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

BACKGROUND: Laser-induced breakdown spectroscopy (LIBS) is a well-recognized analytical technique used for elemental analysis. This method is gaining considerable attention also in biological applications thanks to its ability for spatial mapping and elemental imaging. The implementation of LIBS in the biomedical field is based on the detection of metals or other elements that either naturally occur in the samples or are present artificially. The artificial implementation of nanoparticle labels (Tag-LIBS) enables the use of LIBS as a readout technique for immunochemical assays. However, one of the biggest challenges for LIBS to meet immunoassay readout standards is its sensitivity. RESULTS: This paper focuses on the improvement of LIBS sensitivity for the readout of nanoparticle-based immunoassays. First, the LIBS setup was optimized on photon-upconversion nanoparticle (UCNP) droplets deposited on the microtiter plate wells. Two collection optics systems were compared, with single pulse (SP) and collinear double pulse (DP) LIBS arrangements. By deploying the second laser pulse, the sensitivity was improved up to 30 times. The optimized SP and DP setups were then employed for the indirect detection of human serum albumin based on immunoassay with UCNP-based labels. Compared to our previous LIBS study, the detection limit was enhanced by two orders of magnitude, from 10 ng mL-1 to 0.29 ng mL-1. In addition, two other immunochemical methods were used for reference, based on the readout of upconversion luminescence of UCNPs and absorbance measurement with enzyme labels. Finally, the selectivity of the assay was tested and the practical potential of Tag-LIBS was demonstrated by the successful analysis of urine samples. SIGNIFICANCE AND NOVELTY: In this work, we improved the sensitivity of the Tag-LIBS method by combining new labels based on UCNPs with the improved collection optics and collinear DP configuration. In the instrumental setup optimization, the DP LIBS showed better sensitivity and signal-to-noise ratio than SP. The optimizations allowed the LIBS readout to surpass the sensitivity of enzyme immunoassay, approaching the qualities of upconversion luminescence readout, which is nowadays a state-of-the-art readout technique.

• Klíčová slova
• Double pulse, Human serum albumin, Immunoassay, Laser-induced breakdown spectroscopy, Photon-upconversion nanoparticle, Tag-LIBS,
• MeSH
• imunoanalýza metody   MeSH
• kovy MeSH
• lasery MeSH
• lidé MeSH
• nanočástice * chemie   MeSH
• spektrální analýza metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kovy MeSH

Immunohistochemistry (IHC) and immunocytochemistry (ICC) are widely used to identify cancerous cells within tissues and cell cultures. Even though the optical microscopy evaluation is considered the gold standard, the limited range of useful labels and narrow multiplexing capabilities create an imminent need for alternative readout techniques. Laser-induced breakdown spectroscopy (LIBS) enables large-scale multi-elemental analysis of the surface of biological samples, e.g., thin section or cell pellet. It is, therefore, a potential alternative for IHC and ICC readout of various labels or tags (Tag-LIBS approach). Here, we introduce Tag-LIBS as a method for the specific determination of HER2 biomarker. The cell pellets were labeled with streptavidin-conjugated upconversion nanoparticles (UCNP) through a primary anti-HER2 antibody and a biotinylated secondary antibody. The LIBS scanning enabled detecting the characteristic elemental signature of yttrium as a principal constituent of UCNP, thus indirectly providing a reliable way to differentiate between HER2-positive BT-474 cells and HER2-negative MDA-MB-231 cells. The comparison of results with upconversion optical microscopy and luminescence intensity scanning confirmed that LIBS is a promising alternative for the IHC and ICC readout.

• Klíčová slova
• Immunocytochemistry, Immunohistochemistry, Laser-induced breakdown spectroscopy, Photon-upconversion nanoparticles, Tag-LIBS,
• MeSH
• fluoridy chemie   účinky záření   MeSH
• imobilizační protilátky imunologie   MeSH
• imunohistochemie metody   MeSH
• lidé MeSH
• nádorové biomarkery analýza   imunologie   MeSH
• nádorové buněčné linie MeSH
• nanočástice chemie   účinky záření   MeSH
• receptor erbB-2 analýza   imunologie   MeSH
• spektrální analýza metody   MeSH
• studie proveditelnosti MeSH
• světlo MeSH
• thulium chemie   účinky záření   MeSH
• ytrium chemie   účinky záření   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ERBB2 protein, human MeSH Prohlížeč
• fluoridy MeSH
• imobilizační protilátky MeSH
• nádorové biomarkery MeSH
• receptor erbB-2 MeSH
• sodium yttriumtetrafluoride MeSH Prohlížeč
• thulium MeSH
• ytrium MeSH

Laser-induced breakdown spectroscopy (LIBS) was examined as a novel method for readout of microtiter plate immunoassays involving nanoparticles (NP). The so-called Tag-LIBS technique is a sensitive method for the detection of specific biomarkers. It was applied to the determination of NP labels using nanosecond ablation sampling. The NP labels were examined from the bottom of a standard 96-well microtiter plate. Thanks to the flexibility of LIBS instrumentation, both the plasma emission collection and the focusing optics arrangements can be collinearly arranged. The experiments showed that silver NPs and gold NPs can be readily quantified on the bottom of the microtiter plate. Utilizing this technique, a sandwich immunoassay for human serum albumin using streptavidin-coated AgNP labels was developed. The assay has a 10 ng·mL-1 detection limit which is comparable to the sensitivity of fluorometric readout. The main advantage of this LIBS technique is its wide scope in which it enables a detection of almost any type of NP labels, irrespective to any fluorescence or catalytic properties. Owing to the immediate signal response, the relatively simple instrumentation also enables assay automation. The LIBS capability of multi-elemental analyses makes it a promising and fast alternative to other readout techniques, in particular with respect to multiplexed detection of biomarkers. Graphical abstract Laser-induced breakdown spectroscopy (LIBS) is used as a novel readout method of nanoparticle-based immunoassays in microtiter plates. After formation of sandwich immunocomplex, the analyte concentration is quantified as the signal of Ag nanoparticle labels determined by LIBS.

• Klíčová slova
• Collinear plasma collection, Gold nanoparticles, Laser ablation, Microtiter plate, Sandwich immunoassay, Silver nanoparticles, Streptavidin, Tag-LIBS,
• MeSH
• biologické markery krev   MeSH
• imunoanalýza metody   MeSH
• kovové nanočástice chemie   MeSH
• lasery * MeSH
• lidé MeSH
• lidský sérový albumin analýza   MeSH
• povrchové vlastnosti MeSH
• stříbro chemie   MeSH
• velikost částic MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• lidský sérový albumin MeSH
• stříbro MeSH
• zlato MeSH