Lab-On-A-Chip for Chlorophyll Analysis and Identification of Phytoplankton Taxonomic Groups
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Lab-on-a-chip for chlorophyll analysis and identification of phytoplankton taxonomic groups Denise A. M. Carvalho1, Vânia C. Pinto1, Paulo J. Sousa1, Emilio Fernández2, Luís M. Gonçalves1, Graça Minas1 1 MEMS-UMinho Research Unit, DEI, University of Minho, Campus de Azurém, 4800-058 Guimarães, Portugal 2 Grupo de Oceanografía Biolóxica, Faculty of Marine Science, 36310 Universidade de Vigo, Spain Introduction This work presents the optical properties (absorbance, dispersion and fluorescence) of several phytoplankton species, towards the development of a portable and low-cost lab-on-a-chip able to quantify and identify a number of phytoplankton taxonomic groups. The methodology to select the excitation and detection wavelengths that promotes better identification of phytoplankton is also presented. A proof-of-concept device was fabricated, with LED light excitation at 450 nm and detected at 680 nm by a photodiode, for quantifying the concentration of phytoplankton chlorophyll. A lock-in amplifier was developed and integrated in a portable and low-cost circuit sensor, featuring continuous, autonomous and in-situ underwater measurements. This device has a detection limit of 0.01 µ/L of chlorophyll, in a range above 300 µg/L, with a linear response. Materials and methods Single cell analysis • Six laboratory cultures of non-toxic phytoplankton, representing five Figure shows the results of the size versus complexity obtained with the divisions, were selected for this study. flow cytometer for each of the selected phytoplankton species. The forward • The optical properties of the selected species were obtained with scatter (FS in y-axis of figure) represents the size of cell and the side commercial equipment, namely absorption, fluorescence, and single-cell scatter (SS in y-axis) the regards the granularity and complexity of the cell. flow cytometry. Species Family Order Class Division Flow cell Nannochloropsis Monodopsidaceae Eustigmatales Eustigmatophyceae Ochrophyta gaditana Laser Isochrysis 488nm Isochrysidaceae Isochrysidales Coccolithophyceae Haptophyta Dichroic mirrors galbana Tetraselmis Lens Chlorodendraceae Chlorodendrales Chlorodendrophyceae Chlorophyta Forward scatter 488 DL suecica 488 BK 550 DL Rhodomonas 600 DL Pyrenomonadaceae Pyrenomonadales Cryptophyceae Cryptophyta 645 DL lens 700 DL Skeletonema Skeletonemataceae Thalassiosirales Mediophyceae Bacillariophyta (a) (b) (c) costatum Emiliania Noelaerhabdaceae Isochrysidales Coccolithophyceae Haptophyta huxleyi Alexandrium Pyrophacaceae Gonyaulacales Dinophyceae Miozoa tamarense 1 Phytoplankton species studied. Filter configuration used in the commercial flow cytometer. Results (f) Characterization of microalgae optical properties (d) (e) Absorption and dispersion spectra Size as a function of complexity for each of the phytoplankton species, obtained by flow cytometry: (a) Nannochloropsis gaditana; (b) Isochrysis galbana; (c) Tetraselmis suecica; (d) Rhodomonas lens; Chlorophyll was extracted and the absorbance spectrum from each of the (e) Skeletonema costatum; (f) Emiliania huxleyi. phytoplankton species was studied. Absorbance is higher at two wavelengths, 430 nm and 660 nm. In order to compare absorbance Development and fabrication of the Lab-on-a-chip spectra, the results were normalized at 430 nm (absorbance spectrum of Using the spectral characteristics of each species used in this study, a lab- all species to the same value at this wavelength). Figure shows the on-a-chip for phytoplankton group’s identification was designed. The normalized results. schematic representation of the lab-on-a-chip with integrated fluorescence and scattering detection is showed in figure. Relative absorbance spectrum of each studied phytoplankton species. All curves were normalized to the corresponding absorbance at 430 nm. Excitation / fluorescence spectrum matrix Schematic illustration of the lab-on-a-chip for phytoplankton group’s Figure shows the obtained fluorescence spectrum (emission), for each identification and quantification based on fluorescence detection. excitation wavelength, for three of the presented phytoplankton species, namely Nannochloropsis gaditana (A), Isochrysis galbana (B) and The device performance was assessed, considering the limit of detection, Tetraselmis suecica (C). Fluorescence values where normalized, measurement range and linearity. Thus, the device`s response was initially considering the maximum value measured in the spectrum of each studied calibrated using chlorophyll-a standard solutions with known species. For the toxic species (Alexandrium tamarense), data reported in concentrations in the range of 0.01-300 µg/L. The sensor shows a linear 2 [1] were used. response (R =0.9963) with a limit of detection of 0.01 µg/L using chlorophyll-a standards. (A) (B) (C) (a) Fluorescence spectrum of Nannochloropsis gaditana, with excitation light scanned from 320 nm to 700 nm; (b) Fluorescence spectrum of Isochrysis galbana, with excitation light scanned from 320 nm to 700 nm; (c) Fluorescence spectrum of Tetraselmis suecica, with excitation light scanned from 320 nm to 700 nm. Calibration curve of the chlorophyll sensor using standard solutions. Conclusions • The developed device was able to estimate phytoplankton biomass from in-vivo chlorophyll a measurements, with concentrations ranging from 0 to 300 µg/L, with a detection limit of 0.01 µg/L for extracted chlorophyll and 0.05 µg/L for in-vivo measurements. • This device shows a high potential for oceanographic research, since it allows in-vivo analysis with less time and work without need of complex extraction procedures. References Acknowledgments [1] F. Zhang, J. He, R. Su, and X. Wang, “Assessing Phytoplankton Using a Two- This work was co-financed by the European Regional Development Fund (ERDF) through the Rank Database Based on Excitation- Emission Fluorescence Spectra,” Appl. Interreg V-A Spain-Portugal Programme (POCTEP) 2014-2020, Project N. 0591_FOODSENS_1_E. Spectrosc., vol. 65, no. 1, pp. 1–9, 2011, doi: 10.1366/10-05927. Denise Carvalho want to thanks NORTE 2020 for the BD/ Do*Mar/1017/2016 grant..