Oxygen consumption rate (OCR) is a significant parameter helpful to determine
Determination of oxygen levels is required in many different research fields, such as in environmental analysis, in medical area, and in biotechnology industries, as O2 is an important physiological parameter and a nutrient for the microorganisms, plants, and yeasts used in different applications.
Matching biochips’ readouts with the biological data obtained via traditional methods represents a key point for a correct analysis of the experimental results. Recently, especially in the field of novel drug screening, improved control of cell response plays a pivotal role. For quantitative understanding of these events, microbioreactors are expected opportunities to study cells under simulated physiological microenvironments, enabling spatial and temporal control of cell behaviour. To these aims combining novel structural and sensing devices into whole microsystems is required. The final devices should be minimally cell-invasive, sensitive, and rapid and of low cost, increasing in throughput and decreasing in biological sample and chemical reagents required amount for each assay. Therefore, in this study, a disposable plastic device, for cell microculture (for both suspended and adherent cells), useful in monitoring cellular metabolic state, was developed. By means of an electrochemical sensor, our device detects changes in the [O2] gas passing through perm-selective membrane. The conventional polarographic Clark electrode consists of a Pt cathode and an Ag anode in contact with an electrolytic solution and separated from the measurement sample by an oxygen permeable membrane. Basically, at a constant applied potential, when the gas interchange with the external environment is excluded, cellular activity generates a decrease of the current related to the concentration of dissolved oxygen, which diffuses through the membrane, as described in the following scheme:
In this context, the purpose of this work was to evaluate the efficiency of a new glass/PDMS made microoxygraph device (MOD) for noninvasive electrochemical OCR evaluation even of small cell samples.
Gas permeable and biocompatible soft polymers are suitable for biological applications; particularly, high intrinsic permeability to gases [
Microfabrication processes were performed by means of single-wafer spin processor (Model WS-400°-6 NPP/LITE SHOWN, Laurell Technologies Corporation), Pico Electronic Diener plasma hasher, hot plates, bromograph, and thermal evaporation system. Single-component characterization was carried out by KLA-Tencor, Alpha-Step IQ profilometer, and Bruker Nanoscope VI Multimode Scanning Probe workstation.
Tha assembled device was valued by Autolab, PGSTAT30 potentiostat (Metrohm) controlled by General Purpose Electrochemical System software (GPES).
The detector surface is processed just before starting measurement. Through the microfluidic system, 0.05 M H2SO4 solution was dispensed to remove excess impurities for 2 minutes; this procedure was repeated for three times. After H2SO4 suction, the excess was removed by injection of ultrapure water for three times. Finally, FeCl3 solution was injected to fill the microchannel and allowed to react with the silver film for 50 sec. After cleaning steps, the sensing area was rinsed with ultrapure water; then, 0.1 M KCl electrolyte aqueous solution was dispensed in the microgroove. MOD was connected to potentiostat by micromanipulators aided microtip positioned on the electrode pads (Figure
The presented MOD consists of a three-microelectrode detector on a glass substrate, an electrical insulation layer with a selective opening, and the PDMS microchamber modulus [
Graphical representation of microoxygraph device components’ layout.
Assembled microoxygraph device. Potentiostat connection by micromanipulators (a). Semipermeable PDMS membrane, 80
The fabricated microelectrodes system is shown in Figure
Superficial roughness of FeCl3-treated Ag and Au electrodes.
Superficial roughness average values |
|
|
---|---|---|
Silver layer | ||
0.1 M FeCl3 |
23.13 ± 6.08 SD | 28.4 ± 6.94 SD |
50 mM FeCl3 |
26.27 ± 1.06 SD | 33.27 ± 1.59 SD |
Untreated | 2.01 ± 0.14 SD | 2.52 ± 0.18 SD |
Gold layer | ||
50 mM FeCl3 |
0.92 ± 0.11 SD | 1.18 ± 0.15 SD |
Untreated | 0.95 ± 0.005 SD | 1.22 ± 0.01 SD |
Average values, calculated by Nanoscope software v7.30 (Bruker).
AFM topographic characterization of silver layer. AFM images of untreated silver layer (a); Ag/AgCl layer grown by 0.1 M FeCl3 for 5 min (b) or by 50 mM FeCl3 for 50 sec (c). Scan size, 5
Both parameters’ values show the presence of Ag/AgCl layer; however a rougher particulate layer grew after reaction with 50 mM FeCl3 for 50 sec treatment. A microreference electrode, with higher Ag/AgCl layer thickness as well as silver chloride particles higher surface area, is expected to exhibit increased stability as reported by [
AFM images of PDMS OPM. Scan size of 5.0 (a) or 2.0
Firstly, the electrochemical behavior of electrode system was investigated. Figure
Cyclic voltammogram. Scan rate of 50 mV/sec, in a 0.1 M KCl solution.
