DNA-directed immobilization of horseradish peroxidase onto porous SiO2 optical transducers
© Shtenberg et al.; licensee Springer. 2012
Received: 30 April 2012
Accepted: 11 July 2012
Published: 8 August 2012
Multifunctional porous Si nanostructure is designed to optically monitor enzymatic activity of horseradish peroxidase. First, an oxidized PSi optical nanostructure, a Fabry-Pérot thin film, is synthesized and is used as the optical transducer element. Immobilization of the enzyme onto the nanostructure is performed through DNA-directed immobilization. Preliminary studies demonstrate high enzymatic activity levels of the immobilized horseradish peroxidase, while maintaining its specificity. The catalytic activity of the enzymes immobilized within the porous nanostructure is monitored in real time by reflective interferometric Fourier transform spectroscopy. We show that we can easily regenerate the surface for consecutive biosensing analysis by mild dehybridization conditions.
Nanostructured porous Si (PSi) has emerged as a promising material for optical biosensing applications due to its large internal surface area and tunable optical properties[1–3]. Numerous biosensing applications, including the detection of DNA hybridization, proteins[5, 6], and enzymatic activity[7, 8], have been presented, demonstrating the advantages of these nanosystems in terms of improved detection sensitivity, label-free and real-time rapid analysis. However, the major challenge in designing these biosensors arises from the intrinsic instability of the recognition element during the immobilization procedures onto the transducer’s surface[9–11].
The present work describes a highly versatile approach for reversible enzyme conjugation to a porous SiO2 (PSiO2) surface via DNA-directed immobilization (DDI). This approach has been successfully used for the immobilization of different classes of enzymes onto a range of solid supports, without affecting their biological activity[9, 12]. DDI strategy utilizes the site-selectivity and the high affinity of Watson-Crick base pairing between two complimentary strands. Taking into account that DNA hybridization is a reversible process, the anchored enzyme as well as the biosensor surface can be regenerated and reused for multiple reaction cycles. Herein, we demonstrate this concept for a model enzyme, horseradish peroxidase (HRP), which is one of the most active peroxidases and often used as a powerful tool in biotechnology (e.g., organic synthesis, immune-detection, and wastewater treatment)[13, 14].
Preparation of porous SiO2 nanostructures
Single side polished on the <100 > face oriented and heavily doped p-type Si wafers (0.8 to 1.0 mΩ·cm resistivity, B-doped, from Siltronix Corp., Archamps, France) are electrochemically etched in a 3:1 (v/v) solution of aqueous HF (48%, Merck, Whitehouse Station, NJ, USA) and ethanol (99.9%, Merck) at a constant current density of 385 mA cm−2 for 30 s. Si wafers with an exposed area of 1.33 cm2 are contacted on the backside with a strip of aluminum foil and mounted in a Teflon etching cell; a platinum mesh is used as the counterelectrode. After etching, the surface of the wafer is rinsed with ethanol several times and dried under a dry nitrogen gas. The freshly-etched PSi samples are thermally oxidized in a tube furnace (Thermolyne) at 800°C for 1 h in ambient air, resulting in a PSiO2 layer.
All materials were purchased from Sigma Aldrich Chemicals (St. Louis, MO, USA) unless mentioned otherwise.
Preparation of enzyme-DNA conjugates
To prepare the enzyme-DNA conjugate, 100 μL of a 100-μM solution of 5′-thiol-modified single-stranded oligonucleotide (cD1 sequence) in TE buffer (10-mM Tris and 1-mM EDTA) is mixed with 60 μL 1,4-dithiothreitol (1 M) and incubated overnight at 37°C. HRP type VI (0.92 mg) is dissolved in 200 μL of 50-mM phosphate buffered saline (PBS, pH 7.4) and incubated for 1 h at 37°C with sulfo-succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC) obtained from Thermo Scientific Pierce Protein Biology Products, Rockford, IL USA (2 mg in 60 μL of N,N-dimethylformamide). Both the DNA and the protein reaction mixtures are purified by two consecutive gel-filtration chromatography steps using NAP5 and NAP10 columns (Pharmacia LKB Biotechnology AB, Uppsala, Sweden). The purified DNA and protein solutions are combined and incubated in the dark at room temperature for 3 h. The reaction mixture is concentrated to approximately 500 μL by ultrafiltration (Centricon 30, Millipore Co., Billerica, MA, USA), and the buffer is changed to Tris (20-mM, pH 8.3) during this step. The conjugate is purified by anion exchange chromatography on a MonoQ HR 5/5 column (Pharmacia) using linear gradient over 25 min (AKTATM purifier, Amersham Biosciences, Piscataway, NJ, USA; buffer A 20-mM Tris, pH 8.3 and buffer B 20-mM Tris and 1.5-M NaCl, pH 8.3). The final concentration is determined spectrophotometrically.
