Determination and localization of specific proteins in individual ARPE-19 cells by single cell and laser ablation ICP-MS using iridium nanoclusters as label

Menero-Valdés, Paula; Lores-Padín, Ana; Fernández, Beatriz; Quarles, C. Derrick; García, Montserrat; González‐Iglesias, Héctor; Pereiro, Rosario

doi:10.1016/j.talanta.2022.123974

Cited by 8 publications

(9 citation statements)

References 43 publications

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“…This fact can be attributed to the presence of free MNC-labeled immunoprobes not bound to the proteins. In previous works using quadrupole mass analyzers, such free immunoprobes were successfully discriminated by applying a Poisson threshold or a 5σ threshold. , However, such strategies do not fit the ToF data, and an alternative strategy was proposed to discriminate cell events: only events where Ru was simultaneously detected with at least one of the labels from MNC-labeled immunoprobes were considered.…”

Section: Resultsmentioning

confidence: 99%

“…For the analysis of biomolecules by sc-ICP-MS using metal-labeled antibodies, it would be very convenient to monitor both the elemental label and a cell intrinsic element (e.g., Ca, Cu, Fe, P, etc.) to confirm the integrity of the cells as well as the proper Ab recognition. ,− However, the mass difference between the common endogenous cell elements (low m / z range) typically at very low levels and the metals employed for the labels (e.g., noble metals or lanthanides in a high m / z range) can be a limitation to simultaneously detecting all of them with high sensitivity by sc-ICP-ToF-MS. In our experiments, the simultaneous measurement of an intrinsic element together with the sensitive detection of Ir, Pt, and Au from the MNC-labeled immunoprobes was attempted with the ToF-MS analyzer to obtain a fingerprint of each individual cell.…”

Section: Resultsmentioning

confidence: 99%

“…Fixated cell suspensions were subjected to an immunoassay to simultaneously label the three proteins of interest with the MNC-labeled immunoprobes. The immunoassay protocols used to label HP, MT2, and FPN in ARPE-19 cells were optimized, following the procedure proposed in previous works, , in terms of immunoprobe concentration (referring to the Ab concentration) to ensure the total recognition of the proteins and the number of washing steps to avoid nonspecific interactions. The protocols were independently performed with the three immunoprobes (Anti-h-HP:IrNCs, Anti-h-MT2:PtNCs, or Anti-h-FPN:AuNCs), and optimized Ab concentrations were found as follows: 4 μg mL –1 , 10 μg mL –1 , and 4 μg mL –1 , respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The heterogeneous nature of cells implies that cells of the same line may differ in the levels of their metal and biomolecule expression by up to 2 or 3 orders of magnitude . It has also been reported that such significant cell-to-cell variations may be the origin of several pathologies .…”

Section: Introductionmentioning

confidence: 99%

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Single Cell–ICP–ToF-MS for the Multiplexed Determination of Proteins: Evaluation of the Cellular Stress Response

et al. 2023

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An automated and straightforward detection and data treatment strategy for the determination of the protein relative concentration in individual human cells by single cell−inductively coupled plasma−time-of-flight mass spectrometry (sc-ICP-ToF-MS) is proposed. Metal nanocluster (NC)-labeled specific antibodies for the target proteins were employed, and ruthenium red (RR) staining, which binds to the cells surface, was used to determine the number of cell events as well as to evaluate the relative volume of the cells. As a proof of concept, the expression of hepcidin, metallothionein-2, and ferroportin employing specific antibodies labeled with IrNCs, PtNCs, and AuNCs, respectively, was investigated by sc-ICP-ToF-MS in human ARPE-19 cells. Taking into account that ARPE-19 cells are spherical in suspension and RR binds to the surface of the cells, the Ru intensity was related to the cell volume (i.e., the cell volume is directly proportional to (Ru intensity) 3/2 ), making it possible to determine not only the mass of the target proteins in each individual cell but also the relative concentration. The proposed approach is of particular interest in comparing cell cultures subjected to different supplementations. ARPE-19 cell cultures under two stress conditions were compared: a hyperglycemic model and an oxidative stress model. The comparison of the control with treated cells shows not only the mass of analyzed species but also the relative changes in the cell volume and concentration of target proteins, clearly allowing the identification of subpopulations under the respective treatment.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single Cell–ICP–ToF-MS for the Multiplexed Determination of Proteins: Evaluation of the Cellular Stress Response

