Fabrication and functionalization of picosecond Laser-Induced Graphitic/Graphene (pLIG) materials for biosensor

Abstract

Graphene, a promising material due to its unique electrical, mechanical, and chemical properties are suitable for many applications. A number of techniques have been developed for fabrication of graphene films and among these laser-induced graphitization of polymer is one of the most promising methods for producing graphene/graphite-based films for healthcare diagnosis, supercapacitors, and energy storage. However, reliable fabrication graphene using a laser and repeatable performance after functionalizing it with chemical and biological solutions to reach higher sensitivity is still challenging. Despite a plethora of research on the fabrication and functionalization of graphene, the methods of functionalization and the phenomenon of transferring polyimide or polymeric materials to graphene are poorly understood. In this study, an effort has been made to create graphene films from a polyimide sheet using a picosecond pulsed laser and its application for development of Acute Kidney Injury (AKI) biosensors using a neutrophil gelatinase-associated lipocalin (NGAL) binding aptamer. We report a picosecond pulsed laser-induced graphitized surface-based aptasensor for detecting Neutrophil Gelatinase Associated Lipocalin (NGAL), a promising Acute Kidney Injury (AKI) biomarker. Polyimide surfaces were irradiated with a 1064 nm wavelength laser with a pulse width of 102 ps and 10 kHz repetition frequency to form an electrically conductive surface with a conductivity of 620±10 S/m. Morphological characterization revealed laser-transformed film with closed pore cell foam microstructure. Raman's characterization of the film indicated the presence of multilayer graphene and graphitic materials in the laser-transformed material. In the first approach, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) and N-hydroxy succinimide (NHS) were used as crosslinkers to immobilize aminated aptamer sequences to the carboxylic acid groups on the pLIG surface. In the second approach, 1-Pyrenebutanoic acid succinimide ester (PASE) was used as a linker to immobilize amine functionalized NGAL aptamers on the laser-transformed substrate through π-π stacking interactions with substrate and amine/succinimide ester covalent bonds with aptamer. Aptamer functionalized surfaces were exposed to different concentrations of NGAL proteins in a 10% serum solution. Electrochemical impedance spectroscopy (EIS) was used to determine the impedance changes over a frequency range of 10 Hz to 1 MHz. A distribution of relaxation time-(DRT) based analysis was used to identify the frequency ranges associated with the electrochemical impedance of the sensing surface's macro-, meso-, and micro-pores. The efficacy of the immobilization scheme was determined by comparing the impedance changes on pLIG surfaces functionalized with specific binding and non-specific binding – NGAL protein binding and Ebola virus glycoprotein binding aptamers, respectively. The results suggest that impedance changes associated with macropores on the aptamer immobilized with PBASE chemistry are specific to the presence of NGAL proteins. Surface impedance changes on the exposure to different concentrations of NGAL protein were used to estimate the dissociation coefficient (KD) for NGAL/sensor surface binding to be 7 nM. The limit of detection (LOD) and the limit of quantification (LOQ) of LIG-based biosensors were found at 0.74 nM, and 2.25 nM, respectively. The aptasensors functionalized with PBASE chemistry using superblock buffer can detect NGAL protein with a dissociation constant of approximately 7 nM, limit of quantification (LOQ) of 18 nM, and limit of detection (LOD) of 6 nM, respectively. Finally, we investigated the transferring of Kapton tape to graphene on different substrates to be used in other fields like batteries, supercapacitors, and energy storage.