scientificprotocols authored over 7 years ago
Authors: Priyanka Sharma, V Bhalla, E Senthil Prasad, V Dravid, G Shekhawat & C. Raman Suri
This protocol describes an optimized signal amplification strategy to develop an ultra-sensitive magneto-electrochemical biosensing platform. The new protocol combines the advantages of carbon nanotube (CNT) and reduced graphene oxide (rGO) together with electrochemical bursting of magnetic nanoparticles. The method involves synthesis of gold-iron (Au/Fe) nano-structures functionalized with specific antibodies to be used as nanobioprobes (Ab-Au/Fe). The next step requires the precise designing of the rGO/CNT nanohybrid sensing platform. The combined system offers the enhanced electrochemical properties giving a synergistic effect in electroanalytical performance of the resulting electrode material along with a large number of metal ions (Fe2+) available on electrode demonstrating ultra-high sensitivity of developed assay. This method provides a promising biosensing platform for environmental or clinical applications where sensitivity is a major issue.
Graphene-based nanocomposite films have recently been used as enhanced sensing platform for the development of electrochemical sensors and biosensors because of their unique facile surface modification characteristics and high charge mobility (1-3). Zhang et al., have recently reported a hybrid film consisting of graphene oxide (GO) nanosheets together with the prussian blue films for electrochemical sensing applications (4). In a different approach, an in-situ chemical synthesis approach has been developed to prepare graphene-gold nanoparticles based nanocomposite, demonstrating its good potential as a highly sensitive electrochemical sensing platform (5). A GO sheet consists of two randomly distributed regions namely, aromatic regions with unoxidised benzene rings and regions with aliphatic six-membered rings making it to behave like an amphiphilic molecule (6). The oxygen containing groups render GO sheets hydrophilic and highly dispersible in water, whereas the aromatic regions offer active sites to make it possible to interact with other aromatic molecules through supramolecular interactions. This chemical nature makes GO a unique dispersant to suspend CNTs in water and to develop a new strategy for making graphene/CNT hybrids (7,8). Similarities in structure and physical properties between CNTs and graphene, their hybridization would presumably have useful synergistic effects in biosensing applications (9-11).
Nanometer-sized magnetic particles of iron are potential candidates in catalysis, magnetic separation and biomedical applications (12). However, pure iron nanoparticles are chemically unstable and easily oxidize, which limits their utility in biosensing and other applications. These particles are therefore coated with another inert layer such as metal-oxide (iron oxide), inorganic material (SiO2), and noble metals (gold and silver), thereby making a core–shell nano-structure showing favorable magnetic properties of metal iron while preventing them from oxidation (13). Gold has been one of the potential coating materials owing to its chemical inertness, biocompatibility, non-toxic, and diverse cluster geometries (14). Very recently, inorganic or semiconductor nanoparticles tagged with receptor molecules has generated good interest for electrochemical detection of analyte (15,16). Anodic stripping voltammetry (ASV) has proved to be a very sensitive method for trace determination of metal ions liberated from nanoparticles. Recently, Liu developed multi-QDs functionalized silica nanoparticles based electrochemical amplification platform which dramatically enhanced the intensity of the signal and led to ultrasensitive detection (17). Our previous study reported the use of gold nanoparticles mediated ASV technique based upon oxidative gold nanoparticles dissolution in an acidic solution. The consequent release of large amount of gold (Au) metal ions after dissolution leads to the development of sensitive stripping voltammetry based immunoassay (18). However, it suffers from the use of strongly corrosive and hazardous agents such as HBr/Br2 for the oxidation of gold nanoparticles, which minimizes it’s usage in common lab practices. Although significant achievements have been obtained in this field, the finding of more sensitive, environment friendly convenient assay still attracts increasing interest where sensitivity is a major cause of concern, such as clinically important biomarkers or assaying environmental pollutants. In this protocol, we present a detailed and proven procedure based on metal ions derivatized electrochemical immunoassay format using specific antibody tagged gold-iron (Au/Fe) nanoparticles on reduced graphene oxide-carbon nanotubes (rGO/CNT) modified biosensing platform (19) (Fig. 1).
