A versatile quantitative microdroplet elemental imaging method optimised for integration in biochemical workflows for low-volume samples
Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis of μ-droplets is becoming an attractive alternative for detecting and quantifying elements in biological samples. With minimal sample preparation required and detection limits comparable to solution nebulisation ICP-MS, μ-droplets have substantial advantages over traditional elemental detection, particularly for low volumes, such as aliquots taken from samples required for multiple independent biochemical assays, or fluids and tissues where elements of interest exist at native concentrations not suited to the necessary dilution steps required for solution nebulisation ICP-MS. However, the characteristics of μ-droplet residue deposition are heavily dependent on the matrix, and potential effects on signal suppression or enhancement have not been fully characterised. We present a validated and flexible high-throughput method for quantification of elements in μ-droplets using LA-ICP-MS imaging and matrix-matched external calibrants. Imaging the entire μ-droplet area removes analytical uncertainty arising from the often-heterogenous distribution when compared to radial or bisecting line scans that capture only a small portion of the droplet residue. We examined the effects of common matrices found in a standard biochemistry workflow, including native protein and salt contents, as well as reagents used in typical preparation steps for concurrent biochemical assays, such as total protein quantification and enzyme activity assays. We found that matrix composition results in systemic, concentration-dependent signal enhancement and suppression for carbon, whereas high sodium content has a specific space-charge-like suppression effect on high masses. We confirmed the accuracy of our method using both a certified serum standard (Seronorm™ L1) and independent measurements of analysed samples by solution nebulisation ICP-MS, then tested the specificity and reproducibility by examining spinal cord tissue homogenates from SOD1-G93A transgenic mice with a known molecular phenotype of increased copper- and zinc-binding superoxide dismutase-1 expression and altered copper-to-zinc stoichiometry. The method presented is rapid and transferable to multiple other biological matrices and allows high-throughput analysis of low-volume samples with sensitivity comparable to standard solution nebulisation ICP-MS protocols.
KeywordsBioanalytical methods Biological samples Laser ablation Mass spectrometry/ICP-MS Trace elements Metals/heavy metals
KK was a recipient of the Sigrid Juselius Foundation Postdoctoral Fellowship (Finland). DJH is a NHMRC Career Development Fellow (CDF1) - Industry (1122981) with Agilent Technologies. PJC is an NHMRC Career Development Fellow (CDF2 - 1084927). We wish to the Prize4Life Organization for providing experimental transgenic mice. The Florey Institute of Neuroscience and Mental Health acknowledge the strong support from the Victorian Government and the funding from the Operational Infrastructure Support Grant.
Peter J Crouch and Kai Kysenius designed the project, prepared the samples, and edited the manuscript. Kai Kysenius performed the analyses, prepared the figures, and wrote the manuscript. James B Hilton and Jeffrey R Liddell prepared the samples and edited the manuscript. Dominic J Hare performed the SN-ICP-MS analysis and edited the manuscript. Bence Paul wrote the Iolite analysis code and edited the manuscript.
This study received support from the Jenny Barr Smith, Betty Laidlaw, and Jenny Simko Research Grants from the MND Research Institute of Australia.
Compliance with ethical standards
The use of biological material taken from wild-type C57/B6 and transgenic mice was approved by The University of Melbourne Animal Ethics Committee (approval number 1312908). All procedures were conducted in accordance with National Health and Medical Research Council guidelines.
Conflict of interest statements
Bence Paul receives part of his salary from the sales of the Iolite software. Dominic J Hare receives research and material support from Agilent Technologies through the National Health and Medical Research Career Development Fellowship program. Procypra Therapeutics LLC, Collaborative Medicinal Development LLC, and The University of Melbourne are engaged in development of new therapeutics for ALS using the transgenic animal described herein. The other authors declare that they have no conflict of interest.
- 9.Zhu G, Browner FR. Investigation of experimental parameters with a quadrupole ICP/MS. Appl Spectrosc. 1987;41Google Scholar
- 11.Dyer PE, Karnakis DM, Key PH, Monk P. Excimer laser ablation for micro-machining: geometric effects. In: Fogarassy E, Geohegan D, Stuke M, editors. Laser ablation. Elsevier; 1996.Google Scholar
- 12.Hare DJ, Fryer F, Paul B, Bishop DP, Doble PA. Characterisation of matrix-based polyatomic interference formation in laser ablation-inductively coupled plasma-mass spectrometry using dried micro-droplet ablation and its relevance for bioimaging. Anal Methods. 2016;8:7552–6. https://doi.org/10.1039/C6AY02545E.CrossRefGoogle Scholar
- 16.Nischkauer W, Vanhaecke F, Limbeck A. Self-aliquoting micro-grooves in combination with laser ablation-ICP-mass spectrometry for the analysis of challenging liquids: quantification of lead in whole blood. Anal Bioanal Chem. 2016;408:5671–6. https://doi.org/10.1007/s00216-016-9717-3.CrossRefGoogle Scholar
- 19.Aramendia M, Rello L, Berail S, Donard A, Pecheyran C, Resano M. Direct analysis of dried blood spots by femtosecond-laser ablation-inductively coupled plasma-mass spectrometry. Feasibility of split-flow laser ablation for simultaneous trace element and isotopic analysis (vol 30, pg 296, 2015). J Anal At Spectrom. 2015;30:525. https://doi.org/10.1039/c4ja90069c.CrossRefGoogle Scholar
- 20.Chantada-Vazquez MP, de Becerra-Sanchez C, Fernandez-del-Rio A, Sanchez-Gonzalez J, Bermejo AM, Bermejo-Barrera P, et al. Development and application of molecularly imprinted polymer - Mn-doped ZnS quantum dot fluorescent optosensing for cocaine screening in oral fluid and serum. Talanta. 2018;181:232–8. https://doi.org/10.1016/j.talanta.2018.01.017.CrossRefGoogle Scholar
- 22.Chantada-Vázquez M, Moreda-Piñeiro J, Cantarero-Roldán A, Bermejo-Barrera P, Moreda-Piñeiro A. Development of dried serum spot sampling techniques for the assessment of trace elements in serum samples by LA-ICP-MS. Talanta. 2018;186:169–175. doi: https://doi.org/10.1016/j.talanta.2018.04.049.
