Portable electroanalytical nucleic acid amplification tests using printed circuit boards and open-source electronics

The realization of electrochemical nucleic acid amplification tests (NAATs) at the point of care (POC) is highly desirable, but it remains a challenge given their high cost and lack of true portability/miniaturization. Here we show that mass-produced, industrial standardized, printed circuit boards (PCBs) can be repurposed to act as near-zero cost electrodes for self-assembled monolayer-based DNA biosensing, and further integration with a custom-designed and low-cost portable potentiostat. To show the analytical capability of this system, we developed a NAAT using isothermal recombinase polymerase amplification, bypassing the need of thermal cyclers, followed by an electrochemical readout relying on a sandwich hybridization assay. We used our sensor and device for analytical detection of the toxic microalgae Ostreopsis cf. ovata as a proof of concept. This work shows the potential of PCBs and open-source electronics to be used as powerful POC DNA biosensors at a low-cost.


1.
. List of oligonucleotide sequences and their respective modifications (underlined).

Acronyms
List of abbreviations used in this section:  ADC -Analog-to-Digital Converter  CA -Chronoamperometry  CV -Cyclic Voltammetry  DAC -Digital-to-Analog Converter  EUR -Euro currency  IC -Integrated Circuit  MCU -Microcontroller Unit  N/A -Not Applicable  PCB -Printed Circuit Board  SWV -Square Wave Voltammetry  UART -Universal Asynchronous Receiver-Transmitter  USB -Universal Serial Bus

Hardware Design
This three-electrode (working, counter, reference) potentiostat was designed to be compact, lowcost and easy to replicate and modify. We designed circuitry and PCB layout using free-version of electronics design software Eagle 9.6.2 (Autodesk Inc.). The two-layer PCB boards were fabricated using in-house engraving tool. However PCB designs can be easily submitted to any commercial PCB fabrication service (e.g. EuroCircuits, PCBWay and others.). We choose all components and made design such, that it would be readily replicated in Do-It-Yourself (DIY) manner without need for specialized or expensive equipment. All components can be ordered from common suppliers such as Mouser Inc. PCB assembly requires soldering iron with fine tip, basic soldering supplies, such as solder and flux and tweezers to handle small surface mounted (SMD) components. Figure S1 shows the main components of the potentiostat. Figure S2 shows the circuit diagram of the potentiostat. Figure S3 shows the PCB layout and gives description of connectors to electrodes and USB-to-Serial interface, which was integrated as an independent module, to minimize components to be soldered. Figure S4 shows a picture of the portable potentiostat.    Table S3 lists the details of components. Main components of the potentiostat are: i) microcontroller (MCU) (U4), ii) digital-to-analog converters (DACs) (U2, U5) to generate potential waveforms and iii) Quad (4x) operational amplifier (U3) for both controlling the electrode potential through feedback from the reference electrode and for trans-impedance amplifier to convert the working electrode current into voltage signal, which would be then digitalized by ADC inside of the MCU. For MCU we chose ATMEGA168 due to low-cost and compatibility with Arduino programming (Arduino Nano).

Firmware
Firmware was written using Arduino development environment (IDE). The firmware followed similar structure as used by us in an earlier publication about universal wireless electrochemical detector (UWED) [1], differently from UWED, which communicated with iPhone over Bluetooth, this potentiostat here was designed for computer connection via USB. USB communication was based on serial protocol, where we used USB-to-Serial interface to connect with USART port of the MCU. All commands and replies were based on ASCII text format making it easy to debug using serial console. Firmware implement simple functions to set electrode potential, perform accurately timed potential sweeps (required for CV and SWV The following Table S3 describes all commands available for the potentiostat communication.

Set number of steps in the sweep
This is total number of steps, which will be performed in the sweep, starting from initial potential set with "A" and changing in steps defined by "D" and "E" in intervals defined by

Computer software
The computer software was developed in Visual C# language in the development environment Microsoft Visual Studio 2017 using .NET framework. Software and source code is included in the SI of this article, as well in the GitHub repository [2]. This software combines simple graphical user interface and data acquisition. Interface allows to set linear calibration parameters for the potentiostat, one pair of parameters describing the potential control (mV/dig) and another the current measurement (µA/dig) side of the system. User can choose connection mode, it can be automatic, when potentiostat is the only serial port device connected or can be chosen manually in case multiple serial port device are connected. Once device is connected user can set potential and see the measured current value. There is possibility to perform three types of measurements, chronoamperometry (CA), cyclic voltammetry (CV) and square wave voltammetry (SWV), with sets of parameters defined for each of these methods. Some methods have input text box followed by the text field. In this case the text field shows the actual applied parameter, which is closest possible to the desired parameter. This is due to the digitalization, which do not allow all parameters to be chosen exactly, but only in certain steps. Eventually user can save the data in text files, which can be easily imported to another software for further analysis. Figure S5 shows the main view of the Potentiostat Control Application.

Characterization of electrical performance
We characterized the electrical performance of the potentiostat in two steps, first potential control and thereafter the current measurement side.

Potential control
For calibration and characterization of the potential control we connected the counter and reference electrode together and to the one input terminal of the digital multimeter, while working electrode was connected to the other terminal of the multimeter. Fluke 8808A (5-1/2 digit) multimeter was used for the calibration. Multimeter has in the 2V range resolution 10µV, input resistance >10GΩ and maximum measurement uncertainty 0.46mV, while in the 20V range resolution is 100µV, input resistance 10MΩs and maximum uncertainty 2.1mV. Based on the measurements we determined a linear potential calibration ( Figure S6) Electrode_Potential[V] =DAC_word*a+b with parameters described in Table S4. Figure S6. Potential calibration. Potential vs digital control word applied by potentiostat (left) and deviations from the linearity (right).  Table S5. Open circuit noise was measured, when resistor was removed and working electrode terminal was disconnected (zero current). Calibration and test results are visualized on Figure S7.