Generalizable Electrochemical Biosensors Using DNA-Analyte Conjugates Integrated with Pneumatic Oscillators for Microfluidic Workflow Automation

Abstract

Sensors based on electrochemical (EC) readout offer low cost, miniaturization, and adaptability to the point-of-care (POC). Nonetheless, most EC sensors are specialized to a particular target, and there remains a need for a robust EC biosensor platform for the multitude of biomarkers that are not EC-active, do not undergo enzymatic conversion, or are not suited for potentiometry [1,2]. While aptamer-based EC sensors have been proven for sensing in living animals with temporal resolution of a few seconds [3], most method development has been target-focused, lacking generalizability [4]. Presently, the clinical EC toolbox is a conglomerate of targeted methods, and there is a pressing need to develop a single EC platform amenable to rapid, generalizable, quantitative readout of multiple classes of clinically relevant targets. A direct, generalized EC sensing approach with minimal added reagents or amplification steps is preferred [5,6]. Our group has been working to address this need and expand to more analyte classes for several years, and in 2019 we designed a versatile DNA-nanostructure architecture attached to gold electrode surfaces [6]. In this work, we will discuss our efforts to expand the generalizability of our sensor platform, chiefly through custom synthesis of varied DNA-analyte bioconjugates to incorporate within the DNA-nanostructure, specifically DNA-peptide and DNA-small molecule conjugates. Using the same DNA nano-architecture, sensors have been validated in 98% human serum for a variety of targets, several encompassing the human clinical range—a peptide drug (exendin-4) [7], a larger protein (creatine kinase), and smaller molecules or steroid hormones (testosterone, estradiol, progesterone, cortisol). Overall, this new DNA nanostructure platform provides a generalizable sensor with minimal workflow, direct-readout, and the capability to expand EC sensing to a wide variety of clinically important analytes. These sensors can measure antibodies directly without any reagent addition, and the small-molecule or peptide sensors require just a two-step workflow. While the workflow is minimal, the EC measurements benefit from well-controlled solution pH, ionic strength, etc. As such, automated fluidic handling can enhance the user-friendliness of the sensors while maintaining measurement quality. We will also discuss several advancements in automating microfluidic flow control, leveraging digital circuit analogies to develop on-chip pneumatic circuits [8,9]. Using devices fabricated by inexpensive, resin-based 3D printers, we have developed robust pneumatic oscillators that can control on-chip valves for pumping solutions to the EC sensors. The oscillators are automated and tunable from 0.5 to 100 Hz and can pump multiple solutions on microdevices with only a single vacuum input line. Antibody reagents and samples can be mixed automatically then introduced to the electrode, and buffer washes can be included to improve measurement consistency. Ultimately, by integrating this automated microfluidic control with our generalizable EC sensors in the future, many different analytes should be measurable consistently and in a user-friendly manner.