Nanoparticle-Assisted Analytical Strategies: Pushing the Limits of ICP-MS for Ultrasensitive Detection of Clinical Biomarkers

Clearly, the awesome success of medical science in the development of personalized medicine has occurred because of many achievements, such as the discovery, characterization, identification, and quantification of novel biomarkers for improved patient care. Advances in mass spectrometry (MS) technology, along with high-resolution liquid-phase biomolecule separations and novel bioinformatics tools for large-scale data analysis, have been essential to this progress. At present, one of the major goals in developing clinically effective novel bioanalytical methods for quantifying disease biomarkers is achieving high sensitivity, which is critical for early diagnosis and patient survival.

In this context, the bioanalytical community has made tremendous advances to satisfy the demand for reliable, cost-effective, robust, and powerful detection and monitoring strategies for disease biomarkers based on MS technology. Recent advances in liquid chromatography coupled online to tandem mass spectrometry detection techniques for clinical biomarker discovery and detection have offered much hope for superior patient care, particularly for cancer diagnosis and treatment.¹ However, the complexity, variation, and dynamic range of the biomolecules present in biological samples, along with the extremely low concentration levels that often need to be quantified, are among the major obstacles to using these methods to accurately quantify the biomarker levels. Additionally, molecular MS techniques, which are the most widely used in proteomic studies, use soft ionization sources to preserve molecular information, but these ion sources are not inherently quantitative. In molecular MS, the ion signal intensity is strongly affected not only by the molecular structure of the target biomolecule but also by the sample matrix and solvents used.

As an alternative, more than a decade ago, inductively coupled plasma–mass spectrometry (ICP-MS) was established as a quantitative tool in proteomics (and in biomarker quantification), complementary to soft-ionization MS techniques. ICP-MS offers detection limits in the attomolar range, regardless of the molecular environment of the target element, with a broad linear dynamic range of several orders of magnitude, and simultaneous multi-isotopic analysis is routinely possible. Sensitivity, which is of great importance in bioanalysis, is affected very little by the sample matrix or the chromatographic mobile phase in ICP-MS. These advantages explain the key role that ICP-MS currently plays in biomarker quantification by detecting an elemental label linked to the biomolecule of interest. For that purpose, a commonly used strategy is biomarker recognition with appropriate antibodies tagged with an ICP-MS–detectable element. Additionally, combining differentially elemental-labeled antibodies with ICP-MS detection could allow the design of multiplexed biomarker analytical methods. These special features of modern ICP-MS make it possible to envisage myriad biomedical applications of the technique and guarantee a bright future for this tool in biomarker quantification. In fact, a large variety of ICP-MS-based strategies for biomarker quantification have already been developed over the past two decades.² Moreover, novel ICP-MS/MS instruments could play a critical role in the future use of ICP-MS for proteomics and biomarker quantification. The high selectivity offered by the MS/MS configuration significantly reduced chemical noise and background, enabling virtually interference-free, highly sensitive detection of non-metal atoms naturally present in biomolecules, such as P and S, which can be used as endogenous elemental tags for quantifying biomolecules by ICP-MS.

Biomarkers are typically present at ultralow concentration levels during the early stages of disease progression. Therefore, development of highly sensitive quantification tools is a must for clinical decision-making, allowing not only prompt clinical diagnosis but also making it possible to efficiently evaluate new therapies and to assess the prognosis of disease. The achievement of ultrahigh sensitivity requires the development of innovative approaches that combine different amplification platforms and processes. In this vein, nanotechnology, combined with elemental mass spectrometry, is today a powerful strategy that offers unique opportunities for creating ultrasensitive bioassays.

A simple approach to introduce a high number of ICP-MS–detectable elements per biomarker, thereby amplifying the analytical signal, is to use metal-containing nanoparticles (NPs) as biomarker antibody labels. Of course, such nanoparticle-tagged antibody approaches are more laborious and time-consuming than direct heteroatom-tagged proteomics strategies, and, worse still, they may alter protein properties (for example, by modifying selected antibody recognition capabilities). However, inorganic nanoparticles contain a high number of ICP-MS–detectable atoms ranging from hundreds to tens of thousands of atoms in each nanoparticle. Thus, strong signal amplification could be achieved by detecting the metal atoms labeled on one target, obtaining very low limits of detection.^3,4 Moreover, it is possible to use different inorganic nanoparticles, containing different elements, for labeling different antibodies, enabling multiplexed analysis of many biomarkers in a clinical sample with very high sensitivity and selectivity.

Unfortunately, detection limits achieved with inorganic nanoparticle-tagged immunoassays followed by ICP-MS are still insufficient to detect disease biomarkers at the extremely low levels present in clinical samples during the early stages of disease, necessitating the development of new signal amplification techniques for ultrasensitive detection. For cases where ultrahigh sensitivity is required, novel approaches have recently been reported, based on different amplification processes. One possibility to improve the sensitivity of ICP-MS detection could be the use of larger inorganic particles (microparticles). However, such an approach is not feasible because when larger particles are used, the number of antibodies per inorganic particle increases, and as a result, the signal-per-molecule is lower.