- 24.Pugh JAT, Cox AG, McLeod CW, Bunch J, Whitby B, Gordon B, et al. A novel calibration strategy for analysis and imaging of biological thin sections by laser ablation inductively coupled plasma mass spectrometry. J Anal At Spectrom. 2011;26:1667–73. https://doi.org/10.1039/C1JA10118H.CrossRefGoogle Scholar
- 28.Paul B, Hare DJ, Bishop DP, Paton C, Nguyen VT, Cole N, et al. Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging (vol 6, pg 5383, 2015). Chem Sci. 2016;7:6576. https://doi.org/10.1039/c6sc90060g.CrossRefGoogle Scholar
- 32.Nischkauer W, Vanhaecke F, Bernacchi S, Herwig C, Limbeck A. Radial line-scans as representative sampling strategy in dried-droplet laser ablation of liquid samples deposited on pre-cut filter paper disks. Spectrochim Acta Part B At Spectrosc. 2014;101:123–9. https://doi.org/10.1016/j.sab.2014.07.023.CrossRefGoogle Scholar
- 33.Hilton JB, Mercer SW, Lim NK, Faux NG, Buncic G, Beckman JS, et al. Cu(II)(atsm) improves the neurological phenotype and survival of SOD1(G93A) mice and selectively increases enzymatically active SOD1 in the spinal cord. Sci Rep. 2017;7:42292. https://doi.org/10.1038/srep42292.CrossRefGoogle Scholar
- 38.Hare DJ, Kysenius K, Paul B, Knauer B, Hutchinson RW, O’Connor C, et al. Imaging metals in brain tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS). J Vis Exp. 2017; https://doi.org/10.3791/55042.
- 41.Nischwitz V, Berthele A, Michalke B. Speciation analysis of selected metals and determination of their total contents in paired serum and cerebrospinal fluid samples: an approach to investigate the permeability of the human blood-cerebrospinal fluid-barrier. Anal Chim Acta. 2008;627:258–69. https://doi.org/10.1016/j.aca.2008.08.018.CrossRefGoogle Scholar
- 44.Christensen R. Log-linear models and logistic regression. 2nd ed. Springer-Verlag; 2006.Google Scholar
- 46.May TW, Wiedmeyer RH. A table of polyatomic interferences in ICP-MS. At Spectrosc. 1998;19:150–5.Google Scholar
- 54.ISO 5725-1: Accuracy (trueness and precision) of measurement methods and results — Part 1: General principles and definitions. Geneva 1994.Google Scholar
- 56.Gurney ME. The use of transgenic mouse models of amyotrophic lateral sclerosis in preclinical drug studies. J Neurol Sci. 1997;152 https://doi.org/10.1016/S0022-510X(97)00247-5.
- 58.Roberts BR, Lim NK, McAllum EJ, Donnelly PS, Hare DJ, Doble PA, et al. Oral treatment with CuII(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis. J Neurosci. 2014;34:8021–31. https://doi.org/10.1523/JNEUROSCI.4196-13.2014.CrossRefGoogle Scholar
- 62.Cardoso B, Hare DJ, Bush A, Li Q-X, Fowler C, Masters C et al. Selenium levels in serum, red blood cells, and cerebrospinal fluid of Alzheimer’s disease patients: a report from the Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL). J Alzheimers Dis. 2017;Preprint. doi: https://doi.org/10.3233/JAD-160622.
- 65.Theiner S, Malderen SJM, Acker T, Legin AA, Keppler BK, Vanhaecke F, et al. Fast high-resolution LA-ICP-MS imaging of the distribution of platinum-based anti-cancer compounds in multicellular tumor spheroids. Anal Chem. 2017;89:12641–5. https://doi.org/10.1021/acs.analchem.7b02681.CrossRefGoogle Scholar
- 66.Malderen SJM, Laforce B, Acker T, Nys C, Rijcke M, de Rycke R, et al. Three-dimensional reconstruction of the tissue-specific multielemental distribution within Ceriodaphnia dubia via multimodal registration using laser ablation ICP-mass spectrometry and X-ray spectroscopic techniques. Anal Chem. 2017;89:4161–8. https://doi.org/10.1021/acs.analchem.7b00111.CrossRefGoogle Scholar