Catalytic activity of catalase–silica nanoparticle hybrids: from ensemble to individual entity activity† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04921d Click here for additional data file.

We demonstrate the electrochemical detection and characterization of individual nanoparticle–enzyme hybrids.


SiNP preparation and modification
For all measurements (UV-Vis and electrochemistry), the unmodified SiNPs were used as received and diluted to the desired concentration with ultrapure water from Millipore featuring a resistivity of not less than 18.2 MΩ.cm. The following procedure was used for modifying the SiNP with catalase: 79.5 L of SiNPs were added to 119.7 L of water. Next, 20 L of bovine catalase from stock solution (Sigma, 45 mg/ml) were added. Last, 54.8 L of 50 mM citrate phosphate buffer (pH=5.4) were added as well. The solution was left overnight for incubation at room temperature. Next, the solution was centrifuged (9000 RPM, Eppendorf 5430-R) for 30 min, the supernatant was removed and the pellet was washed with 219 L of water and 55 L of the citrate buffer. The centrifugation and washing process was repeated three times to insure that there is no residual catalase left in solution.

UV-Vis spectroscopy
UV-vis spectroscopy experiments were conducted in citrate-phosphate buffer solution (pH=5.4) using a Shimadzu spectrometer UV-1800 and quartz cells with a 1 cm optical path.

TEM
Silica dioxide nanoparticle characterization was performed using a transmission electron microscope (TEM) JEOL JEM-3000F equipped with an EDX spectrometer with an accelerating voltage of 300 kV. Sample preparation involved drop casting nanoparticle suspensions on holey carbon grids (Agar Scientific) and allowing the samples to dry. A size distribution histogram was plotted from the TEM image analysis of 233 NP (Fig. 1b), using ImageJ software. The mean size and standard deviation of the nanoparticles was estimated using a Gaussian fit (Origin 2015).

NTA
A NanoSight LM10 (NanoSight Limited, Amesbury, UK) was used to carry out nanoparticle tracking analysis. A 500 µl sample of SiNP was syringed into the viewing unit of the NanoSight and a red (638 nm) laser was used to illuminate the particles so they could be tracked. Measurements were recorded at 20 0C. NanoSight's NTA software was used to analyse the size distribution and concentration of the NPs. . For the chronoamperometric measurements a homemade potentiostat was used together with a carbon microelectrode as a working electrode (r = 3.5 m). Before all experiments the electrode was polished using micropolish alumina (Buehler) in the size sequence of 3.0 µm, 1.0 µm and 0.1 µm to a mirror-like finish. Data was recorded with a 4 kHz preamplifier filtered with a built-in passive 100 Hz filter. The properties of the homemade potentiostat were described previously. [1] Impact spikes were analysed using SignalCounter software developed by Dario Omanovic (Centre for Marine and Environmental Research, Ruder Boskovic Institute, Croatia). [2] Fig. S1 (a) NTA of the SiNP-Catalase (b) TEM of bare SiNP and (c) zeta potential of bare and catalase modified SiNP.

Surface coverage of catalase on a SiNP:
The absorption maximum of SiNP/Catalase hybrid ds in solution was at 405 nm and a value of 0.062 was recorded. Using the Beer-Lambert law we can calculate the concentration of bound catalase in solution: Since the concentration of the SiNP in solution was pre-determined to be 0.5 nM, we can estimate the number of catalase enzymes per SiNP to be: The radius of a single SiNP was 59 nm. The radius of catalase is estimated to be 5.12 nm. [3,4] Hence, the maximum number of enzymes that can be loaded on a SiNP can be approximated:

Theoretical calculation of SiNP impact frequency:
The steady-state current at a microdisk electrode of radius r, assuming a simple n electron reduction, is given by where n is the number of electrons transferred, F is the Faraday constant (C mol −1 ), C is bulk concentration (mol cm −3 ), D is diffusion coefficient (cm 2 s −1 ) and f(τ) is a function of time, t (s). A convenient single expression for f(τ) has been obtained from simulation by Shoup and Szabo and shown to correctly predict the current over the entire time domain with a maximum error of less than 0.6%. The Shoup and Szabo expression is: [5] f(τ) = 0.7854 + 0.8863τ −1/2 + 0.2146exp(0.7823τ −1/2 ) where τ = 4Dt/r 2 . Multiplication of this by the Avogadro constant, N A , converts the equation to a form referring to the number of particles. To determine the number of particle impacts expected within a given time, the Shoup-Szabo equation needs to be integrated and this has previously been performed by series expansion. [6] For a 100 pM particles in solution with a radius of 59 nm, the estimated upper value for the average impact frequency is ~ 50 impacts / 10 sec. The theoretical value is about an order of magnitude higher than the experimentally observed impact frequency and can be explained by an irreversible absorption process of the NP hybrids to the insulating glass surrounding the active microelectrode. [7] Theoretical calculation of irreversible two electron reduction of H 2 O 2 : The relation of the peak current (Ip) with the scan rate (υ) can be expected to follow the Randles-Ševčík equation for a two electron fully irreversible process: where I p is the peak current, α=0.3 is the electron transfer coefficient of the rate determining step, n=2 is the number of electrons transferred and assuming 1 st electron transfer is not the rate limiting step, F is the Faraday constant, 2 2 is the diffusion coefficient and equals to 1.71 × 10 −9 2 s -1 for hydrogen peroxide. [8] A is the area of the electrode (r=1.5 mm), [ ] is the hydrogen peroxide concentration, T is the absolute temperature, R is the gas constant and ν is the scan rate.