Design Optimization of a Submersible Chemiluminescent Sensor (DISCO) for Improved Quantification of Reactive Oxygen Species (ROS) in Surface Waters

Abstract

Reactive oxygen species (ROS) are key drivers of biogeochemical cycling while also exhibiting both positive and negative effects on marine ecosystem health. However, quantification of the ROS superoxide (O₂^-) within environmental systems is hindered by its short half-life. Recently, the development of the diver-operated submersible chemiluminescent sensor (DISCO), a submersible, handheld instrument, enabled in situ superoxide measurements in real time within shallow coral reef ecosystems. Here, we present a redesigned and improved instrument, DISCO II. Similar to the previous DISCO, DISCO II is a self-contained, submersible sensor, deployable to 30 m depth and capable of measuring reactive intermediate species in real time. DISCO II is smaller, lighter, lower cost, and more robust than its predecessor. Laboratory validation of DISCO II demonstrated an average limit of detection in natural seawater of 133.1 pM and a percent variance of 0.7%, with stable photo multiplier tube (PMT) counts, internal temperature, and flow rates. DISCO II can also be optimized for diverse environmental conditions by adjustment of the PMT supply voltage and integration time. Field tests showed no drift in the data with a percent variance of 3.0%. Wand tip adaptations allow for in situ calibrations and decay rates of superoxide using a chemical source of superoxide (SOTS-1). Overall, DISCO II is a versatile, user-friendly sensor that enables measurements in diverse environments, thereby improving our understanding of the cycling of reactive intermediates, such as ROS, across various marine ecosystems.

Keywords: chemiluminescent; corals; in situ analysis; ocean sensor; reactive oxygen species; superoxide.

Figure 1

Diagram of DISCO II showing the (A) front and (B) back of the instrument alongside photos of the (C) front and (D) back. Numbers correspond with Table 1 and point to essential components in DISCO II.

Figure 2

Block diagram of DISCO II showing the layout of DISCO and highlighting the function of the key features. The different operating systems are color coded, showing distinction between the fluidics system (blue), electronics system (green), the oil-compensated section (orange), and the tablet and PMT (gray). The other two compartments (pressure-bearing and flooded) are labeled accordingly. The blue arrows associated with the fluidics system indicate the direction of flow.

Figure 3

Raw DISCO II data during extended measurements to test for stability and drift within the instrument both in the (A) laboratory (~30 min) and (B) in situ near a shallow reef (~60 min), PMT settings are default voltage, IT 500ms. Graphs display the PMT counts (left y-axis, black large circles), effluent flow rate (ml min⁻¹, right y-axis, grey small circles labeled “Flow Out”), effluent temperature (°C * 0.1, right y-axis, grey small dashes labeled “Temp Out”), and for graph A only, analyte temperature (°C * 0.1, right y-axis, grey small dashes for line on bottom labeled “Temp In”) throughout the time of day (x-axis). The time when the SOD pump is turned on is labeled and highlighted in a gray vertical bar.

Figure 4

Results from multiple laboratory tests with DISCO, showing the (A) calibration factor (y-axis, pM count⁻¹) and (B) AFSW raw counts (y-axis) across integration time (x-axis, ms) for different voltages (V): 800 (right y-axis, green), 1200 (left y-axis, orange), and default (left y-axis, blue). The box and whisker plot indicates the middle 50 percentile (box), the median (horizontal line), the middle 90 percentile (vertical lines), and the outliers (“o”).

Figure 5

Results from multiple laboratory tests using SOTS-1 across time (x-axis, hours since SOTS-1 was mixed), showing (A) background seawater (BGSW)-normalized raw counts of SOTS-1 and (B) BGSW-normalized concentration of SOTS-1 (pM). Tests were completed using default voltage and different integration times: 100 ms (green), 500 ms (orange), and 900 ms (blue). Points represent the average signal and error bars represent the standard deviation.