The use of core magnetic nanoparticles offers rapid immunocomplex formation on magneto-microtitre plates and their further electrochemical bursting into a large number of Fe2+ ions presented ultra-high sensitivity for diuron detection on SPE. Although this protocol has successfully been implemented for detection of herbicide diuron in environmental samples, yet the success of assay depends on the selection of bioreceptor (antibodies) used with respect to its specificity and sensitivity towards the target molecule.
Experimental design
Ab-Au/Fe synthesis. The Au/Fe nanoparticles were first synthesized by preparing Fe3O4 seeds using modified co-precipitation method20 which were further oxidized to encapsulate with Au shells. Various parameters such as Au/Fe salt concentration and time kinetics of the reaction were optimized to have monodispersed nanoparticles. These gold coated iron oxide particles were separated out from the solutions by using a lab magnet (10 Tesla). High resolution transmission electron microscopy (HR-TEM) was carried out to characterize the surface morphology and elemental mapping of synthesized nanobioprobes. The line mapping and elemental composition studies of the selected nanoparticles confirmed the formation of Fe core and Au shell as single Au/Fe nanostructure (Fig. 2).
Functionalization of synthesized Au/Fe nanoparticles with specific anti-diuron antibody is dependent mainly on pH, ionic strength and hydrophobic attractions besides covalent binding between the gold and sulfur atoms. The ionic strength of antibody solution was kept minimum (10 mM) since the increase in ionic strength effects the reduction of the thickness of the electric double layer over charged surfaces, thus decreasing the electrostatic interactions between antibodies and nanoparticles accompanied by coagulation (21). The minimum amount of protein required to stabilize the nanoparticles was optimized by employing flocculation assay (22). The concentration of protein has a marked tendency for flocculation of nanoparticles in solution. A flocculation assay was designed by taking different concentrations of antibody solutions (0.1–1 mg/ml). 100 μl of each dilution was added to 1 ml of as prepared Au/Fe nanoparticles. After 15 min, flocculation was induced by adding 100 μl of 10% NaCl and absorbance was measured at 580 nm. The characterization of nanobioprobes was done with Dynamic light scattering (DLS), Transmission electron microscopy (TEM), Atomic force microscopy (AFM) and Superconducting quantum interference device (SQUID) (Supplementary Fig. S1 and S2). A fully optimized protocol, both for the Au/Fe nanoparticles synthesis and their functionalization with specific antibodies was developed in this study.
rGO/CNT nanocomposite based biosensing platform. GO was synthesized by the oxidation of exfoliated graphite using modified Hummer’s method (6) requiring ice bath and sonicator (1h, 96% power). Oxidation of GO has marked tendency over single layer GO film formation. Filtrate through anodized aluminium oxide (AAO) membrane with a nominal pore size of 0.02 μm yielded single layer GO thin film. rGO/CNT nanocomposite was prepared using well optimized concentrations of multiwalled CNTs and GO suspension drop-casted on working area of SPE (Fig. 3).
A potential reductive scan from 0 to -1.5 V with the scan rate 0.1 V/s was applied for the electrochemical conversion of rGO/CNT nanocomposites (Supplementary Fig. S3). The thus formed nanohybrid was characterized by Raman spectroscopy and contact angle measurements (Supplementary Figs. S4 & S5). Raman spectroscopy investigated the structural aspects of rGO/CNT modification on SPE. The experimental data was fitted using Microcal Origin 6.1 in order to elucidate the peak position and full width of half-maxima (FWHM) of D, G, and 2D bands. The contact angle measurements further revealed the hydrophilic/hydrophobic character of the modified SPE surface due to the decrease in value of the contact angle after surface modification with rGO/CNT. A large number of hydrophilic (-COOH) groups present in rGO and CNT makes the surface more hydrophilic resulting in reduced contact angle value.
