Ultra-Low-Volume, Real-Time Measurements of Lactate from the Single Heart Cell Using Microsystems Technology

Abstract

The fabrication of microelectrodes integrated within ultra-low-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 two-microelectrode 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.

On-chip acidification rate measurements from single cardiac cells confined in sub-nanoliter volumes

“…Furthermore, single cell analysis in small volumes provides the opportunity for more detailed information on cells that are difficult to culture or are rare, and a more sensitive analysis with a higher temporal resolution than measurements on populations of cells. Single cell studies in oil-sealed restricted volumes on amperometric sensors have shown the release of lactate or purine from single cardiac myocytes (Bratten et al, 1998;Cai et al, 2002); however, this approach does not allow small extracellular volumes, the chemical control, and the perfusion of the extracellular space.…”

Microfluidic sensing: state of the art fabrication and detection techniques

“…The detection methods in microfluidics can be classified into three major types: optical methods, 9, 73-89 electrochemical methods, [90][91][92][93][94][95][96][97][98][99][100][101][102] and mass spectrometry methods. [103][104][105][106][107][108][109][110][111][112][113] Among these methods, optical and electrochemical methods are the most frequently utilized due to their selectivity and sensitivity.…”

Lab-on-a-chip technologies for proteomic analysis from isolated cells

“…For example, the mechanical trapping of cells using microfabricated filters (Carlson et al 1997;Wilding et al 1998) or other physical trapping designs has proved successful, although, in general, it does not offer the versatility of other methods (Rusu 2001;Cai et al 2002). Laser tweezers and magnetic fields have been used to trap, sort and move cells (see Andersson & van der Berg (2003) and Yi et al (2006) and references therein), and droplet-based microfluidics also lends itself naturally to the transport and isolation of single cells (Huebner et al 2007).…”