MULTIPLEX REAL-TIME MONITORING OF CELLULAR METABOLIC ACTIVITY USING A REDOX-REACTIVE NANOWIRE BIOSENSOR

L.C. Hsiung, V. Krivitsky, V. Naddaka, Y.K. Conroy, H. Peretz-Soroka, and F. Patolsky * School of Chemistry, the Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Israel ABSTRACT

We designed a redox-reactive nanowire biosensor for multiplex real-time monitoring of cellular metabolic activities. A field-effect transistor (FET) biosensor modified with 9,10-anthraquinone-2-sulfochloride detected reactive oxygen species (ROS); by ROS’ oxidizing the modified monolayer, surface electron density was reduced to alter measured currents. Likewise, lactate metabolites were converted to peroxide by lactate oxidase (LOX) beforehand for sensing. Significances include: (1) the first nanowire sensing of metabolites in physiological solutions without preprocessing; (2) concentration-dependent sensing responses to H2O2 and lactate were verified to cover physiological concentration ranges; (3) correlating metabolic activities with proliferation rates; and (4) validating pharmaceutical mechanisms of anticancer agents. KEYWORDS: Nanowire Array, Metabolites, H2O2, Multiplex, Real-time Sensing, Field-effect Transistor, Biosensor.

INTRODUCTION

Cellular metabolism influences life and death decisions. Therefore, technologies for monitoring metabolic activity are desirable [1]. The most prevalent technique is mass spectrometry [2] which requires sample preprocessing. Furthermore, electrochemical and fluorescent sensing techniques have sought to combine real-time sensing with multiplex profiling in physiological samples [3]. Moreover, the integration of microfluidic technology and nanotechnology would enable supersensitive detection of metabolites with micro-volume samples [4]. To consummate a biosensing technology for multiplex, real-time profiling of cellular metabolic activity in physiological solutions, we designed a microfluidic biosensor featuring a redox-reactive nanowire FET array to detect ROS, signaling molecules in the regulation of a variety of biological processes, and lactate metabolite, the key metabolite of glycolysis which is highly associated with cancer.

BIOSENSOR DESIGN AND SENSING MECHANISM

The biosensor includes a culture compartment and a sensing compartment (Fig. 1a-b). Cells, reductant (1% v/v N,N-Diethylhydroxylamine, DEHA), and lactate oxidase (LOX) were arranged in the wells of the culture compartment. Then, a solution was introduced from a well to the sensing compartment through a microchannel. Solutions are switched by using solenoids to close or to open underlying channels [5].

A SiNW FET array (Fig. 1c) modified with 9,10-anthraquinone-2-sulfochloride senses ROS and lactate metabolite. Before cellular lactate reaches the FET array, lactate is converted to peroxide by using LOX (Fig. 1d). Then, ROS or consequent H2O2 oxidizes 9,10-dihydroxyanthracene on a FET surface to form 9,10-anthraquinone. This oxidation reaction decreases surface electron density, whereas reductant DEHA reduces 9,10-anthraquinone to 9,10-dihydroxyanthracene to increase surface electron density. Surface electron density varied by oxidation or by reduction changes the measured current.

Figure 1: A nanowire biosensor for multiplex monitoring of metabolites. (a-b) The biosensor. (c-d) A silicon nanowire field-effect transistor array modified with 9,10-anthraquinone-2-sulfochloride senses ROS and metabolites.

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1959 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany

EXPERIMENTAL A PDMS culture compartment with solenoid-actuated valves (Fig. 1a and 1b), for cell culture and control of multiple

solutions, was fabricated using soft lithography. Fabrication of solenoid-activated PDMS valves were described previously [5]. In brief, after the fabrication of the valves, valves were incorporated into the PDMS culture compartment.

