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 coagulation21. The minimum amount of protein required to stabilize the nanoparticles was optimized by employing flocculation assay22. 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 method6 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 transfer24.
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
1│ The Au/Fe nanoparticles were synthesized by first preparing Fe3O4 seeds using modified co-precipitation method25 which are further oxidized to encapsulate with gold shells by following the steps given in Box 1. The synthesized Au/Fe nanoparticles were labeled with anti-diuron antibodies23 (generated in-house) as per the steps followed in Box 2.
Box 1 | SYNTHESIS OF Au/Fe NANOPARTICLES ● TIMING ~1 h 30 min
Dissolve FeCl3 (1.28 M) and FeSO4.7H2O (0.64 M) in 1:2 ratios in deoxygenated water under vigorous stirring in nitrogen environment.
CRITICAL STEP Oxygen-free environment protects the oxidation of iron nano particles/seeds.
Add a solution of 1.5 M NaOH dropwise into the mixture followed by stirring for 40 min.
Black precipitate of magnetite formed which is collected by a permanent magnet. Wash the precipitate with deionized water.
CRITICAL STEP Thoroughly wash the precipitate formed to remove trace amount of NaOH (reducing agent).
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Reconstitute the precipitate 1: 200 dilution in deionized water.
Add sodium citrate (155 mM) slowly to the boiling solution under constant stirring for 15 min.
CRITICAL STEP Boiling of magnetic seeds are important before addition of gold and sodium citrate for the efficient coating of gold over magnetic seeds/nanoparticles.
Add 10 ml of gold chloride (10 mM) immediately into the oxidized magnetic solution on a stirring sonicator to encapsulate the iron nanoparticles with gold shells.
CRITICAL STEP Increase in the Au concentration in the Au/Fe ratio will lead to thicker gold shells thereby affecting the magnetic properties of NPs.
Collect Au/Fe NPs by magnetic separation followed by washings with deionised water and finally reconstitute in 0.5 ml water.
CRITICAL STEP The water used for the synthesis should be de-ionised, pH ~7.0, and having resistivity >18 MΩ-cm to avoid flocculation.
Characterise the synthesised nanoparticles by TEM/EDX. The Figure 2 indicates the inclusion of Fe core and Au shell as single Au/Fe nanostructure on the basis of point and line mapping studies.
Box 2 | LABELING OF Au/Fe NANOPARTICLES ● TIMING ~2h
Prepare antibody solution (1 mg/ml) in PB
Add 100 µl antibody solution in 1 ml Au/Fe solution under mild stirring conditions.
CRITICAL STEP The minimum amount of antibody required to stabilize the NPs is optimized by flocculation assay (see experimental design).
Maintain the pH of NPs solution at 7.4 by adding 0.1 M K2CO3 before adding antibody solution.
Incubate the solution at 37 °C for 2 h followed by centrifugation at 12,000 rpm for 30 min to remove traces of unconjugated antibody.
PAUSE POINT May also be incubated overnight at 4 °C.
Wash the pellet twice with 10 mM Tris (pH 8.0) containing 3% BSA.
CRITICAL STEP The addition of BSA will prevent the aggregation of nanoparticles and will eventually increase the stability of the nanobioprobes.
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- Resuspend the pellet in 1 ml of phosphate buffer (pH 7.4) and store at 4 °C.
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
3│Coat the microtiter ELISA plates with 100 µl of hapten-protein conjugate (10 µg/ml) prepared in carbonate buffer.
4│ Cover the plate with an adhesive plastic sheet and incubate at 37 ⁰C for 2 hours followed by washing with PBST (three times).
PAUSE POINT Incubation can be prolonged to overnight at 4 °C
5│ Block the unbound protein binding sites with 10% defatted skimmed milk (prepared in PBS) for 1 h at 37 °C.
6│ Wash the plates with PBST (three times).
