Chemically Functionalized Phosphorene As a 2D Material Emerging Toward Sensitive Biosensors
Authors: Jakobczyk Pawel , Dettlaff Anna , Brodowski Mateusz , Kowalski Marcin Krzysztof , Ossowski Tadeusz , Bogdanowicz Robert 
Published: May 2020

The interest of phosphorene as 2D material is high due to its excellent mechanical, electrical and optical properties [1]. However, the possibility of its application in the area of biosensors is a result of low cytotoxic cell-viability effects and excellent cytocompatibility [2]. Phosphorene has much higher surface to volume ratio compared to graphene and transition metal dichalcogenides. This due to its “puckered” lattice structure. These properties are advantages in sensing applications [3]. But puckered layer is quite reactive to oxygen and water adsorbed on the surface of phosphorene [4]. One way to change it and improve the ambient stability of layered black phosphorus is to modify the surface so that covalently or non-covalently attached chemical compounds passivate the surface to prevent damage [4, 5].
The biosensing device consists of chemical part and biological unit with unique specifities towards corresponding analytes for example proteins.
In this work the biosensor of bacteria has been prepared in several steps: phosphorene preparation, functionalization with 4-azidobenzoic acid, followed by coating with an antibody layer.
Phosphorene (few layered black phosphorus, FLBP) was prepared from pre-crushed black phosphorus (BP) dispersed in anhydrous dimethylformamide or toluene under an argon atmosphere by the liquid exfoliation. Afterwards, the resultant suspension was centrifuged to remove the residual un-exfoliated particles, yielding supernatant.
We functionalized few layered black phosphorus (FLBP) by direct bonding of the phosphorus atom bearing lone electron pair with nitrogen during reaction with 4-azidobenzoic acid, leading to the formation of P=N double bonds, which passivate the reactive FLBP effectively. A covalent combining few-layered black phosphorus (FLBP) with 4-azidobenzoic acid can function as a bridge between FLBP and biomolecules. The functionalized phosphorene (f-FLBP) results in the possibility of using it as a biosensor platform for the detection of Heamophillus Influenza - one of the most common bacteria that cause infections in humans [6, 7]. It is very important to determine the presence of a given bacterial strain, because nowadays strains resistant to numerous antibiotics are more and more often observed. In the US alone, antibiotic-resistant bacteria cause at least 2 million infections and 23,000 deaths per year, which cause losses of 55-70 billion dollars a year [8].
The efficient functionalization of BP flakes was revealed by infrared spectroscopy. The cyclic voltammetry and electrochemical impedance spectroscopy measurements were carried out in Na2SO4 solution containing reference redox systems: [Fe(CN)6]3−/4−.
Heamophillus Influenza bacteria were detected by the impedimetric method. The changes of the charge transfer resistance (Rct) can be attributed to the selective protein binding on the surface of the electrode. This was preceded by the attachment of antibodies to f-FLBP and obtaining a stable sensory surface. The procedure is shown in Figure 1.
Consequently:
- the benzoic groups enhance electric conductivity delivering fast direct electron transfer.
- antibody-coated functionalized phosphorene were used to detect a bacteria (Haemophilus influenzae).
- modified FLBP can work as surface for determination of organic molecules, e.g., nucleic acid bases or peptides and proteins, while it offers the promising possibility for biosensing.

ACKNOWLEDGEMENTS

This work was supported by the Polish National Science Centre [2016/22/E/ST7/00102]; and the National Centre

for Science and Development [347324/12/NCBR/2017]. The DS funds of the Faculty of Electronics, Telecommunications and Informatics of the Gdansk University of Technology are also acknowledged.

References

[1] Y. Yi, X. Yu, W. Zhou, J. Wang, P. K. Chu, Two-dimensional black phosphorus: Synthesis , modification, properties, and applications, Materials Science and Engineering: R: Reports, 120, 1–33 (2017); doi: 10.1016/j.mser.2017.08.001
[2] H. Fu, Z. Li, H. Xie, Z. Sun, B. Wang, H. Huang, G. Han, H. Wang, P. K. Chu, X-F. Yu, Different-sized black phosphorus nanosheets with good cytocompatibility and high photothermal performance, RSC Adv., 7, 14618-14624 (2017); doi: 10.1039/C7RA00160F
[3] A. Yang, D. Wang, X. Wang, D. Zhang, N. Koratkar, M. Rong, Recent advances in phosphorene as a sensing material, Nano Today, 20, 58-73 (2018); doi: 10.1016/j.nantod.2018.04.001
[4] R. Gusmão, Z. Sofer, M. Pumera, Functional Protection of Exfoliated Black Phosphorus by Noncovalent Modification with Anthraquinone, ACS Nano, 12, 5666-5673 (2018), doi: 10.1021/acsnano.8b01474
[5] C. Joseph, Y. Togawa, N. Shindo, Bacterial and viral infections associated with influenza, Influenza and other Respiratory Viruses, 7, 105–113 (2013); doi: 10.1111/irv.12089
[6] S. K. Morris, W. J. Moss, N. Halsey, Haemophilus influenzae type b conjugate vaccine use and effectiveness, The Lancet Infectious Diseases, 8, 435-443 (2008); doi: 10.1016/S1473-3099(08)70152-X
[8] B. Li, T. J. Webster, Bacteria Antibiotic Resistance: New Challenges and Opportunities for Implant-Associated Orthopaedic Infections, J Orthop. Res., 36, 22-32 (2017); doi: 10.1002/jor.2365

Figure 1