Microscale Electrochemical Platform for Temperature-Dependent Biomolecular Research and Biosensing

Abstract

Introduction Detection of various types of biomolecules, as well as the measurement of biomolecular properties and how those properties change under different conditions, are important processes in applied areas such as drug discovery, biomanufacturing, disease screening, and therapeutic treatment monitoring. While many instrumental techniques characterize biomolecules in free solution, biosensors often use immobilized probes to monitor the presence and properties of biomolecular species.A miniature platform has been designed and fabricated as a prototype to perform temperature-dependent electrochemical analyses on small-volume (< 10 µL) biomolecular samples [1]. Square wave voltammetry signals have been employed to track binding to and conformational changes of immobilized forms of DNA. The fabrication methods for the platforms are scalable to array configurations which could offer an efficient and automated electronic tool for biochemical research and healthcare applications. Fabrication and Features of the Microdevices Each microdevice consists of a planar set of three surface electrodes, integrated components for temperature control and a top-surface sample cell (Figure 1). The devices are produced in wafer runs with techniques that are compatible with producing single units or multi-element arrays with individual addressability. Fabrication begins with 100 mm fused-silica wafers on which serpentine Pt films are deposited for use as both resistive microheaters and platinum resistance thermometers (PRTs). Lithography and etch-back are utilized for processing the sputter-deposited 220 nm Pt layer. A thin (~ 1 µm) high-quality PECVD SiO2 layer is then deposited over the Pt serpentine, and annealed to 800°C, to allow for effective transfer of heat while also providing defect-free electrical insulation between the microheater/PRT and top-surface electrodes. A liftoff process patterns the three planar surface electrodes (of thickness 200 nm over 20 nm Ti) in Pt for the pseudo-reference and counter electrodes, and Au for the working electrode. Data Acquisition For acquisition of temperature-dependent electrochemical profiles, PID-controlled from ~ 10°C to ~ 70°C using the calibrated PRT, two types of mounting assemblies have been employed: one that uses ambient cooling with resistive heating ramps, and one that uses a programmed commercial thermoelectric for cooling and heating [2]. Samples are prepared over the top electrodes within a PDMS containment volume. Biomolecular probes are immobilized to the Au working electrode via thiol attachment, with 6-mercaptohexanol backfill. Methylene blue tags are included on studied biomolecules to enhance the electrochemical (current) signals measured as a function of voltage (and temperature) with square wave voltammetry (Figure 2). All aspects of the electrochemical measurements and temperature control are computer automated. Results and Conclusions A number of sample systems have been used to demonstrate the capabilities of this microscale electronic technology. Single-strand DNA has been immobilized and then the melting temperature, Tm, was determined for binding of a fully complementary strand, one with a single base-pair mismatch, and one involving two mismatches. Tm shifts to lower temperatures by 5.1°C and 7.3°C for the mismatches indicate the ability to detect single nucleotide polymorphisms (SNPs) [1]. The binding of ligands (diaminazene aceturate, DMZ, and proflavine) to surface-immobilized duplex DNA have also been examined (Figure 3) to show their stabilizing influence, as Tm was increased by 11.0°C and 13.5°C for these intercalator and minor-groove binders, respectively [2]. Related measurements are also being performed on other DNA species, including poly-thymine, streptavidin aptamer and G-quadruplexes. Further development of the approach, with incorporation of microfluidics for array formats, offers the opportunity of a sensitive research and sensing tool for efficient analyses of biomolecular interactions and response to chemical and physical stressors, without interference phenomena like those that can be encountered in fluorescence-based techniques.