Hybrid organic/inorganic interfaces as reversible label-free platform for direct monitoring of biochemical interactions
Introduction
The combination of materials of different nature and dimensionalities to form functional elements is an effective approach to address sensing- and biosensing-related challenges (Casalini et al., 2015, Lin et al., 2009, Stern et al., 2007, Zheng et al., 2005). The immobilization of organic layers on nanostructured materials such as silicon nanowires (Ahn et al., 2010, Gao et al., 2010), semiconducting layers (Ashkenasy et al., 2002, Bof Bufon et al., 2011), carbon nanotubes (Lin et al., 2004, Nguyen et al., 2002), high-k oxides films (Bof Bufon et al., 2010, Vervacke et al., 2014, Vervacke et al., 2012) and, more recently, graphene (Liu et al., 2012, Shan et al., 2009) has been recognized as key to produce devices with novel functionalities and improved performance. In sensing and biosensing, additional advantage is achieved by making use of solid-liquid interfaces for the detection of specific analytes. Insulator/electrolyte (Bousse and Bergveld, 1983) interfaces, for instance, have been widely exploited in transistor-like structures for the detection/monitoring of organic solvents (Grimm et al., 2013), heavy metal ions (Cobben et al., 1992) and biomolecules (Cui, 2001), to mention a few. In order to use such interfaces to target specific molecules, receptor elements are immobilized on top of the insulating layer (Berggren et al., 2001, Cui, 2001).
In capacitive sensors, the observation of changes in the solid/liquid interface requires the capacitance of the insulating layer to be as high as possible (Berggren et al., 2001). This strategy is adopted to enhance the capability of measuring the changes on the electrical double layer capacitance as a result of recognition events occurring at the solid/liquid interface. Consequently, to monitor the concentration of specific analytes in this type of devices, which is proportional to the charge density at the interface, the sensor has to operate at low frequencies (<100 Hz) (Taylor and Macdonald, 1987), which is known to be unstable (Maupas et al., 1997). The incorporation of insulating organic molecules on top of conducting substrates has also been investigated and modeled (Finklea et al., 1993, Góes et al., 2012). Nevertheless, the permeability of the organic tail to the plethora of ions present in solution may bring serious instabilities to device operation (Góes et al., 2012).
The understanding of biochemical interactions is also of fundamental importance for both biosensor's development and the identification of biological mechanisms responsible for a set of physiological activities. Reduced glutathione (GSH), for instance, is an ubiquitous tripeptide associated with vital functions in the body, including the regulation of cell's redox system (Martos-Maldonado et al., 2015). The protection of cells against damage occurs via a detoxification process driven by the conjugation of GSH with xenobiotics, which is mediated by the glutathione S-transferase (GST) enzyme (Martos-Maldonado et al., 2015, Martos-Maldonado et al., 2012). In this sense, the GSH/GST pair is key for detecting different exogenous substances, such as herbicides and insecticides (Kapoli et al., 2008), fungicides (Singh et al., 2009) and anticancer drugs (Materon et al., 2014). Furthermore, the GSH concentration, along with the GST expression and activity, have been associated to the neurodegenerative diseases like Parkinson's and Alzheimer's (Liu et al., 2015, Mazzetti et al., 2015). In addition, variable expressions of GST have been correlated to various types of cancer (Chuang et al., 2005), as well as to the capability of cells to respond to anticancer drugs (Townsend and Tew, 2003, Tsuchida and Sato, 1992). In all cited cases, the evaluation methodology consists on comparing healthy and pathological cells, where the relative changes of GSH and GST quantities are of interest.
Complex or labeled methods of analysis, such as radioimmunoassays (Howie et al., 1989), gene chips (Chuang et al., 2005) and ELISA (Daukantienė et al., 2014) are usually employed to follow the probe/target molecule interaction. These methods, however, usually cannot provide real time measurements, are more expensive and bulkier then label-free systems (Tsouti et al., 2011). The development of a label-free GST biosensor, i.e., an analytical device to directly monitor the presence of the target molecule (GST) without requiring additional reagents, usually luminescent and electrochemical compounds, for signal generation, is a relevant case as a proof of concept for the monitoring of biochemical interactions. In the literature, just a very limited number of methods, including surface plasmon resonance (Jung et al., 2006), silicon nanowire field-effect transistors (Lin et al., 2009) and water-gated organic transistors (de Oliveira et al., 2016) have reported the label-free detection of GST. To the best of our knowledge, if all reported GST label-free sensors (Jung et al., 2006, Lin et al., 2009, Martos-Maldonado et al., 2012, Qin et al., 2015) are combined, GST can be monitored in a range of concentration of three orders of magnitude (from 2×10−9 mol L−1 to 4.2×10−6 mol L−1). In addition, most of these devices cannot be reused since they employ innovative but complexe functionalization methods that may lead to difficulties in obtaining reproducible manufacturing/regeneration processes as well as routes for GSH/GST monitoring that are not reversible (Jung et al., 2006, Martos-Maldonado et al., 2012, Qin et al., 2015).
