Background and motivation

Since graphene exhibits innovative mechanical, electrical, thermal and optical properties this two-dimensional material is increasingly attracting attention and it is under active research. Among the various graphene forms with lattice-like nanostructure, graphene oxide (GO) displays advantageous characteristics as a biosensing platform due to its excellent capabilities for direct wiring with biomolecules, heterogeneous chemical and electronic structure, the possibility to be processed in solution and the availability to be tuned as insulator, semiconductor or semi-metal. Moreover, GO bears the photoluminescence property with energy transfer donor/acceptor molecules exposed in a planar surface and even can be proposed as a universal highly efficient long-range quencher, which is opening the way to several unprecedented biosensing strategies. The rational behind the use of GO in optical and electrochemical biosensing is being studied and explored. Taking advantage of the GO properties we are developing simple, sensitive, selective and rapid biosensing platforms based on this emerging advantageous material. [1,2, 3,4]

Graphene as novel platform in quantum dots – based microarray technology

We have discovered that GO is the most powerful acceptor of QDs FRET donors compared with graphite, CNFs and CNTs (Figure 1). The evidenced properties can be of exceptional importance for several kinds of applications in nano-biotechnology. Such a high quenching efficiency of GO may open the way to novel QD-FRET based microarrays and lab-on-a-chip platforms by using of GO as transducing platform. The proposed platform can even be useful for GO quantification (i.e. during their use as labels for DNA, protein or cell sensing) based on its strong quenching of previously spotted QDs. The application of such GO based QD-FRET quenching platforms may bring new advantages in the analytical performance of future biosensing systems beside opening the door to the design of novel sensing strategies with interest for diagnostics, safety and security, environmental and other industrial applications. The QD fluorescence quenching by GO may open unprecedented imaging flexibility and become a general tool for characterizing graphene based materials for several other applications. It can be also extended to several other QDs and fluorescent materials with interest for optoelectronic or biosensing applications. [5]


Nanomaterials-modified microarrays

Turned ON by a pathogen: A highly sensitive pathogen-detection system has been designed and evaluated for the sensing of E. coli bacteria in diverse matrices. It employs antibody–quantum dot (Ab-QD) probes and exploits the extraordinary two-dimensional structure and fluorescence-quenching capabilities of graphene oxide.

Operational concept of the nano-enabled system (illustration not to scale). The system relies on Antibody-Quantum Dots (Ab-QD) microarrays as a pathogen attachment mechanism. Once the pathogen is selectively captured onto the Ab-QD probes (which can be excited with a laser), they are coated with GO platelets that reveal the presence of the pathogen. In the presence of the pathogen, the quenching of the probes is minimal, as they barely interact with GO (ON state), whereas in the absence of the pathogen, the probes are quenched by electrostatic or –π-π stacking interactions between the probes and GO (OFF state). Pending Patent: EP 13188693.9 [6]



Graphene oxide as a pathogen-revealing agent in lateral-flow format

Taking advantage of the aforementioned research, our team has designed a paper-based lateral flow immunoassay for pathogen detection that avoids the use of secondary antibodies and is revealed by the photoluminescence quenching ability of graphene oxide. This device is able to display a highly specific and sensitive performance with a limit of detection of 10 CFU mL−1 in standard buffer and 100 CFU mL−1 in bottled water and milk. This low-cost disposable and easy-to-use device will prove valuable for portable and automated diagnostics applications. [7]


Graphene quantum dots–based sensor for specific pesticide detection

Organic toxic compounds (e.g. less than 1 kDa) are generally challenging to be detected using simple platforms such as biosensors due to their size and difficulty to obtain cost/effective biological or synthetic receptors (e.g. antibodies or aptamers, respectively). We report on the synthesis and characterization of a multifunctional composite material: magnetic silica beads/graphene quantum dots/molecularly imprinted polypyrrole (mSGP). mSGP is engineered to specifically and effectively capture and signal small molecules due to the synergy among chemical, magnetic and optical properties combined with molecular imprinting of tributyltin (291 Da), a hazardous compound, selected as a model analyte. Magnetic and selective properties of the mSGP composite can be exploited to capture and pre-concentrate the analyte onto its surface and its photoluminescent graphene quantum dots, which are quenched upon analyte recognition, are used to interrogate the presence of the contaminant. This multifunctional material enables a rapid, simple and sensitive platform for small molecule detection even in complex mediums such as seawater without any sample treatment. [8]


Interaction of graphene with a Janus-faced fungal protein (the hydrophobin Vmh2 extracted by Pleurotus ostreatus)