et al. 2023

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“…One report employed scICP-MS and LA-ICP-MS as complementary techniques to study intracellular content and distribution of two proteins, Apo E and claudin-1, in individual cells from an in vitro cell model, ARPE-19. 106 The highlights of the work were the achievement of a high transport efficiency for the ARPE-19 cells of 85 ± 9%, by way of a microFAST single cell sample introduction system, and use of Ir-nanoclusters, which contain a large number of Ir atoms and therefore act as an amplification system to increase sensitivity for both the scICP-MS and LA-ICP-MS analysis. The LODs achieved were 0.02 fg per cell for Apo E and 3 ag per cell for claudin 1.…”

Section: Progress With Analytical Techniquesmentioning

confidence: 99%

Atomic spectrometry update: review of advances in the analysis of clinical and biological materials, foods and beverages

Patriarca,

Barlow,

Cross

et al. 2024

J. Anal. At. Spectrom.

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Palladium nanoclusters as a label to determine GFAP in human serum from donors with stroke by bimodal detection: inductively coupled plasma-mass spectrometry and linear sweep voltammetry

Rodríguez-Penedo,

Costa-Rama,

Fernández

et al. 2023

Microchim Acta

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Water-soluble, stable, and monodisperse palladium nanoclusters (PdNCs) were synthesized using NaBH4 as a reductant and lipoic acid as a ligand. PdNCs, measured by high-resolution transmission electron microscopy, showed a round shape and a diameter of 2.49 ± 0.02 nm. It was found that each PdNC contains 550 Pd atoms on average. These PdNCs offer high amplification as a label of biochemical reactions when inductively coupled plasma-mass spectrometry (ICP-MS) is used as a detector. In addition, PdNCs have catalytic activity on electrochemical reactions, allowing detection by linear sweep voltammetry (LSV). As a proof of applicability, a competitive immunoassay based on PdNC labels was developed for the determination of glial fibrillary acidic protein (GFAP) in human serum, comparing ICP-MS and LSV detection. GFAP is a biomarker for differentiating between patients with ischemic stroke (IS) and hemorrhagic stroke (HS). The limit of detection (LoD), corresponding to IC10 (4-parameter logistic curve), was 0.03 pM of GFAP, both by ICP-MS and LSV, being lower than the 0.31 pM LoD provided by the ELISA commercial kit. Using the error profile method, 0.03 pM and 0.11 pM LoDs were obtained respectively by ICP-MS and LSV: LoD is lower by ICP-MS due to the better precision of the measurements. The analyses of human serum samples from IS, HS, and control (CT) donors using PdNC labels and detection by ICP-MS and LSV were validated with a commercial ELISA kit (for CT donors only ICP-MS provided enough sensitivity). Results point out toward the future use of PdNCs as a label in other immunoprobes for the determination of specific proteins requiring very low LoDs as well as the development of electrochemical decentralized methodologies. Graphical Abstract

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Determination and localization of specific proteins in individual ARPE-19 cells by single cell and laser ablation ICP-MS using iridium nanoclusters as label

Cited by 8 publications

References 43 publications

Single Cell–ICP–ToF-MS for the Multiplexed Determination of Proteins: Evaluation of the Cellular Stress Response

Single Cell–ICP–ToF-MS for the Multiplexed Determination of Proteins: Evaluation of the Cellular Stress Response

Atomic spectrometry update: review of advances in the analysis of clinical and biological materials, foods and beverages

Palladium nanoclusters as a label to determine GFAP in human serum from donors with stroke by bimodal detection: inductively coupled plasma-mass spectrometry and linear sweep voltammetry

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