High temporal resolution delayed analysis of clinical microdialysate streams

This paper presents the use of tubing to store clinical microdialysis samples for delayed analysis with high temporal resolution, offering an alternative to traditional discrete offline microdialysis sampling. A model allowing optimal results is described.


Microfluidic biosensor analysis system
The microfluidic biosensing system has been described previously. 3,4 Briefly, it consists of glucose and lactate biosensors housed in a microfluidic chip. Glucose and lactate biosensors were fabricated using combined needle electrodes, made by threading a 50 μm polytetrafluoroethylene (PTFE) insulated platinum/iridium (90%:10%) wire (Advent Research Materials, UK) and a 50 μm polyester insulated silver wire (Goodfellow, UK) through a 27G hypodermic needle. 5 The insulation was removed from the ends of both wires using a flame and the exposed metal was connected to an electrical wire using conductive silver epoxy (RS Components, UK). The needles were filled with epoxy resin (CY1301 and HY1300, Robnor resins) to secure the wires. Once cured, the needles were polished with sandpaper (Buehler, UK) to give a flat surface creating platinum and silver disc electrodes and then sequentially with alumina slurries (1, 0.3 and 0.05 μm). The 50 μm platinum disc formed the working electrode and the silver wire was chloridised to give a 50 μm disc Ag|AgCl reference electrode by dipping the needle tip into potassium dichromate reference solution (BASi, US) for 3s and then into hydrochloric acid (37%) for 20 s to remove the oxide layer from the working and auxiliary electrodes. The stainless steel needle shaft served as the counter electrode. Cyclic voltammetry was used to assess the quality of the working electrode surface. The electrodes were then functionalised in three layers. Initially, the working electrode was coated with poly(m-phenylenediamine) (m-PD) using electropolymerisation to block potential interferences. To do this the needle was placed in a 100 mM solution of m-phenylenediamine in 0.01 M PBS at pH 7.4. The potential was held at 0 V for 20 s, 0.7 V for 20 min to initiate polymerisation and 0 V for 5 min. After electropolymerisation the electrode was rinsed gently with deionised water and cyclic voltammetry was used to verify that the working electrode had been successfully coated. The second step involved dipping the needle tip into a hydrogel layer containing either glucose oxidase or lactate oxidase and placed in the oven at 55°C for 2 hours (using method adapted from Vasylieva et al. 6,7 ). Finally the biosensors were coated with a polyurethane diffusion-limiting film to extend their dynamic range. The biosensors were positioned in a poly(dimethylsiloxane) (PDMS) microfluidic chip 8 as described elsewhere. 3 Biosensors were controlled using in-house potentiostats and a PowerLab 8/35, controlled by LabChart Pro (ADInstruments). Figure S1 illustrates the concept that collection and analysis flow rates can be optimised independently using this methodology. In the example described, microdialysis is carried out at 5 µl/min and dialysate is collected in storage tubing. Subsequent analysis takes place at 1 µl/min, effectively giving a reading every 12 s in real time at the expense of the analysis now taking 5 hours.

High temporal resolution analysis
Electronic Supplementary Material (ESI) for Analyst. This journal is © The Royal Society of Chemistry 2017 Figure SI. Schematic representation of temporal resolution at different flow rates. In this example we imagine an analysis time of 1 min and a sample loop of 0.5 µl. If samples are collected and analysed at 5 μl/min this would give 60 measurements in 1 hour of dialysate (assuming complete loop filling with 2x loop volume). If instead the analysis flow rate is slowed down to 1 μl/min this would give 720 measurements per hour of collected dialysate.

Axial molecular diffusion
Taylor dispersion due to passive diffusion during static storage is negligible; a molecule in solution diffuses a distance of appoximately (where is time in seconds and is the diffusion coefficient. For example, it √2 would take a particle s (equivalent to nearly 2 months) to diffuse 10 cm (assuming is cm 2 s -1 5 × 10 6 1 × 10 -5 at room temperature), equivalent to 1.1 µl volume in FEP tubing (0.12 mm ID).

Microdialysis probe pressure limits
Kiritsis measured the ratio of fluid velocity through the membrane as a function of the pressure gradient across the membrane. This quantity is called . It is related to components of Darcy's Law as shown in Eq. 1.
Because the membrane expands slightly under pressure, increases as pressure increases. The following calculations are for m/(Pa.s), which is the value at kPa (~0.5 atm). The probe used was typical, namely with = 0.9 × 10 -12 ∆ = 50 an inside diameter of 200 µm and a length of 2 mm. As flow rate is just the surface area, , through which fluid flows multiplied by the velocity, For m/(Pa.s), kPa and the area of the probe's membrane ( mm 2 ), we find that the = 0.9 × 10 -12 ∆ = 50 1.3 × 10 -6 flow rate is 3 nl/min. Under many circumstances this is negligible.