L -Lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells using recombinant and nanotechnology approaches

Talanta 144 (2015) 1195–1200

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L-Lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells using recombinant and nanotechnology approaches Maria Karkovska a, Oleh Smutok a, Nataliya Stasyuk a, Mykhailo Gonchar a,b,n a b

Department of Analytical Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine Institute of Applied Biotechnology and Basic Sciences, Rzeszow University, Sokolowska Street 26, Kolbuszowa, Poland

art ic l e i nf o

a b s t r a c t

Article history: Received 14 May 2015 Received in revised form 23 July 2015 Accepted 28 July 2015 Available online 29 July 2015

In the recent years, nanotechnology is the most developing branch due to a wide variety of potential applications in biomedical, biotechnological and agriculture fields. The binding nanoparticles with various biological molecules makes them attractive candidates for using in sensor technologies. The particularly actual is obtaining the bionanomembranes based on biocatalytic elements with improved sensing characteristics. The aim of this investigation is to study the properties of microbial L-lactate-selective sensor based on using the recombinant Hansenula polymorpha yeast cells overproducing flavocytochrome b2 (FC b2), as well as additionally enriched by the enzyme bound with gold nanoparticles (FC b2-nAu). Although, the high permeability of the living cells to nanoparticles is being intensively studied (mostly for delivery of drugs), the idea of using both recombinant technology and nanotechnology to increase the amount of the target enzyme in the biosensing layer is really novel. The FC b2-nAu-enriched living and permeabilized yeast cells were used for construction of a bioselective membrane of microbial L-lactate-selective amperometric biosensor. Phenazine methosulphate was served as a free defusing electron transfer mediator which provides effective electron transfer from the reduced enzyme to the electrode surface. It was shown that the output to L-lactate of FC b2-nAuenriched permeabilized yeast cells is 2.5-fold higher when compared to the control cells. The obtained results confirm that additional enrichment of the recombinant yeast cell by the enzyme bound with nanoparticles improves the analytical parameters of microbial sensor. & 2015 Elsevier B.V. All rights reserved.

Keywords: l-Lactate Microbial biosensor Gold nanoparticles Flavocytochrome b2 Recombinant yeast cells Enzyme-enriched cells Amperometric biosensor

1. Introduction Reliable determination of L-lactate is important in food technology, fermentation and wine industries, as well as in clinical chemistry and sport medicine [1,3]. Among the available methods for assaying of L-lactate there are chromatographic and spectrometric analysis [4,6], however, they are time-consuming, and require laborious sample pre-treatment, as well as a qualified personnel. Biosensors have been widely used in recent years, because of their high selectivity and sensitivity. The most of the enzymes used in the L-lactate-selective microbial biosensor construction (NAD þ depended L-lactate dehydrogenase or lactate oxidase) are costly for routine analysis and require introducing additional con Corresponding author at: Department of Analytical Biotechnology, Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine. E-mail address: [email protected] (M. Gonchar).

http://dx.doi.org/10.1016/j.talanta.2015.07.081 0039-9140/& 2015 Elsevier B.V. All rights reserved.

factor [7]. The whole-cell microbial biosensors are simple and cheap for production due to avoiding the enzyme purification. Also it is important that enzymes are usually more stable in their natural environment inside the cell [8]. There are described only a few microbial biosensors for lactate analysis based on baker's yeast Saccharomyces cerevisiae [9,11], genetically modified H. polymorpha [12,13] and mixed culture of Lactobacillus bulgaricus and Streptococcus thermophilus [14]. Our previous publications [12,13] are devoted to construction of L-lactate-selective biosensors based on thermostable enzyme, Llactate-cytochrome c-oxidoreductase (EC 1.1.2.3; flavocytochrome b2, FC b2). FC b2 is a tetramer with four identical subunits, each consisting of FMN- and heme-binding domains. The main properties of FC b2 are absolutely selectivity to L-lactate and high stability, which excellently fit to requirements for biosensors. The high permeability of living cells to nanoparticles was intensively studied in the recent years due to perspectives of such approaches for controllable drug delivery into organism. The gold nanoparticle (nAu) display a unique combination of chemical

