Monitoring ligand-receptor interactions using molecular wires functionalized sensors

Summary – 2023: We reported the results of our studies related to the development of an electrochemical sensor adapted for the sensitive detection of endogenous hydrogen peroxide. The sensor operates by using signal amplification in square wave voltammetry of the redox marker Methylene Blue, which is grafted onto a peptide structure that exhibits nanozyme activity. The sensor has shown remarkable specificity and sensitivity, with a detection limit of 89 nM and a linear range of 20-200 nM. Our experiments have confirmed the effectiveness of the Methylene Blue-peptide system, which performs two functions as both an electroactive marker and a catalyst for hydrogen peroxide decomposition. The signal amplification system ensures rapid detection, reducing the total analysis time to 1-2 minutes and a significant improvement compared to amperometric detection with immobilized enzymes.

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Summary: The aim of this proposal is to develop a multifunctional molecular-wire supports with conducting properties, designed for ligand immobilization in electrochemical affinity assays. The proposed sensing platform will fast-track the detection and identification of these doping agents banned by the World Antidoping Agency (WADA). This will allow promptly and comprehensive decision-making systems and prevention. The device will be realized as a compact, simple and low-cost platform that may be used, by persons with minimal training.

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Project cod : PN-III-P4-ID-PCE-2020-0998

Acronym: MOLWIRESENS

Contract no. PCE 151/2021

Contract start date: 04.01.2021

Date of conclusion of the contract: 31.12.2023

Duration: 36 months (2021-2023)

Project director: 
Prof. univ. dr. Camelia BALA
e-mail: camelia.bala@chimie.unibuc.ro

Team members:
Lecturer dr. Mihaela Puiu
Assoc. prof. dr. Lucian Rotariu
PostDoc. Lucian-Gabriel Zamfir, PhD
PostDoc. Oana-Maria Dumitrașcu (Istrate), PhD
PhD StudentCristina-Virginia Golobciuc

Objective: The main objective is to develop sensors platforms modified with multifunctional peptide-based molecular-wire with conducting properties, as platform for ligand immobilization in electrochemical affinity and enzymatic assays.

Estimated results:
– Report on peptide sequences with the highest helical content.
– Report on the optimized surface-activation procedures for the deposition of a stable peptide layer.
– Report on peptide bridges with the highest conductive properties.
– Kinetic and affinity modelling of the affinity interaction.

Total value from budget: 1.198.032,00 RON

Total value of the Contract: 1.198.032,00 RON

 

Dissemination

• Publications

Label-free detection of target proteins using peptide molecular wires as conductive supports, M Puiu, LG Zamfir, GM Danila, F Papi, C Nativi, V Mirceski, C Bala, Sensors and Actuators B: Chemical 345 (130416), 2021.

A novel amperometric biosensor based on poly(allylamine hydrochloride) for determination of ethanol in beverages, OM Istrate, L Rotariu, C Bala, Sensors 21(19), 6510, 2021.

Early detection of cannabinoids in biological samples based on their affinity interaction with the growth hormone secretagogue receptor, GM Danila, M Puiu, LG Zamfir, C Bala, Talanta 237 (122905), 2022.

Multi-frequency analysis in a single square-wave chronoamperometric experiment, L. Stojanov, D. Guziejewski, M. Puiu, C. Bala, V. Mirceski, Electrochemistry Communications, 124, 106943, 2021.

Paper based diagnostic platforms and devices, M. Puiu, V Mirceski, C. Bala, Current Opinion in Electrochemistry 100726, 2021.

Amperometric L-lactate biosensor based upon a gold nanoparticles/reduced graphene oxide/ polyallylamine hydrochloride modified screen-printed graphite electrode, O.-M. Istrate, L. Rotariu, C. Bala, Chemosensors, 9(4), 74, 2021.

Assays for Cannabinoids Detection: Are They Amenable to On-Site Screening, Biosensors, M. Puiu, C. Bala, Biosensors, 2022, 12(8), 608.

Trending Methods for Rapid Cannabinoids Detection, M. Puiu, C. Bala Scholarly Community Encyclopedia, Subjects: Chemistry, Analytical; Biochemical Research Methods, Entry Collection: Biopharmaceuticals Technology, 2022, entry 26633

Puiu, C. Bala, Plasmonic Biosensors for Medical Applications In: Narayan, R. (Ed.), Encyclopedia of Sensors and Biosensors, vol. 4. Elsevier, pp. 402–420. 10.1016/B978-0-12-822548- 6.00141-2, ISBN: 9780128225486, 2022 Elsevier Ltd.

Affinity-based biosensors as promising tools in doping control, 6th International conference on analytical and nanoanalytical methods for biomedical and environmental sciences IC-ANMBES 2022, Camelia Bala (Invited Lecture),  8-10 June 2022, Brasov, Romania

Early detection of tumour-associated antigens: Assessment of point-of-care electrochemical immunoassays – Mihaela Puiu, Cristina Nativi, Camelia Bala, TRAC-Trends in Analytical Chemistry, 160, (2023), 116981; (FI 13,1, Q1)

Strategies for Surface Design in Surface Plasmon Resonance (SPR) Sensing – Cristina Virginia Topor, Mihaela Puiu, Camelia Bala, Biosensors, (13), 4, (2023), 465 (FI 5,4, Q1)

Ultrasensitive Detection of Hydrogen Peroxide Using Methylene Blue Grafted on Molecular Wires as Nanozyme with Catalase-like Activity – Mihaela Puiu, Oana-Maria Istrate, Valentin Mirceski, Camelia Bala, Analytical Chemistry. 95, (44), (2023) 16185–16193 (FI 8,008, Q1)

A New Highly Sensitive Electrochemical Biosensor for Ethanol Detection Based on Gold Nanoparticles/Reduced Graphene Oxide/Polyallylamine Hydrochloride Nanocomposite – Oana-Maria Istrate, Camelia Bala, Lucian Rotariu, Biosensors (2023) 13(11), 954 (FI 5,4, Q1)

• Conference participation

Early Detection of Doping Agents based on their Bioaffinity Interactions, Camelia Bala (invited speaker), the 10th International Workshop on Surface Modification for Chemical and Biochemical Sensing (SMCBS’2021), 5 -9th of November 2021, Poland.