Amperometric response reproducibility. Response curves at −0.8 V versus Ag/AgCl RE, obtained by periodic injection (black arrows) and withdrawal (white arrows) of Na2S2O4 solution from microoxygraph chamber inlet. Δ response: 0.54
Repetitively, tests were performed to analyze electrochemical functionality and response time of the three-microelectrode configuration as well as the solidity and reproducibility (strictly related to the potential stability of the reference electrode and the crosstalk effect between the three electrodes of the electrochemical detector [
Experimentally, 0.1 M Na2S2O4 solution was employed as strong oxygen reducing agent to stabilize a zero-oxygen concentration condition. Microoxygraph response was evaluated by periodic injection and suction of Na2S2O4 solution from microoxygraph chamber inlet. In Figure
This behavior is typically due to small amount of oxygen molecules, not reduced by the working sodium dithionite concentration, or by residual oxygen concentration in the electrolyte solution. Once all oxygen dissolved molecules are reduced, the current signal stabilizes at a minimum value, near to zero. Intensity value (± Na2S2O4 solution in the chamber expressed as Δ response) is −0.54
Comparative table of different three-electrode configuration microoxygraphs functional features.
Microoxygraphs | Current work | [ |
[ |
[ |
---|---|---|---|---|
Sample volume | 100 |
d.n.s. | d.n.s. | 200 |
Fluidic system | Yes | No | Yes | No |
Reducing agent [Na2S2O4] | 0.1 M | 0.1 M | 0.1 M | 0.1 M |
Cathode material/thickness (nm) | Gold 50 | Gold 400 | Gold 250 | Gold 250 |
Cathode area (mm2) | 0.2 | 0.04 | 0.0004 | 0.025 |
Membrane material/thickness ( |
PDMS/80 | PDMS/15 | PDMS/20 | |
Δ average current | 0.54 ± 0.06 |
nA (d.n.s.) | 7.09 ± 0.09 nA | 152.18 ± 0.87 nA |
Average 90% response time (sec) | 39.6 | 40 | 13.4 | 4.9 |
Cell cultures were performed in the microchamber of the microoxygraph device. Before cell seeding, the microchamber was sterilized by 70% ethanol and double washed with PBS. The open upper inlet allows sample injection and medium and gas exchange during cell incubation. After 12 h of 3T3 incubation, microelectrodes were quickly subjected to surface cleaning and chlorination treatments. Then, 0.1 M KCl electrolyte solution was injected in microchannel and OCR measurements were performed. A typical OCR
Typical cell OCR test by microoxygraph chip. 3T3 cells (1.8 × 104 cells/microchamber) were seeded in microoxygraph chamber and incubated for 12 h. After DMEM replacement with L15, amperometric measurements were performed. The recorded current is proportional to the dissolved oxygen concentration (directly) and to the cell OCR (inversely). The slope of oxygen concentration with time is defined as the oxygen consumption rate. Blue and red lines evidence the slopes before and after electron transport chain-inhibitor injection, respectively. Black arrow: 20 mM NaN3 injection. The recorded OCR, directly proportional to the number of active cells, is characterized by an initial high speed that slightly decreases after 150 sec. This positive O2 consumption trend is congested by NaN3 cell intoxication (a). Optical image of the 3T3 cells acquired after the recorded metabolic impairment. Plump morphology is observable in mitochondrial toxin damaged cells; cathode is clearly visible under cell culture OPM (b).
In summary, we reported on a disposable fluidic microoxygraph chip, a miniaturized electrochemical system for amperometric analysis of dissolved oxygen. Compared to traditional bench oxygraph, the disposable device displays miniaturization advantages, such as decrease of working volumes (samples, reagents from 200
Comparative features of our and other interesting devices [
Another factor affecting the sensor analytical performance is the working electrode area. The current output is directly proportional to the sensor area. Supposing a uniform current distribution on the working microelectrode surface, in terms of surface charge density, the calculated
Representative biological applications have upheld these evidences. By operating as microculture device too, it first allows cell adhesion and proliferation; then it permits low invasive sensing of cell metabolic activity in different conditions on the same chip. Biocompatible materials (glass and PDMS), utilized to fabricate the microoxygraph device, are advantageous for their transparency properties too, so that, during cell cultures, cells adhesion, spreading, and growth are easily morphologically monitored by a common inverse optical microscope.
The microsystem is suitable for microfluidic biosensor arrays configuration development. Future application for
The authors declare that there is no conflict of interests regarding the publication of this paper.
A. Aloisi and E. Tarentini contributed equally to this work.
This work was partially supported by