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inertness, surface chemistry, size- and shape-dependent electronic and optical properties, which render them ideal for clinical applications. The recent advancements were reported about the nAu usage in vaccine development, gene therapy, enhanced radiotherapy and others [15], however, many relevant issues remain open. They include the molecular mechanisms governing the nanoparticles–cell interactions, the physico-chemical parameters underlying their toxicity to different types of cells, the lack of standard methods and materials, and the uncertainty in the definition of general strategies to develop smart drugs and devices based on nanoparticles [16,18]. The conception of using nanoparticles in biosensor's technology to increase the output is very fresh nowadays. A number of biosensors with immobilized nanoparticle on electrode surface were constructed [19,20]. On the other hand, recently we have reported a few papers describing improvement of analytical parameters of the microbial sensors by genetical manipulation with the yeast cell [12,13,21–23]. The idea of implementation of exogenous enzyme to increase the amount of the target enzyme in the cells to be used in microbial sensors seems to be not highlighted yet. In this paper, the enrichment of the sensing cells by the target enzyme is achieved by a combination of two approaches: (1) on genetic level – by overexpression the corresponding HpCYB2 gene in the recombinant cells; (2) using nanotechnological aapproach – by the transfer of FC b2-bound nanoparticles into the cells.

2. Materials and methods 2.1. Cultivation and permeabilisation of yeast cells Cultivation of the recombinant FC b2-overproducing strain H. polymorpha “tr 1” (gcr1 catX/prAOX_CYB2) [24], was performed in flasks on a shaker (200 rpm) at 28 °C until the middle of the stationary growth phase ( 60 h) in a medium containing (g L  1): (NH4)2SO4 – 3.5; KH2PO4 – 1.0; MgSO4  7H2O – 0.5; CaCl2 – 0.1 supplemented with 0.75% yeast extract. A mixture of glucose (10 g L  1) and L-lactate (2 g L  1) was used as a carbon and energy source. After washing, the cells were suspended in 50 mM phosphate buffer, pH 7.8 containing 1 mM PMSF and 1 mM EDTA followed by drying. Before experiments, the dried yeast cells were re-suspended to 30 mg ml  1 in 50 mM phosphate buffer, pH 7.8, containing 1 mM EDTA. The procedure of the cells permeabilization was following: the same volume of permeabilising reagent (0.85 mM cetyltrimethylammonium bromide) was added to the cell suspension (30 mg ml  1 in 50 mM phosphate buffer, pH 7.8). The resulting solution was incubated at 30 °C in a water bath for 15 min under mixing every 3–4 min. The permeabilized cells were washed by centrifugation (6000 g, 5 min) in 50 mM phosphate buffer, pH 7.8. The precipitated permeabilized cells were re-suspended to 30 mg ml  1 in the same buffer solution and stored on ice at þ4 °C. A half-life of the permeabilized yeast cells in suspension was about three weeks of storage at þ4 °C in freezer. Lyophilized permeabilized cells retain 90% enzymatic activity during storage more than one year. 2.2. Isolation and purification of flavocytochrome b2 L-lactate:cytochrome c-oxidoreductase (flavocytochrome b2) was isolated and purified from the recombinant strain of the thermotolerant yeast H. polymorpha “tr 1” (gcr1 catX/prAOX_CYB2) [24]. The enzyme was purified by ion-exchange chromatography on DEAE-Toyopearl cellulose 650 M [25] to the specific activity of