7│ A competitive inhibition immunoassay format is developed by coating the ELISA plates with DCPU–BSA conjugate by following the steps given in Box 3.
Synthesis of rGO/CNT nanohybrid ● TIMING ~1 h
8│ Synthesize GO by the oxidation of exfoliated graphite using modified Hummer’s method6 from graphite powders using NaNO3, H2SO4, and KMnO4 in an ice bath.
9│ Filter GO through anodized aluminium oxide (AAO) membrane with a nominal pore size of 0.02 μm.
10│ Peel off the thin GO film from the AAO filter after air drying.
CRITICAL STEP Vaccum oven can be used for the complete drying of the nanocomposite.
11│ For preparing rGO/CNT nanocomposite, high aspect ratio (length: 15–30 nm and diameter: ~30 nm) pristine multiwalled CNTs and the above prepared GO in step 5 are dissolved in (1:1) DMF and water.
12│ Sonicate the mixture for 1h at 96% power.
13│ Drop-caste the 5 µl of the suspension on the working area of SPEs followed by incubation in vacuum oven for 1 h at 60 °C.
CRITICAL STEP Optimise the concentration of rGO/CNT nanocomposite on SPE on the basis of maximum current signal response using cyclic votammetry \(CV) technique.
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14│Apply a potential reductive scan from 0 to -1.5 V with the scan rate 0.1 V/s for the electrochemical conversion of rGO/CNT nanocomposites on SPE.
CRITICAL STEP Carefully observe the characteristic reduction peak of rGO/CNT at -0.5V \(Supplementary Fig. S2). If the peak is not observed check the contacts with SPE and repeat the reduction scan.
15│Characterize the thus formed nanohybrid by TEM and Raman spectroscopy. For characterization, samples are prepared electrochemically on Indium tin oxide (ITO) coated glass by applying the potential between 0 to -1.5 V.
16│ Raman spectra of first order scattering (D and G peaks) are observed around 1350 cm-1 and 1600 cm-1 respectively (Supplementary Fig. S4).
17│ Completely dry the samples in vacuum oven for 1h at ~60 ºC. Scrap off the samples from the surface followed by TEM analysis on a carbon coated copper grid (#300 mesh) dropcasted with sample followed by drying in air for 15 min. The micrograph of the nanocomposite display a view of CNT bundles attached to GO layer indicating the formation of rGO/CNT nanocomposite (inset of Fig. 3a).
18│Use the characterized rGO/CNT modified SPE for DPV measurements in the development of immunoassay using varying concentrations of diuron.
Box 3 | IMMUNOCOMPLEX FORMATION AND ASSAY DEVELOPMENT ● TIMING ~45 min
1│ Mix as prepared Ab-Au/Fe nanobioprobes (1:5 dilution) with varying concentrations of diuron (0.01 pg/ml - 1 μg/ml); 50 µl of mixture added into each well of microtiter plate and subsequently incubated for 20 min at RT.
2│A strong magnet kept beneath the plate speed up the immunocomplex formation which is separated.
3│ Wash the immunocomplex formed on the plates with PB.
PAUSE POINT The plates can be stored at 4 ºC.
4│ Dissociate the bound immobilized antibody complex from plate with 0.1N HCl followed by partial neutralization with 0.1 N NaOH to retain pH ~5.2.
5│ Transfer the solution (50 µl) to rGO/CNT modified SPE surface, as prepared in steps 8-14.
6│ Apply a reductive scan (0 to -1.5 V) which will eventually burst Fe2O3 nanoparticles into large number of metal ions (Fe2+) by applying a potential sweep between 0 to -1.6 V vs. Ag/AgCl.
CRITICAL STEP Observe a characteristic broad reductive peak at -0.75 V as shown in inset of supplementary fig. S6.
7│Use differential pulse voltammetry (DPV) at amplitude 50 mV, pulse width 0.2 s, pulse period 0.5 s. using the electrochemical workstation.