Biofunctionalization of PSiO2
The PSiO2 films are first immersed in a solution of 3-glycidoxypropyl(trimethoxy)silane (GPTS) dissolved in toluene (90 mM) for 2 h. The samples are then extensively rinsed with toluene, ethanol, and acetone and dried under a nitrogen gas. The sequence of the capture strand is 5′-amino TCCTGTGTGAAATTGTTATACGCC-3′ (aD1). A solution of the amino-modified probe (100 μM) is applied onto the silanized PSiO2 surface and incubated for 2 h in a humidity chamber, followed by a post-cleaning process. Afterwards, 2.6-μM of the complimentary strand (cD1)-modifiedHRP conjugates are incubated onto the PSiO2 for 30 min. Finally, the PSiO2 samples are rinsed with 50-mM PBS, soaked in the buffer for 20 min, and vigorously rinsed again with PBS to remove any unbounded species from the surface. All processes are carried out at a room temperature (RT), below the melting temperature of the DNA (65.6°C). Mild basic conditions are used for dissociation of the hybridized complex (0.1-M NaOH for 5 min) to allow surface regeneration.
Fluorescent labeling and fluorescence microscopy
Fluorescently labeled (5′-TAMRA-modified) complimentary strand (TAMRA-cD1, 1 μM) is used to confirm DNA hybridization onto the modified PSiO2 surface Following the introduction the fluorescently tagged strand, the PSiO2 samples are characterized using TIRF iMIC microscope, and images are taken using interline transfer CCD (Andor Clara E, Till Photonics, Munich, Germany) and fast filter-based illumination system with 488 nm TIRF laser beam or the oligochrome light. Data are analyzed by LA live acquisition software.
Interferometric reflectance spectra of PSiO2 samples are collected using an Ocean Optics CCD USB 4000 spectrometer (Dunedin, FL, USA) fitted with a microscope objective lens coupled to a bifurcated fiber optic cable. A tungsten light source was focused onto the center of the sample surface with a spot size approximately 1 to 2 mm. Reflectivity data are recorded in the wavelength range of 400 to 1,000 nm, with a spectral acquisition time of 100 ms. Both illumination of the surface and detection of the reflected light are performed along an axis coincident with the surface normal. All the optical experiments are conducted in a fixed cell in order to assure that the sample’s reflectivity is measured at the same spot during all the measurements. All optical measurements are collected in aqueous surrounding. Spectra are collected using a CCD spectrometer and analyzed by applying fast Fourier transform, as previously described[15, 16].
Enzymatic activity of HRP
Ampliflu Red stock solutions are prepared according to the manufacturer’s instructions using 50-mM PBS buffer (pH 7.4). Final concentrations of 1-mM H2O2 and 0.1-mM Ampliflu Red are used to characterize the HRP activity levels. Solutions of 200 μL are placed on the HRP-modified PSiO2 samples, and the reaction progress is monitored for 8 min at RT. The fluorescence values of the reaction product resorufin are recorded at 590 nm, using an excitation wavelength of 530 nm.
Optical sensing of HRP activity
The PSiO2 is washed with 50-mM PBS buffer solution (pH 7.4) for 30 min. Then, 0.8-mM of 4-chloro-1-naphthol in PBS buffer is introduced and continually cycled through the flow cell for 20 min. Finally, 0.16-M H2O2 is added to the cycled solution for HRP surface activation. The reflectivity spectra of the sample are recorded throughout the experiment.
Results and discussion
Biofunctionalization of PSiO2 with DNA-enzyme conjugates
Reflective interferometric Fourier transform spectroscopy
Enzymatic activity assays
Optical detection of enzymatic activity
We demonstrate that DNA-directed protein immobilization is an elegant and facile method for specific and reversible anchoring of a model enzyme, i.e., HRP onto PSiO2 Fabry-Pérot thin films. HRP specific immobilization through Watson-Crick base pairing onto the porous nanostructure is confirmed by both fluorescence microscopy and RIFTS. The surface-immobilized HRP retains relatively high enzymatic activity in comparison to its activity in solution. We show that the catalytic activity of the HRP immobilized within the PSiO2 thin film can be monitored in real time by RIFTS technique. For biosensor design, the DDI method allows the use of small enzyme quantities for monitoring the reaction while allowing both enzyme and surface to be regenerated for subsequent usage. This ‘proof-of-concept’ biosensor scheme can be potentially extended for systematic analysis of the enzyme of interest under unlimited experimental setups.
ES acknowledges the financial support of the NEVET program administered by the Russell Berrie Nanotechnology Institute and by the Marie Curie Reintegration Grant (NANOPACK) administered by the European Community. GS acknowledges the Miriam and Aaron Gutwirth Excellence Scholarship Award. This work was also supported by Weizmann-Technion-KIT Start up grant and DFG-CFN Excellence Initiative Project A5.7.
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