22 U mg  1 and stored as a suspension in 70%-saturated ammonium sulphate, pH 7.8 at þ4 °C before usage. 2.3. Synthesis and functionalisation of gold nanoparticles Gold nanoparticles (nAu) were synthesized by the borohydride reduction method according to [26]. The procedure includes mixing 0.25 mL 10 mM HAuCl4 and 0.6 ml 10 mM sodium borohydride. 7.5 mL 10 mM CTAB was used as a surfactant. The obtained mixture was stirred at 100 °C for 15 min to obtain a winered solution. The nAu were precipitated by centrifugation (9700 g; Hettich Micro-22R centrifuge) during 40 min. The average size of the obtained nAu was characterized by spectrophotometry and corresponds to 20 nm (data not shown). The precipitated nAu were washed twice with water and 10 mM PB, pH 7.5. The obtained nAu were functionalized by amino-groups using aqueous solution of 0.85 mM cystamine and stored at þ 4 °C before usage. The final concentration of functionalized nAu was 0.17 mM. 2.4. FC b2 binding to gold nanoparticles Biofunctionalization of functionalized (cystamine-modified) nAu with concentration 0.17 mM was performed by addition of purified FC b2 with a specific activity 22 U ml  1 in ratio 2:1. The mixture was incubated at þ4 °C overnight. Biofunctionalized nanoparticles (FC b2-nAu) were washed three times by 5 mM PB, pH 7.5. Collection of FC b2-nAu was performed by centrifugation at 9700g for 20 min. The FC b2-activity was analyzed at different stages of biofunctionalization. Finally, the FC b2-nAu demonstrated a specific FC b2 activity of 3.3 U ml  1. 2.5. Enrichment of the yeast cells by FC b2-bound gold nanoparticles The procedure for the enrichment of intact and permeabilized H. polymorpha «tr1» cells was the following: 180 ml yeast cells’ suspension (30 mg ml  1 in 10 mM PB, рН 7.5) were added to 100 ml FC b2-nAu preparation and mixture was incubated for a night at þ4 °C with constant stirring. 180 ml the yeast cells and the same volume of cells modified by nAu without enzyme were incubated in parallel as the reference samples. After night incubation, the three samples of the cells were centrifuged at 670 g for 3 min. The cell's precipitates were washed twice with 10 mM PB, pH 7.5. 2.6. Transmission electron microanalysis The fixation of the samples was performed in 1.5% solution of KMnO4 for 15–20 min at the room temperature. After fixation, the samples were washed 5 fold. The next step was their dehydration in increasing concentration of C2H5OH (50%-, 70-, up to 90%) and finally in absolute ethanol (twice) for 30 min. The samples were fixed in propylepoxide glue during 15 min, then they were transfered in solution of propylepoxide with epoxy (EPON 812) in the ratio of 1:1 for 2–3 h at the room temperature. The transfer was held in pure EPON 812 overnight followed by putting into EPON with polymer during 48 h at 60 °C. Post-fixation of samples was done with 1% OsO4 in cacodylate buffer for 90 min at 0 °C. The slices were obtained using diamond knife in an ultramicrotome UMTP-6 (Sumy, Ukraine). The images were contrasted with lead citrate and carried out by transmission electron microscope PEEM-100 (Sumy, Ukraine) at 75 kV. The final magnification of the micrographs was 10000. Microphotographs were done by photo camera SONY–HG9 (Tokyo, Japan).

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Fig. 1. Transmission Electron Microscopy (TEM) images: intact yeast cells of H. polymorpha “tr 1” (control) (A); gold nanoparticles suspension (nAu) (B); permeabilized yeast cells treated by FC b2-nAu, (C); permeabilized yeast cells enriched by FC b2-nAu, after washing (D).

2.7. SEM with X-ray microanalysis A Scanning Electron Microscope (SEM-microanalyser REMMA102-02, Sumy, Ukraine) supplied with EDAX device was used for morphological analyses of the samples on a graphite rod (diameter – 0.5 cm). The special cover film on the samples with a Butvar solution B-98 (Sigma, St. Louis, MO, USA) in 1.5% chloroform was formed using an ultrasound method. The distance from the last lens of the microscope to the sample (WD) ranged from 17.1 mm to 21.7 mm; the accelerator voltage was in the range from 20 to 40 eV; zooms were from 2,500 to 10,000. 2.8. Biosensors’ preparation and evaluation 2.8.1. Apparatus and techniques Amperometric biosensors were evaluated using constant-potential amperometry in a three-electrode configuration with a Ag/ AgCl/KCl (3 M) reference electrode and a Pt-wire counter electrode. Amperometric measurements were carried out using a potentiostat CHI 1200A (IJ Cambria Scientific, Burry Port, UK)

connected to a personal computer and performed in a batch mode under continuous stirring in a standard 40 ml electrochemical cell at room temperature. Graphite rods (type RW001, 3.05 mm diameter, area 7.3 mm2, Ringsdorff Werke, Bonn, Germany) were used as working electrodes. They were sealed in glass tubes using epoxy glue thus forming disk electrodes. Before sensor preparation, the graphite electrodes were polished with emery paper and cleaned with water in an ultrasonic bath. 2.8.2. Immobilization of the cells by entrapment within a cathodic paint The immobilization procedure was provided using cathode polymer GY 83-0270 0005 (CP) as follows: a few microliters of yeast cell’s suspension in 30 mM PB, pH 7.5 were dropped on the top of the carbon electrode to the final activity of the enzyme on the working electrode area up to 5 U ml  1. After drying for 2 min at room temperature, the cells’ layer was covered with 8 ml 10-fold diluted solution of the cathodic paint GY 83-0270 0005 (pH 5.5). After drying, a well-adhering polymer film was formed.