Magneto-immunoassay optimisation. A competitive inhibition immunoassay format was developed on ELISA plates with in-house generated hapten-protein conjugate and specific bioreceptor (anti-diuron antibody) (23). Concentration of nanobioprobes in the reported ELISA procedure was optimized. Nanobioprobe mediated immunocomplex formed on the plates were washed and acid dissolved for the desorption of nanoparticles from the immobilized antibody by using a mild acid (1N HCl) followed by partial neutralization with 1N NaOH. The electrochemical bursting of Au/Fe nanoparticles to release large number of Fe ions on rGO/CNT modified biosensing platform was optimized in terms of reductive scan (0 to -1.5 V). (Supplementary Fig. 6) monitored by differential pulse voltammetry (DPV) technique. Liberation of the large number of (Fe2+) ions were detected by their oxidation response on rGO/CNT nanostructured electrodes, which possess the enhanced electrochemical response due to the oxygen containing groups leading to rapid electron transfer (24).
Results analysis. Calibration curve for diuron (standard sample concentrations between (0.01 pg/ml to 1 μg/ml) was established based on a semi-log plot method. Data analysis was performed by normalizing the absorbance values using the following formula: % B/B0 = {(I – Iex) / (I0 – Iex)} Where I, I0, and Iex are the relative current intensities of the sample, hapten at zero concentration, and hapten at excess concentration, respectively. The cross reactivity of the generated antibody was calculated by determining half maximal inhibitory concentration (IC50) for diuron and other herbicides, atrazine, 2,4-D, fenuron and linuron (Supplementary Figs. S7 and S8).
Procedure
Synthesis of Ab-Au/Fenanobioprobes ● TIMING ~3 h 30 min
Box 1 | SYNTHESIS OF Au/Fe NANOPARTICLES ● TIMING ~1 h 30 min
Box 2 | LABELING OF Au/Fe NANOPARTICLES ● TIMING ~2h
2│The synthesized Ab-Au/Fenanobioprobes are characterized morphologically by Scanning Transmission Electron Microscope. Further, size profiling of antibody tagged nanoparticles by dynamic light scattering system confirms the binding of antibodies to NPs (Supplementary Fig. S1). SQUID analysis also demonstrates the change in magnetic properties of Au/Fe NPs and their subsequent functionalization with specific antibodies.
CRITICAL STEP For SQUID analysis, the samples should be vacuum concentrated and completely dry.
Development of Magneto-electrochemical immunoassay ● TIMING ~3 h
Synthesis of rGO/CNT nanohybrid ● TIMING ~1 h
Box 3 | IMMUNOCOMPLEX FORMATION AND ASSAY DEVELOPMENT ● TIMING ~45 min
The developed sensing platform combines the advantages of GO and CNT nanohybrid offering enhanced electrochemical properties giving a synergistic effect in electroanalytical performance of the resulting electrode material along with a large number of metal ions (Fe2+) available on electrode which are detected by differential pulse voltammetry technique (Fig. 4a,b). This combined strategy successfully enhanced the immunoassay sensitivity, and thus provides a novel promising platform for environmental or clinical applications where sensitivity is a major issue.
The authors greatly acknowledge NUANCE, NWU, IL for carrying out TEM/EDX imaging. Authors also acknowledge Jiaxing Huang and Laura Cote, NWU, IL for valuable suggestions and discussions on GO synthesis.
Figure 1: Schematic illustration of the optimised nanohybrid biosensing systems
Schematic illustration of the optimised nanohybrid biosensing systems. The method involves synthesis of Au/Fe nanoparticles functionalised with specific antibodies used as nanobioprobes and their subsequent metal ion sensing on rGO/CNT nanostructured electrodes. Microtiter ELISA plates were coated with 100 µl of hapten-protein conjugate (10 µg/ml) prepared in carbonate buffer and subsequently immunocomplex was formed with different concentrations of diuron sample in competitive ELISA approach. Electrochemical bursting of nanoparticles releasing large number of Fe2+ ions presented ultra-high sensitivity for diuron detection on SPE.
