Quantification of Nanoparticles at the Single-cell Level: An Overview About State-of-the-art Techniques and Their Limitations

Dimitri Vanhecke; Laura Rodriguez-Lorenzo; Martin JD Clift; Fabian Blank; Alke Petri-Fink; Barbara Rothen-Rutishauser


Nanomedicine. 2014;9(12):1885-1900. 

In This Article

Chemical Analysis of Quantum Dots, Metal (Oxide) & Magnetic Nanoparticles by Inductively Coupled Plasma Techniques

Size and concentration detection limits and the complex and heterogeneous matrix represent the two major challenges when analyzing NPs in biological systems. Moreover, the discrimination between NPs and their respective dissolved chemical species and their subsequent quantitative separation is not straightforward and relies on the use of several, often coupled analytical methods. ICP analytical techniques are hard-ionization source elemental spectroscopies, which use a radio frequency-induced argon plasma with an average temperature of 5500 K. Samples are readily dried and vaporized, and the elements in the sample are also effectively atomized, ionized and thermally excited, and they can then be detected and quantified with either an emission spectrometer (OES) or a mass spectrometer (MS). Specifically, ICP-OES measures the light emitted at element-specific characteristic wavelengths from thermally excited analyte ions. By contrast, ICP-MS measures the masses of the elements ions generated by the high temperature argon plasma.

ICP-OES[4] and ICP-MS[5] detect the elements of NPs and have been used to determine low amounts of NPs in biological medium, inside cells and to evaluate their biodistribution.[13] Compared to microscopic techniques, both ICP-OES and ICP-MS have much higher throughput, which is particularly interesting for large screening studies, where whole NP libraries are investigated. Typically, ICP-OES systems have two to three orders of magnitude higher detection limits than ICP-MS.[14] In recent years, a large number of studies have been published relying on ICP techniques to detect and quantify the cellular uptake of a wide range of NPs, including metals (e.g., gold [Au], silver [Ag], copper [Cu]),[15–17] oxides, e.g., iron oxide [Fe2O3], zinc oxide [ZnO], titanium dioxide [TiO2]),[18–20] silica dioxide (SiO2),[21] and quantum dots (QDs) NPs[22] under in vitro and in vivo conditions.

Most reports are based on the chemical dissolution of the NPs, which requires profound knowledge of particle properties (e.g., size and state of dispersion in addition to concentration), surface characteristics (e.g., coating material),[21–23] and of the surrounding matrix (e.g., pH, ionic strength, presence of biomolecules such as proteins).[22,24,25] Traditionally, dissolution is accomplished by acidification of the suspension, at room temperature or elevated temperatures.[23] The selection of appropriate acids, however, is rather challenging because the NPs have to be transformed into their ionic forms while the organic matrix (e.g., tissue and cells) must be completely decomposed without any inclusion or adhesion of NPs.[4,5] Metal NPs are easily digested using aqua regia (mixture 1:3 of HNO3 and HCl). However, the analytical determination and characterization of some nanosized metal oxides and silica is not as straightforward as the comparatively 'easy-to-detect metallic' NPs. For example, TiO2 NPs are practically insoluble and therefore it becomes crucial to[4] use a mixture of concentrated hydrofluoric (HF), sulfuric (H2SO4), and/or other acids for dissolution, and[5] to select a suitable solvent for the biological material prior to ICP analysis.[24] Concentrated H2SO4 is regularly used for the dissolution of TiO2 NPs but should be avoided in combination with organic materials due to the possible generation of insoluble sulfides,[25] as the presence of these sulfides can cause adsorption of NPs, which results in poor experimental determinations.[26] Therefore, a mixture including hydrofluoric acid is preferred, however, stringent safety precautions apply.

Fabricius et al.[23] studied the importance of sample preparation of typical inorganic NP suspensions using ICP-MS. They showed that acidified suspensions in general delivered better recoveries than direct measurements; that is, using the undissolved suspensions. Allabashi et al. also clearly demonstrated that the sample matrix is the main factor influencing accuracy and precision.[27] Working with colloidal gold, they show that a preliminary digestion of the gold suspension with aqua regia, is not always necessary. A more recent development is the introduction of single particle ICP-MS, which provides a means of detecting and sizing individual NPs.[28,29] One major challenge remains the differentiation between NPs and their dissolved species. However, NP dissolution can be an important determinant, for example, of NP cytotoxicity. The combinations of separation and quantification techniques are crucial in this context. For example, ultrafiltration coupled with ICP-OES has been widely used to separate and quantify the dissolved ions (e.g., Ag+, Zn2+) from NPs taken up by cells.[18,30] Field flow fractionation (FFF), which is a size-based separation technique applicable to biomolecules, colloids, and bacteria in solution, when interfaced with ICP-MS online is an increasingly popular combination, so that it allows simultaneous particle sizing, characterization of NP interaction with soluble species and analysis in the NPs' original environment.[31]

In addition to the high sensitivity of the method, it is also possible to quantify the cellular internalization of NPs on the subcellular level using ICP-OES as previously shown with HeLa cells and gold NPs (AuNPs) functionalized with the SV40 large T-antigen.[32] The authors showed that ICP-OES could be used to detect NPs inside fractionated cell nuclei, consistent with qualitative investigations of nuclear localization by video-enhanced color differential interference contrast microscopy, demonstrating that this technique can be a valuable tool to add quantitative information to optical and electron imaging methods regarding the intracellular fate of NPs. Another combination approach was proposed by Niehoff et al.[33] who used a combination of laser ablation (LA) and ICP-MS to visualize the distribution of fluorescently encoded NPs in biological tissues. This approach is characterized by a high sensitivity and very low detection limit delivering the best results for electropositive elements (i.e., metal elements), which are characterized by a low natural abundance.[34]