A supramolecular indicator system (cellulose-nanodiamonds-urease) for reusable biochemical detection of urea has been fabricated using sequential immobilization of the components. Modified nanodiamonds (MNDs) were covalently immobilized onto DEAE cellulose granules via the nucleophilic addition reaction. At DEAE cellulose: MND ratio of 4:1 (w/w), up to 110 µg of nanoparticles bound onto 1 mg of the polymer during the addition reaction. Urease was immobilized by covalent conjugation onto the polymer-MND composite with the benzoquinone-activated surface. In comparative experiments, the enzyme was immobilized onto initial polymer granules via nonspecific adsorption and covalent conjugation. However, when these indicator systems were repeatedly used to detect the analyte, the enzyme was considerably inactivated, and that was evidenced by a decrease in the colored product yield. At the same time, the enzyme covalently bound onto the DEAE cellulose-MND composite showed higher functional efficacy and enabled more stable yields of the colored product in repeated urea assays. Comparative experiments with the indicator systems repeatedly used to detect urea at 37 C demonstrated that the enzyme covalently conjugated onto the DEAE cellulose-MND composite showed greater thermo stability, and its activity was reduced at a much slower rate than the activity of the enzyme covalently bound to the polymer. The data obtained in the present study offer the prospect of designing a new type of reusable indicator assay systems (polymer carrier-nanodiamond-biomarker supramolecular systems) for biomedical analytical applications.
Nanodiamond, Cellulose, Composite material, Urease, Indicator system, Urea detection
Nowadays researchers around the world are intensively developing a great number of various types of analytical tools and biosensors such as enzyme-based, tissue-based, DNA biosensors, immunosensors, thermal and piezoelectric biosensors. Biosensors have been applied in many fields such as food industry, fermentation industry, medical field, plant biology sector, marine sector etc. [1-5], because they provide better stability and sensitivity as compared with the traditional methods.
In particular, the efficiency and selectivity of enzymatic catalysis is of great importance in biomedical applications and clinical diagnostics. However, free enzymes have some limits including the loss of catalytic activity after one cycle, low stability, and low activity in the organic phase. They also cannot be separated from the substrate or product [6,7]. The immobilization of enzymes on the carrier can overcome these disadvantages efficiently. Mainly, there are three kinds of immobilization: binding with a support (adsorption, ionic binding, and covalent binding), cross-linking, and entrapment [6,8]. The covalent binding is a practical method for enzyme immobilization: the protection effect of the carrier can enhance the stability of the enzyme, and the leakage of the enzyme can be reduced by increasing the strength of the bonds [9].
Applications of nanomaterials have extended into the field of biomedical sensing expanding the arsenal of currently employed techniques. In particular, nanoparticles of various physical and chemical origins can be an attractive material for developing indicator and diagnostic assay systems as a carrier for immobilizing peptide biomarkers (including enzymes) [10-15]. Along with the fact that a high surface-to-volume ratio offered by nanoparticles provides a significantly higher concentration of the immobilized enzymes than that afforded by immobilization on planar 2D surfaces, the integration of nanoparticles into enzyme carrier structures maintain or even enhance immobilized enzyme performance [16-18].
In this regard, detonation nanodiamond [19,20] are well explored for biomedicine related issues due to their chemical stability, large specific area, facile surface tailoring for specific applications, high adsorption capacity and non-toxicity [21-24]. Along with a high colloidal stability in dispersion media, these properties of modified nanodiamonds (MND) provide for their potential use as a basis for a new class of bioanalytical devices for targeting and quantifying analytes of interest [25-27]. The technology of MND obtaining includes chemical modification of the surface of initial commercial nanodiamonds by NaCl treatment, that reduces the number of inorganic admixtures, e.g. metal ions [24,28]. MND are characterized by high sedimentation stability in aqueous suspensions (hydrosols) and can be separated by differential centrifuging into narrow fractions with various clusters size (nanoparticle aggregates).
In our previous studies, we showed that MNDs can be used to construct systems for biochemical diagnostics and targeted delivery of drugs and new antibacterial preparations [29-31]. The MND-enzyme systems were used in biochemical assays of physiologically significant analytes [29]. The immobilization of the sensing element (nanoparticle-biomarker conjugate) on a solid support can be adapted and advanced the experimental indicator system for clinical analytical applications. The purpose of this study was to construct and test a biochemical assay system for detecting urea in order to provide an experimental basis for designing a reusable indicator systems based on the cellulose-nanodiamond-biomarker supramolecular complexes.