Biological interfacing of graphene has become crucial to improve its biocompatibility, dispersability, and selectivity. However, biofunctionalization of graphene without yielding defects in its sp2-carbon lattice is a major challenge. Here, a process is set out for biofunctionalized defect-free graphene synthesis through the liquid phase ultrasonic exfoliation of raw graphitic material assisted by the self-assembling fungal hydrophobin Vmh2. This protein (extracted from the edible fungus Pleurotus ostreatus) is endowed with peculiar physicochemical properties, exceptional stability, and versatility. The unique properties of Vmh2 and, above all, its superior hydrophobicity, and stability allow to obtain a highly concentrated (≈440–510 μg mL−1) and stable exfoliated material (ζ-potential, +40/+70 mV). In addition controlled centrifugation enables the selection of biofunctionalized few-layer defect-free micrographene flakes, as assessed by Raman spectroscopy, atomic force microscopy, scanning electron microscopy, and electrophoretic mobility. This biofunctionalized product represents a high value added material for the emerging applications of graphene in the biotechnological field such as sensing, nanomedicine, and bioelectronics technologies. [9]



Graphene-based hybrid for enantioselective sensing applications

Chirality is a major field of research of chemical biology and is essential in pharmacology. Accordingly, approaches for distinguishing between different chiral forms of a compound are of great interest. We report on an efficient and generic enantioselective sensor that is achieved by coupling reduced graphene oxide with γ-cyclodextrin (rGO/γ-CD). The enantioselective sensing capability of the resulting structure was operated in both electrical and optical mode for of tryptophan enantiomers (D-/L-Trp). In this sense, voltammetric and photoluminescence measurements were conducted and the experimental results were compared to molecular docking method. We gain insight into the occurring recognition mechanism with selectivity toward D- and L-Trp as shown in voltammetric, photoluminescence and molecular docking responses. As an enantioselective solid phase on an electrochemical transducer, thanks to the different dimensional interaction of enantiomers with hybrid material, a discrepancy occurs in the Gibbs free energy leading to a difference in oxidation peak potential as observed in electrochemical measurements. The optical sensing principle is based on the energy transfer phenomenon that occurs between photoexcited D-/L-Trp enantiomers and rGO/γ-CD giving rise to an enantioselective photoluminescence quenching due to the tendency of chiral enantiomers to form complexes with γ-CD in different molecular orientations as demonstrated by molecular docking studies (Figure 6). The approach, which is the first demonstration of applicability of molecular docking to show both enantioselective electrochemical and photoluminescence quenching capabilities of a graphene-related hybrid material, is truly new and may have broad interest in combination of experimental and computational methods for enantiosensing of chiral molecules [10].

On‐the‐spot immobilization of quantum dots, graphene oxide, and proteins via hydrophobins

Class I hydrophobin Vmh2, a peculiar surface active and versatile fungal protein, is known to self-assemble into chemically stable amphiphilic films, to be able to change wettability of surfaces, and to strongly adsorb other proteins. Herein, a fast, highly homogeneous and efficient glass functionalization by spontaneous self-assembling of Vmh2 at liquid–solid interfaces is achieved (in 2 min) (Figure 7). The Vmh2-coated glass slides are proven to immobilize not only proteins but also nanomaterials such as graphene oxide (GO) and quantum dots (QDs). As models, bovine serum albumin labeled with Alexa 555 fluorophore, anti-immunoglobulin G antibodies, and cadmium telluride QDs are patterned in a microarray fashion in order to demonstrate functionality, reproducibility, and versatility of the proposed substrate. Additionally, a GO layer is effectively and homogeneously self-assembled onto the studied functionalized surface. This approach offers a quick and simple alternative to immobilize nanomaterials and proteins, which is appealing for new bioanalytical and nanobioenabled applications [11].



Water activated graphene oxide transfer using wax printed membranes for fast patterning of a touch sensitive device

We demonstrate a graphene oxide printing technology using wax printed membranes for the fast patterning and water activation transfer using pressure based mechanisms (Figure 8). The wax printed membranes have 50 μm resolution, longtime stability and infinite shaping capability. The use of these membranes complemented with the vacuum filtration of graphene oxide provides the control over the thickness. Our demonstration provides a solvent free methodology for printing graphene oxide devices in all shapes and all substrates using the roll-toroll automatized mechanism present in the wax printing machine. Graphene oxide was transferred over a wide variety of substrates as textile or PET in between others. Finally, we developed a touch switch sensing device integrated in a LED electronic circuit [12].


Graphene/silicon heterojunction schottky diode for vapors sensing using impedance spectroscopy

A graphene(G)/Silicon(Si) heterojunction Schottky diode and a simple method that evaluates its electrical response to different chemical vapors using electrochemical impedance spectroscopy (EIS) are implemented (Figure 9). To study the impedance response of the device of a given vapor, relative impedance change (RIC) as a function of the frequency is evaluated. The minimum value of RIC for different vapors corresponds to different frequency values (18.7, 12.9 and 10.7 KHz for chloroform, phenol, and methanol vapors respectively). The impedance responses to phenol, beside other gases used as model analytes for different vapor concentrations are studied. The equivalent circuit of the device is obtained and simplified, using data fitting from the extracted values of resistances and capacitances. The resistance corresponding to interphase G/Si is used as a parameter to compare the performance of this device upon different phenol concentrations and a high reproducibility with a 4.4% relative standard deviation is obtained. The efficiency of the device fabrication, its selectivity, reproducibility and easy measurement mode using EIS makes the developed system an interesting alternative for gases detection for environmental monitoring and other industrial applications. [13]