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Fig. 2. Characterization of microbial L-lactate-selective electrodes by cyclic voltamperometry. 3.05 mm carbon electrodes modified by permeabilized (A) and FC b2-nAuenriched permeabilized cells (B) of H. polymorpha “tr1” in the absence (dash line) and in the presence (solid lines) of increasing concentrations of L-lactate. Conditions: scan frames  0.4 V to  0.6 V vs Ag/AgCl; scan rate 7 mV s  1 in 50 mM phosphate buffer, pH 7.8.

The prepared microbial electrodes were rinsed by 20 mM PB, pH 7.5 and stored at þ 4 °C before application. All the experiments were carried out independently in triplicates and the reported results were the average of three replicate experiments.

3. Results and discussion For enzyme immobilization on the surface of nAu, there was used purified preparation of FC b2 isolated from the cells of the recombinant strain H. polymorpha “tr1” [24]. Gold nanoparticles (nAu), functionalized by cysteamine (see Section 2.5.) with concentration 0.17 mМ were mixed with the same volume of FC b2 solution with a specific activity 22 U ml  1. The process of FC b2 immobilization on the surface of functionalized nAu is characterized in the Table SI.1 (see Supplementary information (SI)). The yield of the immobilized enzyme on nAu was about 15% that corresponds to 3.3 U ml  1. For additional enrichment of recombinant yeast cells by FC b2bound nAu (FC b2-nAu), there was used the strain H. polymorpha “tr 1” (gcr1 catX/prAOX_CYB2) over-producing FC b2 with a specific activity up to 1.2 U mg  1 of protein in cell-free extracts’ or 4.45 U mg  1 for permeabilized cells. The analysis of total enzymatic activity of FC b2-enriched intact and permeabilized recombinant yeast cells (Sample), as well as reference samples without FC b2-nAu treatment (Control) is represented in Table SI. 2. For intact cells, their treatment by FC b2-nAu resulted in a 1.56fold increased enzymatic activity. For permeabilized cells, the enrichment efficacy was essentially higher (2.33-fold). Thus, specific FC b2-activity of intact cells after FC b2-nAu treatment was increased for 56% and for permeabilized cells – up to 133%. This can be explained by a better permeability of permeabilized cells toward nanoparticles. Morphological characterization of the yeast cells at the different stages of enrichment by FC b2-nAu were carried out using Transmission Electron Microscopy (TEM) (Fig. 1). The image of intact cells of H. polymorpha “tr 1” obtained by TEM was used as a control for analysis of morphological modification of the yeast cells (Fig. 1A). Fig. 1B represents numerous dark points which correspond to nAu. As a result of FC b2-nAu penetration into permeabilized cells and cell wall, the membrane became darker (Fig. 1C). The Fig. 1D displays permeabilized yeast cells enriched by FC b2-nAu, after repeated washing of non-bound bio-nanoparticles. The cellular barriers still keep dark regions that