ObjectivesThe objective of this work was to construct and test a model biochemical assay system for detecting urea in order to provide an experimental basis for designing a reusable indicator systems based on the cellulose-nanodiamond-biomarker supramolecular complexes.
The polymeric carrier (support) for the indicator system was prepared from diethylaminoethyl (DEAE) cellulose granules (Serva, Germany). A sample of DEAE cellulose granules was mixed with deionized (DI) water, prepared using a Milli-Q system (Millipore, U.S.). The suspension was incubated for 12 h at a temperature of 20-22 C to allow the granules to swell. Then, DI water was decanted. The polymeric granules were washed with DI water thrice to remove residual preservatives contained in the commercial DEAE cellulose.
The experiments were conducted using MND with high colloid resistance in hydrosols and average cluster size (nanoparticle aggregates) d50 = 50 nm. Size distribution of MND clusters in hydrosols was measured with a Zetasizer Nano ZS (Malvern Instruments Ltd., U.K.). MND were obtained from commercial nanodiamonds (LTD "Real-Dzerzhinsk", Russia) by chemical modification using original technique described previously [28]. The technique consisted of several stages. First, nanoparticle hydrosol was obtained from nanodiamond powder and deionized water. A modifying agent (NaCl) was added to the obtained suspension; the suspension was mixed and allowed to stay at room temperature for 1 h until precipitation of all particles. The supernatant was removed by decantation; the sediment of nanoparticles was washed with deionized water to remove the agent. After washing hydrosol with MND was differentially centrifuged at various forces (centrifuge Avanti J-E, Beckman Coulter) to isolate MND fractions with different cluster sizes (nanoparticle aggregates). The obtained fraction of MND was exsiccated to obtain dry substance of nanoparticles. We used the hydrosol of MND (1% wt), which was prepared by adding deionized water to the sample of nanoparticles powder.
Urease (EC 3.5.1.5 from jack beans) was purchased from Vektor-Best, Russia. The reagents used in this study to determine urea in blood serum and urine were from the Novokarb kit (Vektor-Best, Russia). The kit contained a urea calibrator (analyte concentration of 5 mmol/L), and solutions of chemical reagents (sodium salicylate, nitroprusside, hypochlorite, and hydroxide). Immobilization was performed using an aqueous solution of urease, which had been prepared by dialyzing the initial enzyme solution against DI water, by ultrafiltration through a 30 kDa cutoff membrane (EMD Millipore Amicon, Germany).
Fabrication of the DEAE cellulose-MND composite materialThe polymeric carrier-nanodiamond-biomarker indicator system was fabricated by sequential immobilization of the components (Figure 1a). The DEAE cellulose-MND composite was prepared using the nucleophilic addition reaction [32]. The aqueous suspension of cellulose granules was mixed with MND hydrogel at a ratio of 4:1 (polymer: nanoparticles, w/w). Hydrochloric acid (Reakhim, Russia) was added to the mixture to reach the final concentration of 5 mM, and the mixture was incubated at a temperature of 20-22 ºC for 1 h under continuous agitation at 180 rpm (Orbital Shaker OS-10, BIOSAN, Latvia). Then, the polymer-nanoparticle composite was collected by centrifugation (Centrifuge 5415R, Eppendorf, Germany) at 2300 g for 5 min at 20 ºC. The settled polymer was washed repeatedly (3 times with DI water, 1 time with a 1 M NaCl solution, and 3 times with DI water) to remove MND particles that remained unbound to the DEAE cellulose granules. Each time, the granules were resuspended in the fresh washing solution and collected by centrifugation as described above. In every phase (after the nucleophilic addition reaction and washing), the supernatants were collected for spectral analysis (an UV-1800 spectrophotometer, Shimadzu, Japan) for the presence of MNDs. The amount of the MNDs unbound to the polymer was determined from the optical density of the initial MND hydrosol and supernatant at a wavelength of 400 nm.
