Design of a multi-sensor platform for integrating extracellular acidification rate with multi-metabolite flux measurement for small biological samples

Abstract

Rates of cellular oxygen consumption (OCR) and extracellular acidification (ECAR) are widely used proxies for mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic rate in cell metabolism studies. However, ECAR can result from both oxidative metabolism (carbonic acid formation) and glycolysis (lactate release), potentially leading to erroneous conclusions about metabolic substrate utilization. Co-measurement of extracellular glucose and lactate flux along with OCR and ECAR can improve the accuracy and provide better insight into cellular metabolic processes but is currently not feasible with any commercially available instrumentation. Herein, we present a miniaturized multi-sensor platform capable of real-time monitoring of OCR and ECAR along with extracellular lactate and glucose flux for small biological samples such as single equine embryos. This multiplexed approach enables validation of ECAR resulting from OXPHOS versus glycolysis, and expression of metabolic flux ratios that provide further insight into cellular substrate utilization. We demonstrate expected shifts in embryo metabolism during development and in response to OXPHOS inhibition as a model system for monitoring metabolic plasticity in very small biological samples. Furthermore, we also present a preliminary interference analysis of the multi-sensor platform to allow better understanding of sensor interference in the proposed multi-sensor platform. The capability of the platform is illustrated with measurements multi-metabolites of single-cell equine embryos for assisted reproduction technologies. However, this platform has a wide potential utility for analyzing small biological samples such as single cells and tumor biopsies for immunology and cancer research applications.

Real-Time Analysis of Oxygen Gradient in Oocyte Respiration Using a High-Density Microelectrode Array

“…Finally, the oxygen consumption rate curves at the cell's surface show 12 fmol/s at t = 0 s and increases to~18 fmol/s at t = 46 s. The high readings towards Y ≤ 60 were possibly due to the oxygen consumption by two COCs located around Y = 20. The measured OCR results are comparable to the results from our previous work and others using various stages of bovine oocytes and embryos, where the OCRs were measured between 5 to 30 fmol/s [11,12,32,[66][67][68]. Furthermore, the OCR results for COCs measured by others from a variety of sources (mouse, bovine, and human) [9,69,70] also show a comparable range (3 to 60 fmol/s) as our measured OCR results.…”

“…The impact of oxygen reduction by the electrodes within a small microfluidic chamber was presented, and possible remedies to minimize the effects were discussed. Furthermore, the system would benefit from electrode surface modifications to improve selectivity to oxygen [11,12,36] and to sense other metabolizing agents (glucose and lactose) [12,32]. Electrode surface modifications would also enable simultaneous neurotransmitter detections [28,71] with high spatial and temporal resolutions.…”

“…In contrast to other methods, the electrochemical method offers a distinct advantage to measure the concentration level of target chemical species without the need for bio-sample pretreatment or an optical probe. Moreover, the electrochemical method has been proven to allow simultaneous detection of multiple chemical contents, such as neurotransmitters [28,29] and metabolic markers [30][31][32].…”

Titanium Dioxide Nanoparticles Induce Cognitive Dysfunction of Rats with Endogenous Protective Mechanism of Increased miR-21-5p

“…2F showed the same trend. In order to further observe the effect of m on cell glycolysis activity, ECAR induced by oligomycin was employed [19,20]. A remarkably decrease in ECAR induced by oligomycin was detected in miR-21-5p-silenced SH-SY5Y cells (shmiR-21-5p) compared with that in the controls under TiO 2 NPs treatment.…”

Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development

“…Recent developments include combining electrochemistry with microfluidic technologies 22 , 23 for embryo-compatible devices. Obeidat and colleagues 24 report detecting oxygen consumption alongside glucose consumption, lactate production and pH in individual equine blastocysts using multiple electrochemical sensors. Extracellular flux analysis using the Seahorse Bioanalyzer (Agilent) has recently been published as a more user-friendly, rapid method of profiling the components of respiration in small groups of oocytes or preimplantation embryos 25 .…”