confirm localization of FC b2-nAu inside the cells. The penetration of FC b2-nAu into of the permeabilized yeast cells was confirmed also by X-ray spectral analysis (Fig. SI 1). The X-ray spectrogram represents the presence of Au0 inside of permeabilized yeast cells that approves penetration of FC b2-nAu into the cells (Kα peak at 2.1 keV is characteristic for Au0). Taken together, the presented data confirm the binding of FC b2-nAu with the cell surface and their accumulation inside the cells. The FC b2-nAu enriched yeast cells were used for construction of microbial biosensor for L-lactate analysis. For evaluation of a prototype of L-lactate selective microbial biosensor, 2 mM phenazine methosulphate (PMS) was used as a free-diffusing redox mediator to establish the electron transfer between electrode and FC b2 located in a native form due to higher expression of CYB2 gene and in bound with nAu form due to penetration of bionanoparticles. It was supposed that PMS can easily diffuse into the permeabilized cell and, after trapping electrons from the reduced enzyme, it is oxidized on the electrode surface (Fig. SI. 2). The efficiency of electron transfer from the reduced FC b2 (in cells) to carbon electrode via PMS for FC b2-nAu-enriched permeabilized yeast cells and contol permeabilized cells (without treatment) were characterized using cyclic voltammetry. The cyclic voltammetry also gives an information about optimal working potential for sufficient PMS oxidation on the graphite rod electrodes (Fig. 2). As shown in Fig. 2B, a twice increased current response on Llactate addition is observed for the sensor based on the FC b2-nAuenriched cells, in comparison to control permeabilized yeast cells (Fig. 2A). A potential þ250 mV vs Ag/AgCl was chosen as optimal working potential for PMS-oxidation. The summarized output of enzymatic oxidation of 3.5 mM L-lactate was estimated as 12 mA for FC b2-nAu-enriched cells and 6 mA for control cells, respectively. The sensor’s outputs to L-lactate in chronoamperometric mode for different types of biorecognizing elements are presented in Fig. 3. The best sensitivity (48.6 A M  1 m  2) was achived for the permeabilized yeast cells enriched by FC b2-nAu which is more than twice compared to the control permeabilized cells without treatment with enzyme–bound nAu (18.7 A M  1 m  2). The control bioelectrode based on intact yeast cells showed a significantly lower sensitivity (4.1 A M  1 m  2). The similar value was obtained for other bioelectrodes constructed using bare nAu-modified intact yeast cells 3.9 A M  1 m  2 (Fig. 3). This negligible difference in

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Fig. 3. Chronoamperometric current response (A) of L-lactate-selective microbial electrode, based on FC b2-nAu-enriched permeabillized yeast cells compared with the controls: (a) intact yeast cells; (b) nAu-modified intact yeast cells; (c) permeabilized yeast cells; (d) FC b2-nAu-enriched permeabilized yeast cells and calibration curves (B) for L-lactate-dependent responses. Conditions: working potential þ 250 mV vs Ag/AgCl, in 50 mM phosphate buffer.

sensitivity evidences the absence of impact of bare gold nanoparticles on sensor output. The lower sensitivity for the two types of bioelectrodes based on intact yeast cells could be explained by diffusion limitations through intact cellular barier. The reproducibility of the developed microbial sensor in the linear frames (from 0.03 up to 2.4 mM L-lactate) was characterized by a low standard deviation (0.95%). A selectivity of the developed sensor was the same compared to earlier described amperometric biosensor based on permeabilized cells of the same yeast strain [12]. The increased sensitivity of the constructed microbial electrode clearly demonstrates that additional enrichment of permeabilized yeast cells by target enzyme immobilized on nanocarriers could be used for construction of microbial biosensors with improved bioanalytical characteristics.

4. Conclusions L-Lactate-selective microbial amperometric sensor based on the recombinant yeast cells of H. polymorpha enriched by FC b2 has been proposed. The enrichment of the cells by the target enzyme was achieved by combination of over-expression of the HpCYB2 gene in the recombinant cells and using nanotechnological approach – transfer of FC b2-bound nanoparticles into the cells. The permeability of the intact and permeabilized yeast cells toward FC b2-nAu was analyzed using TEM and X-ray spectroscopic analysis. The characteristics of the sensors in the absence and presence of FC b2-nAu in the cells have been compared. It was shown that the sensitivity of FC b2-nAu-enriched permeabilized yeast cells to L-lactate is 12-fold higher compared to intact yeast cells and 2.6fold higher than for permeabilized yeast cells. The developed microbial sensor is supposed to be applied for the determination of Llactate in food technology.

Acknowledgements This research was supported in part by NAS of Ukraine in the frame of the Scientific-Technical Program No. 14/26.02.2015 “Sensor systems for medical, ecological and industrial-technological needs: metrological assurance and research exploitation” and Ukrainian-Lithuanian Project No. TAP-LU-03-055/2014 “Investigation of L- and D-lactate: cytochrome с oxidoreductases isolated from the recombinant yeast H. polymorpha and their usage for

construction of amperometric biosensors”.

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2015.07. 081.

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