Urease immobilizationUrease was immobilized on the polymeric granules through nonspecific adsorption and covalent bonding. Covalent bonding was used to immobilize the enzyme onto the DEAE cellulose-MND composite. For the enzyme adsorption, the urease solution was added to the polymeric granule suspension, and the mixture was incubated at a temperature of 20-22 ºC for 1 h under continuous agitation at 180 rpm (Orbital Shaker OS-10). Then, the polymeric granules with the enzyme adsorbed on them were collected by centrifugation (Centrifuge 5415R) under the conditions described in the previous section. The sediment was washed with DI water thrice to remove the residual enzyme, which remained unbound to the granules; each time, the sediment was resuspended in a fresh portion of water and collected by centrifugation. After washing, the granules with the adsorbed urease were resuspended in DI water and used in the investigation. Urease was covalently bonded onto the granules of the polymer and the composite, whose surfaces had been preliminarily activated by benzoquinone (Figure 1a) using the conventional procedure [33-35]. Due to this pretreatment, the subsequent bonding of the proteins (including enzymes) onto the activated support is conducted under relatively mild conditions, preventing protein denaturation [35]. The surface of the cellulose and DEAE cellulose-MND composite granules was activated with benzoquinone, and urease was bonded as described in detail in our previous studies, which were devoted to the construction of the systems for targeted delivery of bioactive substances and biochemical diagnostics based on MNDs and various proteins and enzymes [29,30]. After covalent immobilization of urease, the polymeric and composite granules were collected by centrifugation and washed repeatedly (3 times with a 0.25 M NaCl solution and 1 time with DI water) to remove the enzyme unbound to the support. After washing, the granules with the covalently bonded urease were resuspended in DI water and used in experiments.
Measure of functional activity of the immobilized enzymeThe activity of the enzyme in the experimental supramolecular systems was determined using the urease salicylate-hypochlorite method (modified Berthelot reaction) [36], which yield a colored reaction product in the urea assay. The method consists of two stages (Figure 1b) the enzymatic and the non-enzymatic (chemical) stages. In stage 1, urea is hydrolyzed by urease to form ammonia, which, in stage 2, interacts under alkaline conditions with the chemical components of the reaction mixture to yield a colored product. In the urea assay using the experimental indicator systems, the enzymatic stage was separated from the non-enzymatic one in order to prevent inactivation of the immobilized enzyme by chemical reagents (chiefly sodium hypochlorite and hydroxide) and to prepare reusable indicator systems for determining the analyte. The preliminary experiments showed that even a single run of both reactions in the presence of the indicator systems completely inactivated the immobilized enzyme. In the assay of urease activity, the reaction mixture of a total volume of 250 µl contained 145 µl DI water, 100 µl aqueous suspension of the indicator system (amount of the immobilized enzyme was 30 µg in the reaction), and 5 µl urea calibrator (with analyte concentration in the sample 0.1 mmol/L). All components were mixed and incubated for 5 min at a temperature of 20-22 ºC; then, the systems were collected by centrifugation. The supernatants were collected and mixed with the solutions of the chemical reagents for the non-enzymatic stage, which had been prepared in situ in DI water following the instructions of Vektor-Best. The samples were incubated for 5 min at the temperature mentioned above. The amount of the colored product was determined spectrophotometrically (UV-1800) by measuring the optical density of the samples at a wavelength of 700 nm. The indicator systems intended for reuse were collected by centrifugation after the enzymatic reaction. The supernatants were collected for conducting the chemical reaction and for the spectral determination of the colored product. The sediment was washed twice (1 time with a 125 mM NaCl solution and 1 time with DI water) to remove the residual substrate (urea) and product of the enzymatic reaction (ammonia). Then, the systems were resuspended in DI water, and the analyte was added to start the enzymatic reaction. The thermo stability of the enzyme on the reused indicator systems was estimated by incubating the samples in a thermostat (Thermo bath TB-85, Shimadzu, Japan) at 37 ºC during the enzymatic reaction.
Each measuring experiment was performed in triplicate. Error bars were generated as a standard deviation of the mean from 3 replicates.
Microscopic examinationThe microscopic examination of the experimental supramolecular system was conducted using an AxioImager M2 light microscope (Carl Zeiss, Germany). To visualize the immobilized enzyme, it was labeled with the FITC (fluoresce in isothiocyanate) fluorescent dye (Sigma, U.S.) prepared in dimethyl sulfoxide.
In the present study, the assay system for biochemical detection of urea was constructed to demonstrate the feasibility of constructing polymer-nanodiamond-biomarker supramolecular indicator systems. A composite support for immobilizing the enzyme was constructed from cellulose and MNDs. The comparative experiments showed that the supramolecular system produced by covalent immobilization of urease onto the DEAE cellulose-MND composite had the highest efficacy and enabled the most stable yields of the colored product in repeated analyte assays. The enzyme covalently bound to MNDs in the composite showed higher resistance to damaging factors and greater thermostability. The data obtained in the present study offer the prospect of designing a new type of reusable indicator assay systems (polymer carrier-nanodiamond-biomarker supramolecular systems) for biological and medical analytical applications. Further research is needed to optimize the conditions of constructing supramolecular indicator systems (including the systems on a two-dimensional polymer matrix and 3D polymer constructs) in order to enhance their efficacy and adapt the assay systems for practical use.