Fabrication of the SiNW-FET array [4] began from defining source and drain electrodes of FETs with a multilayer photoresist structure. After exposure and development of the photoresists, the patterns were metallized by e-beam evaporation of Ti/Pd/Ti respectively. Finally, electrodes were insulated with a layer of Si3N4, deposited by plasma-enhanced chemical vapor deposition at 80°C, and a layer of alumina made by atomic layer deposition (ALD).

After fabrication of the SiNW FET array, the chip was chemically modified. Surface modification procedures were as follows: (1) 9,10-anthraquinone-2-sulfochloride was pre-synthesized; (2) the SiNW FET array was coated with (3-aminopropyl)-dimethyl-ethoxysilane [4]; (3) the chip was dipped in a mixture of 9,10-anthraquinone-2-sulfochloride, extra-dry toluene and extra-dry pyridine for 24 hours at room temperature.

A data acquisition system was used to measure the current of a SiNW FET (Ids) induced by surface charges during oxidation by ROS or H2O2. Jurkat cells were cultured with or without 2-deoxy-D-glucose (2DG) in the chip (Fig. 1) in an incubator during measurements. A sample was introduced to the sensing compartment at 20 l min-1. Voltages applied to the drain and source and to the gate were 0.2 V and 0 V, respectively. Current-versus-time signals were recorded at 1-second intervals. After each measurement, reductant DEHA reduced the FET surface (Fig. 1d) for subsequent sensings. For lactate sensing, 0.1 unit/ml of LOX was added to convert lactate to pyruvate and H2O2 beforehand (Fig. 1d). All measurements were performed in phenol red-free serum-added RPMI 1640 medium.

Control experiments, for comparing with sensing of cells, were performed by using dichlorofluorescein assay. Jurkat cells were suspended in phenol red-free RPMI 1640 medium, with 10% FBS and 1% penicillin/streptomycin, at a density of 1 × 106 live cells/ml. Next, cells were removed to obtain cell-free medium. Then, cell-free medium sample was added with 0.5 µM 2’,7’-dichlorofluorescein and loaded into wells of a 96-well black plate at 100 l per well. The plate was prevented from light and incubated at room temperature for 10 minutes, and then the samples were analyzed using a plate reader to determine consequent emission intensities of ROS at 525 nm. Furthermore, concentration of lactate metabolite was also estimated using DCF assay. The main additional procedure was incubating cellular samples with 0.004 unit/ml LOX at 37°C for 5 minutes. Then, DCF was added to the samples, and then the samples were measured. To obtain signals from lactate, readings of LOX-added samples were subtracted by readings of LOX-free samples.

RESULTS AND DISCUSSION

Sensing characteristics of a 9,10-dihydroxyanthracene-modified SiNW FET in response to H2O2 in serum-added medium are presented (Fig. 2a). Importantly, H2O2-specific concentration-dependent sensing capability of the modified SiNW FET was verified. Specifically, the sensing limit to H2O2 was 100 nM, and the sensing response covered the physiological concentration range of H2O2 [6].

In addition, sensing characteristics in response to lactate metabolite are demonstrated(Fig. 2b). Without lactate additives, the signal of a LOX-added sample (the first red curve from the left) was higher than its LOX-free counterpart (the black curve); the difference between the two signals may be due to the high complexity of the serum-added medium. Therefore, we defined the difference as the background signal. To obtain genuine signals from lactate, readings of LOX-added samples were firstly subtracted by readings of LOX-free counterparts―herein, subtracted by the signal of LOX-free blank medium since lactate additives without LOX do not cause any redox to alter measured currents. Finally, the background signal was further subtracted from acquired lactate signals. A corresponding standard curve is presented in the inset. Characteristically, the sensing limit in serum-added medium was 1 M, and the detection range covered the physiological range [7].

(a) (b) Figure 2: Sensing characteristics. (a) H2O2 sensing response. Bases were obtained by flowing a reductant (pH 8.0). (b) Lactate sensing responses. (pH 7.0).

