This paper describes a new biosensor prototype that uses microscale electrodes to measure impedance, pH, and temperature changes caused by bacterial growth <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in-vitro</i> . The prototype uses multiple sensor geometries to optimize sensitivity, and employs custom circuits including an integrated CMOS lock-in amplifier (LIA) for multi-frequency impedance measurement. The gold-plated interdigitated electrodes (AuIDEs), iridium oxide (IrO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> )-based pH electrodes, and snake-shaped gold resistance-temperature detectors (RTDs) electrodes are implemented on a flexible PCB. The custom integrated LIA is designed and fabricated in a 0.18-μm CMOS technology with 1.8 V supply voltage. The overall sensitivity of the LIA is 240 mV/nA with a current detection sensitivity down to 1 pA. A pH measuring circuit is designed using an operational amplifier with a very low input-bias-current. A Wheatstone bridge circuit with a network of resistors and RTD electrode is used to measure RTD resistance changes caused by temperature fluctuations. The whole system is enclosed into a self-contained platform developed to monitor bacterial growth by measuring the impedance as well as the environmental growth parameters such as pH and temperature. The fabricated multi-frequency LIA was employed to test the impedance of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">E. coli</i> cells with different concentrations at multiple frequencies. The results showed that the IDE can measure the impedance variation caused by the bacterial activity with sensitivities of 36.62%, 34.37%, 33.29% and 20.23% at 1, 2, 4, and 10 kHz frequency, respectively. The fabricated pH electrodes showed linearity with a linear regression (R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> ) value of 0.99 and can measure pH changes ranging from 4 to 10, with a sensitivity of 36, 52, and 68 mV/pH for sensing areas of 4 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> , 9 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> , and 16 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> , respectively. The temperature sensor measurements showed that the 4 mm × 4 mm RTD gold-printed temperature electrodes had a linear relationship between resistance and temperature with a R <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> value of 0.996, and the higher sensitivity of 36 (Ω/°C) compared to other tested RTD geometries. The prototype was successfully used to perform bacterial growth measurements <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">in-vitro</i> .
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