The synthesis paths, applications and future for Graphene-encapsulated materials:

Synthesis, applications and trends Graphene-based materials (GBM) are an exceptional type of materials that offer unprecedented application capabilities to the scientific and technologic community. Depending on the encapsulation materials such as drugs, nanoparticles, polymers, oxides and cells inside, the hybrid materials with unprecedented behaviors promises a myriad of advantageous applications, including micro/nanomotors, biosensing platforms, bio/imaging agents, drug delivery systems, potential tumor treatment alternatives, environmental remediation platforms, advanced batteries and novel supercapacitors(Figure 10). We present an overview on graphene-encapsulated materials and their most important synthesis pathways. In addition, we explore the synergistic functionalities provided by these composites and highlight the state-of-the-art related to energy, environmental and bio-applications among others. Finally, we discuss their challenges and future outlooks.[14]

Selected references:

1. Eden Morales-Narváez, Arben Merkoçi, “Graphene oxide as an optical biosensing platform”, Advanced Materials, Adv. Mater. 2012, 24, 3298–3308 (Cover image of the issue).

2. Briza Pérez-López, Arben Merkoçi, “Carbon Nanotubes and Graphene in Analytical Sciences”, Microchim Acta 2012, 179, 1–16.

3. Eden Morales-Narváez, Luis Baptista-Pires, Alejandro Zamora-Gálvez and Arben Merkoçi, “Graphene-based biosensors: Going simple”, Advanced Materials, 2016, In press, DOI: 10.1002/adma.201604905.

4. Eden Morales-Narváez, Lívia Florio Sgobbi, Sergio Antonio Spinola Machado, Arben Merkoçi, “Graphene-encapsulated materials: synthesis, applications and trends”, Progress in Materials Science, 2017, 8, 1–24.

5. Eden Morales-Narváez, Briza Pérez-López, Luis Baptista Pires, Arben Merkoçi, Ultrahigher quantum dot quenching efficiency by graphene oxide in comparison to other carbon structures, Carbon, 50  (2012) 2987–2993.

6. Eden Morales-Narvez, Abdel-Rahim Hassan, and Arben Merkoçi, “Graphene Oxide as a Pathogen-Revealing Agent: Sensing with a Digital-Like Response”, Angwandte Chemie 2013, 52 (51) 13779–13783.

7. Eden Morales-Narváez, Tina Naghdi, Erhan Zor, and Arben Merkoçi, “Photoluminescent Lateral-Flow Immunoassay Revealed by Graphene Oxide: Highly Sensitive Paper-Based Pathogen Detection”,  Anal. Chem., 2015, 87 (16), 8573–8577.

8. Erhan Zor, Eden Morales-Narváez, Alejandro Zamora-Gálvez, Haluk Bingol, Mustafa Ersoz, and Arben Merkoçi, “Graphene Quantum Dots-based Photoluminescent Sensor: A Multifunctional Composite for Pesticide Detection”,  ACS Appl. Mater. Interfaces, 2015, 7 (36), 20272–20279

9. Alfredo M. Gravagnuolo,Eden Morales-Narváez, Sara Longobardi, Everson T. da Silva, Paola Giardina and Arben Merkoçi, “In Situ Production of Biofunctionalized Few-Layer Defect-Free Microsheets of Graphene”, Advanced Functional Materials, 2015, 25 (18), 2771–2779.

10. Erhan Zor, Eden Morales-Narváez, Sabri Alpaydin, Haluk Bingol, Mustafa Ersoz, Arben Merkoçi, “Graphene-based hybrid for enantioselective sensing applications”, Biosensors and Bioelectronics, 2017, 87, 410–416.

11. Alfredo M. Gravagnuolo, Eden Morales-Narváez, Charlene Regina Santos Matos, Sara Longobardi , Paola Giardina and Arben Merkoçi, “On‐the‐Spot Immobilization of Quantum Dots, Graphene Oxide, and Proteins via Hydrophobins”, Advanced Functional Materials, 2015, 25(38) 6084–6092.

12. Luis Baptista-Pires, Carmen C. Mayorga-Martínez, Mariana Medina-Sánchez Helena Montón and Arben Merkoçi, “Water Activated Graphene Oxide Transfer Using Wax Printed Membranes for Fast Patterning of a Touch Sensitive Device”, ACS Nano, 2016, 10 (1), 853–860.

13. Ali Fattah, Saeid Khatami, Carmen C. Mayorga-Martinez, Mariana Medina-Sánchez, Luis Baptista-Pires and Arben Merkoçi , “Graphene/Silicon Heterojunction Schottky Diode for Vapors Sensing Using Impedance Spectroscopy“, Small, 2014, 10 (20), 4193–4199.

14. Eden Morales-Narváez, Lívia Florio Sgobbi, Sergio Antonio Spinola Machado, Arben Merkoçi, “Graphene-encapsulated materials: synthesis, applications and trends”. Progress in Materials Science, 2017,86, 1–24.