Single-particle (digital) immunoassays offer significantly lower limits of detection (LODs) than traditional immunoassays, making them suitable for the detection of low-abundance biomarkers. The most common approach for digital detection is based on counting individual labels. Here, we introduce a novel dot-blot particle-linked immunosorbent assay (PLISA) with digital readout utilizing laser ablation (LA) of photon upconversion nanoparticle (UCNP) labels from the nitrocellulose substrate. Compared to conventional LA, our approach allows desorption of intact nanoparticles and their precise counting by single-particle inductively coupled plasma mass spectrometry (SP ICP MS), thus counting individual UCNP-labeled immunocomplexes. Digital signal processing filters instrument noise and nanoparticle aggregates, minimizing potential errors. The immunoassay and LA SP ICP MS readout were optimized using human serum albumin, a kidney damage biomarker, as a model analyte, obtaining LODs of 0.18 and 0.12 ng/mL for the reference upconversion luminescence (UCL) and LA SP ICP MS readout, respectively. Building upon these optimized conditions, we developed PLISA for prostate-specific antigen, the key prostate cancer biomarker, with LODs of 2.4, 1.4, and 0.3 pg/mL for the UCL, analog, and digital LA SP ICP MS readout, respectively. The LOD in the sub-pg/mL range highlighted the advantage of particle counting and its ability to detect low-abundance biomarkers, as superior performance was achieved compared to the UCL and analog LA ICP MS readout. Finally, clinical serum samples of patients tested for prostate cancer were analyzed, and a strong correlation with the reference electrochemiluminescence method confirmed the potential of LA SP ICP MS for clinical diagnostics.

• MeSH
• Biomarkers analysis   MeSH
• Mass Spectrometry * methods   MeSH
• Immunoassay methods   MeSH
• Laser Therapy * MeSH
• Lasers MeSH
• Humans MeSH
• Serum Albumin, Human * analysis   MeSH
• Limit of Detection MeSH
• Biomarkers, Tumor * blood   analysis   MeSH
• Nanoparticles chemistry   MeSH
• Prostate-Specific Antigen * blood   analysis   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers MeSH
• Serum Albumin, Human * MeSH
• Biomarkers, Tumor * MeSH
• Prostate-Specific Antigen * MeSH

Recent studies have demonstrated the applicability of the "single particle" mode in laser ablation-inductively coupled plasma-mass spectrometry to map and size particles simultaneously. The transport efficiency (TE) is an important parameter in this configuration and affects the detection of individual nanoparticles, reliability of nanoparticle characterization, and related applications. This study introduces a novel method for the precise determination of TE, based on counting upconversion nanoparticles from gels characterized by fluorescent microscopy. The method was found to be most suitable for the 2940-nm laser ablation system, achieving virtually quantitative nanoparticle desorption, with TE primarily governed by ablation cell design and aerosol transport efficiency. With the 213-nm laser, attention had to be paid to incomplete desorption and possible nanoparticle redeposition at low laser fluences to avoid variability in TE measurements. Finally, use of the 193-nm laser-induced nanoparticle disintegration, resulting in elevated baseline noise and lower sensitivity, which prevented the use of this approach for the determination of TE. This study highlights the versatility of the proposed method, while also identifying its limitations, in terms of wavelength and fluence.

• Publication type
• Journal Article MeSH

The detection of a single entity (molecule, cell, particle, etc.) was always a challenging subject. Here we demonstrate the detection of single Ag nanoparticles (NPs) using subatmospheric pressure laser desorption/ionization mass spectrometry (LDI MS). The sample preparation, measurement conditions, generated ions, and limiting experimental factors are discussed here. We detected from 84 to 95% of the deposited 80 nm Ag NPs. The presented LDI MS platform is an alternative to laser ablation inductively coupled plasma mass spectrometry for imaging distribution of individual NPs across the sample surface and has a great potential for multiplexed mapping of low-abundance biomarkers in tissues.

• Publication type
• Journal Article MeSH

This study focuses on mapping the spatial distribution of Au nanoparticles (NPs) by laser desorption/ionization mass spectrometry imaging (LDI MSI). Laser interaction with NPs and associated phenomena, such as change of shape, melting, migration, and release of Au ions, are explored at the single particle level. Arrays of dried droplets containing low numbers of spatially segregated NPs were reproducibly prepared by automated drop-on-demand piezo-dispensing and analyzed by LDI MSI using an ultrahigh resolution orbital trapping instrument. To enhance the signal from NPs, an in source gas-phase chemical reaction of generated Au ions with xylene was employed. The developed technique allowed the detecting, chemical characterization, and mapping of the spatial distribution of Au NPs; the ion signals were detected from as low as ten 50 nm Au NPs on a pixel. Furthermore, the Au NP melting dynamics under laser irradiation was monitored by correlative atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM measurements of Au NPs before and after LDI MSI analysis revealed changes in NP shape from a sphere to a half-ellipsoid and total volume reduction of NPs down to 45% of their initial volume.

• Publication type
• Journal Article MeSH