Bifunctional peptide supports designed for electrochemical signalling-off detection of high molecular weight targets (oral communication), M. Puiu, L-G. Zamfir, V. Mirceski, C. Bala (Bucharest, Romania), European Biosensor Symposium 2021-online, Wildau, Germany, 09 – 12 March, 2021.

Detection of allosteric modulators of growth hormone secretagogue receptor according to their affinity profiles in competitive assays (oral communication), M. Puiu, L.-G. Zamfir, G. M. Danila, C. Bala, XXVIth International Symposium on Bioelectrochemistry and Bioenergetics of the Bioelectrochemical Society-online, Cluj Napoca, Romania, 9-13th of May 2021.

L.-G. Zamfir, M. Puiu, G. M. Danila, C. Bala, Novel conductive peptide molecular materials for electrochemical sensing of biomarkers (flash-poster), XXVIth International Symposium on Bioelectrochemistry and Bioenergetics of the Bioelectrochemical Society-online, Cluj Napoca, Romania, 9-13th of May 2021.

Biosensing platform for sport medicine and doping control, The 10th International workshop on biosensors, Camelia Bala (Invited Lecture), 12-15 November 2022, Dakhla, Marocco.

Affinity-based sensing platform Towards efficient detection of some biomarkers, 20th International Balkan Workshop on Applied Physics and Materials Science, Camelia Bala (Invited Lecture), 12-15 July 2022, Constanta, Romania.

Interface design for improvement of biosensors performance, Camelia Bala (invited lecture), 21st International Balkan Workshop on Applied Physics, 11-14 July 2023 Constanta, Romania.

Peptide-Molecular Wires as Conductive Supports in Electrochemical Bioassays – Mihaela Puiu, Oana-Maria Istrate, Valentin Mirceski, Camelia Bala, 26th Congress of the Society of Chemists and Technologists of Macedonia (SCTM), Ohrid, Macedonia, 20 – 23 Septembrie 2023 (oral presentation)

• Outreach

Presentation of the project results at “Open Days”, University of Bucharest, 12-16 April, 2021(lhttps ://unibuc.ro/facultatea-de-chimie-a-universitatii-din-bucuresti-isi-deschide-virtual-portile-viitorilor-studenti/)

“Open Days”, University of Bucharest, 12-16 April, 2022 (https://www.chimie.unibuc.ro/index.php/avizier/2470-zilele-portilor-deschise-la-facultatea-de-chimie-11-15-aprilie-2022)

ZILELE POR?ILOR DESCHISE la Facultatea de Chimie (online) 15-22 aprilie 2022, în cadrul manifest?rii, au fost prezentate publicului larg, rezultatele acestui proiect (https://www.chimie.unibuc.ro/index.php/avizier/2470-zilele-portilor-deschise-la-facultatea-de-chimie-11-15-aprilie-2022)

“Zilele portilor deschise”, Universitatea din București 3-7 aprilie 2023 (https://chimie.unibuc.ro/index.php/avizier/2710-zilele-portilor-deschise-la-facultatea-de-chimie-3-7-aprilie-2023)

“Open Days”, University of Bucharest, 3-7-16 April, 2023 (https://chimie.unibuc.ro/index.php/avizier/2710-zilele-portilor-deschise-la-facultatea-de-chimie-3-7-aprilie-2023)

SUMMARY – 2021
The report provides a brief description of the current state of the art in the field, the design of the peptide sequences that can function as conductive molecular wires, the immobilization on gold surfaces and the estimation og the kinetic parameters of electron transfer after chemosorption of a redox molecule. The studies have shown that the peptide bridge used mediates the transfer of electrons, having conductive properties far superior to alkylthiols. In this context, peptide molecular strands can be used to develop affinity electrochemical biosensors.

SUMMARY – 2022
The report presents a brief description of the current state of knowledge in the field for the addressed topic: the implementation of affinity pairs A) anti-tumor-associated carbohydrate antigen antibody (anti-α-Tn)/tumor-associated carbohydrate antigen mimetic (α-Tn mimetic) and B) transmembrane receptor/peptide agonist, on conducting molecular wires marked with redox molecules (Methylene Blue), for the development of electrochemical biosensors with signaling-off interrogation (signal decrease when the analyte with high molecular mass binds to the ligand immobilized on the surface of the peptide biosensor). For this purpose, A) the α-Tn mimetic and B) the peptide agonist CJC-1295-DAC of the growth hormone secretagogue receptor (GHS-R1a) were immobilized on the surfaces of gold electrodes. The immobilization of these ligands was monitored by cyclic voltammetry, square wave voltammetry and electrochemical impedance spectroscopy measurements. The chosen affinity format allowed sensitive and selective detection of both anti-α-Tn antibody and GHS-R1a in diluted serum samples.