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24-hour monitoring of metabolic activity of drug-treated Jurkat cells are shown in Figure 3. Measured metabolite levels were normalized by the number of live cells. Relative cell count (Fig. 3b and 3c) is a ratio of the cell count at t=24 hour to the initial cell count. Slightly, a decrease of ROS level of 2DG-treated Jurkat was found at t = 6 h (Fig. 3a). Significantly, ROS levels of drug-treated Jurkat were accumulated after 24-hour treatment, and cell proliferation rates had been reduced (Fig. 3a and 3b). Suggestively, after 2DG-treated cells' attempting to limit ROS level, pro-oxidants could induce oxidative stress [8] to inhibit cell proliferation [9]. Parenthetically, data from control experiments (shown in the insets) agree with the significances detected by the NW biosensor.

Also, the correlation between cellular lactate level and a resultant cell proliferation rate after 24 hours was investigated (Fig. 3c). Lactate metabolite of treated cells was decreased, and a reduced proliferation rate observed. This is consistent with a previous study concluding death receptor-induced apoptosis upregulated by 2DG to inhibit glycolysis [10].

(a) (b)

(c) CONCLUSION

On the whole, the proposed redox-reactive nanowire biosensor was verified for multiplex, real-time profiling of cel-lular metabolic activity in physiological solutions without preprocessing. Feasibly, more metabolites can be detected by using oxidase enzymes to convert them to H2O2. Expectedly, the proposed biosensor would promote the understanding about metabolic networks and requirements of cancers to identify therapeutic targets. REFERENCES [1] N. P. Jones, A. Schulze, “Targeting cancer metabolism--aiming at a tumour's sweet-spot,” Drug Discov. Today, vol.

17, pp. 232-241, 2012. [2] J. L. Griffin, J. P. Shockcor, “Metabolic profiles of cancer cells,” Nat. Rev. Cancer, vol. 4, pp. 551-561, 2004. [3] C. Munoz-Pinedo, N. E. Mjiyad, J. E. Ricci, “Cancer metabolism: current perspectives and future directions,” Cell

Death Dis., vol. 3, pp. e248, 2012. [4] V. Krivitsky, L. C. Hsiung, et al., “Si Nanowires Forest-Based On-Chip Biomolecular Filtering, Separation and

Preconcentration Devices: Nanowires Do it All,” Nano Lett, vol. 12, pp. 4748-4756, 2012. [5] S. E. Hulme, et al., “Incorporation of prefabricated screw, pneumatic, and solenoid valves into microfluidic devic-

es,” Lab Chip, vol. 9, pp. 79-86, 2009 [6] F. Lacy, et al., “Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of

hypertension,” J. Hypertens., vol. 16, pp. 291-303, 1998. [7] P. Wacharasint, et al., “Normal-Range Blood Lactate Concentration in Septic Shock Is Prognostic and Predictive,”

Shock, vol. 38, pp. 4-10, 2012. [8] E. A. Veal, et al., “Hydrogen peroxide sensing and signaling,” Mol. Cell, vol. 26, pp. 1-14, 2007. [9] M. López-Lázaro, “Dual role of hydrogen peroxide in cancer: possible relevance to cancer chemoprevention and

therapy,” Cancer lett., vol. 252, pp. 1-8, 2007. [10] L. A. Pradelli, et al., “Glycolysis inhibition sensitizes tumor cells to death receptors-induced apoptosis by AMP ki-

nase activation leading to Mcl-1 block in translation,” Oncogene, vol. 29, pp. 1641-1652, 2010. CONTACT * F. Patolsky, tel: +972-3- 6408780; fernando@post.tau.ac.il

Figure 3: Monitoring of metabolites. (a) 24-hour monitoring of ROS from Jurkat cells. (b-c) The correlations between levels of metabolites and Jurkat cell proliferation rates after 24 hours. Control experiments in insets were conducted using dichlorofluorescein assay (n ≥ 3 replicates; Student’s t-tests were employed; ** denotes P < 0.01).

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