Microfluidic devices with integrated dual-capacitively coupled contactless conductivity detection to monitor binding events in real time

Abstract

This paper describes the fabrication of microfluidic devices with integrated dual capacitively coupled contactless conductivity detection (C⁴D) for monitoring binding events. The avidin-biotin model was employed to show the feasibility of the C⁴D as a detection mode. The binding event was monitored using microfluidic channels fabricated in poly(dimethylsiloxane) (PDMS). Detection electrodes were fabricated in a three-electrode arrangement by sputtering a Ti/Au/Ti layer over a glass wafer, which were electrically insulated by a SiO₂ layer. The dielectric layer over the sensing electrode area was chemically modified with biotin. After introducing avidin solutions on microchannels, binding events were detected on conductivity sensorgrams by applying a 300-kHz-sinusoidal waveform with amplitude of 2 V (peak-to-peak) to the exciting electrode. The electrical characterization of the modified electrode indicates that the conductivity signal is changed due to the alteration on the dielectric constant of the surface. The limit of detection for avidin was 1.5 μg mL⁻¹, representing 23 nmol L⁻¹.

Introduction

Biomolecular interaction analysis is of foremost importance to the clinical field, once the binding events are essential for a better understanding of basic biological/biochemical processes [1], [2], [3], [4], [5]. To monitor these interactions, a common route involves the immobilization of biomolecules on chemically modified surfaces. The detection of a binding event can involve the use of optical, acoustic, or electrical transducers to indicate the presence of adsorbed biological material on the transducer surface through its mass, dielectric permittivity, conductivity, capacitance, or impedance [1]. The surface plasmon resonance (SPR) technology is the most widely tool employed to this purpose [3], [4], [5]. There are commercially available equipments including the BIAcore^® (Biacore AB, Uppsala, Sweden), Spreeta^® (Texas Instruments, Dallas, USA), and SensíQ^® (SensíQ, Oklahoma City, USA). The cost of SPR equipment is higher than US$ 50,000 and for this reason it is not always in the priority of many researchers. Surface acoustic wave (SAW) and quartz crystal microbalance (QCM) techniques [6], [7], [8] are two competitive alternatives with moderated cost.

In addition to the mentioned technologies, there is a great interest on systems that involve direct electrical signal transduction once they can be more easily integrated with microfluidic channels and then linked to electrical-processing methods in real-time [9], [10]. The overall electrical response contains contributions from the aqueous solution, the biological layers, and any other material that serves as mechanical and electrical support [10]. Electrochemical impedance spectroscopy (EIS) has been extensively explored to characterize the frequency-dependent properties of surfaces modified with biomolecules. Lasseter et al. [10] reported the use of EIS to study the detection of protein binding events on microfluidic devices. Delaney et al. [11] used EIS to measure capacitance in ultrathin chemosensors prepared by molecularly imprinted grafting photopolymerization. Recently, spectroscopic ellipsometry has been also employed to successfully measure the adsorption of proteins to solid surfaces [12], [13].

A new technique called of supercapacitive admittance tomoscopy has been proposed by Gamby et al. [14], [15] to measure the adsorption of biomolecules on a microfluidic sensor surface. The concept of this technique is based on establishing a capacitive coupling between two electrodes placed on one side of a dielectric substrate and the solution in contact with the other side. This contactless impedance technique allowed to measure capacitive response related to the adsorption process of IgG molecules to the channel wall. This technique appears to be quite attractive, however, based on the data reported by the authors, the contribution of the background electrolyte has not been considered, i.e., the analytical response is regarded to the overall impedance variation.

Among all electrochemical detection systems, the capacitively coupled contactless conductivity detection (C⁴D) has gained much popularity due to its instrumental simplicity, low cost, and good versatility when applied in different science fields. Conductivity detection is a simple and universal detection technique, which involves measurement of the conductance between two or four electrodes through which an alternating current is passed and allows convenient detection of ionic species [16], [17]. The contactless detection mode has several advantages over the contact mode, including the absence of problems associated with the interface electrode-solution, effective isolation from high separation voltages (when coupled with electrophoresis systems), a simplified construction and alignment of the detector with microchannels [16], [17], [18]. In the last ten years of C⁴D on chip-based systems, several research groups have reported different applications including bioanalytical or environmental samples, fundamental studies of new geometries or arrangements as well as strategies to improve the detector sensitivity [19], [20], [21]. In addition to the referenced applications, a scanning C⁴D (SC⁴D) mode has also been explored for the characterization of stationary phases on capillaries and microchannels [22], [23], [24].

This paper describes the use of a C⁴D system to monitor binding events on PDMS/glass microfluidic devices. For this purpose, a dual-C⁴D homemade equipment was developed so that one detector is employed to monitor the specific binding event while the other detects all conductivity changes in solution. In this case, the difference in response between both detectors is attributed solely to the binding event. This strategy is highly relevant to express the analytical response only related to binding of biomolecules on immobilized targets. As proof of concept, the avidin-biotin model was used to evaluate the feasibility of the proposed system. Brief results related to the use of a microfluidic biosensor with integrated contactless conductivity transduction have been recently reported in a communication article [25].