Figure from reference 19: Sharma, P., Bhalla, V., Dravid, V., Shekhawat,G., Wu, J., Prasad, E. S., Suri, C. R. Enhancing electrochemical detection on graphene oxide-CNT nanostructured electrodes using magneto-nanobioprobes. Scientific Report 2, 877 (2012).
Figure 2: TEM micrographs of Au/Fe nanoparticles
(a) TEM micrograph of Au/Fe nanoparticles showing the morphology of the synthesized Au/Fe nanoparticles with an approximate dia of ~30 nm (b) The line map curve showing the ratio of Au:Fe found to be nearly 11:1 in a single selected nanoparticle (c) EDX spectra of the whole scan area showing Au LR, Au Lâ, Fe KR, and Fe Kâ lines at 9.8 keV, 11.6 keV, 6.4 keV, and 7.0 keV respectively (d) The whole area mapping analysis of nanoparticles in dark field showing the distribution of Fe and Au in the synthesized nanoparticles. In (e) and (f) pink and red dots represent Fe and Au respectively
Figure from reference 19: Sharma, P., Bhalla, V., Dravid, V., Shekhawat,G., Wu, J., Prasad, E. S., Suri, C. R. Enhancing electrochemical detection on graphene oxide-CNT nanostructured electrodes using magneto-nanobioprobes. Scientific Report 2, 877 (2012).
Figure 3: Cyclic voltammograms of nanocomposite formed on SPE
(a) Cyclic voltammograms (CV) of rGO, CNT and rGO/CNT nanocomposite formed on SPE using 2.5 mM ferrocyanide solution prepared in PBS. Inset of the figure shows the TEM characterization of rGO/CNT nanocomposite. The corresponding CV scans recorded for the redox of small ion (Fe2+) for rGO/CNT showed maximum current signal for anodic and cathodic peak currents for the first reductive scan as compared to GO and CNTs dropcasted individually on separate electrodes and further reduced electrochemically. In figure b, CV scans recorded at different scan rates from 25 to 200 mV/s. The anodic potential shifts more towards the positive potential and the cathodic peak potential shifts in the reverse direction with increase in higher scan rate
Figure from reference 19: Sharma, P., Bhalla, V., Dravid, V., Shekhawat,G., Wu, J., Prasad, E. S., Suri, C. R. Enhancing electrochemical detection on graphene oxide-CNT nanostructured electrodes using magneto-nanobioprobes. Scientific Report 2, 877 (2012).
Figure 4: Magneto-electrochemical immunoassay format using modified SPE
(a) Response curves of rGO/CNT modified SPE The signal response was measured by a differential pulse voltammetry technique at amplitude 50 mV, pulse width 0.2 s, pulse period 0.5 s. (b) Competitive inhibition response curve for diuron at different concentrations from 0.01 pg/ml to 1 µg/ml (a to h). Analysis of the competitive inhibition assay data was performed by normalizing the absorbance (Fig. 4ii). The developed immunoassay showed excellent sensitivity and specificity demonstrating detection limit upto 0.1 pg/ml (sub-ppt) for diuron samples with high degree of reproducibility (n=3)
Figure from reference 19: Sharma, P., Bhalla, V., Dravid, V., Shekhawat,G., Wu, J., Prasad, E. S., Suri, C. R. Enhancing electrochemical detection on graphene oxide-CNT nanostructured electrodes using magneto-nanobioprobes. Scientific Report 2, 877 (2012).
Supplementary document: Supplementary document
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Enhancing electrochemical detection on graphene oxide-CNT nanostructured electrodes using magneto-nanobioprobes. Priyanka Sharma, Vijayender Bhalla, Vinayak Dravid, Gajendera Shekhawat, Jinsong-Wu, E. Senthil Prasad, and C. Raman Suri. Scientific Reports 2() 19/11/2012 doi:10.1038/srep00877
Priyanka Sharma, V Bhalla, E Senthil Prasad & C. Raman Suri, IMTECH
V Dravid & G Shekhawat, NWU, IL
Correspondence to: C. Raman Suri ([email protected])
Source: Protocol Exchange (2012) doi:10.1038/protex.2012.059. Originally published online 6 December 2012.