Anal. Chem. 2002, 74, 908-914
Ultra-Low-Volume, Real-Time Measurements of Lactate from the Single Heart Cell Using Microsystems Technology Xinxia Cai,† Norbert Klauke,† Andrew Glidle,† Peter Cobbold,‡ Godfrey L. Smith,§ and Jonathan M. Cooper*,†
Bioelectronics Research Centre, Department of Electronics and Electrical Engineering, University of Glasgow, Glasgow G12 8LT, U.K., The Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool, L69 3 BX, U.K., and Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, U.K.
The fabrication of microelectrodes integrated within ultralow-volume microtiter chambers for the amperometric determination of metabolites continues to be of interest in the subject of single-cell and high-throughput screening. The microsystem described in this paper consists of a twomicroelectrode sensor with a microfluidic dispensation technology, which is able to deliver both very low titers (6.5 pL) and single heart cells into a low-volume microphotoelectrochemical cell. Devices were fabricated using photolithography and liftoff giving reproducible sensors integrated within high aspect ratio titer chambers (with a volume of 360 pL), made of the photoepoxy SU8. In this paper, the determination of lactate was optimized using an enzyme-linked assay based upon lactate oxidase, involving the amperometric determination of hydrogen peroxide at +640 mV versus an internal Ag|AgCl pseudoreference. The microsystem (including the microfluidic dispensers and structures as well as the microsensor) was subsequently used to measure the lactate content of single heart cells. Dynamic electrochemical measurements of lactate during cell permeabilization are presented. We also show the use of respiratory uncouplers to simulate ischemia in the single myocyte and show that, as expected, the rate of lactate production from the hypoxic heart cell is greater than that within the normoxic healthy myocyte. Microfabrication and micromachining technology has enabled the production of a wide variety of two- and three-dimensional electrode arrays integrated together with microchambers and microchannels.1 The flexibility of both computer-aided design (CAD) and polymer processing methods has resulted in a wide variety of different electrode geometries (typically >2 µm), the vast majority of which have been produced by photolithography. Previously, in developing an integrated sensor technology, we have produced 190-600-pL-sized ultra-low-volume electrochemical * Corresponding author: (fax) +44-141-330-6010; (e-mail) jmcooper@ elec.gla.ac.uk. † Department of Electronics and Electrical Engineering, University of Glasgow. ‡ University of Liverpool. § Institute of Biomedical and Life Sciences, University of Glasgow. (1) Cooper, J. M. Trends Biotechnol. 1999, 17 (6), 226-230.
908 Analytical Chemistry, Vol. 74, No. 4, February 15, 2002
chambers,2-5 which have been used for single-cell measurements of femtomole amounts of purines, including adenosine and inosine, during a variety of metabolic and pathological conditions.4 This previous work has focused on the fabrication of both two- and three-microelectrode devices and has also involved the optimization of a practical analytical protocol, including solutions to the problem of introducing a low volume of fluid into the device. Our current work, described in this paper, not only complements that being performed elsewhere6-11 but also makes significant technical advances over that described previously by us2-5 in providing a method for the picoliter-scale determination of lactate. Furthermore, although commercial microfluidic dispensers are now available, we describe a simple and robust implementation of the technology for automated low-volume microfluidic or singlecell dispensation, which can be assembled at minimal cost. Our research also tackles the additional problem of sensor regeneration and the reproducibility of the measurement in reduced geometric volumes, enabling us to report the real-time production of lactate from the single heart cell. Despite the need to consider the issues of biological representation and variability when single-cell measurements are performed, such assay formats continue to attract significant interest with respect to fundamental and applied studies. For example, when such determinations are being made, there is an ability to deconvolute complex metabolic interactions, providing a method that readily complements other single-cell methodologies, such as patch or voltage clamping or fluorescence imaging. Other significant advantages arising from this format include the speed of the response, the sensitivity of the measurement, and (2) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1997, 69, 253-258. (3) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Chem. Commun. 1998, 471-472. (4) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1998, 70, 1164-1170. (5) Cai, X.; Glidle, A.; Cooper, J. M. Electroanalysis 2000, 12 (9), 631-639. (6) Gavin, P. F.; Ewing, A. G. Anal. Chem. 1997, 69, 3838-3845. (7) Clark, R. A.; Ewing, A. G. Anal. Chem. 1998, 70, 1119-1125. (8) Travis, E. R.; Wightman, R. M. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 77-103. (9) Xin, Q.; Wightman, R. M. Anal. Chem. 1998, 70, 1677-1681. (10) Amatore, C.; Szunerits, S.; Thouin, L.; Warkocz, J. S. Electrochem. Commun. 2000, 2, 353-358. (11) Hartmann, J.; Lindau, M. FEBS Lett. 1995, 363, 217-220. 10.1021/ac010941+ CCC: $22.00
© 2002 American Chemical Society Published on Web 01/19/2002
the ability to “count” the total amount of the metabolite produced by the single cell, as the integral of the i-t transient (as none of the analyte is lost to bulk solution). To our knowledge, lactate has not previously been determined in a real-time, single-cell assay, nor in a microsystems/lab-on-achip format. As a metabolite, being the end product of glycolysis, it is one of the most important cellular metabolites, and is of significant clinical interest. It is produced in small amounts during aerobic respiration and in larger concentrations during anoxia or ischemia (e.g., infarction or cell death). As a model for cellular anoxia, in this study, single myocytes were poisoned by microinjecting carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) into the microchamber. FCCP inhibits oxidative phosphorylation by uncoupling the mitochondrial respiratory chain, thereby simulating the anaerobic conditions, which prevail during heart disease. Simultaneous hydrolysis of ATP also promotes glycolysis, which, under the anaerobic conditions, also leads to an increase in the rate of production of lactate. Measurements of the products of these glycolytic pathways provide further information on the complex time course of metabolic changes, which are often obscured by cell-cell asynchrony in tissue-level measurements (and which will be revealed through single-cell studies). For example, one particular future interest in the measurement of lactate from the single ischemic cell will be to further understand how cell acidosis interferes with calcium ion transients, lactate efflux, and the cell membrane potential. Previously, lactate oxidase (LOx) has been used extensively as part of enzyme-linked electrochemical and spectrophotometric assays for lactate. In the research described in this paper, the enzyme catalyses the oxidation of lactate in the presence of molecular oxygen to produce pyruvate and H2O2 (the peroxide being detected amperometrically; see eqs 1 and 2, below.12-14) In this paper, a two-electrode microamperometric system was developed based on a platinized working microelectrode and an integrated pseudo-Ag|AgCl combined counter and reference electrode. The platinized microelectrode, comprising porous platinum microparticles (platinum black), possesses a very large “internal” surface area and is highly catalytic for many electrochemical detection processes (e.g., with a reduced overpotential for the oxidation of H2O2). The enhanced electrochemical surface area was found to increase the signal/background ratio significantly. Reproducibility after electrode regeneration, which presents a key challenge, was optimized using a dry etch protocol to give devices with a coefficient of variation of