A drastic amplification of the ICP-MS signal is possible by resorting to a controlled immuno-nanoparticle-gold amplification.⁵ Here, ICP-MS signal amplification was achieved for prostate-specific antigen (PSA) quantification by the precipitation of gold ions on the surface of Mn-doped ZnS quantum dots used as tags of a PSA antibody. The nanoparticle tags act as catalytic seeds to promote the reduction of gold ions to metallic gold, which is deposited on the surface of the nanoparticles (NP size amplification) after the immunoassay and before tag detection. Much bigger nanoparticles are thus obtained, and hence, much better sensitivity with ICP-MS detection can be achieved (detection limits below fg/mL levels of the PSA biomarker were reported). In contrast with direct use of big particles (microparticles), this approach does not affect the recognition capabilities of antibodies, because size amplification is carried out once immunorecognition has taken place. In addition, the method showed a very wide dynamic range exceeding eight orders of magnitude, which means that the method could be applied to the determination of biomarkers at many different concentration levels. 

Another approach for the improvement of ICP-MS sensitivity is nanoparticle analysis by single particle ICP-MS (spICP-MS). In spICP-MS, time-resolved transient signals induced by the flash of ions generated by the ionization of the inorganic nanoparticle upon entering the plasma can reflect the concentrations of the NPs. Thus, sp-ICP-MS enables quantification of nanoparticle-tagged immunocomplexes by measuring the frequency of transient ion signals. Based on this principle, single-nanoparticle analysis offers great potential for developing extremely sensitive bioassays without the need for signal amplification.⁶

From this very brief overview, it can be concluded that the synergistic combination of ICP-MS detection and the selectivity of nanoparticle-tagged antibodies currently provides an outstanding tool for the robust quantification of disease biomarkers with extremely high sensitivity. Looking ahead, it is clear that the design and implementation of novel amplification strategies using inorganic nanoparticles, in combination with ICP-MS detection, will yield bioanalytical methods capable of detecting biomarkers at extremely low concentrations, constituting an indispensable tool in clinical chemistry.

Finally, I would like to congratulate the Spectroscopy team on this celebration of the 35th year of publishing! Through the hard work and dedication of its editorial board, journal staff, and all contributors and sponsors, the journal has become a leading publisher of contributions dealing with spectroscopic techniques and their applications in life sciences, environmental analysis, technology, industry, and academia. I am pleased and honored to participate in this special issue, and I am confident that the next 35 years will witness the further growth of Spectroscopy and its importance within the spectroscopy community.  

References

1. Simpson, K. L.; Whetton, A. D.; Dive, C. Quantitative Mass Spectrometry-Based Techniques for Clinical Use: Biomarker Identification and Quantification. J. Chromatogr. B 2009, 877, 1240–1249. DOI: 10.1016/j.jchromb.2008.11.023
2. Cid-Barrio, L.; Calderon-Celis, F.; Abasolo-Linares, P.; Fernandez-Sanchez, M. L.; Costa-Fernandez, J. M.; Ruiz Encinar, J.; Sanz-Medel, A. Advances in Absolute Protein Quantification and Quantitative Protein Mapping Using ICP-MS. Trends Anal. Chem. 2018, 104, 148–159. DOI: 10.1016/j.trac.2017.09.024
3. Liu, R.; Wu, P.; Yang, L.; Hou, X.; Lv, Y. Inductively Coupled Plasma Mass Spectrometry-Based Immunoassay: A Review. Mass Spectrom. Rev. 2014, 33, 373–393. DOI: 10.1002/mas.21391
4. Liu, Z.; Li, X.; Xiao, G.; Chen, B.; He, M.; Hu, B. Application of Inductively Coupled Plasma Mass Spectrometry in the Quantitative Analysis of Biomolecules With Exogenous Tags: A Review. Trends Anal. Chem. 2017, 93, 78–101. DOI: 10.1016/j.trac.2017.05.008
5. Garcia-Cortes, M.; Ruiz Encinar, J.; Costa-Fernandez, J. M.; Sanz-Medel, A. Highly Sensitive Nanoparticle-Based Immunoassays With Elemental Detection: Application to Prostate-Specific Antigen Quantification. Biosens. Bioelectron. 2016, 85, 128–134. DOI: 10.1016/j.bios.2016.04.090
6. Hu, J.; Deng, D.; Liu, R.; Lv, Y. Single Nanoparticle Analysis by ICPMS: A Potential Tool for Bioassay. J. Anal. At. Spectrom. 2018, 33, 57–67. DOI: 10.1039/C7JA00235A

Jose M. Costa-Fernandez, PhD, is a full professor of analytical chemistry in the Department of Physical and Analytical Chemistry at the University of Oviedo, in Oviedo, Spain. Direct correspondence to jcostafe@uniovi.es