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
• biologické markery analýza   MeSH
• hmotnostní spektrometrie * metody   MeSH
• imunoanalýza metody   MeSH
• laserová terapie * MeSH
• lasery MeSH
• lidé MeSH
• lidský sérový albumin * analýza   MeSH
• limita detekce MeSH
• nádorové biomarkery * krev   analýza   MeSH
• nanočástice chemie   MeSH
• prostatický specifický antigen * krev   analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• lidský sérový albumin * MeSH
• nádorové biomarkery * MeSH
• prostatický specifický antigen * MeSH

Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1-14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.

• Klíčová slova
• antibody, dot-blot, protein p53,
• MeSH
• europium chemie   MeSH
• hmotnostní spektrometrie MeSH
• imunoblotting MeSH
• kadmium chemie   MeSH
• kovové nanočástice chemie   MeSH
• kvantové tečky chemie   MeSH
• lasery MeSH
• lidé MeSH
• limita detekce MeSH
• monoklonální protilátky chemie   farmakologie   MeSH
• nádorový supresorový protein p53 antagonisté a inhibitory   MeSH
• stříbro chemie   MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• europium MeSH
• kadmium MeSH
• monoklonální protilátky MeSH
• nádorový supresorový protein p53 MeSH
• stříbro MeSH
• TP53 protein, human MeSH Prohlížeč
• zlato MeSH