose oxidase (GOx), polyarginine (Polyarg) or concanavalin A (ConA); and gold surfaces (Au) modified with reduced graphene oxide (RGrO) covalently modified with chitosan.
Experimental
MWCNTs were modified with Av, ConA and Polyarg by ultrasonication for a given time: 0.50 mg/mL MWCNTs in 1.00 mgmL-1 Av + 2.00 mgmL-1 GOx (MWCNTs-Av-GOx); 1.50 mg/mL MWCNTs in 2.00 mg/mL ConA (MWCNTs-ConA) or 0.75 mg/mL MWCNTs in 1.00 mg/mL Polyarg (MWCNTs-Polyarg). GrO (0.50 mg/mL) was covalently functionalized with 1.00 mg/mLchitosan (CHIT) followed by chemical reduction (RGO-CHIT). The analytical platforms were obtained either by drop-coating the GCEs with the corresponding dispersion, MWCNTs-Av-GOx (GCE/MWCNTs-Av-GOx), MWCNTs-Polyarg (GCE/MWCNTs-Polyarg) and MWCNTs-ConA (GCE/MWCNTs-ConA) or by self-assembling of RGrO-CHIT at Au modified with 3-mercaptopropane sulfonate(MPS), (Au/MPS/RGrO-CHIT).
Results and Conclusions
The enzymatic biosensors were prepared by immobilization of: I) biotinylated horseradish peroxidase (biot-HRP) at GCE/MWCNTs-Av-GOx to develop an amperometric biosensor for the micromolar detection of glucose (linear range between 25 and 750 µM, sensitivity of (4.8 ± 0.3) µAmM-1 and a detection limit of 1.2 μM) with successful recovery percentages in a pharmaceutical product (Nasal spray “Allennys”, (2.7 ± 0.1) mg/dose vs a reported value of (2.75 mg/dose, and milk samples (“La Serenísima”), (2.1 ± 0.1) g mL-1 vs. the reported value of 1.9 gmL-1); II)Alcohol dehydrogenase at GCE/MWCNTs-Polyarg containing the NAD+ accumulated at the surface for the submicromolar amperometric detection of ethanol without needing of external enzymatic mediator (linear range between 5.0x10-6 and 1.0x10-4 M, sensitivity of (1487 ± 6) µA M-1, and a detection limit of 0.65 µM) with efficient applications in different alcoholic beverages; III) biotinylated GOx at GCE/MWCNTs-Av followed by the electrodeposition of Ru nanozyme to obtain a sensitive psuedobienzymatic amperometric glucose biosensor (linear range from 2.0 x 10-5 M to 1.23 x 10-3 M, sensitivity of (0.343 ± 0.002) µA mM-1, and a detection limit of 3.3 µM) with successful applications for the determination of glucose in different beverages (Gatorade®, Red bull® and Pepsi® samples); III) GOx and HRP at GCE/MWCNTs-ConA to build bienzymatic glucose biosensors through the site-specific anchoring of both glycoenzymes, HRP and GOx, for the amperometric detection of glucose at submicromolar concentrations with practical applications in a blood human serum with excellent correlation with the laboratory results ((0.84 ± 0.04) g/L vs the laboratory value of 0.87 g/L). The proposed arquitectures present the advantages that the functionalizing agents made possible not only the efficient exfoliation of the MWCNTs but also give to them special properties: Polyarg allowed the highly successful accumulation of NAD+ at the electrode surface eliminating the usual problem of incorporating the enzymatic mediator in the measurement solution, and avidin and concanavalin A worked as specific anchoring site of the biorecognition element. The association of MWCNTs-Av-biotGOx with the nanozyme Ru allowed to obtain an innovative hybrid material with improved properties compared to the individual components due to the combined properties and/or synergistic effect of the individual materials.
The genosensor for the quantification of microRNA-21 was obtained by covalent attachment of NH2-DNA probe sequence at Au/MPS/RGrO-CHIT followed by the hybridization in phosphate buffer solution pH 7.40. The hybridization event was label-free detected using Electrochemical Impedance Spectroscopy from the charge transfer resistances of the redox indicator potassium ferrocyanide/potassium ferricyanide. Micro-RNA-21 was detected at picomolar levels, with successful recoveries in human blood serum, urine and saliva samples.
The proposed MWCNTs-proteins dispersions represent an interesting and innovative alternative to build multifunctional nanoarquitectures for the development of different biosensors just by changing the biotinylated biomolecule in the case of GCE/MWCNTs-Av or the glycobiomolecule anchored at GCE/MWCNTs-ConA. The correct selection of the transduction scheme according to the graphenaceous material incorporated within the biosensor and the critical design of the biorecognition platform allowed the successful detection of microRNA-21, opening the doors to future biomarkers sensing development.
References
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[2]G.A. Rivas, M.C. Rodríguez, M.D. Rubianes, F.A. Gutierrez, M. Eguílaz, P.R. Dalmasso, E.N. Primo, C. Tettamanti, M.L. Ramírez, A. Montemerlo, P. Gallay, C. Parrado, Carbon nanotubes-based electrochemical (bio)sensors for biomarkers, Appl. Mater. Today. 9 (2017) 566–588. doi:10.1016/j.apmt.2017.10.005.
[3] F. Gutierrez, M. D. Rubianes, G. A. Rivas, F. Gutierrez, M. D. Rubianes, G. A. Rivas, Anal. Chim. Acta, 1065 (2019) 1. doi.org/10.1016/j.aca.2019.03.022.