In this work, we demonstrate a label-free, reversible and highly sensitive hybrid organic-inorganic biosensor to monitor GSH/GST at a wide range of GST concentration (from 200 pmol L−1 to 2 µmol L−1). The sensor's architecture and operation rely on the combination of an ultra-thin high-k dielectric layer (3.3 nm Al2O3) with the functional bioactive tail to monitor variations of the net charge at the hybrid solid/liquid interface caused by the GSH/GST biospecific interaction. Here, the organic-inorganic combination allowed us to overcome two critical drawbacks in capacitive biosensors: (a) the instabilities arising from low frequency operation (Maupas et al., 1997, Taylor and Macdonald, 1987) and (b) the high ionic permeability of the biorecognition layer, which may lead to electrical instabilities and irreproducible measurements (Berggren et al., 2001). The conformational Al2O3 coating of metallic electrodes is capable of suppressing eventual leakage currents and enables the device to continuously operate in aqueous buffered medium.
The immobilization of biomolecules in biosensors and related biotechnological applications commonly utilizes functionalization agents bearing thiol and silane groups (Lin et al., 2009, Casero et al., 2002). Here, we employed an alternative functionalization route capable of delivering high quality self-assembled phosphonic acid monolayers (SAM) chemically bonded to the Al2O3 nanocoating. Such a layer is part of the organic functional tail of our device that allows the GSH immobilization. Once the functionalized device is exposed to the GST in aqueous phosphate buffer solution (PBS), the GSH/GST reversible interaction can be precisely monitored. The very same biosensor was successfully employed to quantify the GST concentration from 200 pmol L−1 to 2 μmol L−1; the broadest linear operation range reported so far to this enzyme. For comparison, even combining all label-free devices reported in literature, our device outperforms their operation range in one order of magnitude. In addition, to the best of our knowledge, we also achieved the lowest detectable concentration of GST (200 pmol L−1) among such devices (Jung et al., 2006, Lin et al., 2009). From the manufacturing point of view, the fabrication process is compatible with standard microfabrication techniques and the scaling-up production is simple and straightforward. Finally, the devices exhibited a stable operation, with the possibility of being constantly regenerated for further use.
Section snippets
Fabrication of the biosensor's inorganic structure
The inorganic part of the device comprises nickel interdigitated electrodes, prepared by standard photolithography and thin-film deposition processes (see Section 1 and Fig. S1 in the Supplementary material for details), coated with a conformational Al2O3 insulating layer. Therefore, the device's electrodes are protected from PBS used for the device functionalization and characterization. The gap between electrodes (~21 µm) is filled with PBS. In such a configuration, the device electrical
Evaluation of the oxide/liquid interface capacitance
Fig. 1c shows the measured capacitance (Cp) as a function of the Al2O3 thickness for devices immersed in PBS. Considering the equivalent circuit presented in Fig. 1b, the measured capacitance (Cp) can be fit according:where ε0 is the permittivity of the free space and A the electrode's area. The specific capacitance of the electrical double layer (Cdl/A) was found ~19 µF/cm2, in agreement with previously reported values (Bard and Faulkner, 2000). Here, it is worth
Conclusion
In this work, we demonstrate the fabrication and characterization of a reversible label-free hybrid organic-inorganic nanostructured biosensor for the direct evaluation of biochemical interactions. The device's manufacture process combines standard microfabrication techniques with self-assembly methods, highlighting the synergy between top-down and bottom-up approaches. The device performance also evidences the potentiality of nanostructured hybrid systems, which in our case involves materials
Acknowledgements
The authors acknowledge Angelo Luiz Gobbi, Maria Helena de Oliveira Piazzetta, Paulo Zambrozi Júnior e Rui César Murer for technical support during the microfabrication process. We also acknowledge Rafael Furlan de Oliveira for the fruitful discussions and suggestions. CAPES, CNPq (Project 483550/2013-2), FAPESP (Project 2013/22127-2) and FAPESP (Project 2014/25979-2) for the financial support.
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