3-D printed microfluidics for rapid prototyping and testing of electrochemical, aptamer-based sensor devices under flow conditions

Abstract

We demonstrate the ability to rapidly prototype and fabricate an epoxy-embedded electrode platform and microfluidic device suitable for using electrochemical biosensors under flow conditions. We utilize three-dimensional (3-D) printing to rapidly prototype molds to fabricate epoxy-embedded electrodes in addition to molds for rapid prototyping of PDMS microfluidic components. We characterize the bare gold epoxy-embedded electrodes using ferricyanide as a redox indicator and then characterize the performance of an adenosine triphosphate (ATP) specific electrochemical, aptamer-based (E-AB) sensor. We then incorporate the ATP specific E-AB sensors into the microfluidic device to study and take advantage of the dynamic response this class of sensor offers. We were able to flow varying concentrations of target analyte and monitor the dynamic response of the sensors to the changing concentration. This work demonstrates the ability to rapidly prototype E-AB sensors under flow conditions using 3-D printing which can lead to rapid and affordable point-of-care or fieldable applications where dynamic measurements of concentration, specificity and sensitivity and multiplex detection are necessary.

Keywords: 3-D printed microfluidic prototypes; Aptamer-based sensors; Dynamic concentration measurement; Electrochemical; Epoxyembedded electrodes.

Figure 1.. Epoxy-embedded electrodes fabrication scheme

The epoxy-embedded electrode setup comprises three gold working electrodes (500 mm diameter), an Ag/AgCl wire reference electrode (500 mm diameter) and a platinum counter electrode (500 mm diameter). a) A “base” mold is printed using Formlabs flexible resin, which is a mixture of (meth)acrylated monomers and oligomers and photoinitiators, with five wells to hold the electrode wires in place during epoxy curing. b) A wire holder printed using a clear resin secures the wires such that polishing the face of the epoxy will yield disk-shaped microelectrode. c) The templated, Armstrong C-7 epoxy and Armstrong Activator A mixture is cured for 2 hours at 74°C and polished to yield 5, in-plane disk electrodes. Schematic of the epoxy-embedded gold of working microelectrodes, Ag/AgCl wire electrode and platinum counter electrode. (Right) SEM micrograph of an epoxy-embedded 500 μm gold electrode.

Figure 2.. Cyclic voltammograms of epoxy-embedded electrodes

Cyclic voltammogram of 5 mM ferrocyanide with 0.1 M KCl at 100 mV/sec demonstrate a well-sealed, functioning epoxy embedded electrode with peak currents as expected. More specifically, the peak current is measured to be 2.19 μA +/− 0.02 μA which is similar to 2.25 μA predicted.

Figure 3.. Epoxy-embedded gold microelectrodes support electrochemical, aptamer-based (E-AB) sensor performance

(Top) E-AB sensors are fabricated on the gold working electrodes using a nucleic acid sequence that is specific for ATP. (Bottom Left) The E-AB sensor responds as expected. SWV without target (green) and with target (red), yields a signal increase in the presence of 1 mM ATP. That signal is rapidly regenerated upon rinsing the sensor surface with buffer (blue). Calibration curves show a fitted with a Langmuir-like isotherm provide a K_d = 78 μM.

Figure 4.. Epoxy-embedded electrode microfluidic device and their electrochemical response under flow

3-D printed Y-shaped mold (top). PDMS microfluidic device reversibly sealed on the epoxy embedded electrodes (middle). Cyclic voltammograms of 1 mM potassium ferricyanide with 0.1 M KCl flowing at varying flow rates and the corresponding electrochemical response. The current increases with increasing flow rates and the cyclic voltammograms begin to exhibit a steady state-like current response (bottom) likely due to the increase flow rate and mass transfer to the electrode surface.

Figure 5:. Scan rate and flow rate experiments to show diffusion limited and transport-limited current

Scan rate experiment performed using 1 mM potassium ferricyanide with 0.1 M KCl show diffusion limited regime at no flow and slow flow rates when plotted as current vs square root of scan rate (V/s) depicted by the linear relationship described by the Randles-Sevick equation (top and middle). Under fast flow rates, according to the Levich equation the transport-limited current is a function of the cube root of flow rate so it results in a linear graph when plotted as Ip vs V_f^1/3 (bottom).

Figure 6.. ATP specific E-AB sensor response to varying concentrations of ATP under flow conditions

The ATP specific E-AB sensors fabricated on the epoxy embedded electrodes were interrogated with different concentrations of ATP target displaying dynamic response to changes in concentration in tris buffer (Left, maroon) and in fetal bovine serum (FBS, Right, blue). Our platform provides the capability to dynamically measure changes in concentration of analytes of interests in buffers as well as relevant biological matrices.

Figure 7.. Dynamic multiplex detection of ATP and Insuling inside a microchannel.

The ATP and Insulin specific E-AB sensors were fabricated on our epoxy-embedded electrodes and adhered to our PDMS microfluidic device and challenged with the respective targets of 500 μM ATP and 20 μM insulin at a flow rate of 10 μLmin-1. As shown above, the sensors respond specifically to their target when it is flowed through the channel with the ATP E-AB sensor showing 90 % signal change to 500 μM ATP and the insulin E-AB sensor responding with ~40 % signal-off to 20 μM of insulin.