Laser-induced breakdown spectroscopy (LIBS) is a promising technique for the readout of immunochemical assays utilizing indirect detection of labels (Tag-LIBS), typically based on nanoparticles. We have previously demonstrated that Tag-LIBS immunoassay employing yttrium-based photon-upconversion nanoparticles (UCNPs) can reach sensitivity similar to commonly used enzyme and fluorescence immunoassays. In this study, we report on further increasing the sensitivity of UCNP-based Tag-LIBS immunoassay by employing magnetic microbeads (MBs) as the solid phase in the determination of cancer biomarker prostate-specific antigen. Due to the possibility of analyte preconcentration, MBs enabled achieving a limit of detection (LOD) of 4.0 pg·mL-1, representing two orders of magnitude improvement compared with equivalent microtiter plate-based assay (LOD of 460 pg·mL-1). In addition, utilizing MBs opens up the possibility of an internal standardization of the LIBS readout by employing iron spectral lines, which improves the assay robustness by compensating for LIBS signal fluctuations and bead-bound immunocomplexes lost throughout the washing steps. Finally, the practical applicability of the technique was confirmed by the successful analysis of clinical samples, showing a strong correlation with the standard electrochemiluminescence immunoassay. Overall, MB-based Tag-LIBS was confirmed as a promising immunoassay approach, combining fast readout, multiplexing possibilities, and high sensitivity approaching upconversion luminescence scanning while avoiding the requirement of luminescence properties of labels.

• Klíčová slova
• Double-pulse LIBS, Magnetic microparticle, Photon-upconversion nanoparticle, Prostate-specific antigen, Tag-LIBS,
• MeSH
• imunoanalýza metody   MeSH
• lasery * MeSH
• lidé MeSH
• limita detekce * MeSH
• mikrosféry MeSH
• prostatický specifický antigen * analýza   imunologie   krev   MeSH
• spektrální analýza metody   MeSH
• ytrium chemie   účinky záření   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• prostatický specifický antigen * MeSH
• ytrium MeSH

Dot-blot immunoassays are widely used for the user-friendly detection of clinical biomarkers. However, the majority of dot-blot assays have only limited sensitivity and are only used for qualitative or semiquantitative analysis. To overcome this limitation, we have employed labels based on photon-upconversion nanoparticles (UCNPs) that exhibit anti-Stokes luminescence and can be detected without optical background interference. First, the dot-blot immunoassay on a nitrocellulose membrane was optimized for the quantitative analysis of human serum albumin (HSA), resulting in a limit of detection (LOD) of 0.19 ng/mL and a signal-to-background ratio (S/B) of 722. Commercial quantum dots were used as a reference label, reaching the LOD of 4.32 ng/mL and the S/B of 3, clearly indicating the advantages of UCNPs. In addition, the potential of UCNP-based dot-blot for real sample analysis was confirmed by analyzing spiked urine samples, reaching the LOD of 0.24 ng/mL and recovery rates from 79 to 123%. Furthermore, we demonstrated the versatility and robustness of the assay by adapting it to the detection of two other clinically relevant biomarkers, prostate-specific antigen (PSA) and cardiac troponin (cTn), reaching the LODs in spiked serum of 9.4 pg/mL and 0.62 ng/mL for PSA and cTn, respectively. Finally, clinical samples of patients examined for prostate cancer were analyzed, achieving a strong correlation with the reference electrochemiluminescence immunoassay (recovery rates from 89 to 117%). The achieved results demonstrate that UCNPs are highly sensitive labels that enable the development of dot-blot immunoassays for quantitative analysis of low-abundance biomarkers.

• MeSH
• biologické markery * krev   moč   analýza   MeSH
• imunoanalýza metody   MeSH
• kvantové tečky chemie   MeSH
• lidé MeSH
• lidský sérový albumin analýza   moč   MeSH
• limita detekce * MeSH
• nanočástice * chemie   MeSH
• prostatický specifický antigen * krev   analýza   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery * MeSH
• lidský sérový albumin MeSH
• prostatický specifický antigen * MeSH

The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.

• Klíčová slova
• antifouling biointerface, biomarker, digital readout of assay, rolling circle amplification, single molecule detection, surface plasmon resonance, surface plasmon-enhanced fluorescence,
• MeSH
• jednovláknová DNA * MeSH
• lidé MeSH
• povrchová plasmonová rezonance * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jednovláknová DNA * MeSH

Photon upconversion is an intensively investigated phenomenon in the materials sciences due to its unique applications, mainly in biomedicine for disease prevention and treatment. This study reports the synthesis and properties of tetragonal LiYbF4:Tm3+@LiYF4 core@shell nanoparticles (NPs) and their applications. The NPs had sizes ranging from 18.5 to 23.7 nm. As a result of the energy transfer between Yb3+ and Tm3+ ions, the synthesized NPs show intense emission in the ultraviolet (UV) range up to 347 nm under 975 nm excitation. The bright emission in the UV range allows for singlet oxygen generation in the presence of hematoporphyrin on the surface of NPs. Our studies show that irradiation with a 975 nm laser of the functionalized NPs allows for the production of amounts of singlet oxygen easily detectable by Singlet Oxygen Sensor Green. The high emission intensity of NPs at 800 nm allowed the application of the synthesized NPs in an upconversion-linked immunosorbent assay (ULISA) for highly sensitive detection of the nucleoprotein from SARS-CoV-2, the causative agent of Covid-19. This article proves that LiYbF4:Tm3+@LiYF4 core@shell nanoparticles can be perfect alternatives for the most commonly studied upconverting NPs based on the NaYF4 host compound and are good candidates for biomedical applications.

• Klíčová slova
• Covid-19 diagnosis, NIR to UV upconverting nanoparticles, Reactive oxygen species, Singlet oxygen,
• MeSH
• COVID-19 * diagnóza   MeSH
• imunoanalýza MeSH
• lidé MeSH
• nanočástice * MeSH
• SARS-CoV-2 MeSH
• singletový kyslík MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• singletový kyslík MeSH